Cancer Patent

Abstract
Novel indolocarbazole derivatives potentially useful for the treatment of dementias characterized by tau hyperphosphorylation [Alzheimer's disease (AD), frontal lobe degeneration (FLD), argyrophilic grains disease, subacute sclerotizing panencephalitis (SSPE) as a late complication of viral infections in the CNS], and cancer.

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Inventors: Roder; Hanno (Ratingen, DE), Lowinger; Timothy B. (Nishinomiya, JP), Brittelli; David R. (Branford, CT), VanZandt; Michael C. (Guilford, CT)
Assignee: Bayer Corporation (Pittsburgh, PA)

Appl. No.: 09/109,131
Filed: July 2, 1998
Claims

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We claim:

1. A composition of matter comprising: (Formula I) ##STR27## wherein Z is O or 2H, R.sub.1 is H, OH, CO.sub.2 R.sub.9, CONHR.sub.9, CH.sub.2 OR.sub.9, or CONR.sub.9 R.sub.10 ;

R.sub.2 is H or OH; R.sub.3 is H or OH; R.sub.4 is H or OH;

R.sub.5 is H, OH, NR.sub.9 R.sub.10, NHCOR.sub.9, OCOR.sub.9, OCR.sub.9, halide, COOR.sub.9, or CONR.sub.9 R.sub.10 ;

R.sub.6 is H, OH, NR.sub.9 R.sub.10, NHCOR.sub.9, OCOR.sub.9, OCR.sub.9, halide, COOR.sub.9, or CONR.sub.9 R.sub.10 ;

R.sub.7 is H, OH, O or halide;

R.sub.8 is H, OH or halide; or R.sub.7 and R.sub.8 together form .dbd.O;

R.sub.9 is an alkyl of 1-6 carbons, a cycloalkyl of 3-6 carbons or H;

R.sub.10 is an alkyl of 1-6 carbons, a cycloalkyl of 3-6 carbons or H.

2. The composition of matter of claim 1, wherein Z is O; R.sub.1 is OH, CO.sub.2 R.sub.9, CONHR.sub.9, CH.sub.2 OR.sub.9 ; R.sub.4 is H; R.sub.5 is H; R.sub.6 is H; and R.sub.8 is H.

3. The composition of matter of claim 1, wherein Z is O; R.sub.1 is CO.sub.2 CH.sub.3 or CONHCH.sub.3 ; R.sub.2 is H; R.sub.3 is OH; R.sub.4 is H; R.sub.5 is H; and R.sub.6 is H.

4. The composition of matter of claim 1, wherein the composition of matter is selected from the group consisting of: ##STR28##

5. A pharmaceutical composition comprising: the composition of matter of claim 1, and

the pharmaceutically-acceptable carrier.

6. A pharmaceutical composition comprising:

the composition of matter of claim 2, and

a pharmaceutically-acceptable carrier.

7. A pharmaceutical composition comprising:

the composition of matter of claim 3, and

a pharmaceutically-acceptable carrier.

8. A pharmaceutical composition comprising:

the composition of matter of claim 4, and

a pharmaceutically-acceptable carrier.

9. The pharmaceutical composition of claim 5 in an oral dosage form.

10. The pharmaceutical composition of claim 6 in an oral dosage form.

11. The pharmaceutical composition of claim 7 in an oral dosage form.

12. The pharmaceutical composition of claim 8 in an oral dosage form.

13. A composition of matter comprising ##STR29## wherein, Z=O or 2H; R.sub.13, R.sub.13 ‘=H or OP; and R.sub.14 =O, H, OH or OP. Preferably, Z is O and R.sub.13 and R.sub.13 ‘ are H.

14. The composition of claim 13 wherein Z=O and R.sub.13 and R.sub.13 ‘=H.
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Description

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FIELD OF THE INVENTION

Novel indolocarbazole derivatives potentially useful for the treatment of dementias characterized by tau hyperphosphorylation [Alzheimer's disease (AD), frontal lobe degeneration (FLD), argyrophilic grains disease, subacute sclerotising panencephalitis (SSPE) as a late complication of viral infections in the CNS], and cancer.

BACKGROUND OF THE INVENTION

Several dementias, most importantly Alzheimer’s disease (AD), are characterized by the formation of intracellular aggregates consisting of the microtubule-associated protein tau, termed neurofibrillary tangles (NFT). The importance of this biochemical abnormality for the clinical syndrome of dementia is illustrated by essentially three facts: (I) there is a close correlation between the state of dementia and the extent and density of NFT in various parts of the cortex [e.g., Bancher C. et al. (1993) Neurosci. Lett. 162, 179-182)]; (ii) individual neurons containing NFT in the cell body and/or the neurites are morphologically degenerating, i.e., lose synaptic connections and eventually die [Braak E. et al. (1994) Acta Neuropathol. 87, 554-567; Callahan L. M. et al., (1995) Neurobiol. Aging 16, 311-314]; (iii) a certain density of NFT in various otherwise unrelated dementias is always associated with dementia, without exception.

The tau protein contained in NFT is severely hyperphosphorylated [Goedert M. et al. (1995) Neurobiol. Aging 16, 325-334; Hasegawa M. et al. (1996) FEBS Lett. 384, 25-30]. This abnormal phosphorylation renders the protein incompetent to retain its original function, i.e., stabilization of the microtubule cytoskeleton, which is of fundamental importance for the integrity of a neuron [Iqbal K. et al. (1994) FEBS Lett. 349, 104-108; Garver T. D. et al. (1996) J. Neurosci. Res. 44, 12-20]. This explains the paucity of intact microtubules in AD brains. Phosphorylation alone is responsible for this effect, as dephosphorylation restores the abilities of tau.

Because of a relationship between tau phosphorylation, cytoskeletal destabilization, synaptic loss and neuronal degeneration, and ultimately dementia, it would be therapeutically desirable to have pharmaceutical means to interfere with the pathological process of tau hyperphosphorylation.

The characteristics of hyperphosphorylated tau in NFT suggest that the protein kinase ERK2 is responsible for the pathological tau modification in AD [Drewes G. et al. (1990) EMBO J. 11, 2131-2138; Roder H. M. et al. (1993) Biochem. Biophys. Res. Commun. 193, 639-647]. ERK2 may exist in an abnormally activated state in AD [Roder H. M. et al. (1995) J. Neurochem. 64, 2203-2212). Inhibition of ERK2 has therefore been suggested as a point of interference to prevent tau hyperphosphorylation, and ultimately to stop NFT formation in neurons.

AD-like tau hyperphosphorylation can be induced in several cellular models (including brain slices), converting tau into a phosphorylation state indistinguishable from tau phosphorylated by ERK2 in vitro. The most convincing cellular models involve PP2A inhibition [Sautier, P. E. et al., Neurodegeneration 3, 53-60 (1994); Harris K. A. et al., Ann. Neurol. 13, 77-87 (1993)].

However, compounds which inhibit ERK2 and thereby prevent AD-like tau hyperphosphorylation in biological model systems, have previously not been disclosed. Such compounds can be expected to affect processes of neurofibrillary degeneration, tied to tau hyperphosphorylation, in a beneficial manner.

The protein kinases of the ERK family, often termed MAP-kinases, have also been implicated in a variety of important cellular regulation events outside the CNS, such as growth, differentiation and inflammation [e.g., Sale E. M. et al., EMBO J. 14, 674-684 (1995); Pages G. et al., Proc. Natl. Acad. Sci. USA 90, 8319-8323 (1993); Cowley S. et al., Cell 77, 841-852 (1994)]. Consequently, aberrant ERK activation has been implicated in several diseases characterized by loss of growth and differentiation control. In some tumors constitutive ERK activation is associated with cellular transformation due to dominant (activating) mutations in signal transduction proteins or viral proteins interfering with ERK inactivators [Sontag E. et al., Cell 75, 887-897 (1993); Leevers S. J. and Marshall C. J., EMBO J. 11, 569-574 (1992); Gallego G. et al., Proc. Natl. Acad. USA 89, 7355-7359 (1992); Gupta S. K. et al., J. Biol. Chem. 267, 7987-7990 (1992)].

The use of the disclosed kinase inhibitors for cancer is also indicated by their ability to inhibit cdc2 kinase. The role of cdc2 and homologous (cdks) kinases in cell cycle control is very well appreciated [Norbury C., and Nurse P., Annu. Rev. Biochem. 61, 441-470 (1992)]. Regulation of these enzymes is essential for both commitment to cell cycle from the resting state (START), and ordered transition through several phases of the cell cycle. The need for regulation is reflected in the existence of numerous positive and negative regulatory features of cdks, such as cyclin subunits, inhibiting (Thr) and activating (Tyr) phosphorylations, and endogenous peptide inhibitors.

Because of this central role of cdks in control of cell cycle and proliferation, they are considered as attractive drug targets for cancer therapies [e.g., Filguera de Azevedo W. et al., Proc. Natl. Acad. Sci. USA 93, 2735-2740 (1996)].

DESCRIPTION OF RELATED ART

Indolocarbazole derivatives structurally related to the invention compounds have been described in the literature. The majority of these compounds are derived from the natural product K252a. The production and isolation of K252a was first published by Kase, et al. [J. of Antibiotics 39, 1059 (1986)]. Subsequent structure elucidation of K252a, b, c and d were reported in the same year by Yasuzawa et al. [J. of Antibiotics 39, 1072 (1986)]. Since the original disclosure and structure elucidation, K252a has been shown to be active in a variety of enzyme and cell-based assays. In particular, these compounds have demonstrated potent protein kinase C (PKC) activity. The most common uses claimed include: cancer, EP 0 323 171 (priority date Dec. 24, 1987), EP 0 643 966 (priority date Mar. 3, 1993), U.S. Pat. No. 4,923,986 (priority date Mar. 9, 1987), U.S. Pat. No. 4,877,776 (priority date Dec. 24, 1987), WO 94 27982 (priority date May 28, 1993); neurodegenerative disorders, WO 95 07911 (priority date Sep. 16, 1993), WO 94 02488 (priority date Jul. 24, 1992), antimicrobial [Prudhomme et al., J. Antibiotics 47, 792 (1994)], and hypertension [Hachisu et al., Life Sciences 44, 1351 (1989)]. ##STR1##

In general, prior art compounds related to the invention are derived from K252a and contain the basic core structure where a tetrahydrofuran moiety is attached to the aglycone forming two glycosidic bonds. Modifications of the K252a core structure include additional substituents on the lactam and indole portions, and modifications of the a-hydroxy ester. The tetrahydrofuran oxygen in the core structure limits the opportunities for further modification.

SUMMARY OF THE INVENTION

Incorporation of a carbon at the tetrahydrofuran oxygen position of the K252a core structure significantly alters the core structure by removing the two glycosidic bonds and replacing the electron rich disubstituted atom with an electronically more neutral tetra-substituted moiety. This change also provides additional opportunities to incorporate functional groups that may enhance properties such as potency, selectivity, stability, toxicity, bioavailability, etc. which can result in an improved biological profile and consequently, a better therapeutic agent.

Compounds containing this important modification are completely inaccessible via synthetic methods used to prepare compounds of the prior art.

According to one aspect of the invention, a composition of matter is provided having the formula of Formula I, as follows: ##STR2## wherein Z is O or 2H (in which case the double bond is two single bonds), R1 is H, OH, CO.sub.2 R9, CONHR9, CH.sub.2 OR9, or CONR.sub.9 R.sub.10 ;

R2 is H or OH; R3 is H or OH; R4 is H or OH;

R5 is H, OH, NR.sub.9 R.sub.10, NHCOR.sub.9, OCOR.sub.9, OCR.sub.9, halide, COOR.sub.9, or CONR.sub.9 R.sub.10 ;

R6 is H, OH, NR.sub.9 R.sub.10, NHCOR.sub.9, OCOR.sub.9, OCR.sub.9, halide, COOR.sub.9, or CONR.sub.9 R.sub.10 ;

R.sub.7 is H, OH, O or halide;

R8 is H, OH, halide or nothing (when R7 is O);

R9 is an alkyl of 1-6 carbons, a cycloalkyl of 3-6 carbons or H;

R10 is an alkyl of 1-6 carbons, a cycloalkyl of 3-6 carbons or H.

In certain preferred embodiments, Z is O; R1 is OH, CO.sub.2 R9, CHNHR9 or CH.sub.2 OR9; R4 is H; R5 is H; R6 is H; and R8 is H. In other preferred embodiments, Z is O; R1 is CO.sub.2 CH.sub.3 or CONHCH.sub.3 ; R2 is H; R3 is OH; R4 is H; R5 is H; and R6 is H. The most preferred compositions of matter are: ##STR3##

According to another aspect of the invention, pharmaceutical compositions are provided. The pharmaceutical compositions include the compositions of matter described above, together with a pharmaceutically acceptable carrier. The preferred pharmaceutical compositions are as described above. Particularly preferred pharmaceutical compositions are those formulated in an oral dosage form.

In some embodiments, the pharmaceutical composition contains the composition of matter in an amount effective for inhibiting abnormal hyperphosphorylation associated with a dementia. In other embodiments, the pharmaceutical composition contains the composition of matter in an amount effective for inhibiting a cdk kinase, such as cdc2 kinase. In still other embodiments, the pharmaceutical composition contains the composition of matter in an amount effective to inhibit cell proliferation, and in certain embodiments to inhibit cancer cell proliferation by cancer cells expressing abnormal amounts of a cdk kinase.

According to another aspect of the invention, a method is provided for inhibiting in a subject a kinase which binds a compound of Formula I. A compound of Formula I is administered to a subject in need of such treatment in an amount effective to inhibit in the subject the kinase activity. Preferred compounds are as described above. In one embodiment, the subject has a dementia and the compound is administered in an amount effective to inhibit abnormal hyperphosphorylation characteristic of the dementia. The dementia can be, among other things, Alzheimer’s disease and the compound can be administered in an amount effective to inhibit phosphorylation activity of ERK2 which is characteristic of abnormal tau hyperphosphorylation in Alzheimer’s disease.

According to another aspect of the invention, a method is provided for treating a subject having a cancer which expresses abnormal levels of cdk kinase activity. The method involves administering to a subject in need of such treatment a compound of Formula I in an amount effective to inhibit the cdk kinase activity. In some embodiments, the kinase is cdc2 kinase. The preferred compounds are as described above.

According to another aspect of the invention, intermediates for preparing the compounds of Formula I are provided. The intermediates are described in detail in the text below. Particularly important intermediates are those numbered 13, 19 and 24.

According to still another aspect of the invention, a method is provided which involves the use of a compound of Formula I in the preparation of a medicament. In particular embodiments, the medicament is for treating a dementia (e.g. Alzheimer’s disease), a proliferative disorder (e.g. a cancer). These and other aspects of the invention are described in greater detail below.

According to another aspect of the invention, intermediates for manufacturing the above compounds are provided. These are compositions of matter comprising: ##STR4## wherein, Z.dbd.O or 2H (in which case the double bond is two single bonds);

R.sub.11, R.sub.12 .dbd.H, ##STR5## except that when R.sub.11 is not H, then R.sub.12 is H and when R.sub.12 is not H, then R.sub.11 is H;

R.sub.13, R.sub.13 ‘.dbd.H or OP’, and R.sub.14 is H or OP. Preferably, Z is O and R.sub.13 and R.sub.13 ‘ are H. Most preferable the composition of matter is compound 14. P is a protecting group. Preferred Ps for OP are benzyl- and t-butyl-dimethyl sylyl. Most preferably the composition of matter is compound 12, 13, 18, 19, 23 or 24.

Other compositions of matter are ##STR6## Wherein, Z.dbd.O or 2H; R.sub.13 and R.sub.13 ‘.dbd.H or OP; and R14.dbd.O, H, OH or OP.

Preferably, Z is O and R.sub.13 and R.sub.13 ‘ are H. Most preferably the composition of matter is compound 14.

Mixtures of the foregoing compounds including isomeric mixtures also are contemplated.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a drawing of a blot on okadaic acid stimulated cells showing that compound CIII prevent tau hyperphosphorylation caused by okadaic acid.

FIG. 2. Western-blot comparison of tau from human SY5Y cells and from neonatal rat brain in various states of phosphorylation with PHF-tau from AD-brain. FIG. 2A shows human SY5Y cells. FIG. 2B shows neonatal rat brain. FIG. 2C shows PHF-tau from AD-brain.

FIG. 3 is a drawing of a pair of gels showing neonatal rat tau phosphorylation in vitro by PK40 without and with prior dephosphorylation by PP2B.

FIG. 4 is a drawing of a blot showing that a compound similar to CIII as an inhibitor of ERK2 prevents abnormal AD-like hyperphosphorylation in a SY5Y cell model system.

FIG. 5. Prevention of AD-like tau hyperphosphorylation in adult rat hippocampal brain slices. In an experimental paradigm similar to SY5Y cells tau hyperphosphorylation is prevented by CII at similar doses as in SY5Y cells. Note that the results with AP422, currently the most specific criterion for AD-like tau hyperphosphorylation, are identical to those with the commonly used mAb AT8, indicating that ERK2 alone is responsible for all okadaic acid induced changes in tau phosphorylation because AT8 but not AP422 reactivity can be induced by kinases other than ERK2.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to certain novel indolocarbazole derivatives that contain a cyclopentane core structure and may be medicinally useful for the treatment of a variety of disorders, including certain cancers and neurodegenerative disorders. The compounds have ERK2 and/or cdk, and in particular, cdc2, inhibitory activity.

The compounds of the invention, including the preferred compounds have been described above. An aspect of the invention is the replacement of the oxygen molecule of the tetrahydrofuran portion of certain prior art molecules (K252a and analogs) with a carbon atom. Such a class of materials was not available prior to the present invention which also provides a synthetic procedure for preparing this class of materials. The procedure also forms an aspect of the invention. The procedure for making the class of materials is described in detail below in the Examples section.

The most preferred compounds of the invention are indicated and named below:

CI. 9,12-Methano-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-I][1,6]benzodiaz ocine-1,3(2H)-dione,9,10,11,12-tetrahydro-8,10,11-trihydroxy-(8.alpha.,9.al pha.,10.alpha.,11.alpha.,12.alpha.).

CII. 9,12-Methano-1H-diindolo[1,2,3-fg:3',2'0,1'-kl]pyrrolo[3,4-I][1,6]benzodia zocine-10-carboxamide, 2,3,9,10,11,12-hexahydro-10-hydroxy-N-methyl-1,3-dioxo-(9.alpha.,10.beta., 12.alpha.).

CIII. 9,12-Methano-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-I][1,6]benzodiaz ocine-10-carboxylic acid, 2,3,9,10,11,12-hexahydro-10-hydroxy-1,3-dioxomethyl ester, (9.alpha.,10.beta.,12.alpha.).

The invention also involves intermediates for manufacturing the above compounds. The intermediates are described above. Mixtures including isomeric mixtures also may result depending upon the symmetry of the starting molecule. Such mixtures are within the scope of the invention.

To prepare the full range of compounds of the invention, only the chemistry described below, together with chemistry well known to those of ordinary skill in the art is required. In particular, modifications of the core structures can be accomplished using routine chemistry such as that used to make similar modifications to k252a, as detailed in WO94/02488, WO94/27982, WO94/04541 and numerous other U.S. patents and published applications showig derivatives of k252a.

A subject as used herein means humans, primates, horses, cows, pigs, sheep, goats, dogs, cats and rodents.

The pharmaceutical preparations of the invention are administered to subjects in effective amounts. An effective amount means that amount necessary to delay the onset of, inhibit the progression of, halt altogether the onset or progression of or diagnose the particular condition being treated. In general, an effective amount for treating a dementia is that amount necessary to affect favorably abnormal hyperphosphorylation characteristic of the dementia. In one embodiment, the effective amount is that amount necessary to affect favorably abnormal tau hyperphosphorylation associated with Alzheimer’s disease. In general, an effective amount for treating cancer will be that amount necessary to favorably affect mammalian cancer cell proliferation in-situ. When administered to a subject, effective amounts will depend, of course, on the particular condition being treated; the severity of the condition; individual patient parameters including age, physical condition, size and weight; concurrent treatment; frequency of treatment; and the mode of administration. These factors are well known to those of ordinary skill in the art and can be addressed with no more than routine experimentation. It is preferred generally that a maximum dose be used, that is, the highest safe dose according to sound medical judgment.

A variety of administration routes are available. The particular mode selected will depend, of course, upon the particular condition being treated, the particular drug selected, the severity of the condition being treated and the dosage required for therapeutic efficacy. The methods of this invention, generally speaking, may be practiced using any mode of administration that is medically acceptable, meaning any mode that produces effective levels of the active compounds without causing clinically unacceptable adverse effects. Such modes of administration include oral, rectal, sublingual, topical, nasal, transdermal, intradermal or parenteral routes. The term “parenteral” includes subcutaneous, intravenous, intramuscular, or infusion. Oral routes are preferred.

Dosage may be adjusted appropriately to achieve desired drug levels, locally or systemically. Generally, daily oral doses of active compounds will be from about 0.01 mg/kg per day to 1000 mg/kg per day. It is expected that IV doses in the range of about 1 to 1000 mg/m.sup.2 per day will be effective. In the event that the response in a subject is insufficient at such doses, even higher doses (or effective higher doses by a different, more localized delivery route) may be employed to the extent that patient tolerance permits.

The compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the conjugates of the invention into association with a carrier which constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the compounds into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.

Compositions suitable for oral administration may be presented as discrete units such as capsules, cachets, tablets, or lozenges, each containing a predetermined amount of the active compound. Other compositions include suspensions in aqueous liquors or non-aqueous liquids such as a syrup, an elixir, or an emulsion.

Other delivery systems can include time-release, delayed release or sustained release delivery systems. Such systems can avoid repeated administrations of the active compounds of the invention, increasing convenience to the subject and the physician. Many types of release delivery systems are available and known to those of ordinary skill in the art. They include polymer based systems such as polylactic and polyglycolic acid, polyanhydrides and polycaprolactone; nonpolymer systems that are lipids including sterols such as cholesterol, cholesterol esters and fatty acids or neutral fats such as mono-, di and triglycerides; hydrogel release systems; silastic systems; peptide based systems; wax coatings, compressed tablets using conventional binders and excipients, partially fused implants and the like. In addition, a pump-based hardware delivery system can be used, some of which are adapted for implantation.

A long-term sustained release implant also may be used. “Long-term” release, as used herein, means that the implant is constructed and arranged to deliver therapeutic levels of the active ingredient for at least 30 days, and preferably 60 days. Long-term sustained release implants are well known to those of ordinary skill in the art and include some of the release systems described above. Such implants can be particularly useful in treating solid tumors by placing the implant near or directly within the tumor, thereby affecting localized, high-doses of the compounds of the invention.

When administered, the formulations of the invention are applied in pharmaceutically acceptable compositions. Such preparations may routinely contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic ingredients. When used in medicine the salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of the invention. Such salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulphuric, nitric, phosphoric, maleic, acetic, salicylic, p-toluene sulfonic, tartaric, citric, methane sulfonic, formic, malonic, succinic, naphthalene-2-sulfonic, and benzene sulfonic. Also, pharmaceutically acceptable salts can be prepared as alkaline metal or alkaline earth salts, such as sodium, potassium or calcium salts.

Suitable buffering agents include: acetic acid and a salt (1-2% W/V); citric acid and a salt (1-3% W/V); and phosphoric acid and a salt (0.8-2% W/V).

Suitable preservatives include benzalkonium chloride (0.003-0.03% W/V); chlorobutanol (0.3-0.9% W/V); parabens (0.01-0.25% W/V) and thimerosal (0.004-0.02% W/V).

Suitable carriers are pharmaceutically-acceptable carriers. The term pharmaceutically-acceptable carrier means one or more compatible solid or liquid filler, dilutants or encapsulating substances which are suitable for administration to a human or other animal. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions are capable of being commingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction which would substantially impair the desired pharmaceutical efficacy. Carrier formulations suitable for oral, subcutaneous, intravenous, intramuscular, etc. can be found in Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa. and in the numerous prior art patents relating to K252a and its analogs.

The compounds useful in the invention may be delivered with other therapeutic agents. In the case of cancer, the compounds would be delivered separately or in the form of anti-cancer cocktails. An anti-cancer cocktail is a mixture of any one of the compounds of this invention with another anti-cancer agent such as an anti-cancer drug, a cytokine, and/or supplementary potentiating agent(s). The use of cocktails in the treatment of cancer is routine. In this embodiment, a common administration vehicle (e.g., pill, tablet, implant, injectable solution, etc.) could contain both the compounds useful in this invention (described above) and the anti-cancer drug and/or supplementary potentiating agent.

Thus, cocktails of non-Formula I compounds and Formula I compounds are contemplated. Non-Formula I anti-neoplastic compounds include:

Antineoplastic: Acivicin; Aclarubicin; Acodazole Hydrochloride; Acronine; Adozelesin; Aldesleukin; Altretamine; Ambomycin; Ametantrone Acetate; Aminoglutethimide; Amsacrine; Anastrozole; Anthramycin; Asparaginase; Asperlin ; Azacitidine; Azetepa; Azotomycin; Batimastat; Benzodepa; Bicalutamide; Bisantrene Hydrochloride; Bisnafide Dimesylate; Bizelesin; Bleomycin Sulfate; Brequinar Sodium; Bropirimine; Busulfan; Cactinomycin; Calusterone; Caracemide; Carbetimer; Carboplatin; Carmustine; Carubicin Hydrochloride; Carzelesin; Cedefingol; Chlorambucil; Cirolemycin ; Cisplatin; Cladribine; Crisnatol Mesylate; Cyclophosphamide ; Cytarabine ; Dacarbazine; Dactinomycin; Daunorubicin Hydrochloride; Decitabine; Dexormaplatin; Dezaguanine; Dezaguanine Mesylate; Diaziquone; Docetaxel; Doxorubicin; Doxorubicin Hydrochloride; Droloxifene; Droloxifene Citrate; Dromostanolone Propionate; Duazomycin; Edatrexate; Eflornithine Hydrochloride ; Elsamitrucin; Enloplatin; Enpromate; Epipropidine; Epirubicin Hydrochloride; Erbulozole; Esorubicin Hydrochloride; Estramustine; Estramustine Phosphate Sodium; Etanidazole; Ethiodized Oil I 131; Etoposide; Etoposide Phosphate; Etoprine; Fadrozole Hydrochloride; Fazarabine; Fenretinide; Floxuridine; Fludarabine Phosphate; Fluorouracil; Flurocitabine; Fosquidone; Fostriecin Sodium; Gemcitabine; Gemcitabine Hydrochloride; Gold Au 198 ; Hydroxyurea; Idarubicin Hydrochloride; Ifosfamide; lmofosine; Interferon Alfa-2a; Interferon Alfa-2b ; Interferon Alfa-n1; Interferon Alfa-n3; Interferon Beta- I a; Interferon Gamma- I b; Iproplatin; Irinotecan Hydrochloride ; Lanreotide Acetate; Letrozole; Leuprolide Acetate ; Liarozole Hydrochloride; Lometrexol Sodium; Lomustine; Losoxantrone Hydrochloride; Masoprocol; Maytansine;

Mechlorethamine Hydrochloride; Megestrol Acetate; Melengestrol Acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate Sodium; Metoprine; Meturedepa; Mitindomide; Mitocarcin; Mitocromin; Mitogillin; Mitomalcin; Mitomycin; Mitosper; Mitotane; Mitoxantrone Hydrochloride; Mycophenolic Acid; Nocodazole; Nogalamycin; Ormaplatin; Oxisuran; Paclitaxel; Pegaspargase; Peliomycin; Pentamustine; Peplomycin Sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone Hydrochloride; Plicamycin; Plomestane; Porfimer Sodium; Porfiromycin; Prednimustine; Procarbazine Hydrochloride; Puromycin; Puromycin Hydrochloride; Pyrazofurin; Riboprine; Rogletimide; Safingol; Safingol Hydrochloride; Semustine; Simtrazene; Sparfosate Sodium; Sparsomycinl, Spirogermanium Hydrochloride; Spiromustine; Spiroplatin; Streptonigrin; Streptozocin; Strontium Chloride Sr 89; Sulofenur; Talisomycin; Taxane; Taxoid; Tecogalan Sodium; Tegafur; Teloxantrone Hydrochloride; Temoporfin; Teniposide; Teroxirone; Testolactone; Thiamiprine; Thioguanine; Thiotepa; Tiazofurin; Tirapazamine; Topotecan Hydrochloride; Toremifene Citrate; Trestolone Acetate; Triciribine Phosphate; Trimetrexate; Trimetrexate Glucuronate; Triptorelin; Tubulozole Hydrochloride; Uracil Mustard; Uredepa; Vapreotide; Verteporfin; Vinblastine Sulfate; Vincristine Sulfate; Vindesine; Vindesine Sulfate; Vinepidine Sulfate; Vinglycinate Sulfate; Vinleurosine Sulfate; Vinorelbine Tartrate; Vinrosidine Sulfate; Vinzolidine Sulfate; Vorozole; Zeniplatin; Zinostatin; Zorubicin Hydrochloride. Other anti-neoplastic compounds include: 20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor-1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; irinotecan; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotrophin; monophosphoryl lipid A+myobacterium cell wall sk; mopidamol; multiple drug resistance gene inhibitor; multiple tumor suppressor 1-based therapy; mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; 06-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormiaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel analogues; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum-triamine complex; porfimer sodium; porfiromycin; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RII retinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginone B 1; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single chain antigen binding protein; sizofiran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stem cell inhibitor; stem-cell division inhibitors; stipiamide; stromelysin inhibitors; sulfinosine; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thalidomide; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene dichloride; topotecan; topsentin; toremifene; totipotent stem cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribine; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; vitaxin; vorozole; zanoterone; zeniplatin; zilascorb; zinostatin stimalamer.

Anti-cancer Supplementary Potentiating Agents: Tricyclic anti-depressant drugs (e.g., imipramine, desipramine, amitryptyline, clomipramine, trimipramine, doxepin, nortriptyline, protriptyline, amoxapine and maprotiline); non-tricyclic anti-depressant drugs (e.g., sertraline, trazodone and citalopram); Ca.sup.++ antagonists (e.g., verapamil, nifedipine, nitrendipine and caroverine); Calmodulin inhibitors (e.g., prenylamine, trifluoroperazine and clomipramine); Amphotericin B; Triparanol analogues (e.g., tamoxifen); antiarrhythmic drugs (e.g., quinidine); antihypertensive drugs (e.g., reserpine); Thiol depleters (e.g., buthionine and sulfoximine) and Multiple Drug Resistance reducing agents such as Cremaphor EL. The compounds of the invention also can be administered with cytokines such as granulocyte colony stimulating factor.

The conjugates of the invention also are useful, in general, for treating mammalian cell proliferative disorders other than cancer, including psoriasis, actinic keratosis, etc.

General Preparative Methods

The compounds of the invention may be prepared by use of known chemical reactions and procedures. Nevertheless, the following general preparative methods are presented to aid the reader in synthesizing the inhibitors. More detailed procedures for particular examples are presented below in the experimental section.

In the general methods, the following generic descriptions apply. The group designated P represents a protecting group. It may be appreciated by one skilled in the art that a variety of different protecting groups may be used to protect a potentially reactive functional group (e.g., imide nitrogen, hydroxyl, carboxycylic acid) and that the particular choice will depend upon the reaction conditions required to prepare a given target compound. A description of such protecting groups may be found in: Protective Groups in Organic Synthesis , Second Edition, T. W. Green and P. G. M. Wuts, John Wiley and Sons, New York, 1991.

The group designated X represents a leaving group. It is well-known to those skilled in the art that several different functional groups such as halides, mesylates, tosylates and triflates may serve as leaving groups. It is also known that the choice of a particular leaving group typically depends on such factors as the reactivity of the nucleophile, stability of the compound and ease of synthesis. It is understood that in cases where R represents a potentially reactive functional group such as an alcohol or an amine, appropriate protection and deprotections steps may be required. It is also understood that all variable groups of these methods are as described in the generic description if they are not specifically defined below. When a variable group with a given symbol (i.e., R4) is used more than once, each of these groups may be independently varied within the range of the definition of that symbol.

General Method A The compounds of the invention where the cyclopentane ring is cis-dihydroxylated anti to the indolocarbazole moiety (R.sup.1,R.sup.2, Formula I=–OH) are conveniently prepared by method A. The first key step in the process involves the alkylation of the protected indolo[2,3-a]carbazole moiety with a suitable cyclopentane (ene) electrophile. The protected indolo[2,3-a]carbazole moiety is conveniently prepared using methods described in the literature [Lowinger, T. B. et al., Tetrahedron Lett. 36, 8383 (1995), P=paramethoxy benzyl]. Electrophile 11 where R.sup.14 =–H and X=OMs can be prepared from commercially available 3-acetoxy-cyclopentene-2-ol by treatment with methanesulfony chloride and triethylamine. Derivatives with R.sup.14 .noteq.H can be prepared using standard methods known to those skilled in the art. Treatment of the protected indolo[2,3-a]carbazole with a base like Cs.sub.2 CO.sub.3 or NaH in a polar parotic solvent like DMF followed by addition of the alkylating agent (11) provides the desired monoalkylated material. Conversion of 12 to alcohol 13 can be accomplished by a variety of methods well-known to those skilled in the art. One method involves a transesterification reaction where the acetate moiety is transferred to an alcoholic solvent by treatment with catalytic NaCN. Cyclization of alcohol 13 to form the 7-membered ring of 14 can be carried out using triphenylshosphine and diethyl azodicarboxylate in a reaction known as the Mitsunobo reaction. An excellent review of this chemistry is described in Organic Reactions 42, 335 (1992). Subsequent oxidation of 14 to diol 15 can be accomplished by an OSO.sub.4 catalyzed cis-hydroxylation. The oxidation reaction is conveniently carried out using a catalytic amount of OSO.sub.4 with a reoxidant such as N-methyl morpholine N-oxide (NMO) in an aqueous tetrahydrofuran (THF) or acetone solution. Similar oxidation using other metal-like Manganese and Ruthenium can also be used. The method used to remove the protecting group P from intermediate 15 will depend on the particular group used.

Deprotection of 15 where P is p-methoxybenzyl can be accomplished by treatment with trifluoroacetic acid (TFA) at elevated temperatures. Addition of a cation scavenger like anisole to the reaction mixture often results in higher yields. Those skilled in the art will appreciate that different protecting groups may be required depending on the reactivity of the various R groups. ##STR7## General Method B

The compounds of the invention where the cyclopentane ring is cis-dihydroxylated anti to the indolocarbazole moiety (R.sup.1,R.sup.2, Formula I=–OH) and R.sup.14 =hydroxyl or a substituent derived from the hydroxyl group are conveniently prepared by method B. The first key step in the process involves the alkylation of the protected indolo[2,3-a]carbazole moiety with a suitable electrophile (17). The protected indolo[2,3-a]carbazole can be prepared using methods described in the literature [Lowinger T. B. et al., Tetrahedron Lett. 36, 8383 (1995), P=paramethoxy benzyl]. Alkylation of the protected indolo[2,3-a]carbazole moiety with mesylate 17 (Johnson et al. . . .) using a base like NaH or Cs.sub.2 CO.sub.3 in a polar parotic solvent like DMF provides the mono-alkylated product 18. Deprotection of the acetonide moiety using standard hydrolysis conditions provide dialcohol 19. Dialcohol 19 can be converted to the cyclized product 20 by hydroxyl directed epoxidation and subsequent intramolecular alkylation, or cyclization using Mitsunobu conditions followed by cis-hydroxylation using O.sub.S O.sub.4. Deprotection of 20 where P is p-methoxybenzyl can be accomplished by treatment with trifluoroacetic acid (TFA) at elevated temperatures. Addition of a cation scavenger like anisole to the reaction mixture often results in higher yields. Those skilled in the art will appreciate that different protecting groups may be required depending on the reactivity of the various R groups. ##STR8## General Method C

The compounds of the invention with an .alpha.-hydroxy carboxyl moiety as illustrated in Scheme 3 are conveniently prepared using method C. The first key step in the process involves alkylation of the protected indolo[2,34-a]carbazole moiety with a wuitable cyclopentene electrophile. Protected indolo[2,3-a]carbazole moiety 10 is conveniently prepared using methods described in the literature [Lowinger T. B. et al., Tetrahedron Lett. 36, 8383 (1995) P=paramethoxy benzyl]. Electrophile 22 can be prepared from cyclopentene-3-ol by treatment with methanesulfonyl chloride and triethylamine. Cyclopenpene-3-ol can be prepared according to the procedures described in J. Org. Chem. 32, 4138 (1967). Treatment of the protected indolo[2,3-a]carbazole with a base like Cs.sub.2 CO.sub.3 or NaH in a polar parotic solvent like DMF followed by addition of the alkylating agent (22) provides the desired mono-alkylated material 23. Subsequent activation of the double bond can be accomplished by an OsO.sub.4 with a reoxidant such as N-methyl morpholine N-oxide (NMO) in an aqueous THF or Acetone solution. Cyclization of diol 24 using Mitsunobu conditions provides the bis alkylated adduct 25. A recent review of this Mitsunobu chemistry can be found in Organic Reactions, 42, 335 (1992). Oxidation of alcohol 25 to ketone 26 can be accomplished using a wide variety of reagents and reaction conditions well-known to those skilled in the art. One common method involves the use of chromium based reagents like pyrdinnium chlorochromate (PCC) in an parotic solvent such as methylenechloride. A wide variety of nucleophiles may be added to the ketone moiety in a stereoselective manner. To generate an a-hydroxy carboxyl group it is convient to add carboxylic acid anion equivalent. A general review of this methodology is described in “Unpoled Synthons”, Hase T. A., Ed.; John Wiley & Sons, 1987. One example of a carboxylic acid anion equivalent is an ortho thioformyl carbanion [e.g., LiC(SMe).sub.3 ]. This nucleophile is conveniently prepared by treating tris(methylthio)methane with a strong base like n-BuLi. In general, the addition of the nucleophile to the ketone occurs opposite to the aglycone moiety. The thiocarboxylic acid orthoester is easily hydrolyzed by a lewis acid like boron trifluoride etherate or mercury (II) oxide. Either an ester or a carboxylic acid can be obtained from the orthoester depending on the reagents used in the hydrolysis. The methyl ester (28, Q=OMe) is conveniently obtained by treating the orthoester with mercury (II) chloride and mercury (II) oxide in aqueous methanol. The corresponding carboxylic acid (28, Q=OH) can be obtained by treatment with boron trifluoride etherate in an aqueous THF solution. Once formed, the carboxylic acid can be used as an intermediate to prepare amides (28, Q=NHMe) via a coupling reagent like carbonyldiimidizole (CDI). These procedures are well-known to those skilled in the art. The method used to remove the protecting group P from intermediate 28 will depend on the particular group used. Deprotection of 28 where P is p-methoxybenzyl (PMB) can be accomplished by treatment with trifluoroacetic acid (TFA) at elevated temperatures. Addition of a cation scavenger like anisole to the reaction mixture often results in a higher yielding reaction. Those skilled in the art will appreciate that different protecting groups may be required depending on the reactivity of the various R groups. ##STR9##

EXAMPLES

Example 1

Synthesis of 9,12-Methano-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-I][1,6]benzodiaz ocine-1,3(2H)-dione,9,10,11,12-tetrahydro-10,11-dihydroxy-(9.alpha.,10.alph a.,11.alpha.,12.alpha.)

In order to develop more compounds having ERK2 inhibiting activity a series of synthetically modified derivatives of K252a were prepared. The preparation of four such compounds in which the preferential inhibition of PK40 over PKC/PKA was maintained by a margin of at least 2-3 orders of magnitude is described in Examples 1-4.

It is believed that these ATP analogs act as inhibitors of PK40(ERK2) by binding to the ATP binding site on PK40. PK40 seems to be particularly susceptible to inhibition by ATP analogs, resulting in similar selectivity to K252a and ATP itself. ##STR10## Step 1. A solution of (1S,4R)-cis-4-acetoxy-2-cyclopentene-1-ol (53 mg, 0.37 mmol) and triethylamine (0.77 mL, 0.55 mmol) in a mixture of benzene (0.8 mL) and pentane (0.8 mL) was cooled to -5.degree. C. -0.degree. C. and treated with methanesulfonyl chloride (0.043 mL, 0.56 mmol) at a rate such that the temperature remained below 0.degree. C. A white precipitate was observed.

In a separate round bottom flask, a solution of aglycone [prepared using the protocols described in Tetrahedron Letters 36, 8383 (1995)] (319 mg, 0.72 mmol) in DMF (6.0 mL) was cooled to -5.degree. C.-0.degree. C. for one hour, the reaction mixture was quenched with brine and extracted with ethyl acetate. The organic phase was dried over Na.sub.2 SO.sub.4, filtered and concentrated in vacuo. Purification by MPLC (silica, 50-100% CH.sub.2 Cl.sub.2 -hexanes) gave the target compound (55 mg, 22-26%) as a yellow solid. .sup.1 H NMR (DMSO-d.sub.6) .delta. 12.19 (s, 1H), 9.19 (d, J=2.7 Hz, 1H), 9.10 (d, J=2.7 Hz, 1H), 7.78-6.87 (m, 10H), 6.81 (m, 1H), 6.51 (m, 1H), 6.36 (m, 1H), 6.13 (m, 1H), 4.82 (s, 2H), 3.69 (s, 3H), 2.68 (m, 2H), 2.10 (s, 3H); MS (FAB-LSIMS) m/z (relative intensity) 569 (M+, 44), 508 (32), 462 (100), 444 (50), 429 (30); TLC: R.sub.f 0.4 (silica, 7% EtOAc-hexanes); MP>200.degree. C. ##STR11## Step 2. A solution of the acetate from step 1 (30 mg, 0.05 mmol) and sodium cyanide (10 mg, 0.2 mmol) in ethanol (2.0 mL) was heated at reflux until no starting material was observed by TLC (2 h). The mixture was concentrated in vacuo, washed in water (20 mL) and extracted with EtOAc (20 mL). The organic extract was dried over Na.sub.2 SO.sub.4 and concentrated to give a yellow oil. Purification by MPLC (silica, 0-15% EtOAc-CH.sub.2 Cl.sub.2) afforded the target alcohol (27 mg, 90%) as an orange powder. .sup.1 H NMR (DMSO-d.sub.6) .delta. 12.17 (s, 1H), 9.19 (d, J=2.6Hz, 1H), 9.10 (d, J=2.5 Hz, 1H), 7.78-6.87 (m, 10H), 6.79 (m, 1H), 6.28 (m, 2H), 5.25 (m, 2H), 4.83 (s, 2H), 3.68 (s, 3H), 2.54 (m, 2H); TLC (silica, 10% EtOAc-CH.sub.2 Cl.sub.2). ##STR12## Step 3. The alcohol from step 2 was added to a solution of diethyl azodicarboxylate (65.1 mg, 0.46 mmol) and triphenylphosphine (141 mg, 0.54 mmol) in tetrahydrofuran (4.0 mL). After stirring overnight at room temperature, the reaction was quenched with brine and extracted with EtOAc. The organic phase was dried over Na.sub.2 SO.sub.4, filtered and concentrated in vacuo. The resulting brown oil was purified by MPLC (silica, 20-30% EtOAc-hexanes) to give the cyclized product (60mg, 31%) as a yellow powder. .sup.1 H NMR (DMSO-d.sub.6) .delta. 9.07 (s, 1H), 9.04 (s, 1H), 8.02-6.87 (m, 10H), 6.41 (s 2H), 6.22 (m, 2H), 4.83 (s 2H), 3.68 (s, 3H), 3.15 (m, 1H), 2.71 (m, 1H); TLC: Rf 0.75 (silica, 50% EtOAc-hexanes). ##STR13## Step 4. A solution of the imide from step 3 (49.1 mg, 0.096 mmol) in anisole (0.68 mL) was stirred at room temperature for fifteen minutes and cooled to over 0.degree. C. Over the next twenty minutes, trifluoroacetic acid (6.8 mL) was added to the solution. After allowing the orange mixture to warm to room temperature, the solution was heated to reflux overnight. After removing the solvent in vacuo, the resulting brown oil was washed with saturated. aq. NaHCO.sub.3 (20 mL) and extracted with EtOAc (25 mL). The organic phase was dried over Na.sub.2 SO.sub.4, filtered and concentrated in vacuo. Purification of the resulting oil via flash chromatography (silica, 0-10% EtOAc-CH.sub.2 Cl.sub.2) gave the deprotected imide as an orange powder (34.9 mg, 93%). .sup.1 H NMR (DMSO-d.sub.6) .delta. 11.06 (s, 1H), 9.06 (s, 1H), 6.22 (d, J=2.2 Hz, 2H), 3.12 (m, 1H), 2.70 (m, 1H); MS (FAB-LSIMS) m/z (relative intensity) 390 (M+H, 60), 369 (32), 347 (62), 319 (30), 305 (18) 293 (22), 277 (100), 267 (18), 254 (28), 241 (14), 207 (14); TLC: R.sub.f 0.50 (5% EtOAc-CH.sub.2 Cl.sub.2). MP>230.degree. C. ##STR14## Step 5–Preparation of Example 2. A solution of the imide from step 4 (14.4 mg, 0.04 mmol) and N-methylmorpholine (0.2 mL) in tetrahydrofuran (0.4 mL) was treated with osmium tetroxide (0.1 mL, 1.0 M in THF) and stirred at room temperature for one hour (until no starting material remained by TLC, EtOAc. The reaction mixture was quenched with NaHSO.sub.3 (1.5 mL, 2 M aqueous solution) and stirred vigorously for 1 hour. The solution was diluted with brine and extracted with EtOAc. The organic phase was dried over Na.sub.2 SO.sub.4 and concentrated in vacuo. The resulting yellow oil was purified by HPLC (0-3% MeOH-chloroform) to afford the target diol as a red-orange powder (9.5 mg, 61%). .sup.1 H NMR (DMSO-d.sub.6) .delta. 11.05 (s, 1H), 9.05 (s, 1H), 9.02 (s, 1H), 7.85 (s, 1H), 7.65 (m, 2H), 7.39 (m, 2H), 5.51 (m, 2H), 5.39 (m, 2H), 4.06 (s, 2H), 3.27 (m, 1H); 2.40 (m 1H). MS (FAB-LSIMS) m/z (relative intensity) 424 (M+H, 34), 381 (24), 362 (12), 310 (16), 185 (42), 121 (72), 93 (100), 55 (50); TLC: R.sub.f 0.2 (EtOAc–); MP>230.degree. C.

Example 2

Synthesis of 9,12-Methano-1H-diindolo[1,2,3-fg:3',2',1'-kl] pyrrolo[3,4-I[1,6]benzodiazocine-10-carboxylic acid, 2,3,9,10,11,12-hexahydro-10-hydroxy-1,3-dioxomethyl ester, (9.alpha.,10.beta.,12.alpha.). ##STR15## Step 1. A solution of cyclopentene-3-ol [prepared using the protocols described in J. Org. Chem. 32, 1967, 4138] (2.1 g, 25.0 mmol) and triethylamine (3.60 mL, 25.8 mmol) in CH.sub.2 Cl.sub.2 (15.0 mL) was cooled to 0.degree. C. and treated with methanesulfonyl chloride (1.9 mL, 24.5 mmol) at a rate such that the temperature remained below 0.degree. C. After warming to room temperature and stirring for two hours, the reaction mixture was quenched with brine (40 mL) and extracted with CH.sub.2 Cl.sub.2 (90 mL). The organic extract was dried over Na.sub.2 SO.sub.4, filtered and concentrated in vacuo. Purification by MPLC (silica, 15-40% EtOAc-hexanes) gave the desired mesylate as a pale yellow liquid (3.74 g, 92%). .sup.1 H NMR (CDCl.sub.3) .delta. 5.74 (m 2H), 5.38 (m, 1H), 3.02 (s, 3 H, 2.84-2.63 (m, 4H); TLC: R.sub.f 0.4 (40% EtOAc-hexanes). ##STR16## Step 2. A solution of the protected aglycone [prepared using the protocols described in Tetrahedron Lett. 36, 1995, 8383] (3.72 g, 8.3 mmol) in dimethylformamide (50 mL) was heated to 60-65.degree. C. and treated with cesium carbonate (10.9 g, 33.3 mmol). The resulting dark red mixture was stirred for 30 min. Over the next four hours, the mesylate from step 1 (4.04 g, 24.9 mmol) was added in 500 mg portions and mixture stirred for two days at 65-70.degree. C. After cooling to room temperature, the reaction mixture was quenched with brine (300 mL) and extracted with EtOAc (300 mL). The organic phase was dried over Na.sub.2 SO.sub.4, filtered and concentrated in vacuo. Purification by flash chromatography (silica, 25-25% CH.sub.2 Cl.sub.2 -hexanes) gave the desired mono alkylated product as an orange powder (2.0 g, 47%). .sup.1 H NMR (DMSO-d.sub.6) .delta. 12.13 (s, 1H), 9.21 (d, J=2.6 Hz, 1H), 9.09 (d, J=2.7 Hz), 7.77-6.87 (m, 1H), 6.07 (s, 2H), 4.79 (s, 2H), 3.68 (s, 3H), 3.25-3.17 (m, 2H), 3.01-2.93 (m, 2H); MS (FAB-LSIMS) m/z (relative intensity) 511 (M+, 20), 419 (14), 391 (30), 378 (64) 363 (54), 255 (8); TLC: R.sub.f 0.5 (60% EtOAc-hexanes). ##STR17## Step 3. A solution of the cyclopentene intermediate from step 2 (1.67 g, 3.26 mmol), and 4-methylmorpholine-N-oxide (60% aqueous solution, 0.55 mL, 5.31 mmol) in tetrahydrofuran (39 mL) was treated with OsO.sub.4 (3.91 mL, 0.1 M in THF, 0.12 eq) and stirred overnight. After quenching mixture with aqueous 2.0 M sodium bisulfite solution and stirring for thirty minutes, the solution was extracted with EtOAc (150 mL) and washed with brine (300 mL). The organic phase was dried over Na.sub.2 SO.sub.4, filtered and concentrated in vacuo. Purified by flash chromatography (silica 0-10% MeOH-EtOAc) gave the target compound as a orange-yellow solid (1.43 g, 80%). .sup.1 H NMR (DMSO-d.sub.6) .delta. 12.04 (s, 1H), 9.23 (d, J=2.7 Hz, 1H), 9.10 (d, J=2.7 Hz, 1H), 7.80-6.87(m, 10H), 6.11 (m, 1H), 4.81 (m, 4H), 4.74 (m, 2H), 3.68 (s, 3H); MS (FAB-LISMS) m/z (relative intensity) 545 (M+, 18), 438 (40), 338 (10), 255 (8); TLC: R.sub.f 0.2 (5% MeOH-chloroform). ##STR18## Step 4. A solution of the diol intermediate from step 3 (277 mg, 0.42 mmol) and triphenylphosphine (383 mg, 1.46 mmol) in tetrahydrofuran (26 mL) was treated with diethyl azodicarboxylate (0.16 mL, 0.98 mmol) at a rate such that the resulting orange-red color of the reaction mixture was allowed to return to its initial yellow color. After the addition was completed, the mixture was stirred for two days and subsequently heated to reflux for one day. The reaction mixture was quenched with brine (100 mL) and extracted with EtOAc (160 mL). The organic phase was dried over Na.sub.2 SO.sub.4, filtered and concentrated in vacuo. Purification of the resulting oil by MPLC (silica, 0-20% EtOAc-CH.sub.2 Cl.sub.2) afforded the cyclized product as an orange powder (165 mg, 75%). .sup.1 H NMR (DMSO-d.sub.6) .delta. 9.13 (d, J=2.7 Hz, 1H), 9.08 (d, J=2.7 Hz, 1H), 8.04-6.96 (m, 10H), 5.98 (m, 1H), 5.70 (m, 1H), 5.41 (m, 1H), 4.89 (s, 2H), 4.29 (m, 1 5H), 3.77 (s, 3H), 3.22 (m, 1H), 2.68 (m, 1H), 2.45 (m, 1H), 2.05 (m, 1H); MS (FAB-LSIMS) m/z (relative intensity) 527 (M+, 32), 420 (62); TLC:R.sub.f 0.5(30% EtOAc-CH.sub.2 Cl.sub.2). ##STR19## Step 5. A solution of pyridiniumn chlorochromate (89 mg, 0.41 mmol) in CH.sub.2 Cl.sub.2 (2.0 mL) was treated with the alcohol from step 4 (140 mg, 0.27 mmol) as a solution in CH.sub.2 Cl.sub.2 (18 mL). A second portion of pyridinium chlorochromate (40 mg) was added to the brown mixture. After stirring the mixture for 2 hours, the solution was filtered through a short pad of silica gel and concentrated in vacuo. Purification by flash chromatography (silica, 80-100% CH.sub.2 Cl.sub.2 -hexanes) afforded the target ketone as a yellow powder (108 mg, 77%). .sup.1 H NMR (DMSO-d.sub.6) .delta. 9.05 (d, J=2.6 Hz, 1H), 9.03 (d, J=2.4 Hz, 1H), 7.98-6.86 (m, 1H), 6.14 (m, 1H), 5.58 (m, 1H), 4.82 (s, 2H), 3.68 (s, 3H), 3.44 (m, 1H), 3.22 (m, 1H), 3.00 (M, 1H), 2.50 (m, 1H); MS (FAB-LSIMS) m/z (relative intensity) 525 (M+, 14), 418 (30), 2181 (34), 185 (100), 147 (38), 121 (66); TLC: R.sub.f 0.6 (20% EtOAc-CH.sub.2 Cl.sub.2). ##STR20## Step 6. A solution of tris(methylthio)methane (0.16 mL, 1.15 mmol) in tetrahydrofuran (3.0 mL) was cooled to -78.degree. C. and treated with n-butyl lithium (0.59 mL, 1.6 M, 0.94 mmol). After stirring for twenty minutes, a solution of the ketone from step 5 (199 mg, 0.38 mmol) in tetrahydrofuran (6.0 mL) was added to the reaction mixture and stirred for two hours. After quenching with a saturated ammonium chloride solution (10 mL) and warming to room temperature, the reaction mixture was diluted with water (10 mL) and extracted with EtOAc (90 mL). The organic layer was separated, dried over Na.sub.2 SO.sub.4 and concentrated in vacuo. Purification of the resulting oil by flash chromatography (silica, 20% EtOAc-hexanes) provided the addition product as a yellow solid (67 mg, 26%). .sup.1 H NMR (DMSO-d.sub.6) .delta. 9.06 (m, 2H), 8.17-6.87 (m, 10H), 5.74 (m, 1H), 5.05 (s, 1H), 4.85 (s, 2H), 3.69 (s 3H), 3.01 (m, 2H), 2.17 (s, 9H); MS (FAB-LSIMS) m/z (relative intensity) 679 (M+, 12), 572 (22), 488 (24), 310 (100), 284 (86); TLC: R.sub.f 0.8 (50% EtOAc-hexanes). ##STR21## Step 7 A solution of the thiocarboxylic acid orthoester intermediate from step 6 (50 mg, 0.074 mmol) in tetrahydrofuran (5.0 mL) was treated with methanol (12.0 mL), water (1.0 mL), mercury (II) oxide (84 mg, 0.39 mmol) and mercury (II) chloride (236 mg, 0.87 mmol). The reaction mixture was heated to reflux. The solution was diluted with brine (30 mL) and extracted with CH.sub.2 Cl.sub.2 (60 mL). The organic phase was dried over Na.sub.2 SO.sub.4, filtered and concentrated in vacuo. Purification by flash chromatography (silica, 0-3% EtOAc-CH.sub.2 Cl.sub.2) gave the target ester as an orange solid (18 mg, 42%). .sup.1 H NMR (CDCl.sub.3) .delta. 9.25 (m, 2H), 7.60-7.40 (m, 8H), 6.88-6.85) (m, 2H), 5.61 (m, 1H), 5.40 (m, 1H), 4.93 (s, 2H), 4.03 (s, 3H), 3.77 (s, 3H), 3.21 (m, 2H), 2.88 (m, 1H), 2.77 (m, 1H), 1.95 (m, 1H); MS (FAB-LSIMS) m/z (relative intensity) 585 (M+, 8), 478 (8), 277 (11), 185 (100); TLC: R.sub.f 0.15 (50% EtOAc-hexanes). ##STR22## Step 8. Preparation of Example 3. A solution of the ester intermediate from step 7 (18 mg, 0.03 mmol) was dissolved in anisole (0.3 mL) over fifteen minutes and subsequently treated with trifluoroacetic acid (2.7 mL). The reaction mixture was heated to reflux for 2 hours (until no starting material remained by TLC, 5% EtOAc-CH.sub.2 Cl.sub.2). The solution was concentrated in vacuo. Purification by flash chromatography (silica, 10-20% EtOAc-CH.sub.2 Cl.sub.2) afforded the target imide as a yellow solid (11.0 mg, 77%). .sup.1 H NMR (DMSO-d.sub.6) .delta. 11.04 (s, 1H), 9.03 (m, 2H), 7.90-7.33 (m, 6H), 5.81 (m, 1H), 5.71 (m, 1H), 5.54 (s, 1H), 3.83 (s, 3H), 3.08 (m, 2H), 2.76 (m, 1H), 1.71 (m, 1H); .sup.13 CNMR (DMSO-d.sub.6) .delta. 175.3 (C.dbd.O), 171 (C.dbd.O imide), 170, (C.dbd.O imide,), 142.0, 140.0, 129.7, 128.4, 126.8, 126.7, 124.4, 121.3, 121.3, 121.2, 120.4, 120.2, 119.6, 119.5, 119.4, 115.3, 110.4, 109.7, 81.1 (COH), 61.7 (CHN), 55.2 (CHN), 52.07 (OCH3), 45.3, (CH2), 39.0 (CH2); MS (FAB-LSIMS) m/z (relative intensity) 466 (M+H, 14), 423 (6), 185 (28), 93 (100); TLC: R.sub.f 0.2 (5% EtOAc-CH.sub.2 Cl.sub.2); MP>230.degree. C.

Example 3

Synthesis of 9,12-Methano-1H-diindolol ,2,3-fg:3′,2′,1′-kl]pyrrolo[3,4-I][1,6]benzodiazocine-1,3,10(2H,9H)-trone, 11,12-dihydro. ##STR23## Step 1. Preparation of Example 4. A solution of the ketone for step 5 of example 2 (15 mg, 0.029 mmol) was dissolved in anisole (0.3 mL) over fifteen minutes and subsequently treated with trifluoroacetic acid (2.7 mL). The mixture was heated to reflux temperatures until no starting material was detected by TLC (10% EtOAc-CH.sub.2 Cl.sub.2). The solution was concentrated under reduced pressure and purified by flash chromatography (silica, 0-10% EtOAc-CH.sub.2 Cl.sub.2) to afford the target ketone (8.9 mg, 77%) as a yellow solid. .sup.1 H NMR (CDCl.sub.3) .delta. 9.16 (m, 2H), 7.68-7.24 (m, 7H), 5.90 (m, 1H), 5.23 (m, 1H), 3.32 (m, 1H), 3.14-2.93 (m, 2H), 2.52 (m, 1H); MS (FAB-LSIMS) m/z (relative intensity) 405 (M+H, 12), 354 (12), 324 (18), 224 (18), 191 (52); TLC: R.sub.f 0.4 (10% EtOAc-CH.sub.2 Cl.sub.2); MP>225.degree. C.

Example 4

Synthesis of 9,12-Methano-1H-diindolo[1,2,3-fg:3',2',1'-kl]pyrrolo[3,4-I][1,6]benzodiaz ocine-10-carboxamide,2,3,9,10,11,12-hexahydro-10-hydroxy-N-methyl-1,3-dioxo -(9.alpha.,10.beta.,12.alpha.). ##STR24## Step 1. A solution of the thiocarboxylic acid ortho ester intermediate from step 6 of example 2 (28 mg, 0.04 mmol) in 20% H.sub.2 O-tetrahydrofuran (1.3 mL) was treated with mercury (II) oxide (45 mg, 0.21 mmol) and boron trifluoride diethyl etherate (0.073 mL, 0.59 mmol). The reaction mixture was stirred for two hours at room temperature [until only one major spot was seen by TLC (2:3:95 acetic acid-methanol-CH.sub.2 Cl.sub.2)], diluted with water (10 mL) and extracted with EtOAc (20 mL). The organic phase was dried over Na.sub.2 SO.sub.4, filtered and concentrated in vacuo. Purification by flash chromatography (silica, 0-10% methanol-CH.sub.2 Cl.sub.2) afforded the target acid as an orange solid (76%, 18.0 mg). MS(FAB-LSIMS) m/z (relative intensity) 571(M+, 8), 464 (14), 381 (32), 330 (88), 181 (100); TLC:R.sub.f 0.3 (2:3:95 acetic acid-methanol-CH.sub.2 Cl.sub.2). ##STR25## Step 2. A solution of carboxylic acid from step 1 (20 mg, 0.034 mmol) in tetrahydrofuran (2.5 mL) was cooled to 0.degree. C. and treated with 1,1′-carbonyldiimidazole (60 mg, 0.37 mmol) and stirred for ten minutes. A solution of approximately 50% methylamine-tetrahydrofuran (2 mL) was quickly added to the reaction. After five minutes, no starting material was observed by TLC (20% EtOAc-CH.sub.2 Cl.sub.2) and the reaction mixture was quenched with a saturated citric acid solution (3.0 mL). After warming to room temperature, the solution was diluted with brine (15 mL) and extracted with EtOAc (20 mL). The organic phase was dried over Na.sub.2 SO.sub.4, filtered and concentrated in vacuo. Purification by flash chromatography (silica, 0-20% EtOAc-CH.sub.2 Cl.sub.2) gave the target methyl amide (7.0 mg, 35%). MS (FAB-LSIMS) m/z (relative intensity 584 (M+, 6), 477 (8), 253 (8), 169 (84), 132 (30), 85 (100); TLC:R.sub.f 0.3 (20% EtOAc-CH.sub.2 Cl.sub.2). ##STR26## Step 3. Preparation of example 5. A solution of the protected methyl amide from step 2 (7.0 mg, 0.01 mmol) was dissolved in anisole (0.5 mL) over ten minutes and subsequently treated with trifluoroacetic acid (4.5 mL). The mixture was heated to reflux for eight hours [no starting material was observed by TLC (50% EtOAc-CH.sub.2 Cl.sub.2)]. The mixture was concentrated in vacuo and purified by flash chromatography (silica, 10-20% EtOAc-CH.sub.2 Cl.sub.2) to afford the methyl amide as a orange powder (3.4 mg, 61%). .sup.1 H NMR (DMSO-d6) .delta. 11.03 (s, 1HO, 9.04 (m, 2H), 7.94-7.32 (m, 7H), 5.81 (m, 1H), 5.59 (s, 1H), 5.41 (1H) 3.25-3.05 (m, 2H), 2.66 (d, J=1.5 Hz, 3H), 2.64 (m, 1H), 1.74 (m, 1H); MS (FAB-LSIMS) m/z (relative intensity) 465 (M+H, 14), 361 (82), 346 (38), 322 (100), 315 (38); TLC:R.sub.f 0.4 (50%EtOAc-CH.sub.2 Cl.sub.2); MP>230.degree. C.

Example 5

Preparation and Analysis of Kinases for Inhibitor Assays.

Preparation of Kinases:

Preparation of PKC: PKC was purified from rat brain using the method of Woodgett J. R. and Hunter T., J. Biol. Chem 262, 4836-4843 (1987).

Preparation of cAMP-dependent Kinase: The catalytic subunit of bovine heart PKA was obtained commercially from Sigma.

Preparation of cdc2 Kinase: Human cdc2 kinase was prepared from nocodazole-arrested HeLa cells according to Marshak D. M. er al., J. Cell Biochem., 45, 391-400 (1991).

Preparation of ERK2: Recombinant human ERK2 with a N-terminal histidine (his) tag was prepared as follows: An ERK2 cDNA clone was amplified from a human frontal cortex library by PCR with primers matching the published human sequence [Gonzales F. A. et al, FEBS Lett. 304, 170-178 (1992)]. The histidine tag was introduced by site-directed mutagenesis. The cDNA was cloned into a pET-14b (Novagen) vector and transfected into the E. coli lysogen strain B121pLysS. Single colony transformants were grown in LB medium containing 35 mg/ml chloramphenicol to maintain pLysS and 50 mg/ml kanamycin to an O.D..sub.600 OF 0.6. The culture was then induced with 0.4 mM IPTG for 4 hours. The expressed ERK2 protein was then analyzed on a 10% SDS PAGE by both coomassie blue staining and anti-ERK Western blotting. Bacterial pellets from 0.5-1 [cultures were freeze-thawed at -78.degree. C. and homogenized by ultra-sonication for 3 min in 15 ml Ni.sup.2+ -column buffer (20 mM Tris HCL pH 7.9, 0.5 M NaCl, 5 mM imidazole, Novagen). After centrifugation at 35,000.times. g for 30 min supernatants were loaded onto a 1 ml Ni.sup.2+ charged resin (Novagen). After washing with column buffer containing 60 mM imidazole the ERK2 protein was eluted with column buffer containing 1 M imidazole. ERK2 containing fractions were identified by SDS-PAGE and dialyzed into Mono Q A-buffer (25 mM Tris HCL, pH 7.5 25 mM NaCl, 1 mM EDTA). The dialysate was loaded onto a HR5/5 Mono Q FPLC column (Pharmacia) and eluted with a 30 ml gradient from Mono Q A-buffer to the same buffer containing 250 mM NaCl (Mono Q B-buffer) at 1 ml/min collecting 30 fractions. Fractions #19-20 and #27-28 typically contained the peak amounts of two ERK2 conformers, as identified by western blotting. Only the first fraction was applied to HR 5/5 Phenylsuperose FPLC (Pharrnacia) and eluted with a 15 ml gradient from 25 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM DTT to the same buffer containing 25 mM NaCl and 60% ethylene glycol with a flow rate from 0.5 ml/min decreasing to 0.1 ml/min at the end of the gradient. Homogeneous ERK2 typically eluted after 13-14 ml.

For activation of ERK2 to the active PK40 form about 1 mg of purified histidine tagged ERK2 was mixed with 25 ml of CM-Sepharose eluate fraction prepared from bovine brain extracts according to Roder H. M. et al., J. Neurochem. 64, 2203-2212 (1995), adjusted to 2 mM Mg.sup.2 +/0.5 mM ATP and incubated for 2 hrs at 37.degree. C. The mixture was dialyzed twice into 11 each of Ni.sup.2+ column binding buffer (20 mM Tris, pH 2.9, 500 mM NaCl, 5 mM imidazole) to remove traces of DTT and loaded onto 0.3 mL Ni.sup.2+ charged resin (Novagen) at 25 mL/hr. After washing with 10 mL column buffer followed by 4 mL column buffer containing 40 mM imidazole homogeneous activated PK40.sup.erk2 was eluted with 4 mL column buffer containing 1 M imidazole. The product was dialyzed extensively into 10 mM HEPES, pH 7.0, 1 mM EDTA to remove imidazole and traces of Ni.sup.2+, and finally into 10 mM HEPES, pH 7.0, 1 mM EDTA, 1 mM DTT.

Assays for Kinase Activity:

PK40.sup.erk2 was assayed in 50.mu. 25 mM HEPES, pH 7.0, 1 mM MgCl.sub.2, 1 mM DTT, 0.25 mM ATP, 1 mg/mL BSA using 15-30 ng of PK40.sup.erk2 and 0.1 mg/mL myelin basic protein (Sigma) as a substrate.

PKC was assayed in 50 .mu.l 25 mM HEPES, pH 7.0, 10 mM MgCl.sub.2, 2 mM CaCl.sub.2, 1 mM EDTA, 1 mM DTT, 0.25 mM ATP, 0.2 mg/mL phosphatidylserine, using 20 ng PKC and 0.08 mg/mL histone III-S as a substrate.

The catalytic subunit of PKA was assayed in 50 .mu.l 25 mM HEPES, pH 7.0, 10 mM MgCl.sub.2, 1 mM EDTA, 1 mM DTT, using 70 ng PKA and 0.1 mg/mL human recombinant tau protein as a substrate.

cdc2 kinase was assayed in 50 .mu.l 25 mM HEPES, pH 7.0, 1 mM Mg.sup.2+, 0.25 mM ATP (150-300 cpm/pmole), 1 mM DTT, using 0.5 ng cdc2 kinase, 5 .mu.g human recombinant tau as substrate, and 0.1 mg/mL BSA a as carrier.

Determination of Potency of Kinase Inhibitors

Enzyme, substrate and inhibitor were preincubated for 5-10 min at 4.degree. C. in assay buffers containing a final concentration of 2% DMSO before initiating the reaction with 0.25 mM .UPSILON..sup.2 P-ATP. Samples were incubated for 30 min at 37.degree. C. and reactions were terminated with 10% trichloracetic acid/2% sodium pyrophosphate (TCA/PPA), followed by filtration over a glass fiber filtermat (type A) with a cell harvester (Tomtec). The filtermats were washed twice for several hours with TCA/PPA until all background radioactivity was removed. Precipitable counts were quantitated directly on the filtermat with a microbeta scintillation counter system (Wallac-Pharmacia). Inhibitor data were subjected to curve fitting and IC.sub.50 values were calculated from these curves using the GraphIt program.

Example 6

Determination of Potency of Inhibitors to Prevent AD-like Tau Hyperphosphoalylation in SY5Y Cell Model

In vitro, and presumably also in vivo, tau is a substrate for multiple kinases. The main problem to evaluate inhibitors specifically interfering with the AD-like hyperphosphorylation of tau is to distinguish clearly between normal and abnormal phosphorylation of tau in model systems. In the cell line SY5Y comparisons can be made with tau associated with tangles from human AD brain, because of its human origin. As in fetal brains, SY5Y cells express only one of the 6 splice isoforms of tau, simplifying the survey of tau phosphorylation states.

Methods

SKNSH-SY 5Y cells were plated on fibronectin-coated 6 well (30 mm.sup.2) culture dishes (Biocoat.RTM., Collaborative Biomedical Products, Inc.) and grown to confluence in 5 mL of 50% D-MEM/50% F-12 Nutrient Mixture (Ham) supplemented with 15% heat-inactivated bovine serum (JRH Bioscience), 0.1 mM non-essential amino acids solution, 2 mM glutamine and pen/strep/fungizone (GibcoBRL Life Technologies, Inc.). Cell culture medium was changed every 48 hours.

For drug testing, cells were routinely pretreated with inhibitors in 1 mL (30 mm.sup.2) for 60 min at concentrations of 30 nM, 100 nM, 300 nM, 1 mM, 3 mM and 10 mM. Compound stocks were all at 10 mM in DMSO and dilutions were made in DMSO. Cells were then treated with 1 .mu.M okadaic acid (ammonium salt; LC Laboratories, dissolved at 1 mM in DMSO) for 90 min. All experiments, including controls, contained a final concentration of between 0.5 and 1% DMSO.

Cells were detached from 30 mm.sup.2 plates and suspended into 1 mL of ice-cold PBS by gentle trituration, transferred into microcentrifuge tubes and sedimented for 12 seconds at 14,000.times. g. The supernatant was removed and cells were lysed in 250 .mu.l cold homogenization buffer (50 mM MES, pH 5.8, 5 mM sodium pyrophosphate, 50 mM p-nitrophenylphosphate, 1 .mu.M okadaic acid, 2 mM Na-orthovandate, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 10 glycerol, 10 .mu.leupeptin, 1 .mu.M pepstatin, 1 mg/mL aprotinin, 10 .mu.M chymostatin, 1 mM PMSF, and 1% TRITON.RTM..times.100) and vortexed briefly to aid in lysis. Cell debris was removed by centrifugation at 14,000.times. g for 5 minutes at 4.degree. C. and cell supernatants were analyzed by anti-ERK2 and anti-tau Western-blotting as described below.

25 mL of total cell lysate was run on a 10% tris-glycine polyacrylamide gels (Novex, 1.5 mm.times.10 well) at 100 volts for 2.5 hors and Western-blotted on nitrocellulose (Novex) overnight at 23 volts or 1.5 hrs at 100 V in transfer buffer [Towbin et al. Proc. Natl. Acad. Sci. USA 76, 4350-4354 (1979]) at 4.degree. C. Blots were analyzed for ERK2 and phosphotyrosine immunoreactivity with anti-ERK1+2 (Z033;Zymed Laboratories, Inc.; 1:5,000) and anti-phosphotyrosine (4G10; Upstate Biotechnology Inc.; 1:1,000) mAbs. Blots were also analyzed for phosphorylation-sensitive Tau immuno-reactivity with mAb Tau-1 (Boehringer Mannheim; 1:5,000) and phosphorylation dependent mAb AT8 against FHF-tau (Biosource International; 1:200). Total Tau populations were detected by Tau-1 immunoblotting after treating blots for 16 hrs at 37.degree. C. with 100 units/mL alkaline phosphatase (Gibco BRL) in 5 mL of 50 mM Tris-HCl (pH 8.5), 0.1 mM EDTA. All blots were developed using ECL (enhanced chemiluminescence) Western blotting protocol (Amersham Life Science) with horseradish peroxidase-linked sheep anti-mouse secondary antibody and analyzed on Kodak X-OMAT AR scientific imaging film. Films were scanned into Adobe Photoshop and imported into NIH Image 1.44, where densitometric analysis was performed. Changes in tau phosphorylation were assessed by normalizing densitometrically determined mAb Tau-1 immunoreactivities in cell extracts to Tau-1 reaction after dephosphorylation on the blot. Ratios of Tau-1 reactivity prior to and after dephosphorylation were expressed in % relative to the ratio obtained from control cells not treated with okadaic acid (100%). Proteins isolated form neonatal rat brain were used for comparison in some experiments.

Results

The potency of the compounds of the invention tested, including CII and CIII, in the in vitro kinase assay was well correlated with their inhibitory activity of tau hyperphosphorylation in the cell. Moreover, each of the compounds demonstrated a correlation between inhibition of ERK2 and tau hyperphosphorylation.

Correlation of potencies of inhibition of PK40 activity in vitro, and of OA-induced ERK2 and tau phosphorylation in SY5Y cells. ERK2 phosphorylation was quantified as the ratio of low mobility/total ERK2, densitometrically determined from ERK2 Western-blots (e.g. of FIG. 1). Tau hyperphosphorylation was expressed as densitometrically measured Tau-1 immunoreactivity normalized to total Tau-1 reactivity after unmasking of the epitope by phosphatase treatment of the blots.

In FIG. 2, tau from untreated SY5Y cells is compared to the completely hyper/dephosphorylated state (accomplished by treatment in vitro with PK40(ERK2) and phosphatase 2B, respectively), and to PHF-tau from AD brains with regard to Tau-1 immunoreactivity and electrophoretic mobility. Tau proteins were analyzed as isolated from tissue in comparison to tau exhaustively dephosphorylated with PP2B calcineurin), or hyperphosphorylated in vitro with PK40. Western-blots were stained with mAb Tau-l (FIG. 2A, B, upper panels) or AT8 (FIG. 2C, lanes 4-6). Relative gel mobilities and loading were visualized by Tau-l after complete unmasking of the epitope by phosphatase treatment on the blot (FIGS. 2A, B, lower panels; FIG. 2C, lanes 1-3). The phosphorylation of fetal tau appears to be similar to SY5Y tau. In either case hyperphosphorylation by PK40 completely abolishes residual Tau-1 reactivity and induces a small additional mobility shift. By these criteria SY5Y tau hyperphosphorylated in vitro by PK40 is indistinguishable from tau hyperphosphorylated in situ after okadaic acid induction, and from PHF-tau. Soluble fractions of PHF-tau were extracted from purified PHF by water or SDS.

FIG. 2 shows that in SY5Y cells most of the potential Tau-l reactivity is already masked by phosphorylation, and the electrophoretic mobility of tau is close to maximally retarded. By the criteria of FIG. 2, the phosphorylation state of tau in SY5Y cells does not appear to be substantially different from tau in neonatal rat brains (FIG. 2C). This probably applies to tau from adult brains as well, as newer data avoiding post-mortem artifacts in isolating tau argue against the previously held notion that the fetal phosphorylation state is higher than in the adult state.

Hyperphosphorylation with PK40(ERK2) in vitro does induce a small but detectable change in tau properties as isolated from SY5Y cells. Only in this state the electrophoretic mobility of tau matches exactly the gel mobility of the corresponding pathologically phosphorylated splice isoform extracted from tangles (FIG. 2C). In cells, the same abnormal phosphorylation state can be induced by inhibition of protein phosphatase 2A with okadaic acid.

Example 7

Methods

Neonatal rat tau hyperphosphorylated in vitro by PK40 with or without prior dephosphorylation by PP2B. Equal amounts of purified 32P-hyperphosphorylated tau samples were digested with trypsin, and peptides were analyzed by 2D electrophoresis. The results are shown in FIG. 3. Labeling of peptides was quantified by counting (cpm displayed for each spot). Comparison of total cpm showed that dephosphorylation liberated only about 1/5th of the available ERK2 sites.

Results

In order to demonstrate that the small changes in immunochemical and gel mobility properties observed in the data presented herein is useful and a relevant model for assessing the large AD-like hyperphosphorylation effects which occur the degree of dephosphorylation/hyperphosphorylation of tau in neonatal rat cells was observed. The small change of tau associated with abnormal AD-like phosphorylation in vitro and in cells does not necessarily reflect a small change in the phosphorylation state. As shown in FIG. 3 the degree of hyperphosphorylation of tau from fetal/neonatal rat brains by PK40(ERK2) which were not pre-dephosphorylated is only about 20% lower than the degree of dephosphorylation observed when tau is dephosphorylated completely prior to hyperphosphorylation. In addition, the two-dimensional phosphopeptide maps of tau in this comparative study are qualitatively indistinguishable (FIG. 3).

Example 8

Inhibitors of PK40 Prevent Abnormal AD-like Hyperphosphorylation.

Methods

Compound CIII prevents ERK2 phosphorylation and tau hyperphosphorylation in a correlated fashion (FIG. 4). Compared to control cells (lane C) 1 .mu.M okadaic acid induced ERK2 phosphorylation/activation, as shown by a small gel mobility shift of ERK2 (lane OA) and induction of reactivity with a mAb sensitive to the double phosphorylation of the regulatory Thr-Glu-Tyr motif of ERK2 (anti-active ERK2). Both effects were prevented by .ltoreq.1 .mu.M compound CIII (IC50 at about 1 .mu.M, complete at 10 .mu.M). Highly correlated with the effect on ERK2 was the prevention of OA induced tau hyperphosphorylation, as tracked by elimination of Tau-1 reactivity and prevention of a small gel mobility shift typical of AD-like tau. Note that at 10 .mu.M, with ERK2 activation completely arrested, the tau phosphorylation state (including the phosphoisoform pattern) remains unaltered compared to normal phosphorylation in control cells.

Prevention of tau hyperphosphorylation by the preferred compound CIII (FIG. 1). Okadaic acid at 1 .mu.M induced the complete elimination of the Tau-1 epitope (upper panel) as in PHF-tau of AD. The shift in electrophoretic mobility corresponding to human PHF-tau was visualized by phosphatase treatment of duplicate Western-blots (lower panel) to recover the masked Tau-1 epitope. The compound CIII prevents the tau hyperphosphorylation in a dose dependent manner. At fully effective doses (>1 .mu.M) tau remained in a phosphorylation state similar to the normal state in control cells (lane C). Tau in normal cells not treated by okadaic acid is phosphorylated to a substantial degree; this normal phosphorylation was apparently not affected by CIII. The ratio of densitometrically measured Tau-1 signal over the Tau-1 signal after dephosphorylation, a normalizing measure of the total tau population, formed the basis for quantitative analysis to determin IC.sub.50 values.

Results

Inhibitors of PK40(ERK2), exemplified by CIII, indeed prove capable of preventing abnormal AD-like hyperphosphorylation in a SY5Y cell model system. FIG. 1 shows that increasing concentrations of CIII prevent the okadaic acid provoked hyperphosphorylation of tau. This protective effect is highly correlated with the prevention of the activating phosphorylation of ERK2 in the same cells. By binding to ERK2, CIII is able to both inhibit the activity of ERK2 as well as its activation (either via autophosphorylation or via another kinase), with both effects essentially eliminating cellular tau hyperphosphorylating activity. Moreover, the normal cellular phosphorylation state of tau is not affected by CIII in the same concentration range, demonstrating a case of cellular selectivity (not shown).

Example 9

Determination of Potency of Inhibitors to Prevent AD-like Tau Hyperphosphorylation in Rat Hippocampal Brain Slices

Methods

Adult male Long-Evans rats were subjected to CO.sub.2 anesthesia and sacrificed by decapitation. Brains were rapidly removed (<2 min) and whole hippocampus was dissected using a blunt spatula. Hippocampi were cut into 450 mM slices using a McIlwain tissue chopper and placed into ice cold low Ca.sup.2+ Krebs-Bicarbonate buffer (pH 7.) of the following composition in mM: NaCl, 124; KCL, 3.33; CaCl.sub.2, 0.01; KH.sub.2 PO.sub.4, 1.25; MgSO.sub.4 1.33; nAhco.sub.3, 25.7; D-glucose, 10; HEPES, 20. The slices were separated and placed, 5-8 per tube, into 5 mL of low Ca.sup.2+ buffer and incubated for at least 30 min at 33-34.degree. C. with water saturated oxygenation (95% O.sub.2, 5% CO.sub.2). After 30 min the solution was replaced with buffer containing a physiological level of Ca.sup.2+ (1.3 mM) and incubated for an additional 30 min.

After a total equilibration period of at least 1 hr, the slices were pretreated with vehicle or inhibitor at concentrations ranging from 30 nM to 10 .mu.M for 1 hr, and then exposed to either vehicle or okadaic acid fora 90 min. After treatment, the buffer was removed and the slices were sonicated for 10-20 sec in 500 .mu.l of homogenization buffer (100 mM KH.sub.2 PO.sub.4, pH 6.5, 2 mM EGTA, 2 mM EDTA, 1 .mu.M okadaic acid and the following protease inhibitors: aprotinin (10 .mu.g/ml); leupeptin (10 .mu.M); chymostatin (40 .mu.M); PMSF (100 .mu.M) and pepstatin (6 .mu.g/ml).

Following sonication, the samples were centrifuged 16,000.times. g for 30 min and the supernatants were removed. After boiling of the supernatants for 5 mn at 100.degree. C. the concentration of protein was determined by the BCA assay (Pierce) using BSA as standard and samples were normalized to equal protein concentration.

Aliquots of the heat stable supernatants were separated on 10% SDS-PAGE and Western-blotted with phosphorylation-sensitive tau mAb Tau-1 and PHF-tau mnAb AT8 as described for SY5Y studies. Blots were developed by an ECL kit (Amersham Life Science). AT8 immunoreactivity was quantitated on Kodak X0OMAT AR film using a Biorad imaging densitometer GS 670, the strongest signals not exceeding an O.D. of 12.

Results

Freshly isolated hippocampal brain slices from adult rats were used for similar experiments under conditions more relevant to the brain (FIG. 5). Again, okadaic acid induced AD-like tau hyperphosphorylation, while CII prevented it with the same IC50 as in SY5Y cells (0.1 .mu.M).

Okadaic acid induced reactivity with the novel phosphorylation dependent mAb AP422. This response was inhibited at the same dose as the response with the more conventional mAb AT8 (FIG. 5), indicating a single tau hyperphosphorylating activity. In vitro reactivity of tau with this mAb can only be induced by ERK2, but not other candidate tau kinases (e.g. cdk, GSK3), providing an independent criterion that ERK2 is the relevant drug target.

The intensity of AP422 reactivity induced by PK40(ERK2) in vitro matches that of isolated PHF-tau from AD-brain (not shown). In contrast, even with the most conservative precautions to avoid post-mortem dephosphorylation in rat brains, AP422 reactivity is completely absent in normal adult tau. This suggests that tau hyperphosphorylation in AD is qualitatively abnormal, and does not involve enhanced activity of normal kinases, but rather the pathological activation of ERK2 as an abnormal tau kinase.

Prevention of AD-like tau hyperphosphorylation in adult rat hippocampal brain slices. In an experimental paradigm similar to SY5Y cells tau hyperphosphorylation is prevented by derivative CII at similar doses as in SY5Y cells.

Note that the results with AP422, currently the most specific criterion for AD-like tau hyperphosphorylation, are identical to those with the commonly used mnAb AT8, indicating that ERK2 alone is responsible for all okadaic acid induced changes in tau phosphorylation.

TABLE 1 ______________________________________ Properties of Preferred Compounds as Inhibitors of ERK2 (PK40), Activation of ERK2, cdc2, and Tau Hyperphosphorylation in Biological Models of PHF-tau formation (IC.sub.50 values in .mu.M) CII CIII ______________________________________ PK40 (ERK2) 0.044 >>30 cdc2 0.044 3.3 PKA 0.65.sup.a) >100 PKC 0.65.sup.a) >100 Inhibition in SY5Y 0.57 5.7 cells.sup.b) of ERK2 activation tau hyperphos. 0.58 3.6 Inhibition of tau 0.18 0.9 hyperphos. in brain slices.sup.b) ______________________________________ .sup.a) = partial inhibition only .sup.b) = means of triplicate determinations .sup.c) = concomitant inhibition of normal tau phosphorylation

Abstract
A method for screening individuals at risk for prostate cancer progression is disclosed. The method is useful for evaluating cells from patients at risk for recurrence of prostate cancer following surgery for prostate cancer. Specifically, the method uses specific Markovian nuclear texture factors, alone or in combination with other biomarkers, to determine whether the cancer will progress or lose organ confinement. In addition, methods of predicting the development of fatal metastatic disease by statistical analysis of selected biomarkers is also disclosed. The invention also contemplates a method that uses a neural network to analyze and interpret cell morphology data. Utilizing Markovian factors and other biomarkers as parameters, the network is first trained with a sets of cell data from known progressors and known non-progressors. The trained network is then used to predict prostate cancer progression in patient samples.
Government Interests

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The government may own rights in the present invention pursuant grant number P50-CA58236-02 from the National Institutes of Health.
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Claims

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What is claimed is:

1. A method of predicting prostate cancer progression, comprising:

(a) obtaining prostate cells from a subject;

(b) analyzing predictive parameters in the prostate cells, wherein the predictive parameters are nuclear morphometric descriptors, including: object sum optical density, picograms of DNA, contrast, correlation, sum average, sum variance, difference variance, difference entropy, information measure B, product moment, standard deviation, and DNA ploidy; and

(c) predicting cancer progression by statistical analysis of the predictive parameters, where the statistical analysis is logistic regression, discriminate analysis, recursive partitioning, neural network, or classification and regression tree analysis.

2. The method of claim 1, wherein the nuclear morphometric descriptors are selected from the group consisting of object sum optical density, object shape, picograms of DNA, contrast, correlation, inverse difference moment, sum average, sum variance, difference variance, difference entropy, information measure B, product moment, standard deviation, and DNA ploidy.

3. The method of claim 1, wherein the nuclear morphometric descriptors are selected from the group consisting of object sum optical density, object size, object shape, picograms of DNA, angular second moment, contrast, correlation, difference moment, inverse difference moment, sum average, sum variance, sum entropy, entropy, difference variance, difference entropy, information measure A, information measure B, maximal correlation coefficient, coefficient of variation, peak transition probability, diagonal variance, diagonal moment, second diagonal moment, product moment, triangular symmetry, blobness, standard deviation, and DNA ploidy.

4. The method of claim 1, wherein the predictive parameters further include Post-op Gleason.

5. The method of claim 4, wherein the predictive parameters further include PD-41 antigenic expression and Her-2-neu antigenicity.

6. The method of claim 1, wherein the nuclear morphometric descriptors are Markovian nuclear texture features.

7. The method of claim 1, wherein the statistical analysis is univariate or multivariate analysis.

8. The method of claim 7, wherein the statistical analysis is multivariate analysis.

9. A method for predicting the recurrence of prostate cancer following radical prostatectomy comprising the steps of:

(a) obtaining prostate cells from a subject;

(b) generating nuclear morphometric descriptors for the cells, including: object sum optical density, picograms of DNA, contrast, correlation, sum average, sum variance, difference variance, difference entropy, information measure B, product moment, standard deviation, and DNA ploidy; and

(c) predicting the recurrence of prostate cancer in the cell samples by statistical analysis of the nuclear morphometric descriptors, where the statistical analysis is logistic regression, discriminate analysis, recursive partitioning, neural network, or classification and regression tree analysis.

10. The method of claim 9, wherein the statistical analysis is multivariate statistical analysis.

11. The method of claim 10, further comprising statistical analysis of predictive parameters selected from the group consisting of Post-op Gleason, nuclear roundness variance, PD-41 antigenic expression and Her-2-neu antigenic expression.

12. The method of claim 11, wherein the nuclear morphometric descriptors are Markovian nuclear texture features.

13. A method of predicting the occurrence of fatal metastatic prostate disease comprising the steps of:

(a) obtaining prostate cells from a subject;

(b) generating nuclear morphometric descriptors for the cells, including: object sumn optical density, picograms of DNA, contrast, correlation, sum average, sum variance, difference variance, difference entropy, information measure B, product moment, and standard deviation; and

(c) predicting the occurrence of fatal metastatic prostate disease by statistical analysis of the nuclear morphometric descriptors.

14. The method of claim 13, further comprising statistical analysis of predictive parameters selected from the group consisting of post-op Gleason DNA ploidy, PD-41 antigenic expression, or Her-2-neu antigenic expression.

15. The method of claim 14, wherein the predictive parameter is Her-2-neu antigenic expression.

16. A method of predicting the progression of prostate cells from a normal state to a malignant state comprising the steps of:

(a) obtaining prostate cells from a subject;

(b) generating nuclear morphometric descriptors for the cells;

(c) analyzing selected cell biomarkers; and

(d) predicting the progression of the cells by using multivariate statistical modeling of the nuclear morphometric descriptors and the selected biomarkers.

17. The method of claim 16, wherein the selected biomarkers are PD-41 antigenic expression and Her-2-neu antigenic expression.

18. The method of claim 16, wherein the nuclear morphometric descriptors include Markovian texture features.

19. A method of determining prostate cancer progression comprising: (a) providing a neural network;

(b) training the neural network using predictive parameters, obtained from prostate cells known to progress and a set of predictive parameters obtained from prostate cells known not to progress, the predictive parameters comprising nuclear morphometric descriptors;

(c) analyzing dredictive parameters in tumor cells of an individual having an unknown state of cancer progression; and

(d) predicting cancer progression in cells of the individual having an unknown state of cancer progression using the predictive parameters and the trained neural network.

20. The method of claim 19, wherein the nuclear morphometric descriptors are selected from the group consisting of object sum optical density, object shape, picograms of DNA, contrast, correlation, inverse difference moment, sum average, sum variance, difference variance, difference entropy, information measure B, product moment, standard deviation, and DNA ploidy.

21. The method of claim 19, wherein the nuclear morphometric descriptors are selected from the group consisting of object sum optical density, object size, object shape, picograms of DNA, angular second moment, contrast, correlation, difference moment, inverse difference moment, sum average, sum variance, sum entropy, entropy, difference variance, difference entropy, information measure A, information measure B, maximal correlation coefficient, coefficient of variation, peak transition probability, diagonal variance, diagonal moment, second diagonal moment, product moment, triangular symmetry, blobness, standard deviation, and DNA ploidy.

22. The method of claim 19, wherein the neural network is of the back propagation type.

23. The method of claim 19, wherein the neural network is of a hybrid type.

24. The method of claim 19, wherein the prognostic parameters of step (b) further include post operative Gleason score, PD-41 antigenic expression, or Her-2-neu antigenic expression.

25. The method of claim 1, 9 or 13, further comprising analyzing one or more nuclear morphometric descriptors selected from the group consisting of object size, object shape, angular second moment, difference moment, inverse difference moment, sum entropy, entropy, information measure A, maximal correlation coefficient, coefficient of variation, peak transition probability, diagonal variance, diagonal moment, second diagonal moment, triangular symmetry, perimeter, DNA index, density, average optical density, feret X, feret Y, maximum diameter, minimum diameter and elongation.

26. The method of claim 16 or claim 19, wherein the nuclear morphometric descriptors are selected from the group consisting of object sum optical density, object size, object shape, picoprams of DNA, angular second moment, contrast, correlation, difference moment, inverse difference moment, sum average, sum variance, sum entropy, entropy, difference variance, difference entropy, information measure A, information measure B, maximal correlation coefficient, coefficient of variation, peak transition probability, diagonal variance, diagonal moment, second diagonal moment, product moment, triangular symmetry, standard deviation, DNA ploidy, perimeter, DNA index, density, average optical density, feret X, feret Y, maximum diameter, minimum diameter and elongation.

27. A method of predicting the progression of prostate cancer comprising the steps of:

(a) obtaining prostate cells from a subject;

(b) analyzing predictive parameters from the prostate cells, the predictive parameters including nuclear morphometric descriptors;

(c) utilizing statistical analysis to determine multivariately significant nuclear morphometric descriptors to calculate a quantitative nuclear grade; and

(d) predicting the probability of progression in the patient by statistical analysis of the quantitative nuclear grade.

28. The method of claim 27, further including the steps of analyzing predictive parameters from the prostate cells including utilizing statistical analysis to determine univariately significant patient derived pathology and clinical information variables that contribute to a multivariate model solution and predicting the probability of progression in the patient by further statistical analysis of the quantitative nuclear grade and urivariately significant patient derived pathology and clinical information variables.

29. The method of claim 27, wherein the nuclear morphometric descriptors include Markovian nuclear texture features.

30. The method of claim 13 or claim 16, wherein the statistical analysis is logistic regression, discriminate analysis, recursive partitioning, classification and regression tree analysis, or neural network.

31. The method according to claim 17, wherein the patient derived pathology and clinical information variables are selected from the group consisting of post operative Gleason score, serum PSA, PD-41 antigenic expression, or Her-2-neu antigenic expression.
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Description

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BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to fields of computer-assisted quantitative image analysis and methods to classify cells related to cancer progression. More specifically, it concerns methods to detect patients at risk for progression following radical surgery that have been diagnosed with localized prostate cancer by current state of the art clinical pathology and clinical laboratory methodology. Additionally, it concerns using the same approach to predict organ confined disease status using pre-treatment information extracted from the core biopsy materials.

2. Description of the Related Art

Prostate cancer is diagnosed in 100/100,000 white males and in 70.1/100,000 black males in the United States. It is the second leading cause of male cancer deaths and the most commonly diagnosed cancer in men in the United States representing 21% of all newly diagnosed cancers. In 1993 an estimated 165,000 men in the United States were diagnosed with clinically apparent prostate cancer and 35,000 will succumb to the disease. The age-specific increase in incidence achieves a maximum of 1000/100,000 in men >75 years of age. The lifetime risk of developing clinical prostate cancer in the U.S. is 8.7% for white and 9.4% for black Americans with a lifetime risk of dying being 2.6% and 4.3% respectively. The risk of developing prostate cancer has risen 42.6% since 1975 as compared to an increase of only 26% in risk of developing lung cancer for that same time period. Approximately 65% of prostate cancers are clinically localized at the time of diagnosis and potentially curable with standard surgical techniques, yet only 50% of men are found to have disease confined to the prostate at the time of surgery. Pack and Spitz (Pack R. and Spitz M. A. The Cancer Bulletin, 45:384-388, 1993), reviewing the epidemiology of prostate cancer, indicated several definable risk factors such as age, race, dietary fat consumption, vasectomy, and familial aggregation with at least a two-fold increased risk for first generation relatives of men with prostate cancer (rare autosomal dominant inheritance). These causal correlations, though impressive, can not yet explain the complex etiology, biologic heterogeneity, and rapidly increasing incidence of this disease, and await further investigations of genetic, epigenetic and environmental factors.

The mortality rate for prostate cancer has been steadily increasing over the past 40 years and will continue to do so as our population ages. This clinically evident disease represents only the tip of the iceberg in that nearly 30 percent of all men over age 50 harbor a silent microscopic form of latent prostate cancer. Current early detection methods are increasing the numbers of this latent form of cancer identified, which now represent more than 11 million cases within the male population in the United States, and growth rate studies indicate that these tumors appear to grow very slowly and the great majority should remain clinically silent.

Recent advancements in transrectal ultrasonography and the development of a serum based assay (prostate specific antigen, PSA) for early detection has caused the diagnosis of premalignant neoplasias as well as prostate cancer to increase at an alarming rate. Many of these newly diagnosed neoplasias could represent the non-aggressive, potentially latent form of the disease that may never have become clinically evident if followed without therapy. Unfortunately, no accurate and specific methods presently exist to distinguish the more potentially aggressive form of prostate cancer from the latent form of the disease; thus most patients diagnosed are presently treated as though they had the aggressive form of the disease. At present, the factors to be considered in assessing cancer progression are estimates and significance of tumor volume, pre- and post-operative histological grading of cancer and high grade intraepithelial neoplasia, clinical and pathological stage, and serum prostate specific antigen (PSA) to predict biological aggressiveness of prostate cancer. These techniques generally have only marginal predictive value.

It is well accepted that the epigenetic and genetic transformation of a normal prostatic epithelial cell to a cancer cell with progression to a metastatic phenotype requires multiple steps. The development of methods to quantify accurately these changes in order to better predict tumor aggressiveness has been the subject of much experimental work in prostate cancer.

Diamond and associates (Diamond et al., Prostate, 3:321, 1982; Diamond et al., J. Urol., 128:729, 1982) were the first to employ a simple nuclear shape factor (nuclear roundness) to describe the shape of cancerous nuclei for patients with stage B1 and B2 (Whitmore-Jewett staging) prostate cancer and accurately predicted outcome for these patients. Since then several investigators have used this method to predict prognosis for patients with various stages of prostate cancer. More recently, Partin et al. (Partin et al., Cancer 70:161-168, 1992.19) used a multivariate analysis of the variance of nuclear roundness, clinical stage, Gleason score, and the patients age to predict disease free survival among a group of 100 post-operative patients with localized prostate cancer. The use of chromatin texture feature data extracted from either H&E or Feulgen stained sections correlate well to classification of malignant cells. However, the sensitivity of Markovian texture measurements is complicated by the level of pixel gray level resolution (grain). Dawson et al. (Dawson et al., Analytical and Quantitative Cytology and Histology, 15:227-35, 1993.47) used a CAS-100 Image Analysis System and software to measure 22 Markovian texture features at 20 levels of pixel resolution (grain) and found ten features that discriminated chromatin patterns in breast cancer images captured by the CAS-100. Markovian analysis is a method based on determining gray-level transition probabilities and it allows discrimination among different nuclear texture features; the value for each feature depending on the level of grain resolution for each measurement.

Christen et al. (Christen et al., Analytical Quant. Cytol. Histol., 15: 383-388, 1991) have applied a linear discriminant statistical model analysis of shape, size and texture features of H&E stained prostate nuclei to a high efficiency, 93% correct classification of normal and abnormal cells. More recently, Irinopoulou et al. (Irinopoulou et al., Analytical and Quantitative Cytology and Histology, 15: 341-44, 1993) employed Feulgen stained nuclei and a computer-assisted image analysis system to characterize digitized images (512.times.512 pixels, with 256 possible gray tone levels) from twenty-three patients with Stage B carcinoma of the prostate followed for at least three years. Using five chromatin texture features and discriminant analysis methodology, these patients could be divided into those with a good and poor prognosis.

In spite of the progress made in evaluating the progression of prostate cancer, it is evident that improvements are needed in the accuracy of such determinations. A particular advantage would be realized by the development of methods that provide for accurate and reproducible statistical analysis of prognostic variables to maximize the aggregate positive predictive value while simultaneously reducing false negatives and false positives.

SUMMARY OF INVENTION

The present invention provides new and improved methods for determining the biological potential for progression of treatable, localized prostate cancers using core biopsies, fine needle aspirates, or radical prostatectomy specimens in order to: (1) better evaluate which patients have tumors that need any treatment, (2) determine the prognosis of patients with prostate cancer pathologically localized to the gland, after surgery, so that adjuvant treatment of those patients with a high probability of disease progression might begin earlier in the natural course of the disease, and finally, (3) provide more objective means to select patients for chemoprevention trials using dietary modifications, retinoids, and hormonal manipulation (i.e. 5-alpha reductase inhibitors).

For the purposes of this invention, progression is defined as recurrence of disease post-treatment (surgery or irradiation), for example in the case of prostate cancer, as determined by PSA elevation, clinical evidence of local or regional tumor recurrence, distant metastasis, or death. Organ confined disease status is defined as prostate cancer that is still contained within the prostate gland and has not invaded the prostatic capsule.

In certain embodiments of the invention, a statistically analyzed combination that includes quantitative nuclear image features selected from pathologically important tissue sections and appropriately selected biomarkers provides an aggregate positive predictive value with negligible false negatives and false positives that exceeds current conventional pathological methods. In addition, this approach affords the probability of identifying additional potential utilities for biomarkers to identify progressors with pathologically defined low to moderate Gleason score (.ltoreq.6) as being of higher risk due to the presence of such biomarkers. Also, select biomarkers served to identify tumors that have extended beyond the prostate gland (non-organ confined disease). The aggregate positive predictive power of the model, all parameters (i.e. quantitative nuclear image features and biomarkers), was achieved using clinically and pathologically well defined, long term (>7 years follow-up) retrospective patient samples. The ultimate goal of this invention is to apply these predictive capabilities to prostate biopsies (both progression and organ confined disease status) and/or post-surgery prostatectomy samples (progression risk only).

This invention provides a method to collect nuclear images and extract all relevant nuclear morphometric descriptors (NMD’s), including size, shape, texture (Markovian analysis), and DNA content features. Additionally, other phenotypic cancer cell properties were assessed through the use of antibody probes for cellular biomarkers (e.g. Her-2/neu, Feulgen DNA stain, PD-41 (prostate mucin antigen), etc.). The NMD’s combined with the biomarkers can then be analyzed to construct a non-parametric, non-linear mathematical model (e.g. Applying statistical methods such as logistic regression; discriminate analysis (Bayesian classifier or Fischer analysis); recursive partitioning methods (Classification and Regression Trees, or CART); or neural networks (both standard and proprietary)), that can yield a single predictive probability for prostate cancer progression or organ confinement, with or without conventional pathological grading. The pathologically significant areas are identified by an expert trained in the identification of abnormal cells and tissue architecture associated with malignancies of the prostate. Such abnormalities may be present in core biopsies, fine needle aspirates, or radical prostatectomy specimens that have been fixed using methods that preserve the antigenicity of the biomolecules of diagnostic significance, cellular architecture, and integrity of the deoxyribonucleic acid (DNA) or chromatin.

According to the present invention, a method of predicting prostate cancer progression or organ confined disease status is provided, comprising the steps of first obtaining a clinical sample from a subject, then analyzing cell nuclei from areas selected by pathology experts and collecting the NMD’s as well as phenotypic cellular biomarker information, and thirdly, predicting prostate cancer progression or organ confined disease status using non-parametric statistical analysis of the data. Cell sampling for image analysis involves the selection of intact cell nuclei representative of the worst state of differentiation as well as, when present, well to moderately differentiated cancer cells. This provides a measure of tumor heterogeneity often present in prostate cancer. It is suggested that at least 50% of the cells analyzed be of the worst state of cellular differentiation present in the clinical sample, and that the remainder of the cancer cells analyzed represent the well to moderately differentiated cell population, if present, in the clinical sample.

In certain embodiments, the resulting data includes biomarker results (e.g. including but not limited to Her-2/neu antigenicity, PD-41 positivity, and DNA ploidy) and NMD’s, calculated based on nuclear size and shape, as well as texture features derived by nearest neighbor relational analysis of individual pixel gray levels to mathematically determine several features (e.g. object sum, picograms of DNA, contrast, correlation, sum average, sum variance, difference variance, difference entropy, information measure B, product moment, and standard deviation). For the purposes of this invention, these parameters are collectively referred to as prognostic parameters.

Clinical samples obtained from patients at risk for recurrence of prostate cancer following prostatectomy are analyzed and values generated for various prognostic parameters, including the nuclear morphometric descriptors (NMD’s), Her-2/neu antigenicity, PD-41 (prostate mucin antigen) positivity above a 5% cutoff, and nuclear roundness variance (NRV-DynaCell.TM.). Summary statistics from the NMD’s (e.g. standard deviation and variance) and raw data from the biomarkers are analyzed using logistic regression. The logistic regression may be applied in a univariate or multivariate mode. As used herein, multivariate analysis means that several univariately significant independent variables are jointly regressed on the same dependent variable. A dependent variable refers to a clinical outcome (e.g. Progression as determined by PSA elevation, local or regional tumor recurrence, distant metastasis, or death), or a pathological disease state (e.g. organ confinement status). Based upon significance levels, the statistical program selects only those univariately significant independent variables that contribute to the correct prediction of the dependent variable (e.g. progression or organ confined disease status). Notable is the fact that future changes in the model, such as measurement of NMD’s (e.g. different magnifications for collection, improved camera resolution, etc.) and or additional biomarkers, may change the parameters needed and only improve upon the predictive power of the models by small percentages so it may approach 100%.

As used herein, a receiver operating characteristic (ROC) curve plots an independent variable’s sensitivity (true positive fraction) on the y-axis against 1-specificity (the false positive fraction) on the x-axis as the cutoff value for a predicted positive observation is varied. A positive observation means that the predicted probability is greater than or equal to an investigator selected cutoff value. The ROC curve or plot is useful for determining the sensitivity, specificity, and negative and positive predictive values of a single test or a multiparameter test. In addition, the ROC curve can be used to establish the optimum threshold cutoff for a continuous variable. When quantitating the area under a ROC curve, an area of 1.0 means a perfect predictive value, while an area of 0.5 means no predictive value and is due to random chance alone.

For the purposes of the invention, sensitivity is the fraction of observed positive cases that are correctly classified and equals: true positives.div.{true positives+false negatives}. The positive predictive value equals: true positives.div.{true positives+false positives}.

Specificity is the fraction of observed negative cases that are correctly classified and equals: true negatives.div.{true negatives+false positives}. The negative predictive value equals: true negatives+{true negatives+false negatives}.

As used herein, Markovian analysis means a process by which an image (pattern space) is transformed into a transitional-probability space. The Markovian approach to texture measurement treats images as stochastic processes in the form of discrete Markovian fields, yielding matrices of gray-level transition probabilities. These probabilities are arrays of numbers that describe digital images in terms of the probability of occurrence of different gray levels and sequences of gray levels. The current invention uses 22 texture parameters calculated from such matrices.

Texture is an important visual feature for many pattern recognition tasks. As used herein, texture describes the interdependent characteristics of pixels within a neighboring area. Regular texture has more or less periodic patterns, while random texture is best described by its “coarseness”. A texture parameter is a local statistic, meaning that the statistic is computable from a set of neighboring picture points in a neighborhood that is small compared to the number of picture points in the entire region.

A Markovian matrix of the present invention is constructed using a cell nucleus image captured in the QDA Morphology mode of the CMP v3.0 software on a CAS-200 Image Analysis System. Using the QDA Morphology mode of CMP v3.0 software allows the measurement and calculation of the features listed in Table I. CMP v3.0 calculates 22 different Markovian nuclear descriptors based upon the gray-level transition probabilities of Feulgen stained nuclei. The step size selected may range from 1 pixel to 256 pixels. The step size defines the size of the “grain” or picture point in number of pixels that is to be compared to the neighboring picture points. Each cell nucleus image is normalized by partitioning the image into eight equally frequent gray level ranges, each range consisting of an equal number of pixels. This normalization process is done by first plotting each individual pixel optical density (gray level) that is above an operator set threshold against the number of pixels. This plot is divided into eight equally frequent gray-level ranges (optical density ranges); each range containing an equal number of pixels (FIG. 1A and FIG. 1B). This yields a normalized cell nucleus image consisting of pixels with gray-level values ranging from 0-7.

TABLE I ______________________________________ Nuclear Morphometric Descriptors Measured Using CMP v3.0 in the QDA Morphology Mode ______________________________________ 1. (OBSD) Object Sum OD 2. (OBSZ) Object Size 3. (OBSH) Object Shape 4. Picograms of DNA 5. (TXA001) Angular Second Moment 6. (TXB001) Contrast 7. (TXC001) Correlation 8. (TXD001) Difference Moment 9. (TXE001) Inverse Difference Moment 10. (TXF001) Sum Average 11. (TXG001) Sum Variance 12. (TXH001) Sum Entropy 13. (TXI001) Entropy 14. (TXJ001) Difference Variance 15. (TXK001) Difference Entropy 16. (TXL001) Information Measure A 17. (TXM001) Information Measure B 18. (TXN001) Maximal Correlation Coeff. 19. (TXO001) Coefficient of Variation 20. (TXP001) Peak Transition Probability 21. (TXQ001) Diagonal Variance 22. (TXR001) Diagonal Moment 23. (TXS001) Second Diagonal Moment 24. (TXT001) Product Moment 25. (TXU001) Triangular Symmetry 26. (TXV001) Blobness 27. (TXW) Standard Deviation 28. Cell Classification (1 = Hypodiploid, 2 = Diploid, 3 = S-Phase, 5 = Tetraploid, 6 = Hyperploid) ______________________________________ NOTE: Values 5-25 are grain dependent Markovian texture features. Grain may be looked at as a measurement in pixels of the width of an average sized object. The grain values for all of these measurements were set to 1.

As a further step, an 8.times.8 gray-level transition matrix is constructed from the normalized cell nucleus image by comparing the gray-levels of neighboring picture points (e.g. if a given picture point has a normalized value of 4, and its neighboring picture point has a normalized value of 3, an entry is made in the matrix at location Row-4 and Column-3, and all of the entries at this location are summed). This matrix is then transformed into an 8.times.8 conditional gray-level transition probability matrix by dividing every matrix entry by the total number of pixels in the cell nucleus image. This “Markovian” probability matrix (Equation 1) is then used to compute the 22 Markovian texture features (Table I). ##EQU1## where each matrix element P.sub.L (i/j) is defined as the conditional probability of gray-level i occurring L picture points after gray-level j occurs, where L is defined as the step size (or size in pixels of the picture point).

More recently, JVB Imaging (Elmhurst, Ill.) has written a software application called ILM Morphometry v1.0 that can be applied to listmode files (*.ILM) generated using a CAS-200 Image Analysis System. This program therefore allows the measurement and calculation of the same features as the CMP v3.0 software (Markovian and DNA Content features) as well as eight additional features (listed in Table II). The inventors have already tested this new software on the patient sample reported in this invention and obtained similar statistical model performance as with the CMP v3.0 software.

TABLE II ______________________________________ Nuclear Morphometric Descriptors Measured Using ILM Morphometry v1.0 ______________________________________ 1. Object Sum Optical Density 2. Object Size 3. Object Shape 4. Picograms of DNA 5. Angular Second Moment 6. Contrast 7. Correlation 8. Difference Moment 9. Inverse Difference Moment 10. Sum Average 11. Sum Variance 12. Sum Entropy 13. Entropy 14. Difference Variance 15. Difference Entropy 16. Information Measure A 17. Information Measure B 18. Maximal Correlation Coefficient 19. Coefficient of Variation 20. Peak Transition Probability 21. Diagonal Variance 22. Diagonal Moment 23. Second Diagonal Moment 24. Product Moment 25. Triangular Symmetry 26. Standard Deviation 27. Cell Classification (1 = Hypodiploid, 2 = Diploid, 3 = S-Phase, 5 = Tetraploid, 6 = Hyperploid) 28. Perimeter 29. DNA Index 30. Density 31. Average Optical Density 32. Feret X 33. Feret Y 34. Maximum Diameter 35. Minimum Diameter 36. Elongation ______________________________________ NOTE: Values 5-25 are grain dependent Markovian texture features. Grain may be looked at as a measurement in pixels of the width of an average sized object. The grain values for all of these measurements were set to 1.

In exemplary embodiments of the invention, consideration must be given to several parameters, such as cell selection, magnification, pixel shape and size, camera resolution (number of pixels in the x and y dimension, e.g. number of pixels per .mu.m.sup.2), and Markovian step size, which can significantly alter nuclear morphometric descriptor outputs (FIG. 2). The NMD’s have been demonstrated in this invention to be significant independent variables in the prediction of tumor progression and organ confined disease status. As previously indicated, the method for cell selection is critical because of the need to sample biologic tumor heterogeneity. Additionally, the total number of NMD’s required to predict an outcome is decreased as the magnification increases (Table III; also see FIGS. 3-6), as well as significant changes in the individual NMD’s required. The latter is due to an increase in the number of pixels per .mu.m.sup.2 that would enhance the resolution of calculations for the NMD’s. Also notable, the predictive power for all outcomes using the NMD component of the model increases as the magnification increases.

TABLE III ______________________________________ Magnification Effects (40.times. vs. 63.times.) upon Number of Nuclear Morphometric Descriptors needed to Accurately Predict Progression in a Subset of 10 Progressors and 10 Non-Progressors ______________________________________ 40.times. 63.times. CMP v3.0* JVB v1.0** CMP v3.0* JVB v1.0** ______________________________________ P Value Cutoff 0.45 0.50 0.25 0.70 Sensitivity 90.00% 90.00% 100.00% 90.00% Positive Pred. 90.00% 100.00% 76.92% 100.00% Value Specificity 90.00% 100.00% 70.00% 100.00% Negative Pred. 90.00% 90.91% 100.00% 90.91% Value # False 1 0 3 0 Positives # False 1 1 0 1 Negatives Area Under 0.9400 0.9500 0.9400 0.9600 ROC Curve ______________________________________ 5 Markovian & Sum O.D, NMD’s 6 Markovian Area, 5 Markovian 4 Markovian in Model & Area perimeter & Area & Sum O.D. ______________________________________ # Concordant 3 3 NMD’s # Discordant 4 5 3 2 NMD’s ______________________________________ *Number of CMP Nuclear Morphometric Descriptors (NMD’s) = 28 **Number of JVB Nuclear Morphometric Descriptors (NMD’s) = 36

In other embodiments, either non-parametric statistical methods, or standard or proprietary neural networks were used to validate the utility of NMD’s and biomarkers for the prediction of two possible outcomes, progression and organ confined disease status. Using a clinically and pathologically well-defined retrospective patient sample diagnosed with localized prostate cancer, logistic regression methods were applied on a well defined patient sample (n=124) (Table IV) to determine which NMD’s and biomarkers were capable (e.g. statistically significant) of predicting either progression or organ confined disease status.

LOGISTIC REGRESSION PROGRESSION MODEL

The invention applies logistic regression to select the univariately significant variables for progression (Table V) using the STATA.TM. statistical software package (STATA.TM. command: logistic). Next, these univariately significant variables are multivariately assessed using backwards stepwise logistic regression (STATA.TM. command: swlogis) to determine which independent variables (e.g. NMD’s (CMP or JVB), Gleason Score, Nuclear Roundness Variance, and biomarkers) are retained to predict progression (Tables VI, Via, VII, and VIIa).

TABLE IV __________________________________________________________________________ JHH-1 PATIENT SAMPLE __________________________________________________________________________ Average Age: 59.6 .+-. 6.4 years [40-87 yrs] Non-progressors: 74(60%) First operation: Jun-’75 Progressors: 50(40%) Last Operation: Jun-’91 Avg. Time to Prog: 3.62 .+-. 2.1 years Time to assess: 6.6 .+-. 3.1 years [1-15 yrs] Local Recurrence: 11(9%) Clin. Stage A1,A2 6% Distant Mets: 5(4%) Clin. Stage B1,B2 94% __________________________________________________________________________ GLEASON GRADING PATHOLOGY DIAGNOSIS SCORE PRE-OP POST-OP __________________________________________________________________________ STAGE T1b 2(2%) FCP+ = 89(72%) 2 0% 1% STAGE T1c 1(1%) FCP- = 35(28%) 3 1% 0% STAGE T2a 72(58%) ECP+ = 52(42%) 4 11% 1% STAGE T2b 45(36%) ECP- = 72(58%) 5 25% 27% STAGE T2c 4(3%) OC NO = 95(77%) 6 41% 28% OC Yes = 29(23%) 7 19% 36% SM+ = 52(42%) 8 2% 6% SM- = 72(58%) 9 0% 1% SV+ = 0 LN+ = 0 __________________________________________________________________________

TABLE V ______________________________________ Univariate Analysis for Progression Prediction using STATA .TM. Logistic Regression Statistical Significance Independent Variable (p Value) ______________________________________ Post Operative Gleason Score p .ltoreq. 0.00001 Nuclear Roundness Variance* p .ltoreq. 0.00001 Best CAS-200 CMP v3.0 Nuclear Morphometric p .ltoreq. 0.00001 Descriptors Best CAS-200 JVB v1.0 Nuclear Morphometric p .ltoreq. 0.00001 Descriptors CAS-200 DNA Ploidy – 1 (C-DNA1) p = 0.0080 CAS-200 DNA Ploidy – 10 (C-DNA10) p = 0.0274 CAS-200 DNA Ploidy – JIE (JHHDNA10) p = 0.0109 Her-2/neu Antigenicity: Focal, Diffuse, Negative p = 0.0147 (H2NFDN) PCNA Antigenicity p = 0.1600** PD-41 Antigenicity above a 5% cutoff p = 0.3045** ______________________________________ *Measured using DynaCell .TM. system at Johns Hopkins Hospital **Not Statistically Significant. Must be less than 0.0500 to be statistically significant.

TABLE VI __________________________________________________________________________ Univariate/Multivariate Analysis for Prostate Cancer Progression {N = 124 Radical Prostatectomy Specimens} Progressors/Non-Progressors: 50/74 Statistical Analysis of JHH-1 Database using Univariately Significant STDEV & VAR Statistics for CMP Texture Features, Biomarkers, Post Gleason, % JHH-NRV in Progression Prediction M N K L CMP CMP CMP CMP Nuclear Nuclear J Nuclear Nuclear Des- Des- H I CMP Des- Des- criptors criptors D2 CMP CMP Nuclear criptors criptors (n = 10)* (n = 13)* CMP Nuclear Nuclear Des- (n = 12)* (n = 12)* Bio- Bio- C Nuclear Des- Des- criptors Bio- Bio- markers markers Bio- Des- criptors criptors (n = 13)* markers markers (n = (H2NFDN) A B markers criptors (n = 13*) (n = 10)* JHH-NRV (n = (n = 2)*** 2)**** JHH-NRV Post GL JHH-NRV (n = 3)** (n = 12)* Post GL JHH-NRV Post GL 2)**** Post GL JHH-NRV Post GL __________________________________________________________________________ Positive Cutoff 0.50 0.35 0.40 0.35 0.40 0.40 0.50 0.45 0.45 0.45 0.50 (p>=#) SENSITIVITY 78.00% 86.00% 62.00% 86.00% 84.00% 86.00% 94.00% 78.00% 86.00% 92.00% 96.00% Pos Predictive 73.58% 78.18% 54.39% 65.15% 85.71% 84.31% 97.92% 73.58% 84.31% 88.46% 100.00% Value SPECIFICITY 81.08% 83.56% 64.86% 68.92% 90.54% 89.04% 98.63% 81.08% 89.19% 91.78% 100.00% Neg Predictive 84.51% 89.71% 71.64% 87.93% 89.33% 90.28% 96.00% 84.51% 90.41% 94.37% 97.33% Value % False 26.42% 21.82% 45.61% 34.85% 14.29% 15.69% 2.08% 26.42% 15.69% 11.54% 0.00% Positives % False 15.49% 10.29% 28.36% 12.07% 10.67% 9.72% 4.00% 15.49% 9.59% 5.63% 2.67% Negatives Area Under 0.8262 0.8975 0.7108 0.8557 0.9154 0.9556 0.9885 0.8857 0.9368 0.9726 0.9915 ROC Curve Significance <0.00001 <0.00001 0.0009 <0.00001 <0.00001 <0.00001 <0.00001 <0.00001 <0.00001 <0.00001 __________________________________________________________________________ <0.00001 *NOTE: n = number of CMP Nuclear Descriptors which survived the model, either Std. Deviation or Variance or both statistics. **NOTE: Surviving Biomarkers are CDNA1, H2NFDN, and PD415 ***NOTE: Surviving Biomarkers are CDNA1 and H2NFDN ****NOTE: Surviving Biomarkers are H2NFDN and PD415

TABLE VIa ______________________________________ Feature D1 D2 H I J K L M N ______________________________________ 1 X X X X X X 2 X X X X X X X 3 X X X X X X X X 4 X X X X X X X 5 6 X X X X X X X X 7 X X X X X X X X X 8 X X X 9 10 X X X X X X X X X 11 X X X X X X X 12 13 14 X X X X X X X X X 15 X X X X X X X X X 16 17 X X X X X X X X X 18 19 20 21 22 X X X X X X X X X 23 X 24 25 26 27 X X X X X X X X X 28 ______________________________________

TABLE VII __________________________________________________________________________ Univariate/Multivariate Analysis for Prostate Cancer Progression {N = 124 Radical Prostatectomy Specimens} Progressors/Non-Progressors: 50/74 Statistical Analysis of JHH-1 Database using Univariately Significant STDEV & VAR Statistics for JVB Texture Features, Biomarkers, Post Gleason, & JHH-NRV in Progression Prediction N K L M JVB JVB JVB JVB Nuclear J Nuclear Nuclear Nuclear Des- H I JVB Des- Des- Des- criptors D2 JVB JVB Nuclear criptors criptors criptors (n = 14)* JVB Nuclear Nuclear Des- (n = 17)* (n = 17)* (n = 17)* Bio- C Nuclear Des- Des- criptors Bio- Bio- Bio- markers Bio- Des- criptors criptors (n = 15)* markers markers markers (n = 2)*** A B markers criptors (n = 16*) (n = 17)* JHH-NRV (n = (n = 3)** (n = 3)** JHH-NRV Post GL JHH-NRV (n = 3)** (n = 19)* Post GL JHH-NRV Post GL 2)**** Post GL JHH-NRV Post GL __________________________________________________________________________ Positive Cutoff 0.50 0.35 0.40 0.40 0.50 0.40 0.50 0.45 0.50 0.50 0.50 (p>=#) SENSITIVITY 78.00% 86.00% 62.00% 86.00% 86.00% 96.00% 98.00% 88.00% 88.00% 92.00% 98.00% Pos Predictive 73.58% 78.18% 54.39% 76.79% 86.00% 94.12% 98.00% 81.48% 88.00% 95.83% 98.00% Value SPECIFICITY 81.08% 83.56% 64.86% 82.43% 90.54% 95.89% 98.63% 86.49% 91.89% 97.26% 98.63% Neg Predictive 84.51% 89.71% 71.64% 89.71% 90.54% 97.22% 98.63% 91.43% 91.89% 94.67% 98.63% Value % False Positives 26.42% 21.82% 45.61% 23.21% 14.00% 5.88% 2.00% 18.52% 12.00% 4.17% 2.00% % False 15.49% 10.29% 28.36% 10.29% 9.46% 2.78% 1.37% 8.57% 8.11% 5.33% 1.37% Negatives Area Under 0.8262 0.8975 0.7108 0.9300 0.9462 0.9866 0.9934 0.9503 0.9589 0.9885 0.9948 ROC Curve Significance <0.00001 <0.00001 0.0009 <0.00001 <0.00001 <0.00001 <0.00001 <0.00001 <0.00001 <0.00001 <0.00001 __________________________________________________________________________ *NOTE: n = number of JVB Nuclear Descriptors which survived the model, either Std. Deviation or Variance or both statistics. **NOTE: Surviving Biomarkers are CDNA1, H2NFDN, and PD415 ***NOTE: Surviving Biomarkers are CDNA1 and H2NFDN ****NOTE: Surviving Biomarkers are H2NFDN and PD415

TABLE VIIa ______________________________________ Feature D1 D2 H I J K L M N ______________________________________ 1 X X X X X X X X 2 X X X X X X X X X 3 X X X X X X X X X 4 X X X X X X X X 5 6 7 X X X X X X X 8 X X X X X X 9 10 X X X X X X X X X 11 X X X X X X X X X 12 13 14 X X X X X X X 15 X X X X X X X X 16 17 X X X X X X X 18 19 20 21 22 X X X X X X 23 X 24 X X X X X X X X X 25 26 X X X X X X 27 28 X X X X X X X 29 X X X X X X 30 X X X X X X X 31 X X X X X X 32 33 34 X X X X X X X X X 35 X X X X X X X X 36 ______________________________________

In another embodiment, the ability of Her-2/neu antigenic expression to identify high risk sub-populations of well to moderately differentiated Gleason grades (2-6) as well as high Gleason grades (.gtoreq.7) is clearly demonstrated in FIG. 19. Additionally, it was demonstrated that non-diploid DNA ploidy status selected out a subset of well to moderately differentiated Gleason grades (2-6) that were at risk for progression (FIG. 20).

LOGISTIC REGRESSION ORGAN CONFINEMENT MODEL

This invention also applies logistic regression to select the univariately significant variables for organ confinement (Table VIII) using the STATA.TM. statistical software package (STATA.TM. command: loogistic). Next, these univariately significant variables are multivariately assessed using backwards stepwise logistic regression (STATA.TM. command: swlogis) to determine which independent variables (e.g. NMD's (CMP or JVB), Gleason Score, Nuclear Roundness Variance, and biomarkers) are retained to predict organ confinement (Tables IX, Ixa, X, and Xa).

TABLE VIII ______________________________________ Univariate Analysis for Organ Confined Disease Status Prediction using STATA .TM. Logistic Regression Statistical Significance Independent Variable (p Value) ______________________________________ Post Operative Gleason Score p .ltoreq. 0.00001 Nuclear Roundness Variance* p = 0.0073 Best CAS-200 CMP v3.0 Nuclear Morphometric p = 0.0005 Descriptors Best CAS-200 JVB v1.0 Nuclear Morphometric p = 0.0003 Descriptors CAS-200 DNA Ploidy - 1 (C-DNA1) p = 0.0703** CAS-200 DNA Ploidy - 10 (C-DNA10) p = 0.0546** CAS-200 DNA Ploidy - JIE (JHHDNA10) p = 0.0499 Her-2/neu Antigenicity: Focal, Diffuse, Negative p = 0.0023 (H2NFDN) PCNA Antigenicity p = 0.1330** PD-41 Antigenicity p = 0.0198 ______________________________________ *Measured using DynaCell .TM. system at Johns Hopkins Hospital **Not Statistically Significant. Must be less than 0.0500 to be statistically significant.

TABLE IX __________________________________________________________________________ Univariate/Multivariate Analysis for Prostate Cancer Organ Confinment {N = 124 Radical Prostatectomy Specimens} Organ Confined/Non-Organ Confined: 29/95 Statistical Analysis of JHH-1 Database using Univariately Significant STDEV & VAR Statistics for CMP Texture Features, Biomarkers, Post Gleason, & JHH-NRV in Organ Confinment Prediction N CMP K L M Nuclear CMP CMP CMP Des- J Nuclear Nuclear Nuclear criptors H I CMP Des- Des- Des- (n = 14)* D2 CMP CMP Nuclear criptors criptors criptors Bio- CMP Nuclear Nuclear Des- (n = 12)* (n = 14)* (n = 12)* markers C Nuclear Des- Des- criptors Bio- Bio- Bio- (n = Bio- Des- criptors criptors (n = 14)* markers markers markers 2)**** A B markers criptors (n = 11*) (n = 13)* JHH-NRV (n = (PD41:5) (n = 3)** JHH-NRV Post GL JHH-NRV (n = 3)** (n = 10)* Post GL JHH-NRV Post GL 2)*** Post GL JHH-NRV Post GL __________________________________________________________________________ Positive Cutoff 0.20 0.30 0.25 0.30 0.25 0.25 0.50 0.40 0.50 0.35 0.50 (p>=#) SENSITIVITY 86.21% 58.62% 55.17% 72.41% 82.76% 82.76% 68.97% 68.97% 75.86% 75.86% 72.41% Pos Predictive 35.21% 39.53% 34.04% 52.50% 54.55% 57.14% 83.33% 66.67% 78.57% 62.86% 75.00% Value SPECIFICITY 51.58% 72.34% 67.37% 80.00% 78.95% 80.85% 95.74% 89.47% 93.65% 86.17% 92.55% Neg Predictive 92.45% 85.00% 83.12% 90.48% 93.75% 93.83% 90.91% 90.43% 92.71% 92.05% 91.58% Value % False Positives 64.79% 60.47% 65.96% 47.50% 45.45% 42.86% 16.67% 33.33% 21.43% 37.14% 25.00% % False 7.55% 15.00% 16.88% 9.52% 6.25% 6.17% 9.09% 9.57% 7.29% 7.95% 8.42% Negatives Area Under 0.733 0.6618 0.6973 0.8635 0.9180 0.8833 0.9219 0.8831 0.9397 0.9028 0.9413 ROC Curve Significance <0.00001 0.0073 0.0026 0.0005 <0.00001 0.0017 <0.00001 0.0015 <0.00001 0.006 <0.00001 __________________________________________________________________________ *NOTE: n = number of CMP Nuclear Descriptors which survived the model, either Std. Deviation or Variance or both statistics. **NOTE: Surviving Biomarkers are CDNA1, H2NFDN, and PD415 ***NOTE: Surviving Biomarkers are H2NFDN and PD415 ****NOTE: Surviving Biomarkers are CDNA1 and PD415

TABLE IXa ______________________________________ Feature D1 D2 H I J K L M N ______________________________________ 1 X X X X X X X X X 2 3 4 X X X X X X X X X 5 6 X X X X X X X X X 7 8 X X X X X X 9 10 X X X X X X X X X 11 X X X X X X X X X 12 13 X X X X X X X 14 X X X X X X X 15 X X X X X X X X X 16 X X X X X X X X X 17 X X X X X X X 18 19 20 21 22 X X X X X X X 23 X 24 X X X X X X X X X 25 26 X X X X X X X X 27 X 28 ______________________________________

TABLE X __________________________________________________________________________ Univariate/Multivariate Analysis for Prostate Cancer Organ Confinment {N = 124 Radical Prostatectomy Specimens} Organ Confined/Non-Organ Confined: 29/95 Statistical Analysis of JHH-1 Database using Univariately Significant STDEV & VAR Statistics for JVB Texture Features, Biomarkers, Post Gleason, & JHH-NRV in Organ Confinment Prediction N K L M JVB JVB JVB JVB Nuclear J Nuclear Nuclear Nuclear Des- H I JVB Des- Des- Des- criptors D2 JVB JVB Nuclear criptors criptors criptors (n = 16)* JVB Nuclear Nuclear Des- (n = 16)* (n = 15)* (n = 16)* Bio- C Nuclear Des- Des- criptors Bio- Bio- Bio- markers Bio- Des- criptors criptors (n = 15)* markers markers markers (n = 3)** A B markers criptors (n = 15*) (n = 16)* (dropped) (n = (n = 2)*** (n = 2)*** (dropped) Post GL JHH-NRV (n = 3)** (n = 15)* Post GL JHH-NRV Post GL 2)*** Post GL JHH-NRV Post GL __________________________________________________________________________ Positive Cutoff 0.20 0.30 0.25 0.35 0.40 0.40 0.40 0.40 0.40 0.40 0.40 (p>=#) SENSITIVITY 86.21% 58.62% 55.17% 86.21% 79.31% 79.31% 79.31% 82.76% 93.10% 82.76% 93.10% Pos predictive 35.21% 39.53% 34.04% 64.10% 71.88% 65.71% 71.88 75.00% 81.82% 72.73% 81.82% Value SPECIFICITY 51.58% 72.34% 67.37% 85.26% 90.53% 87.23% 90.53 91.58% 93.68% 90.43% 93.68% Neg Predictive 92.45% 85.00% 83.12% 95.29% 93.48% 93.18% 93.48% 94.57% 97.80% 94.44% 97.80% Value % False Positives 64.79% 60.47% 65.96% 35.90% 28.13% 34.29% 28.13% 25.00% 18.18% 27.27% 18.18% % False 7.55% 15.00% 16.88% 4.71% 6.52% 6.82% 6.52% 5.43% 2.20% 5.56% 2.20% Negatives Area Under 0.7330 0.6618 0.6973 0.9143 0.9170 0.9156 0.9470 0.9336 0.9670 0.9384 0.9655 ROC Curve Significance <0.00001 0.0073 0.0026 0.0003 <0.00001 0.0003 <0.00001 <0.00001 <0.00001 <0.00001 <0.00001 __________________________________________________________________________ *NOTE: n = number of JVB Nuclear Descriptors which survived the model, either Std. Deviation or Variance or both statistics. **NOTE: Surviving Biomarkers are CDNA1, H2NFDN, and PD415 ***NOTE: Surviving Biomarkers are H2NFDN and PD415

TABLE Xa ______________________________________ Feature D1 D2 H I J K L M N ______________________________________ 1 X X X X X X X X X 2 3 4 X X X X X X X X X 5 6 X X X X X X X X X 7 8 X X X X X X X X X 9 10 X X X X X X X X X 11 X X X X X X X X X 12 13 X X X X X X X X 14 X X X X X X X X X 15 X X X X X X X X X 16 X X X X X X X X X 17 X X X X X X X X X 18 19 20 21 22 X X X X X X X X X 23 X 24 X X X X X X X X X 25 26 X X X X X X 27 28 29 X 30 X X X X X X X X X 31 X X X X X X X X X 32 33 34 35 36 ______________________________________

TABLE Xb __________________________________________________________________________ JHH1 METASTATIC SUBSET n = 13 Total Model (JVB JVB NMD JVB NMD Progression Predicted Predicted Total Model Model L) Progression Progression (Model L) Predicted Ex- DNA Her- Pro- Outcome Predicted Outcome PCode OC PROG PostGL posure Local Distant PD41 Ploidy 2/neu batility (Cutoff = 0.40) Probabilities (Cutoff = __________________________________________________________________________ 0.50) 45 0 1 9 7 0 1 + Aneuploid + 0.9988 1 0.9645 1 100 0 1 6 7 1 1 + Aneuploid + 0.3499 0 0.1498 0 101 0 1 6 1 1 0 + Aneuploid + 0.0917 0 0.4268 0 105 0 1 8 8 1 0 + Aneuploid + 0.7284 1 0.9763 1 149 1 1 6 1 1 0 - Aneuploid + 0.9767 1 0.5812 1 200 0 1 7 5 1 0 + Aneuploid - 0.7355 1 0.8157 1 210 0 1 8 6 1 0 + Diploid + 0.9997 1 0.9999 1 260 0 1 7 1 1 1 - Aneuploid + 0.8009 1 0.8254 1 359 0 1 7 2 1 0 - Diploid + 1.0000 1 0.9906 1 410 0 1 7 4 0 1 + Diploid + 0.6481 1 0.6127 1 498 0 1 7 3 1 0 + Diploid + 0.7657 1 0.9685 1 699 0 1 8 3 1 0 + Aneuploid + 0.8843 1 0.9999 1 753 0 1 7 2 0 1 - Aneuploid + 1.0000 1 1.0000 1 __________________________________________________________________________ OC: 1 = Organ Confined Disease, 0 = NonOrgan Confined Disease PROG: 1 = Disease Progression, 0 = NonProgression of Disease PostGL: Combined PostOperative Gleason Score Exposure: The time in years that it took for disease progression. Local: 1 = Metastasis to surrounding tissue (i.e. lymph nodes and seminal vesicles), 0 = No local metastasis. Distant: 1 = Metastasis to distant site (i.e. bone marrow), 0 = No distan metastasis. Predicted Outcomes: 1 = Prostate cancer predicted to progress, 0 = Prostate cancer predicted not to progress.

In another embodiment, the ability of PD-41 antigenic expression to identify non-organ confined disease status was evaluated. Positive PD-41 antigenic expression was found in 67 of 95 (71%) patients with non-organ confined disease. Additionally, Her-2/neu antigenic expression was also shown to strongly correlate to non-organ confined disease status. Positive Her-2/neu antigenic expression was found in 80 of 95 (84%) patients with non-organ confined disease.

Finally, a select subgroup of patients that developed fatal metastatic disease (n=13) were a evaluated for DNA ploidy, PD-41 and Her-2/neu antigenic expression (Table Xb). DNA aneuploidy was observed in 9 of 13 (69%) and positive PD-41 antigenic expression was found in 9 of 13 (69%) patients that recurred with fatal metastatic disease. Additionally, Her-2/neu antigenic expression was shown to strongly correlate to fatal metastatic disease. Positive Her-2/neu antigenic expression was found in 12 of 13 (92%) patients that recurred with fatal metastatic disease. These data clearly support the potential value of these biomarkers for early identification of patients at high risk for fatal metastatic disease.

METHODS TO OBTAIN PATIENT-SPECIFIC RESULTS

Logistic Regression Method--STATA.TM. provides a command (logit, an estimated maximum-likelihood logit model) that provides the weighted coefficients for the statistically significant independent variables used in the multivariate model and the model constant. The general formulas for calculating the predictive index and predictive probability are as follows:

Predictive Index (xb)=(.beta..sub.0 +.beta..sub.1 var(1)+.beta..sub.2 var(2)+ - - - +.beta..sub.n var(n))

Predictive Probability (p)=e.sup.xb /(1+e.sup.xb)

Where:

.beta..sub.0 =Formula Constant

.beta..sub.1 through .beta..sub.n =Weight factors for variables 1 through n

var(1) through var(n)=Independent variables being used in logistic regression model.

The final calculation of the predictive probability provides a patient-specific value, between 0 and 1, for the probability of a specific outcome (e.g. progression or organ confined disease status). The threshold value (cutoff) for the predicted probability is selected based upon the results of the ROC curves. Table XI illustrates patient specific NMD and multi-parameter (combined) predictive probabilities calculated using this method.

TABLE XI __________________________________________________________________________ Use of Logistic Regression (logit) to Predict Patient-Specific Outcomes Combined Morphometry Parameters Predicted Case Predictive Predictive Outcome I.D. PD41-5 C-DNA1 H2NFDN PostGL Probability Probabilities (Cutoff: 0.50) __________________________________________________________________________ 33 + Diploid Focal 5 0.14 0.02 0 34 - Ab: >S + G2M Diffuse 7 0.92 0.93 1 149 – Aneuploid Focal 6 0.98 0.58 1 9952 – Hypodiploid Diffuse 4 0.09 0.00 0 __________________________________________________________________________

Neural Networks–The first network configuration to be considered was a standard multilayer sigmoidal network with a single hidden layer. The neural network input layer consisted of either 15, 28, or 30 input nodes to accommodate the input data set of either 15, 28, or 30 measurements (NMD’s and Gleason Score). The activation function in each hidden layer and output layer neuron is sigmoid. Different network configurations (number of hidden layer neurons) with various training termination conditions were tested. FIG. 29 illustrates the neural network configuration used in this study. It was found, for the given training set, that the neural network classifier works best with 20 hidden layer neurons and when the training is terminated at approximately 1000 iterations.

The second network configuration tested consisted of a single hidden layer as well. However, the non-linearity function used was the sinusoidal function (proprietary Hybrid network). The output layer neurons still used the sigmoidal transfer function. It had the same structure as the first network (see FIG. 29). A number of different frequencies were tested to find the best combination. The best frequency was found to be F=0.2.

All networks (standard and hybrid with 15, 28, or 30 inputs) were tested using the ten different combinations of randomly selected test (18) and training (106) cases. The threshold value (cutoff) used in all cases was 0.5. Table XII summarizes the results using the standard sigmoidal neural networks applied to the n=124 patient sample described in this invention (Tables XIIa, b, & c). Notable is the fact that this network degrades as the number of input features is increased from 15 to 28 to 30. Please note that the 28 and 30 feature networks did not undergo pre-selection using logistic regression methods. The best performing network was the one labeled “15 Input Features”, where the features were pre-selected based upon statistical significance using logistic regression methods. Therefore, the use of statistical methods to pre-select statically significant features improves network performance. Also it is evident from the variation in the predictive rates among the ten trained networks that if the number of patients is increased, the performance of the network should be significantly improved.

TABLE XII ______________________________________ Sigmoidal Neural Network Comparisons: JHH-1 (n = 124) Progression Predictive Rates Network # 15 Feature NN 28 Feature NN 30 Feature NN ______________________________________ 1 78% 61% 67% 2 83% 61% 61% 3 89% 50% 50% 4 67% 83% 67% 5 67% 72% 72% 6 83% 78% 67% 7 89% 78% 72% 8 78% 78% 72% 9 72% 78% 78% 10 89% 72% 72% Mean 79% 71% 68% Standard Error 3% 3% 2% Median 81% 75% 70% Mode 78% 78% 72% Standard Deviation 9% 10% 8% Variance 1% 1% 1% ______________________________________

TABLE XIIa ______________________________________ Sigmoidal Neural Network: 15 Input Features Predictive Total % Error % Error Network # Rate Error Progressors Non-Progressors ______________________________________ 1 78% 22% 33% 11% 2 83% 17% 33% 0% 3 89% 11% 22% 0% 4 67% 33% 55% 11% 5 67% 33% 55% 11% 6 83% 17% 22% 11% 7 89% 11% 22% 0% 8 78% 22% 33% 11% 9 72% 28% 33% 22% 10 89% 11% 11% 11% Mean 80% 21% 32% 9% Standard Error 3% 3% 4% 2% Median 81% 20% 33% 11% Mode 89% 11% 33% 11% Standard Deviation 9% 9% 14% 7% Variance 1% 1% 2% 0% ______________________________________

TABLE XIIb ______________________________________ Sigmoidal Neural Network: 28 Input Features Predictive Total % Error % Error Network # Rate Error Progressors Non-Progressors ______________________________________ 1 61% 39% 55% 22% 2 61% 39% 55% 22% 3 50% 50% 67% 33% 4 83% 17% 33% 0% 5 72% 28% 44% 11% 6 78% 22% 33% 11% 7 78% 22% 33% 11% 8 78% 22% 33% 11% 9 78% 22% 44% 0% 10 72% 28% 44% 11% Mean 71% 29% 44% 13% Standard Error 3% 3% 4% 3% Median 75% 25% 44% 11% Mode 78% 22% 33% 11% Standard Deviation 10% 10% 12% 10% Variance 1% 1% 1% 1% ______________________________________

TABLE XIIc ______________________________________ Sigmoidal Neural Network: 30 Input Features Predictive Total % Error % Error Network # Rate Error Progressors Non-Progressors ______________________________________ 1 67% 33% 44% 22% 2 61% 39% 67% 11% 3 50% 50% 67% 33% 4 67% 33% 67% 0% 5 72% 28% 44% 11% 6 67% 33% 44% 22% 7 72% 28% 44% 11% 8 72% 28% 33% 22% 9 78% 22% 44% 0% 10 72% 28% 44% 11% Mean 68% 32% 50% 14% Standard Error 2% 2% 4% 3% Median 70% 31% 0% 11% Mode 72% 28% 0% 11% Standard Deviation 8% 8% 12% 10% Variance 1% 1% 2% 1% ______________________________________

Table XIII illustrates the results for the Hybrid neural network using the n=124 patient sample described in this invention (Tables XIIIa, b, & c). The same observations as made for the standard sigmoidal network above apply to the Hybrid neural network. In conclusion, the use of appropriately trained standard or Hybrid neural networks can be used to predict patient specific outcomes (e.g. progression or organ confined disease status).

Classification and Regression Trees (CART)–Application of recursive partitioning methods using the SYSTAT CART.TM. for DOS v1.02 (Evanston, Ill.) software program was performed. This method is another example of a non-parametric statistical classifier. The use of this method yields similar classification results using the well defined patient sample (n=124), and can generate a patient specific outcome using a trained CART. Those experienced in the art of non-parametric statistical classifiers realize that several other such methods exist and can be applied to achieve this same end.

TABLE XIII ______________________________________ Hybrid Neural Network Comparisons: JHH-1 (n = 124) Progression Predictive Rates Network # 15 Feature NN 28 Feature NN 30 Feature NN ______________________________________ 1 67% 72% 67% 2 67% 56% 67% 3 83% 56% 56% 4 67% 56% 67% 5 72% 67% 78% 6 89% 72% 72% 7 89% 67% 78% 8 78% 72% 67% 9 72% 72% 67% 10 78% 67% 78% Mean 76% 68% 70% Standard Error 3% 1% 2% Median 75% 67% 67% Mode 67% 67% 67% Standard Deviation 9% 4% 7% Variance 1% 0% 0% ______________________________________

TABLE XIIIa ______________________________________ Hybrid Neural Network: 15 Input Features Predictive Total % Error % Error Non- Network # Rate Error Progressors Progressors ______________________________________ 1 67% 33% 44% 22% 2 67% 33% 55% 11% 3 83% 17% 22% 11% 4 67% 33% 44% 22% 5 72% 28% 44% 11% 6 89% 11% 22% 0% 7 90% 11% 22% 0% 8 78% 22% 33% 11% 9 72% 28% 33% 22% 10 78% 22% 22% 22% Mean 76% 24% 34% 13% Standard Error 3% 3% 4% 3% Median 75% 25% 33% 11% Mode 67% 33% 22% 22% Standard Deviation 9% 9% 12% 9% Variance 1% 1% 1% 1% ______________________________________

TABLE XIIIb ______________________________________ Hybrid Neural Network: 28 Input Features Predictive Total % Error % Error Non- Network # Rate Error Progressors Progressors ______________________________________ 1 72% 28% 33% 22% 2 56% 44% 55% 33% 3 56% 44% 67% 22% 4 56% 44% 67% 22% 5 67% 33% 44% 22% 6 72% 28% 44% 11% 7 67% 33% 55% 11% 8 72% 28% 33% 22% 9 72% 28% 44% 11% 10 67% 33% 55% 11% Mean 66% 34% 50% 19% Standard Error 2% 2% 4% 2% Median 67% 33% 50% 22% Mode 67% 28% 55% 11% Standard Deviation 7% 7% 12% 7% Variance 1% 1% 1% 1% ______________________________________

TABLE XIIIc ______________________________________ Hybrid Neural Network: 30 Input Features Predictive Total % Error % Error Non- Network # Rate Error Progressors Progressors ______________________________________ 1 67% 33% 44% 22% 2 67% 33% 44% 22% 3 56% 44% 78% 11% 4 67% 33% 55% 11% 5 78% 22% 33% 11% 6 72% 28% 33% 22% 7 78% 22% 22% 22% 8 67% 33% 44% 22% 9 67% 33% 55% 11% 10 78% 22% 33% 11% Mean 70% 30% 44% 17% Standard error 2% 2% 5% 2% Median 67% 33% 44% 17% Mode 67% 33% 44% 22% Standard Deviation 7% 7% 16% 6% Variance 0% 0% 2% 0% ______________________________________

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIG. 1A and FIG. 1B. Normalization plot of each individual pixel optical density (gray level), divided into eight equally frequent gray-level ranges (optical density ranges); each range containing an equal number of pixels.

FIG. 2. Parameters including cell selection, magnification, camera resolution (pixel size, pixel shape and number of pixels), and Markovian step size that can significantly alter nuclear morphometric descriptor outputs.

FIG. 3. Identification of progressors (subset of 20 cases) using standard deviation and variance of best CMP 40.times. nuclear morphometic descriptors. This figure shows the predictive power using the combined CMP NMD’s measured with a 40.times. objective. Using 7 different CMP NMD’s, a ROC curve was produced with an area under the curve of 94%.

FIG. 4. Identification of progressors (subset of 20 cases) using standard deviation and variance of best JVB 40.times. nuclear morphometric descriptors. This figure shows the predictive power using the combined JVB NMD’s measured with a 40.times. objective. Using 8 different JNB NMD’s, a ROC curve was produced with an area under the curve of 95%.

FIG. 5. Identification of progressors (subset of 20 cases) using standard deviation and variance of best CMP 63.times. nuclear morphometric descriptors. This figure shows the predictive power using the combined CMP NMD’s measured with a 63.times. objective. Using 6 different CMP NMD’S, a ROC curve was produced with an area under the curve of 94%.

FIG. 6. Identification of progressors (subset of 20 cases) using standard deviation and variance of best JVB 63.times. nuclear morphometric descriptors. This figure shows the predictive power using the combined JVB NMD’s measured with a 63.times. objective. Using 5 different JVB NMD’s, a ROC curve was produced with an area under the curve of 96%.

FIG. 7. Post-operative Gleason score significance in the prediction of prostate cancer progression. This figure illustrates the predictive power of Post Operative Gleason Score as a single independent variable to predict progression. A ROC curve was produced with an area under the curve of 82.62%. Please refer to Columrrn A of Table VI.

FIG. 8. Post-operative Gleason score significance in the prediction of prostate cancer progression (0=predicted not to progress; 1=predicted to progress). This figure demonstrates the ability of the Post Operative Gleason Score alone to stratify progressors and non-progressors using a Kaplan-Meier Survival Recurrence) Curve. This is the pathologic standard against which all models are tested.

FIG. 9. Nuclear roundness variance significance in the prediction of prostate cancer progression. This figure illustrates the predictive power of Nuclear Roundness Variance (as measured by the DynaCell System at 1 00.times.) to predict progression. A ROC curve was produced with an area under the curve of 89.75%. Please refer to Column B of Table VI.

FIG. 10. Nuclear roundness variance significance in the prediction of prostate cancer progression (0=predicted not to progress; 1-predicted to progress). This figure demonstrates the ability of Nuclear Roundness Variance alone to stratify progressors and non-progressors using a Kaplan-Meier Survival (Recurrence) Curve.

FIG. 11. 12 CMP nuclear descriptors found to be significant in the prediction of prostate cancer progression. This figure illustrates the predictive power of the 12 CMP NMD’s to predict progression. A ROC curve was produced with an area under the curve of 85.57%. Please refer to Colurm D2 of Table VI.

FIG. 12. 12 CMP nuclear descriptors found to be significant in the prediction of prostate cancer progression (0=predicted not to progress; 1=predicted to progress). This figure demonstrates the ability of the 12 CMP NMD’s to stratify progressors and non-progressors using a Kaplan-Meier Survival (Recurrence) Curve.

FIG. 13. 13 CMP nuclear descriptors, Her-2/neu staining, nuclear roundness variance, and post-op Gleason found to be significant in the prediction of prostate cancer progression. This figure illustrates the predictive power of the 13 CMP NMD’s, 1 biomarker, NRV, and Gleason Score combined to predict progression. A ROC curve was produced with an area under the curve of 99.15%. Please refer to Column N of Table VI.

FIG. 14. 13 CMP nuclear descriptors, Her-2/neu staining, nuclear roundness variance, and post-op Gleason found to be significant in the prediction of prostate cancer progression (0=predicted not to progress; 1=predicted to progress). This figure demonstrates the ability of the 13 CMP NMD’s, 1 biomarker, NRV, and Gleason Score combined to stratify progressors and non-progressors using a Kaplan-Meier Survival (Recurrence) Curve.

FIG. 15. 19 JVB nuclear descriptors found to be significant in the prediction of prostate cancer progression. This figure illustrates the predictive power of the 19 JVB NMD’s to predict progression. A ROC curve was produced with an area under the curve of 93%. Please refer to Column D2 of Table VII. Also, please note the difference in the number of features required for JVB NMD’s as well as an increase the predictive power of the model as compared to CMP NMD’s in FIG. 10.

FIG. 16. 19 JVB nuclear descriptors found to be significant in the prediction of prostate cancer progression (0=predicted not to progress; 1=predicted to progress). This figure demonstrates the ability of the 19 JVB 3 NMD’s to stratify progressors and non-progressors using a Kaplan-Meier Survival (Recurrence) Curve. Please note the difference in the ability of JVB NMD’s to stratify progressors and non-progressors as compared to CMP NMD’s in FIG. 11.

FIG. 17. 14 JVB nuclear descriptors, 2 biomarkers, nuclear roundness variance, and post-op Gleason found to be significant in the prediction of prostate cancer progression. This figure illustrates the predictive power of the 14 JVB NMD’s, 2 biomarkers, NRV, and Gleason Score combined to predict progression. A ROC curve was produced with an area under the curve of 99.48%. Please refer to Column N of Table VII.

FIG. 18. 14 WB nuclear descriptors, 2 biomarkers, nuclear roundness variance, and post-op Gleason found to be significant in the prediction of prostate cancer progression (0=predicted not to progress; 1=predicted to progress). This FIG. demonstrates the ability of the 14 JVB NMD’s, 2 biomarkers, NRV, and Gleason Score combined to stratify progressors and non-progressors using a Kaplan-Meier Survival (Recurrence) Curve.

FIG. 19. Stratification of progressors among well to moderately differentiated prostate cancers using Her-2/neu antigenic expression (Kaplan-Meier Survival (Recurrence) Curve).

FIG. 20. Stratification of progressors among well to moderately differentiated prostate cancers using DNA ploidy cytometry (Kaplan-Meier Survival (Recurrence) Curve).

FIG. 21. Post-operative Gleason score significance in the prediction of organ confinement status. This figure illustrates the predictive power of Post Operative Gleason Score to predict organ confined disease status A ROC curve was produced with an area under the curve of 73.3%. Please refer to Column A of Table IX. Note the lower predictive value of this independent variable as compared to the same variable used to predict progression (see FIG. 6).

FIG. 22. Nuclear roundness variance significance in the prediction of organ confinement status. This figure illustrates the predictive power of Nuclear Roundness Variance to predict organ confined disease status. A ROC curve was produced with an area under the curve of 66.18%. Please refer to Column B of Table IX. Once again note the much lower predictive value of this independent variable compared to its contribution in prediction progression (see FIG. 9).

FIG. 23. 10 CMP nuclear descriptors found to be significant in the prediction of organ confinement status. This figure illustrates the predictive power of the 10 CMP NMD’s to predict organ confined disease status. A ROC curve was produced with an area under the curve of 86.35%. Please refer to Column D2 of Table IX. Note the significant improvement of the CMP NMD’s alone as compared to NRV alone (FIG. 22) in the prediction of organ confined disease status.

FIG. 24. 12 CMP nuclear descriptors, 3 biomarkers, and nuclear roundness variance found to be significant in the prediction of organ confinement status. This figure illustrates the predictive power of the 12 CMP NMD’s, 3 biomarkers, and NRV to predict organ confined disease status. A ROC curve was produced with an area under the curve of 90.28%. Please refer to Column M of Table IX.

FIG. 25. 15 JVB nuclear descriptors found to be significant in the prediction of organ confinement status. This figure illustrates the predictive power of the 15 JVB NMD’s to predict organ confined disease status. A ROC curve was produced with an area under the curve of 91.43%. Please refer to Column D2 of Table X. Note the improvement of predictive power when using JVB NMD’s alone as compared to CMP NMD’s alone (FIG. 22) in the prediction of organ confined disease status.

FIG. 26. 15 JVB nuclear descriptors and post-op Gleason found to be significant in the prediction of organ confinement status. This figure illustrates the predictive power of the 15 JVB NMD’s and Post Operative Gleason score to predict organ confined disease status. A ROC curve was produced with an area under the curve of 94.7%. Please refer to Column H of Table X.

FIG. 27. 16 JVB nuclear descriptors, 3 biomarkers, nuclear roundness variance (DROPPED), and post-op Gleason found to be significant in the prediction of organ confinement status. This figure illustrates the predictive power of the 16 JVB NMD’s, 3 biomarkers, and Post Operative Gleason score to predict organ confined disease status- A ROC curve was produced with an area under the curve of 96.55%. Please refer to Column N of Table X. Note that NRV was dropped as a significant independent variable in this model.

FIG. 28A and FIG. 28B. DNA Classification scheme for prostate image analysis. Normal range: Diploid: S-Hase+G2M<10% of cells studies; Out of normal range: Hypodiploid: DNA Index<0.70 >S+G2M: 11-21% of cells studies (includes hyperploidy); Abnormal range>S+G2M: >22% of cells studied; aneuploid: >10% of cells studied; tetraploid: >16% of cells studied.

FIG. 29–Neural network configuration.

DETAILED DESCRIPTION

The invention, in its broadest sense, is a method for predicting organ confined disease status or the potential for progression of prostate cancer following radical surgery using either non-parametric statistical analysis methods or neural networks. The parameters assessed by these methods include, but are not limited to, cellular biomarkers and nuclear morphometric descriptors. The invention provides a method to collect nuclear images and extract all relevant shape, size, Markovian texture, and DNA content features important to construction of a mathematical method that gives a single predictive probability for prostate cancer progression or organ localization, with or without pathological grading. The texture features utilized in the present invention are set forth in Table I (CMP v3.0) and Table II (JVB v1.0). It is recognized that in predicting the probability of prostate cancer progression and organ localization, prognostic variable factors other than those listed may be used within the scope and spirit of the present invention.

Also embodied in the present invention is the use of a trained neural network to provide a single predictive probability for prostate cancer progression or organ localization given any number of inputs. The multi-layer perceptron network of the present invention is a feed-forward network with one or more hidden layers of neurons between the input and output layers. Using this architecture, many shortcomings of the single layer perceptron are avoided. However, because of the added complexity, the convergence theorem and weight adjustment procedure suggested by Rosenblatt is not applicable. An alternate procedure called “back propagation” has been independently developed by Werbos (Werbos, Ph.D. Thesis, Harvard University, 1974), Parker (Parker, Innovation Report, 581-664, File 1, Office of Technology Licensing, Stanford University, October, 1982), and Rumelhart (see Rumelhart et al., Parallel Distributed Processing Explorations in the Microstructures of Cognition Vol. 1, Foundations, MIT Press, Cambridge, Mass., 1988). This procedure is effective and allows for efficient use of multi-layer perceptrons. But the procedure does not guarantee convergence to the global minima at all times. Also, it requires a large number of training iterations in order to learn a given set of transformations.

Because of the problems associated with back propagation, it is of interest to modify the weight adjustment procedure and/or the model developed by Rosenblatt to enable single-layer perceptrons to solve problems such as XOR problems. In this work, a modified perceptron is utilized. The modified perceptron used is a multiple threshold perceptron that is capable of solving XOR problems. This modified perceptron is obtained by changing the non-linearity function. Unlike previous efforts in developing multiple threshold perceptrons, the perceptron of the present invention is capable of handling both binary and analog inputs. The procedure requires fewer number of iterations to develop appropriate input to output transformations when compared to back propagation.

For the purposes of this invention, the following clinical and pathological staging criteria is used. The use of other criteria does not depart from the scope and spirit of the invention.

TO–No evidence of Prostatic tumor.

T1–Clinically inapparent tumor, non-palpable nor visible by imaging.

T1a–Tumor is incidental histologic finding with three or fewer microscopic foci. Non-palpable, with 5% or less of TURP chips (trans-urethral resected prostate tissue) positive for cancer.

T1b–Tumor is incidental histologic finding with more than three microscopic foci. Non-palpable, with greater then 5% of TURP chips (trans-urethral resected prostate tissue) positive for cancer.

T1c–Tumor is non-palpable, and is found in one or both lobes by needle biopsy diagnosis.

T2–Tumor is confined within the prostate.

T2a–Tumor present clinically or grossly, limited to the prostate, tumor 1.5 cm or less in greatest dimension, with normal tissue on at least three sides. Palpable, half of 1 lobe or less.

T2b–Tumor present clinically or grossly, limited to the prostate, tumor more than 1.5 cm in greatest dimension, or in only one lobe. Palpable, greater than half of 1 lobe but not both lobes.

T2c–Tumor present clinically or grossly, limited to the prostate, tumor more than 1.5 cm in greatest dimension, and in both lobes. Palpable, involves both lobes.

T3–Tumor extends through the prostatic capsule. T3a–Palpable tumor extends unilaterally into or beyond the prostatic capsule, but with no seminal vesicle or lymph node involvement. Palpable, unilateral capsular penetration.

T3b–Palpable tumor extends bilaterally into or beyond the prostatic capsule, but with no seminal vesicle or lymph node involvement. Palpable, bilateral capsular penetration.

T3c–Palpable tumor extends unilaterally and or bilaterally beyond the prostatic capsule, with seminal vesicle and/or lymph node involvement. Palpable, seminal vesicle or lymph node involvement.

T4–Tumor is fixed or invades adjacent structures other than the seminal vesicles or lymph nodes.

T4a–Tumor invades any of: bladder neck, external sphincter, rectum.

T4b–Tumor invades levator muscles and/or is fixed to pelvic wall.

The following examples are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent techniques discovered by the inventor to function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes c an be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXAMPLE I

DNA Staining Procedure Using CAS Quantitative DNA Staining Kit (Elmhurst, Ill.; Catalog #102300-01)

Preparation of Feulgen Stain Solution:

Place 90 ml of Type I H.sub.2 O in a volumetric flask and add 10 ml of 1N HCL. Place a stir bar in a 125 ml Erlenmeyer flask and add the above solution. Add 1 vial of DNA stain reagent to the flask while stirring the solution. Place a rubber stopper in the flask, and stir the contents for at least 1 hour. This Feulgen stain solution should be filtered through a Whatman No. 1 filter immediately before staining of the specimen.

Preparation of Feulgen Rinse Solution:

Place 285 ml of Type I H.sub.2 O in a 500 ml graduated cylinder and add 15 ml of 1N HCL. Pour this solution into a 500 ml bottle. Immediately before rinsing, place 1 vial of DNA rinse reagent into the bottle and mix the contents by swirling. This solution is stable for 2-3 hours.

Preparation of Calibration Slides:

To prepare the control cells, place two (2) CAS calibration slides (Elmhurst, Ill.; Catalog #102202-00) in 10% neutral buffered formalin for 30 minutes at room temperature. The calibration slides are touch-prep rat hepatocytes that have a known shape, size, and DNA amount. Next, rinse the CAS calibration slides in running deionized H.sub.2 O for 5 minutes.

Preparation of Tissue Samples:

The 5 .mu.m formalin fixed, paraffin embedded, tissue sections are first placed on Probe-On.TM. Plus microscope slides. Place the slides in Hemo-De for 1 minute at 45.degree. C., and then drain the Hemo-De from the slides with the aid of absorbent paper. This step is repeated three (3) more times.

Next, place the specimen slides in absolute ethanol for 1 minute at room temperature, and then drain the alcohol from the slides with the aid of absorbent paper. Repeat this step one (1) more time.

Finally, place the specimen slides in PBS (pH 7.6) with 0.1% Triton X-100 for 10 seconds at room temperature, and then drain the PBS from the slides with the aid of absorbent paper. Repeat this step one (1) more time.

Feulgen Staining Procedure:

Place the slides (CAS calibration slides and specimen slides) in 5N HCL for 1 hour at room temperature. Next, place all of the slides in the Feulgen stain solution for 1 hour at room temperature (stir while staining). Drain the Feulgen stain solution and rinse the slides in the Feulgen rinse solution for 30 seconds at room temperature, followed by rinsing the slides in Feulgen rinse solution for 5 minutes at room temperature, followed by rinsing the slides in Feulgen rinse solution for 10 minutes at room temperature. The slides are then rinsed in running deionized H.sub.2 O for 5 minutes. Destaining is done in 1 acid alcohol for 5 minutes at room temperature. This is followed by dipping the slides in 95% ethanol 10 times, followed by dipping the slides in absolute ethanol 10 times, followed by finally dipping the slides in xylene 10 times. Place a cover slip on the slides using a toluene or xylene based mounting media.

EXAMPLE II

Collection and Processing of CAS-200 CMP v3.0 Nuclear Morphometric Descriptors (40.times. Objective)

The morphometry data from the radical prostatectomy specimens is captured using the Cell Measurement Program v3.0 (CMP v3.0) software from a CAS-200 Image Analysis System. First, a study is set up in CMP v3.0 using the QDA Morphology Mode. The QDA Morphology Mode of CMP v3.0 allows the measurement of the Sum O.D., size, shape, cell class, and the 22 Markovian texture features (a step size of 1 was used in this invention) for each cell (see Table I), as well as the generation of a DNA histogram through the use of the QDA v3.0 software program on the CAS-200 Image Analysis System. Once the study is set up, the CMP v3.0 program (under the QDA Morphology Mode) activates the QDA v3.0 program, and the optical system is calibrated using the CAS calibration slides that were stained with the specimen slides. At least 20 calibration cells are measured, with a calibration peak percent coefficient of variation (% C.v.) of less than 2.0%. (NOTE: If the % C.V. is greater than 2.0%, a problem has occurred in the staining process.) Next, at least 125 cancer cells are analyzed using the method described in Example IV, and the cell nuclear images captured from each 5 .mu.m Feulgen stained tissue section, with all of the sum O.D., size, shape, and Markovian texture measurements being saved to a CMP v3.0 vector (*.VEC) file. The nuclear cell images and DNA content information are saved to a QDA v3.0 listmode (*.ILM) file. The CMP vector file (*.VEC) is then converted to a Lotus 1-2-3 file (*.WK1) using the CMP Exporting Utility (a feature of the CMP v3.0 software). The DNA content information contained in the listmode file is extracted with specially written software and saved to a comma delimited text file. The Lotus 1-2-3 file (*.WK1) is then transferred to a 486 PC equipped with Windows v3.1 and Excel v5.0 for Windows, and an Excel v5.0 macro file is used to convert the Lotus 1-2-3 file (*.WK1) into separate Excel v5.0 files (*.XLS) for each case, each file containing the following information for every cell captured from that particular specimen: the sum O.D, size, shape, cell class, 22 Markovian texture features, and DNA content; (referred to collectively as CMP Nuclear Morphometric Descriptors, or CMP NMD’s). Each Excel v5.0 file (*.XLS) also contains the means, standard deviations, variances, minima, and maxima for each CMP NMD. In addition, the macro creates a summary file containing the above statistics for each sMP NMD from every case.

EXAMPLE III

Collection and Processing of JVB ILM Morphometry v1.0 Nuclear Morphometric Descriptors

The morphometry data from radical prostatectomy specimens is captured from the saved listmode files (*.ILM) using the JVB ILM Morphometry v1.0 software program, which allows the measurement and calculation of up to 36 different features. The listmode files (*.ILM) are created using the QDA v3.0 software from a CAS-200 Image Analysis System. The optical system is calibrated using the CAS calibration slides that were stained with the specimen slides by measuring at least 20 calibration cells, with a calibration peak percent coefficient of variation (% C.V.) of less than 2.0%. (NOTE: If the % C.V. is greater than 2.0%, a problem has occurred in the staining process.) Next, at least 125 cancer cells are analyzed using the method described in Example IV, and the cell nuclear images captured from each 5 .mu.m Feulgen stained tissue section. The DNA content information and cell nuclear images are saved to a listmode (*.ILM) file. The listmode files (*.ILM) are then transferred to a 486 PC equipped with Windows v3.1 and Excel v5.0 for Windows, and converted using the JVB ILM Morphometry v1.0 program into 36 measurements (collectively referred to as JVB Nuclear Morphometric Descriptors, or JVB NMD’s), which are contained in a Microsoft Access Database file (*.MDB). These 36 measurements include the sum O.D., size, shape, DNA content, 22 Markovian texture features, and nuclear shape features (see Table II). The Microsoft Access Database file (*.MDB) is then converted to an ASCII comma delimited file (*.CSV) using a conversion feature of the JVB ILM Morphometry v1.0 program. Finally, using Excel v5.0, an Excel v5.0 macro file is used to convert the ASCII comma delimited file (*.CSV) into separate Excel v5.0 files (*.XLS) for each case, each file containing the JVB NMD’s for every cell captured from that particular specimen. Each Excel v5.0 file (*.XLS) also contains the means, standard deviations, variances, minima, and maxima for each JVB NMD. In addition, the macro creates a summary file containing the above statistics for each JVB NMD from every case.

EXAMPLE IV

Cancer Cell Selection Method

The inventors used a cell selection process for the radical prostatectomy specimens that seemed to introduce the least amount of bias and took into account the heterogeneity of prostate cancer tumors. The tumor area must first be identified by an expert pathologist. Once the tumor area(s) have been identified, a minimum of 25 image fields and a maximum of 5-6 cells per image field must then be analyzed and the cell nuclear images captured. The cells selected may not be overlapping, and they may not contain any “holes”, which is the result of an improper background threshold setting. Sample the entire circled tumor area. The best way to do this is to mentally partition the circled tumor area into four separate quadrants, and then measure a minimum of 6-7 image fields per quadrant. In each quadrant, select image fields from the “worst” (e.g. Highest grade) cancer areas. (NOTE: The “worst” area in each quadrant may vary from low grade, well differentiated cancer to high grade, poorly differentiated cancer. Just be sure to measure from the “worst” area in each of the four quadrants.) Once you have collected the required number of cells, save the DNA information and nuclear images to a listmode file.

EXAMPLE V

Analysis of CAS-200 DNA Histograms

The DNA histograms were interpreted and classified by three different methods by the consensus of five individuals.

The three different methods employed cut-offs based upon the results of a DNA Consensus meeting held at Prautz Neck, ME in 1992 (Shankey, T. V. et al. Cytometry 14:497-500, 1993). The histograms were interpreted by four different individuals, and a consensus DNA ploidy classification agreed upon. The classification methods are as follows: (See FIG. 28A and FIG. 28B)

DNA-1=(0) Diploid; (1) ONR: Hypodiploid; (2) ONR: >S+G2M (11-21%); (3) Abnormal: >S+G2M (.gtoreq.21%); (4) Abnormal: Aneuploid; and (5) Abnormal: Tetraploid

DNA-IO=(0) Diploid and ONR: Hypodiploid; (1) ONR: >S+G2M (11-21%); (2) Abnormal: >S+G2M (.gtoreq.21%), Aneuploid, and Tetraploid

DNA-10=(0) Normal and Out of Normal Range; (1) Abnormal

The three different methods employed cut-offs determined by the inventors. The histograms were interpreted, and the classification methods are as follows:

JHHDNA=(0) Diploid; (1) Tetraploid; (2) Aneuploid

JHHDNA10=(0) Diploid; (1) Non-Diploid (i.e. Tetraploid and Aneuploid)

JHH%>2N=Percentages of S-Phase and Tetraploid fractions combined from ploidy determinations.

For statistical analysis, each classification scheme coded every subclass as a result for each patient (i.e. CDI DNA Ploidy: Diploid=0, Hypodiploid=1, ONR: >S+G2M=2, Tetraploid=5, Normal=0, Abnormal=1; JHH DNA Ploidy: Diploid=0, Tetraploid=1, Non-Diploid=1, etc.). The JHH%>2N classification method used the percentage as a result for each patient. These coded results were used for statistical analysis.

EXAMPLE VI

Nuclear Roundness Factor Measurement and Calculation of Variance

Definition of Nuclear Roundness Factor

The nuclear roundness factor represents a dimensionless, size-invariant shape descriptor that is based on the ratio of the calculated radius for the measured perimeter divided by the calculated radius for the measured area of the nucleus. This descriptor yields a low value of 1.00 for a perfect circle and increases as the shape of the nucleus deviates from circularity. In mathematical terms:

Solve Equations for the Radius

Substitute Radius Equations into Nuclear Roundness Factor Equation

The variance in the nuclear roundness (NRV) was calculated using the following formula: ##EQU2## n=Number of cells measured j=The j.sup.th cell

Y.sub.j =The nuclear roundness factor of the j.sup.th cell

Y=The average or mean nuclear roundness factor for all of the cells

Measurement of Nuclear Roundness using the DynaCELL.TM. System

Histologic tissue sections (5-6 .mu.m) were cut from re-embedded paraffin blocks of radical prostatectomy specimens. Multiple sections were cut (thirty sections per specimen), and one set of slides from each specimen were stained with Hematoxylin and Eosin (Sakura Diversified Stainer, Model DRS-601) and Feulgen stain (Cell Analysis Systems, Elmhurst, Ill.). The H&E staining procedure was performed on sections #1, 10, 20, and 30 for purposes of pathology review to confirm the presence of cancer for additional biomarker studies. All pathologic radical prostatectomy specimens were assigned Gleason scores (sum). The nuclear roundness factor measurements were performed using the H&E sections. A total of 150 cancerous nuclei from the primary tumor were analyzed with a Zeiss inverted IM microscope (Carl Zeiss, Inc., Thornwood, N.Y.) equipped with a Zeiss Planochromatic 100.times. oil emersion objective, giving a total magnification of 2440.times.. The nuclear images were digitized and analyzed with the DynaCELL.TM. Motility Morphometry Measurement workstation (JAW Associates, Inc., Annapolis, Md.). In this invention, the nuclear roundness variance measurement is the only calculation used from the DynaCELL.TM. Motility Morphometry Measurement software.

EXAMPLE VII

Utilization of Increased Magnification (63.times.) to Reduce the Number of NMD’s Required to Predict Progression

Using a subset of the original patient sample (10 progressors and 10 non-progressors), measurements were conducted as in Examples II & III, except that instead of using the normal 40.times. objective, a 63.times. objective lens was used. The data and statistics obtained using the 63.times. objective were analyzed and compared to the data and statistics obtained using the 40.times. objective. Table III summarizes the results of the statistical analysis using the 40.times. and 63.times. data to predict prostate cancer progression in the subset of 20 patients. Please note that the total number of NMD’s required to predict an outcome is decreased as the magnification increases (Table III; also see FIGS. 3-6), as well as significant changes in the actual individual NMD’s utilized in the model.

EXAMPLE VIII

Immunochemical Staining for Her-2/neu (c-erbB2)

Her-2-neu (c-erbB2) monoclonal antibody (Ab-3, OP-15) was provided by Oncogene Sciences Inc. (Uniondale, N.Y.) as a gift. The SuperSensitive MultiLink.TM. kit (BioGenex Inc., Ca.), which employs the strep-avidin biotin complex (ABC) alkaline phosphatase (AP) labelling method, was used for monoclonal antibody detection. All staining was performed with the MicroProbe.TM. manual staining system (Fisher Scientific, Pittsburgh, Pa.) that utilizes capillary action vertical staining principles. Incubation for the monoclonal antibody was 4.degree. centigrade overnight. Briefly, the staining procedure includes first preparing the immunostaining reagents as follows:

Immunostaining Reagent Preparation

PBS pH 7.6 with 0.1% Triton X-100

Place 450 ml of Type I H.sub.2 O into a 500 ml graduated cylinder. Then add one envelope of Coulter PBS Buffer Reagent (Coulter Source, Marietta, Ga.) to the type I water while stirring. Adjust the pH to 7.6 with approximately 20 drops of 1 N NaOH (a plastic transfer pipet is useful in adding the NaOH). Pipette 500 .mu.l of Triton X-100 to the solution. Next, Adjust the volume of the solution to 500 ml with Type I H.sub.2 O.

PBS pH 7.6 with 0.5% Triton X-100

Place 450 ml of Type I H.sub.2 O into a 500 ml graduated cylinder. Then add one envelope of Coulter PBS Buffer Reagent to the type I water while stirring. Adjust the pH to 7.6 with approximately 20 drops of 1 N NaOH (a plastic transfer pipet is useful in adding the NaOH). Pipette 2.5 ml of Triton X-100 to the solution. Adjust the volume of the solution to 500 ml with Type I H.sub.2 O.

1M Levamisole Stock Solution

Measure 241 mg (0.241 g) of levamisole (Sigma) using an analytic balance. Place the levamisole into a 1.5 ml microcentrifuge tube containing 1 ml of Type I H.sub.2 O. Mix the contents with the aid of a vortex mixer. Store the solution at 4.degree. C. until it is used.

5% Nonfat dry milk with PBS pH 7.6 0.1% Triton X-100, 0.05% thimerosal

Place 5 grams of nonfat dry milk in a Erlenmeyer flask containing 100 ml PBS pH 7.6 with 0.1% Triton.RTM. X-100. Then, add 0.05 g of thimerosal and mix the solution by stirring. Store 5 ml aliquots of the solution at -80.degree. C. Upon thawing, the solution should be stored at 4.degree. C. Do not use this solution if it has been stored at 4.degree. C. for longer than 5 days.

0.5% Nonfat dry milk with PBS pH 7.6 0.1% Triton X-100

Pipette 100 .mu.l 5.0% nonfat dry milk with PBS pH 7.6 0.1% Triton X-100, 0.05% thimerosal into a 1.5 ml microcentrifuge tube or 10 ml test tube containing 900 .mu.l of PBS pH 7.6 with 0.1% Triton X-100. Mix the solution with the aid of a vortex. The solution should be stored at 4.degree. C. Do not use this solution if it has been stored at 4.degree. C. for longer than 5 days.

C-Neu, Her-2/Neu (1:40)

Pipette 875 .mu.l of PBS pH 7.6 with 0.1% Triton X-100 into a 1.5 ml microcentrifuge tube or 10 ml test tube. Pipette 100 .mu.l 5.0% nonfat dry milk with PBS pH 7.6 with 0.1% Triton X-100 to the tube and mix the solution with the aid of a vortex. Then, pipette 25 .mu.l of C-Neu (ab-3) to the tube and mix with the aid of a vortex. The antibody should be added last to the solution.

Normal Mouse Serum Control (1:1000)

Pipette 899 .mu.l of PBS pH 7.6 with 0.1% Triton X-100 into a 1.5 ml microcentrifuge tube or 10 ml test tube. Pipette 100 .mu.l 5.0% nonfat dry milk with PBS ph 7.6 with 0.1% Triton X-100 to the tube and mix the solution with the aid of a vortex. Pipette 1 .mu.l Normal Mouse Serum (Dako) to the solution and mix with the vortex. The normal mouse serum should be added last to the solution.

Mouse IgG1 Isotypic Control (1:200)

Pipette 895 .mu.l of PBS pH 7.6 with 0.1% Triton X-100 into a 1.5 ml microcentrifuge tube or 10 ml test tube. Pipette 100 .mu.l 5.0% nonfat dry milk with PBS ph 7.6 with 0.1% Triton X-100 to the tube and mix the solution with the aid of a vortex. Pipette 5 .mu.l Mouse IgG1 Isotypic Control (Coulter) to the solution and mix with the vortex. The Mouse IgG1 Isotypic Control should be added last to the solution.

2.degree.Ab (Biotinylated anti-mouse IgG, Multilink)

Comes premixed in the BioGenex Large Volume MultiLink Kit

Label (Streptavidin/Alkaline Phosphatase)

Comes premixed in the BioGenex Large Volume MultiLink Kit

Fast Red Chromogen Solution

Pipette 5 .mu.l of 1.0 M Levamisole to the 5 ml vial of Naphthol Phosphate in Tris Buffer. Add one Fast Red Tablet to the solution and vortex until the tablet is completely dissolved. This solution must be used immediately after preparation. *Levamisole is added to the Fast Red Solution to block endogenous alkaline phosphatase activity.

The Her-2/neu antigenicity was then scored. The scoring method assessed the amount of staining area within the “dotted cancer zone” as either negative (0), focal (1), or diffuse (2), and the intensity of the staining was scored as 0-4+, ranging from negative (0) to strong red color (4+) resulting from the AP red substrate reaction (see FIG. 28A and FIG. 28B).

EXAMPLE IX

Immunochemical Staining for PD-41

The PD-41 (Prostate Mucin Antigen) monoclonal antibody was provided by Dr. George Wright at Eastern Virginia Medical School under a materials transfer agreement. The SuperSensitive MultiLink.TM. kit (BioGenex Inc., Ca.), which employs the strep-avidin biotin complex (ABC) alkaline phosphatase (AP) labelling method, was used for monoclonal antibody detection. All staining was performed with the MicroProbe.TM. manual staining system (Fisher Scientific, Pittsburgh, Pa.) that utilizes capillary action vertical staining principles. Incubation for the monoclonal antibody was 370 centigrade for 15 minutes. Briefly, the staining procedure includes first preparing the immunostaining reagents as in Example X, except with the following changes:

Immunostaining Reagent Preparation

PD-41 (15 .mu.g/ml)

Place 800 .mu.l of PBS pH 7.6 with 0.1% Triton X-100 in a 1.5 ml microcentrifuge tube. Add 100 .mu.l 50 milk to the tube and mix the contents with the aid of a vortex mixer. Then, add 100 .mu.l of PD-41 to the tube and mix the contents with the aid of a vortex mixer.

CAS Red Chromogen Solution

Add 900 .mu.l of Type I H.sub.2 O to a 1.5 ml microcentrifuge tube. Add the 1 .mu.l of 1 M levamisole to the tube. Then, add 100 .mu.l of CAS red substrate concentrate and mix the contents with the aid of a vortex mixer. Add 45 .mu.l of CAS red chromogen concentrate (always add this ingredient last) to the solution and mix the contents with the aid of a vortex mixer. This solution must be used immediately after preparation.

The PD-41 antigenicity was then scored. The scoring method employed the number of positive staining ducts divided by the total number of ducts in the “dotted cancerous zone”. The percentages of positively staining ducts was used as a patient result.

PD-41 Background

Monoclonal antibody PD-41, a mouse IgG.sub.1k, was first described by Beckett et al. (Beckett, ML, Lipford, GB, Haley, Cl, Schellhammer, PF and Wright, GL. Monoclonal Antibody PD41 Recognizes an Antigen Restricted to Prostate Adenocarcinomas. Cancer Res. 51:1326-1333, 1991) by its reactivity to an prostate adenocarcinoma-restricted mucoprotein known as prostate mucin antigen (PMA). The target PMA, an O-linked oligosaccharide-associated protein with a molecular weight of >400 kd in prostate cancer patient seminal plasma, has not been demonstrated to recognize mucins at other organ sites. Wright et al. (Wright, GL, Beckett, ML et al. Mucins as biomarkers of prostate carcinoma. J. Urol. 149:450A, 1993) demonstrated immunoperoxidase immunoreactivity of PD-41 with in 100% of primary, 71% of metastatic carcinomas and under 1% of normal and benign prostatic tissues, including BPH.

An independent study of 95 prostate needle core biopsy paraffin-embedded sections showed PD-41 reactivity in ductal epithelia and/or prostatic glandular secretions within 56% (53/95) of prostate tumor specimens (Marley, GM, Veltri, RW, Patton, KP and Wright, GL. Histochemical Expression of a Unique Prostate Mucin Antigen from Core Biopsies. Proc. Amer. Assoc. Cancer Res. 34:28, 1993). When Gleason score or DNA ploidy were employed as stratification parameters, PD-41 proved to be an independent factor of prognostic value. Clinical follow-ups of 61% of this cohort confirmed that PD-41 expression acted as an independent marker of tumor aggressiveness (Veltri, RW et al., recent CDI unpublished data).

Abstract
A brassier for reducing breast cancer includes cups for holding the mammary glands. Each of the cups has a slot formed therein for accommodating a papilla. Each slot is longitudinally elongated to accommodate for variances in the longitudinal location of the papilla from woman to woman. By positioning the papilla within the slots, the papillae are not subject to pressure and compression which inflict trauma on the breasts. The brassier for reducing trauma may include cup linings disposed within the cups. Each lining has an opening formed therein for accommodating a papilla. Each lining may also have protective material disposed about the opening to provide protection for the papilla when received within the opening.

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Inventors: Yonchar; Jack (Los Angeles, CA)
Appl. No.: 09/169,514
Filed: October 9, 1998
Claims

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What is claimed is:

1. A brassier for reducing breast cancer, comprising:

a cup for holding a mammary gland; and

a slot formed in said cup for accommodating a papilla;

said slot being elongated longitudinally;

whereby said slot accommodates for variances in areolar location in a longitudinal direction.

2. A brassier for reducing breast cancer, comprising:

a cup for holding a mammary gland;

a slot formed in said cup for accommodating a papilla; and

a lining received in said cup and including an opening for accommodating a papilla;

said slot being elongated longitudinally;

whereby said slot accommodates for variances in areolar location in a longitudinal position.

3. A brassier as claimed in claim 2 wherein said lining further comprises protective material disposed about said opening.

4. A brassier as claimed in claim 3 wherein said protective material is substantially resilient.

5. A brassier as claimed in claim 3 wherein said protective material is foam rubber.

6. A brassier as claimed in claim 2 wherein said opening is substantially circular.

7. A brassier as claimed in claim 2 wherein said lining is configured to be disposed substantially over a mammary gland.

8. A brassier as claimed in claim 2 wherein said lining is attached to said cup along a periphery of said lining.

9. A brassier as claimed in claim 2 wherein said lining is made from spandex.

10. A brassier for reducing breast cancer, comprising:

a cup for holding a mammary gland;

a slot formed in said cup for accommodating a papilla; and

a plurality of grommets formed along sides of said slot and a lace received in said grommets;

said slot being elongated longitudinally;

whereby said slot accommodates for variances in areolar location in a longitudinal direction and said slot is adjustable in width.
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Description

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FIELD OF THE INVENTION

The present invention relates to feminine garments and, more particularly, to brassieres with medical applications.

BACKGROUND OF THE INVENTION

With reference to FIG. 1, the mammary glands of a woman secrete milk and are accessory glands of the generative system. The glands are two hemispherical eminences lying within the superficial fascia and situated on the front and sides of the chest. Each gland extends from the second rib above to the sixth rib below, and from the side of the sternum to near the mid-axillary line. The weight and dimensions of the mammary glands differ at different periods of life and in different individuals. Before puberty the mammary glands are small in size, but enlarge as the generative organs become more completely developed. The glands increase during pregnancy and especially after delivery, and become atrophied in old age. The left gland is generally a little larger than the right. The deep or inner surface of each is nearly circular, flattened, or slightly concave, and has its long diameter directed upward and lateralward toward the axilla (which is the pyramidal space situated between the upper lateral part of the chest and the medial side of the arm). Each gland is separated from the fascia covering the Pectoralis major, Serratus anterior, and Obliquus externus abdominis by loose connective tissue. The subcutaneous surface of the mammary gland is convex and presents, just below the center, a small conical prominence, or papilla 10.

The mammary papilla or nipple 10 is a cylindrical or conical eminence situated about the level of the fourth intercostal space. It is capable of undergoing erection from mechanical stimulation, a change mainly due to the contraction of its muscular fibers. Its surface is wrinkled and provided with secondary papillae, and it is perforated by from 15 to 20 orifices, the apertures of lactiferous ducts 12. The base of the mammary papilla 10 is surrounded by an areola 14. Near the base of the papilla 10, and upon the surface of the areola 14, are numerous large sebaceous glands, the areolar glands, which become much enlarged during lactation, and present the appearance of small tubercles beneath the skin. The mammary papilla 10 consists of numerous vessels, intermixed with plain muscular fibers, which are principally arranged in a circular manner around the base.

The mammary gland consists of gland tissue; of fibrous tissue, connecting its lobes; and of fatty tissue in the intervals between the lobes. The subcutaneous surface of the mammary gland presents numerous irregular processes which project toward the skin and are joined to it by bands of connective tissue. It consists of numerous lobes, which are composed of lobules 16, connected together by areolar tissue, blood vessels, and ducts. The smallest lobules consist of a cluster of rounded alveoli, which open into the smallest branches of the lactiferous ducts 12. These ducts unite to form larger ducts, and these end in a single canal, corresponding with one of the chief subdivisions of the gland. The number of excretory ducts varies from 15 to 20, which are termed the tubuli lactiferi. They converge toward the areola 14, beneath which they form dilatations or ampullae 18, which serve as reservoirs for the milk, and, at the base of the papillae 10, become contracted and pursue a straight course to its summit, perforating it by separate orifices considerable narrower than the ducts themselves.

The fibrous tissue of the mammary glands invests the entire surface of the mamma. Bands of fibrous tissue traverse the gland and connect the overlying skin to the underlying pectoral fascia. These constitute the ligaments of Cooper. The fatty tissue 20 covers the surface of the gland, and occupies the interval between its lobes. It usually exists in considerable abundance, and determines the form and size of the gland. However, there is no fat immediately beneath the areola 14 and papilla 10.

Trauma is inflicted by conventional brassieres by constriction and pressure applied to the papillae and the lactiferous tubules, ampullae, and lobules. Aggravation to the breast is furthered by friction of conventional brassieres on the papillae and areolae during activities such as walking and exercising, and while lying on the breast while sleeping.

Conventional brassieres encase the entire breast and compress and constrain the papillae and areolar area where the lactiferous tubules are located. The compression of the papillae and the tubules are irritating factors that inflict trauma on the breasts and may contribute to the formation of cancer.

In view of the foregoing, there is a need in the art for a brassier which reduces trauma to the breasts, particularly trauma to the papillae and areolae.

BRIEF SUMMARY OF THE INVENTION

The present invention provides brassier which reduce the trauma subjected to breasts, particularly trauma to the papillae and areolae. Such trauma reduction may contribute to the reduction of breast cancer caused by irritation and compression of conventional brassieres on the breasts.

In accordance with one aspect of the present invention, a brassier includes cups for holding mammary glands. Each of the cups has a slot formed therein for accommodating a papilla. Each slot is longitudinally elongated. Because of the elongated configuration, the slots are able to accommodate for variances in the longitudinal location of the papilla from woman to woman. By positioning the papilla within the slots, the papillae are not subject to pressure and compression which inflict trauma on the breasts.

According to another aspect of the invention, the brassier of the present invention may include cup linings respectively disposed within the cups. Each lining has an opening formed therein for accommodating a papilla. Each lining may also have protective material disposed about the opening to provide protection for the papilla when received within the opening. The protective material may be resilient or compressive material such as foam rubber. The protective material further reduces trauma which may be inflicted by tight-fitting garments or during sporting activities. In addition, the linings are preferably made from resilient material, which further enhances the shock-reducing functionality of the brassier to reduce trauma.

Other objects, features, and advantages of the present invention will become apparent to those skilled in the art from a consideration of the following detailed description taken in conjunction with the accompanying.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

FIG. 1 is a perspective view of a mammary gland shown in partial section;

FIG. 2 is a perspective view of an exemplary embodiment of a brassier for reducing breast cancer in accordance with the present invention;

FIG. 3 is a perspective view of another exemplary embodiment of a brassier for reducing breast cancer in accordance with the present invention;

FIG. 4 is a plan view of a cup lining of the present invention;

FIG. 5 is a cross-sectional view of the cup lining taken along line 5–5 of FIG. 4; and

FIG. 6 is a perspective view of another exemplary embodiment of a brassier for reducing breast cancer in accordance with the present invention.

DETAILED DESCRIPTION OF THE INVENTION

Referring more particularly to the drawings, an exemplary brassier 30 for reducing breast trauma and cancer produced in accordance with the teachings of the present invention is illustrated in FIG. 2. The brassier 30 generally includes cups 32 for holding mammary glands. Each of the cups 32 has a slot 34 formed therein for accommodating a papilla. Each slot 34 is generally elongated and extends longitudinally across the cup 32. As shown in FIG. 2, exemplary brassier 30 may also include a chest strap 36 and shoulder straps 38, as known in the art.

Because of the elongated configuration, the slots 34 are able to accommodate for variances in the longitudinal location of the papilla from woman to woman. The slots 34 may also be sufficiently wide to accommodate for variances in the lateral location of the papilla from woman to woman. However, the lateral position is predominantly dictated by the cups 32 in aligning the papillae respectively within the slots 34. By positioning the papilla within the slots 34, the papillae are not subject to pressure and compression. Accordingly, a more natural situation is effected.

Referencing FIGS. 3, 4, and 5, exemplary brassier 30 may also include cup linings 40 respectively disposed within the cups 32. (For clarity, only one lining 40 is shown in FIG. 3, and the lining 40 is shown separated from the cup 32.) Each lining 40 has an opening 42 formed therein for accommodating a papilla. Each lining 40 may also have protective material 44 disposed about the opening 42. The protective material 44 provides protection for, and, thereby, further reduces trauma to, the papilla when received within the opening 42. The protective material 44 may be resilient or compressive material such as foam rubber.

Each lining 40 is preferably attached to a respective cup 32 along a periphery 46 thereof by stitching. Accordingly, the lining 40, as well as the protective material 44, is independently movable with respect to the cup 32. This feature of the brassier 30 provides enhanced shock-reducing functionality, thereby further reducing trauma. In addition, the linings 40 are preferably made from resilient or stretchable material such as lycra, further enhancing the shock-absorbing feature of the brassier 30. Alternatively, each lining 40 may be a separate or detached element of the brassier 30, or may be attached only along a portion of the periphery 46. Releasable fasteners such as hook-and-eye fasteners may also be used to attach the linings 40 to the cups 32.

The opening 42 may be formed completely through the lining 40 or may be formed as a recess with the lining 40 extending over an outer side thereof, as shown in FIG. 5. With the lining 40 covering the outer side of the opening 42, any irritation to the papillae caused by a garment is eliminated. In addition to the substantially circular configuration shown in the drawings, the openings 42 may be elongated or oval. As particularly shown in FIG. 4, the lining 40 is preferably cup shaped to complement the shape of the cup 32 and to substantially cover a mammary gland.

Referencing FIG. 6, exemplary brassier 30 may also include lacing 48 received in a plurality of grommets 50 formed around each slot 32. Accordingly, the width of each slot 32 may be adjusted by the user according to a preferred level of support.

In a commercial embodiment of the invention, the cups 32 may be sized as known in the art. The slots 34 may range in lateral width upwards from about 3/8 inch, and may range in longitudinal length from about 5/8 inch to, for example, about 5 inches, for the average-figure woman.

Those skilled in the art will understand that the present invention is not limited to the specifically illustrated and described embodiments above. The scope of the present invention is determined by the terms of the appended claims and their legal equivalents, rather than by the examples described above.

Abstract
The invention provides a wide range of methods and compositions for detecting and treating cervical cancer in an individual. Specifically, the invention provides target cervical cancer-associated proteins, which permit a rapid detection, preferably before metastases occur, of cervical cancer. The target cervical cancer-associated protein, may be detected, for example, by reacting the sample with a labeled binding moiety, for example, a labeled antibody capable of binding specifically to the protein. The invention also provides kits useful in the detection of cervical cancer in an individual. In addition, the invention provides methods utilizing the cervical cancer-associated proteins either as targets for treating cervical cancer or as indicators for monitoring of the efficacy of such a treatment.

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Inventors: Keesee; Susan K. (Harvard, MA), Obar; Robert (Walpole, MA), Wu; Ying-Jye (Framingham, MA)
Assignee: Matritech, Inc. (Newton, MA)

Appl. No.: 08/989,045
Filed: December 11, 1997
Parent Case Text

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REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No. 08/705,660, filed Aug. 30, 1991 now U.S. Pat. No. 5,858,683.
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Claims

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What is claimed is:

1. A method of screening for cervical cancer in a human, the method comprising:

(a) obtaining a sample isolated from said human; and

(b) detecting in said sample the presence of a protein characterized as being detectable at a higher level in a cervical cancer cell than in a normal cervical cell and comprising an amino acid sequence selected from the group of sequences consisting of SEQ ID NO: 1; SEQ ID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6; SEQ ID NO:7; SEQ ID NO:8; SEQ ID NO:9; and SEQ ID NO: 10, which if present is indicative of cervical cancer in said human.

2. The method of claim 1, wherein said protein comprises the amino acid sequence set forth in SEQ ID NO: 1.

3. The method of claim 1, wherein said protein comprises the amino acid sequence set forth in SEQ ID NO:2.

4. The method of claim 1, wherein said protein comprises the amino acid sequence set forth in SEQ ID NO:3.

5. The method of claim 1, wherein said protein comprises the amino acid sequence set forth in SEQ ID NO:4.

6. The method of claim 1, wherein said protein comprises the amino acid sequence set forth in SEQ ID NO:5.

7. The method of claim 1, wherein said protein comprises the amino acid sequence set forth in SEQ ID NO:6.

8. The method of claim 1, wherein said protein comprises the amino acid sequence set forth in SEQ ID NO:7.

9. The method of claim 1, wherein said protein comprises the amino acid sequence set forth in SEQ ID NO:8.

10. The method of claim 1, wherein said protein comprises the amino acid sequence set forth in SEQ ID NO:9.

11. The method of claim 1, wherein said protein comprises the amino acid sequence set forth in SEQ ID NO:10.

12. The method of claim 1, wherein said sample is a tissue or body fluid sample.

13. The method of claim 1, wherein said sample is a biopsy sample.

14. The method of claim 1, wherein said sample is a cervical cell sample.

15. The method of claim 1, wherein said sample is a Papanicolaou smear.

16. A method of screening for cervical cancer in a human, the method comprising the steps of:

(a) contacting a sample derived from said human with a binding moiety that binds specifically to a cervical cancer-associated protein to produce a binding moiety-cervical cancer-associated protein complex, wherein said binding moiety is selected from the group consisting of an antibody, an antibody fragment and a biosynthetic antibody binding site, and wherein said binding moiety binds specifically to a protein comprising the amino acid sequence set forth in SEQ ID NO: 10; and

(b) detecting the presence of said complex, which if present is indicative of the presence of cervical cancer in said human.

17. The method of claim 16, wherein said cervical cancer-associated protein is further characterized as being present at a higher amount in a human cervical cancer cell than in a normal human cervical cell, as determined by two dimensional gel electrophoresis.

18. The method of claim 16, wherein said sample is a tissue or body fluid sample.

19. The method of claim 16, wherein said sample is a biopsy sample.

20. The method of claim 16, wherein said sample is a Papanicolaou smear.

21. The method of claim 16, wherein said sample is a cervical cell sample.

22. The method of claim 16, wherein said binding moiety is an antibody.

23. The method of claim 22, wherein said antibody is a monoclonal antibody.

24. The method of claim 22, wherein said antibody is labeled with a detectable moiety.

25. The method of claim 23, wherein said monoclonal antibody is labeled with a detectable moiety.

26. The method of claim 1, wherein absence of a detectable amount of said protein is indicative of the absence of cervical cancer.

27. The method of claim 1, further comprising the additional steps of (c) measuring an amount of said protein in said sample and (d) comparing the amount of said protein in said sample with the amount of said protein in a prior sample previously obtained from said human, wherein an increae in amount of said protein in said sample relative to the amount of said protein in said prior sample is indicative of progression of said cervical cancer.

28. The method of claim 16, wherein absence of a detectable amount of said complex is indicative of the absence of cervical cancer.

29. The method of claim 16, further comprising the additional steps of (c) measuring an amount of said protein in said sample and (d) comparing the amount of said protein in said sample with the amount of said protein in a prior sample previously obtained from said human, wherein an increase in amount of said protein in said sample relative to the amount of said protein in said prior sample is indicative of progression of said cervical cancer.
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Description

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FIELD OF THE INVENTION

The present invention relates generally to methods and compositions for the detection of cervical cancer. More specifically, the present invention relates to cervical cancer-associated proteins which act as cellular markers useful (i) in detecting cervical cancer, and (ii) as molecular targets for cervical cancer therapy.

BACKGROUND OF THE INVENTION

Cancer of the uterine cervix is one of the most common malignancies in women and remains a significant public health problem throughout the world. In the united States alone, invasive cervical cancer accounts for approximately 19% of all gynecological cancers (Miller et al. (1993) in “Surveillance Epidemiology, and End Results Program cancer Statistics Review: 1973-1990″, NIH Pub. No. 93-2789, Bethesda, Md.: National Cancer Institute). In 1996, it is estimated that there will be 14,700 newly diagnosed cases and 4900 deaths attributed to this disease (American Cancer Society, Cancer Facts & Figures 1996, Atlanta, Ga.: American Cancer Society, 1996). In many developing countries, where mass screening programs are not widely available, the clinical problem is more serious. Worldwide, the number of new cases is estimated to be 471,000 with a 4 year survival rate of 40% (Munoz et al. (1989) “Epidemiology of Cervical Cancer” in “Human Papillomavirus”, New York, Oxford Press, pp 9-39; and National Institutes of Health, Consensus Development Conference Statement on Cervical Cancer, Apr. 1-3, 1996).

The precursor to cervical cancer is dysplasia, also known in the art as cervical intraepithelial neoplasia (CIN) or squamous intraepithelial lesions (SIL) (Brinton et al. (1992) “Epidemiology of Cervical Cancer: Overview” in “The Epidemiology of Cervical Cancer and Human Papillomavirus”, Lyon, France: International Agency for Research on Cancer; and Tabbara et al. (1992) “The Bethesda classification for squamous intraepithelial lesions: histologic, cytologic and viral correlates”, Obstet. Gynecol. 79: 338-346). While it is not understood how normal cells become transformed, the concept of a continuous spectrum of histopathological change from normal, stratified epithelium through CIN to invasive cancer has been widely accepted for many years (see, for example, Mitchell et al (1994) “The natural history of cervical intraepithelial neoplasia: an argument of intermediate endpoint biomarkers”, Cancer Epidmiol. Biomark. Prev. 3: 619-626). A large body of epidemiological and molecular biological evidence has been gathered that establishes human papillomavirus (HPV) infection as a causative factor in cervical cancer (Munoz et al. (1992) in “The Epidemiology of Human Papillomavirus and Cervical Cancer”, IRAC publication no. 119, Lyon France: Int. Agency for Research on Cancer, pp 251-261). HPV is found in 85% or more of squamous cell invasive lesions, which represent the most common histologic type seen in cervical carcinoma (Cox et al. (1995) Baillierre ’s Clin. Obstet Gynaecol. 91-37). Additional cofactors include, for example, oncogenes activated by point mutations, and chromosomal translocations of deletions (Spandidos etal. (1989)J. Pathol. 157: 1-10).

Cytological examination of Papanicolaou-stained cervical smears (also referred to as Pap smears) currently is the method of choice for detecting cervical cancer. Despite the historical success of this test, concerns have arisen regarding its ability to predict reliably the behavior of same preinvasive lesions (Ostor et al. (1993) Int. J. Gynecol. Pathol. 12: 186-192; and Genest et al. (1993) Human Pathol. 24: 730-736). The identification of a cervical cancer-associated tumor marker for reliably detecting early onset of cervical cancer and/or providing early prognostic information will greatly aid the management of cervical cancer.

All eukaryotic cells have a nucleus containing DNA, or chromatin, which is organized by an internal protein scaffolding known as the nuclear matrix (NM). The nuclear matrix was first described in 1974 by Berezney et al. (Berezney et al. (1974) Biochem. Biophys. Res. Commun., 60: 1410-1417). Penman et al. describe a method for selectively extracting insoluble interior nuclear matrix proteins and their associated nucleic acids from cells and determining the particular cell type by analyzing the proteins by two-dimensional gel electrophoresis (see for example, U.S. Pat. No. 4,882,268, issued Nov. 21, 1989, and U.S. Pat. No. 4,885,236, issued Dec. 5, 1989, the disclosures of which are incorporated herein by reference).

The nuclear matrix is believed to be involved in a wide variety of nuclear functions fundamental to the control of gene expression. For a general review see, for example, Fey et al. (1991) Crit. Rev. Euk. Gene Express. 1: 127-143. Tissue-specific nuclear matrix proteins have been identified in the rat, mouse and human. Fey et al. (1986) Proc. Natl. Acad. Sci. USA 85: 121-125; Stuurman et al. (1990) J. Biol. Chem. 265: 5460-5465; and Getzenberg et al. (1990) Mol. Endocrinol. 4: 1336-1342. Changes in the presence or absence of specific nuclear matrix proteins have been associated with cellular transformation and differentiation (Bidwell et al. (1993) Proc. Natl. Acad. Sci. USA 90: 3162-3166; Brancolini et al. (1991) Proc. Natl. Acad. Sci. USA 88: 6936-6940; and Greenfield et al. (1991) Proc. Natl. Acad. Sci. USA 88:11217-11221).

Several recent studies using similar methodology have identified tumor-specific nuclear matrix proteins in cancers of the prostate (Partin et al. (1993) Cancer Res. 53: 744-746), breast (Khanuja et al. (1993) Cancer Res. 53: 3394-3398), colon cancer (Keesee et al. (1994) Proc. Natl. Acad. Sci. USA 91: 1913-1916), bone (Bidwell et al. (1994) Cancer Res. 54: 28-32), bladder (Getzenberg et al. (1996) Cancer Res. 56: 690-694) and the larynx (Donat et al. (1996) Otolaryngol. Head Neck Surg. 114: 387-393). Molecular characterization of the specific nuclear matrix proteins, however, remains poorly defined, due to the low abundance of these proteins in the cell and their generally insoluble character.

There is, however, a need in the art for specific, reliable markers that are expressed differentially in normal and cancerous cervical tissue and that may be useful in the detecting cervical cancer or in the prediction of its onset. Accordingly, it is an object of this invention to provide cervical cancer-associated molecules which are useful as markers for the early and/or rapid detection of cervical cancers in an individual. It is another object of this invention to provide methods for detecting cervical cancers in an individual. It is another object of the invention to provide methods and compositions for treating cervical cancers in an individual and for monitoring the efficacy of such a treatment in the individual.

SUMMARY OF THE INVENTION

The invention provides a variety of methods and compositions for detecting and/or prognosing cervical cancer in a tissue or body fluid sample of an individual. The invention is based, in part, upon the discovery of cervical cancer-associated proteins which are present at detectable levels in cervical cancer cells, but which are not detectable in normal cervical cells, as determined by two-dimensional gel electrophoresis.

In one aspect, the invention provides a method for detecting cervical cancer in a human. The method comprises the step of detecting the presence of a cervical cancer-associated protein in a tissue or body fluid sample of the human thereby to indicate the presence of a cervical cancer or a precursor of a cervical cancer. The cervical cancer-associated protein is characterized as having a molecular weight of from about 44,900 Daltons to about 69,400 Daltons, as determined by standard polyacrylamide gel electrophoresis techniques and an isoelectric point of from about 5.1 to about 6.6 as determined by standard isoelectric focusing techniques. In addition, the cervical cancer-associated protein is further characterized as being a non-chromatin protein which is detectable at a higher level in a human cervical cancer cell than in a normal human cervical cell, as determined by two-dimensional gel electrophoresis. It is contemplated, however, that the accuracy and/or reliability of the method may be further enhanced by detecting the presence of a plurality of cervical cancer-associated proteins in the preselected tissue or body fluid sample.

As used herein, the term “cervical cancer” is understood to mean any cancer or cancerous lesion associated with cervical tissue or cervical cells and, in addition, includes precusors to cervical cancer, for example, dysplasia (also known in the art as a cervical intraepithelial neoplasia or a squamous intraepithelial lesion).

As used herein, the term “cervical cancer-associated” molecules refers to molecules originating from and isolatable from a cervical cancer cell or cells, and substantially neither originating from nor isolatable from a normal cervical cell or cells. As used herein, the term “cervical cancer-associated protein” is understood to mean any protein which is detectable at a higher level in cervical cancer cells than in normal cervical cells, as determined by two-dimensional (2-D) gel electrophoresis. It is not necessary that the target molecule or target protein be unique to a cervical cancer cell; rather it is preferred that the target molecule or protein has a signal to noise ration high enough to discriminate between samples originating from a cervical cancer tissue or body fluid and samples originating from normal cervical tissue or body fluid.

In a preferred embodiment, methods of the invention comprise the step of detecting one or more cervical cancer (CvC) associated proteins, referred to herein as CvC-1 through CvC-5, which can be purified or co-purified using nuclear matrix protein purification methodologies, well known and thoroughly documented in the art. See, for example, Fey et al. (1986) Proc. Natl. Acad. Sci, USA 85: 121-125, the disclosure of which is incorporated herein by reference. As used herein, the term “nuclear matrix protein” is understood to mean any non-cytoskeletal, non-lamin, non-chromatin protein that (i) is isolated from mammalian cell nuclei, (ii) is resistant to solubilization from the nuclei in 0.25M ammonium sulfate, (iii) remains in solution following dialysis into physiological buffer from 8M urea and (iv) is detectable on a silver stained two-dimensional electrophoresis gel. Accordingly, one or more of the resultant cervical cancer-associated proteins may be further defined as being a nuclear matrix protein.

In a preferred embodiment, methods of the invention may comprise the step of detecting the protein CvC-1, a protein having a molecular weight of about 69,400 Daltons, as determined by polyacrylamide gel electrophoresis, and a pI of about 5.8, as determined by isoelectric focusing techniques. Alternatively, the methods of the invention may comprise the step of detecting the protein CvC-2, a protein having a molecular weight of about 53,800 Daltons, as determined by polyacrylamide gel electrophoresis, and a pI of about 5.5, as determined by isoelectric focusing techniques. Alternatively, the methods of the invention may comprise the step of detecting the protein CvC-3, a protein having a molecular weight of about 47,900 Daltons, as determined by polyacrylamide gel electrophoresis, and a pI of about 5.6, as determined by isoelectric focusing techniques. Alternatively, the methods of the invention may comprise the step of detecting the protein CvC-4, a protein having a molecular weight of about 46,000 Daltons, as determined by polyacrylamide gel electrophoresis, and a pI of about 5.1, as determined by isoelectric focusing techniques. Alternatively, the methods of the invention may comprise the step of detecting the protein CvC-5, a protein having a molecular weight of about 44,900 Daltons, as determined by polyacrylamide gel electrophoresis, and a pI of about 6.6, as determined by isoelectric focusing techniques.

In another preferred embodiment, the methods of the invention may comprise the step of detecting a cervical cancer-associated protein which comprises a continuous amino acid sequence selected from the group consisting of: SEQ ID NO.: 1; SEQ ID NO.: 2; SEQ ID NO.: 3; SEQ ID NO.: 4; SEQ ID NO.: 5; SEQ ID NO.: 6; SEQ ID NO.: 7; SEQ ID NO.: 8; and SEQ ID NO.: 9. Alternatively, the method of the invention may comprise the step of detecting a cervical cancer-associated protein having the amino acid sequence set forth in SEQ ID NO.: 10, commonly referred to in the art as IEF SSP 9502. See, for example, Honore et al. (1994) Gene 151: 291-296, the disclosure of which is incorporated herein by reference.

In another preferred embodiment, the methods of the invention may comprise the step of detecting a cervical cancer-associated protein which comprises a continuous amino acid sequence selected from the group consisting of: SEQ ID NO.: 11; SEQ ID NO.: 12; SEQ ID NO.: 13; SEQ ID NO.: 14; SEQ ID NO.: 15; SEQ ID NO.: 16; and SEQ ID NO.: 17. Alternatively, the method of the invention may comprise the step of detecting a cervical cancer-associated protein having the amino acid sequence set forth in SEQ ID NO.: 18, and commonly referred to in the art as Cytokeratin 17. See, for example, Troyanovsky et al. (1992) J. Biol. Biol. 59: 127-137, the disclosure of which is incorporated herein by reference.

In another preferred embodiment, the methods of the invention may comprise the step of detecting a cervical cancer-associated protein which comprises a continuous amino acid sequence selected from the group consisting of: SEQ ID NO.: 19; SEQ ID NO.: 20; SEQ ID NO.: 21; SEQ ID NO.: 22; SEQ ID NO.: 23; SEQ ID NO.: 24; and SEQ ID NO.: 25. Alternatively, the method of the invention may comprise the step of detecting a cervical cancer-associated protein having the amino acid sequence set forth in SEQ ID NO.: 26, commonly referred to in the art as TDP-43. See, for example, Ou et al. (1995) J. Virol. 69: 3584-3596, the disclosure of which is incorporated herein by reference.

In another preferred embodiment, the methods of the invention may comprise the step of detecting a cervical cancer-associated protein which comprises a continuous amino acid sequence selected from the group consisting of: SEQ ID NO.: 27; SEQ ID NO.: 28; SEQ ID NO.: 29; SEQ ID NO.: 30; SEQ ID NO.: 31; SEQ ID NO.: 32; and SEQ ID NO.: 33. Alternatively, the method of the invention may comprise the step of detecting a cervical cancer-associated protein having the amino acid sequence set forth in SEQ ID NO.: 34, commonly referred to in the art as Nup358. See, for example, Wu et al. (1995) J. Biol. Chem. 270: 14209-14213, the disclosure of which is incorporated herein by reference.

In another preferred embodiment, the methods of the invention may comprise the step of detecting a cervical cancer-associated protein which comprises a continuous amino acid sequence selected from the group consisting of: SEQ ID NO.: 35; SEQ ID NO.: 36; SEQ ID NO.: 37; SEQ ID NO.: 38; SEQ ID NO.: 39; SEQ ID NO.: 40; SEQ ID NO.: 41; SEQ ID NO.: 42; SEQ ID NO.: 43; SEQ ID NO.: 44; and SEQ ID NO.: 45. Alternatively, the method of the invention may comprise the step of detecting a cervical cancer-associated protein having the amino acid sequence set forth in SEQ ID NO.: 46, commonly referred to in the art as lamin A. See, for example, Fisher et al. (1986) Proc. Natl. Acad. Sci. USA. 83: 6450-6454, the disclosure of which is incorporated herein by reference.

The methods of the invention may be performed on any relevant tissue or body fluid sample. For example, methods of the invention may be performed on cervical tissue, more preferably cervical biopsy tissue, and most preferably on Pap smears. Alternatively, the methods of the invention may be performed on a human body fluid sample selected from the group consisting of: blood; serum; plasma; fecal matter; urine; vaginal secretion; spinal fluid; saliva; ascitic fluid; peritoneal fluid; sputum; and breast exudate. It is contemplated, however, that the methods of the invention also may be useful in assays for metastasized cervical cancer cells in other tissue or body fluid samples.

Marker proteins associated with a cervical cancer in a tissue or body fluid sample may be detected using any of a number of assay methods available in the art. In one embodiment, for example, the marker cervical cancer-associated protein may be reacted with a labeled binding moiety capable of specifically binding to the marker protein thereby to produce a labeled complex of the binding moiety and the marker protein. The labeled complex thereafter may be detected, using conventional methodologies well known in the art. Detection of the presence of the labeled complex may provide an indication of the presence of the cervical cancer cells or pre-cancerous cells in the individual being tested. As used herein, the term “binding moiety” is understood to mean any binding partner capable of specifically binding to a cervical cancer-associated protein with a binding affinity greater than about 10.sup.5 M.sup.-1. As used herein the terms “specifically binding”, “specifically bound” and “binds specifically” refer to a binding interaction with a binding affinity of greater than about 10.sup.5 M.sup.-1. As used herein, the binding moiety is labeled with a detectable moiety, for example, a radioactive, fluoroscopic, spectroscopic, or enzymatic label, using techniques well known in the art.

It is appreciated that, binding moieties which interact and bind specifically with the target protein, may be designed using conventional methods well known in the art. In the invention, the binding moiety can be an antibody, for example, a monoclonal or a polyclonal antibody. Monoclonal antibodies are preferred. It is contemplated, however, that other useful binding moieties useful in the practice of the instant invention may include, for example, biosynthetic antibody binding sites, also referred to in the art as BABS or sFv’s, and antibody fragments, for example, Fv, Fab, Fab’ and (Fab’).sub.2 fragments. Procedures for preparing, testing, and labeling BABS and antibody fragments are well known in the art, and so are not discussed in detail herein.

In another embodiment, one or more marker proteins in a sample may be detected by first isolating the proteins from the sample, and then separating the proteins by two-dimensional gel electrophoresis to produce a characteristic two-dimensional gel electrophoresis pattern. The gel electrophoresis pattern then may be compared with a standard, for example, a standard gel pattern obtained from a data base of gel electrophoresis patterns. Thus, in another embodiment, the invention provides electrophoresis gel patterns or electropherograms of cervical cancer-associated proteins which are useful in detecting a cervical cancer in an individual.

The cervical cancer-associated proteins of the invention can be purified or co-purified from cervical cancer cells using nuclear matrix protein isolation procedures, such as those disclosed in U.S. Pat. No. 4,885,236 and U.S. Pat. No. 4,882,268, the disclosures of which are incorporated herein. Alternatively, the marker proteins, once identified and characterized may be isolated from the sample by any of a range of protein purification protocols well known to those skilled in the art, such as affinity chromatography, to yield isolated proteins. As used herein, the term “isolated” is understood to mean substantially free of undesired, contaminating proteinaceous material.

Furthermore, the skilled artisan may produce nucleic acid sequences encoding the entire isolated marker protein, or fragments thereof, using methods currently available in the art (see, for example, Maniatis et al., eds. (1989) “Molecular Cloning: A Laboratory Manual,” C.old Spring Harbor Press). For example, an isolated cervical cancer-associated protein may be sequenced using conventional peptide sequencing protocols, and then oligonucleotide hybridization probes designed for screening a cDNA library. The cDNA library then may be screened with the resultant oligonucleotide to isolate full or partial length cDNA sequences which encode the isolated protein.

Furthermore, the skilled artisan, using the methodologies described in U.S. Pat. Nos. 4,885,236 and 4,882,268 may isolate from a cell sample a nucleic acid molecule having a sequence capable of recognizing and being specifically bound by a cervical cancer-associated protein. In such a procedure, the soluble proteins are separated from the nucleus and cytoskeleton by extracting mammalian cells with a non-ionic detergent solution at physiological pH and ionic strength. The insoluble protein and nucleic acids then are digested with DNAase and then eluted with a buffered ammonium sulfate solution to yield a nucleic acid molecule capable of recognizing and being specifically bound by a cervical cancer-associated protein. Any remaining proteins then are separated from the target nucleic acid molecule.

Detection of the aforementioned nucleic acid molecules thus can serve as an indicator of the presence of cervical cancer and/or metastasized cervical cancer in an individual. Accordingly, in another aspect, the invention provides another method for detecting cervical cancer in a human. The method comprises the step of detecting the presence of a nucleic acid molecule in a tissue or body fluid sample thereby to indicate the presence of a cervical carcinoma in the individual. The nucleic acid molecule is selected from the group consisting of (i) a nucleic acid molecule comprising a sequence capable of recognizing and being specifically bound by a cervical cancer-associated protein, and (ii) a nucleic acid molecule comprising a sequence encoding a cervical cancer-associated protein. As defined herein, the cervical cancer-associated protein is characterized as being selected from the group consisting of (i) a protein having a molecular weight of about 69,400 Daltons and an isoelectric point of about 5.8; (ii) a protein having a molecular weight of about 53,800 Daltons and an isoelectric point of about 5.5; (iii) a protein having a molecular weight of about 47,900 Daltons and an isoelectric point of about 5.6; (iv) a protein having a molecular weight of about 46,000 Daltons, and an isoelectric point of about 5.1; and (v) a protein having a molecular weight of about 44,900 Daltons and an isoelectric point of about 6.6, wherein in each example, the molecular weight is determined by standard polyacrylamide gel electrophoresis techniques and the isoelectric point is determined by standard isoelectric focusing techniques, and wherein the cervical cancer-associated protein is further characterized as being a non-chromatin protein which is detectable at a higher level in a human cervical cancer cell than in a normal human cervical cell, as determined by two-dimensional gel electrophoresis.

A target nucleic acid molecule in a sample may be detected, for example, by Northern blot analysis by reacting the sample with a labeled hybridization probe, for example, a .sup.32 P labeled oligonucleotide probe, capable of hybridizing specifically with at least a portion of the nucleic acid molecule encoding the marker protein. Detection of a nucleic acid molecule either encoding a cervical cancer-associated protein or capable of being specifically bound by a cervical cancer-associated protein, thus can serve as an indicator of the presence of a cervical cancer in the individual being tested.

In another aspect, the invention provides a kit for detecting the presence of cervical cancer or for evaluating the efficacy of a therapeutic treatment of a cervical cancer. Such kits may comprise, in combination, (i) a receptacle for receiving a human tissue or body fluid sample from the individual, (ii) a binding partner which binds specifically either to an epitope on a marker cervical cancer-associated protein or a nucleic acid sequence encoding at least a portion of the marker cervical cancer-associated protein, (iii) means for detecting the binding of the binding partner with either the cervical cancer-associated protein or the nucleic acid sequence encoding at least a portion of the cervical cancer-associated protein, and (iv) a reference sample.

In one embodiment of the kit, the binding moiety binds specifically to a cervical cancer-associated protein selected from the group of proteins further defined as having: a molecular weight of about 69,400 Daltons and an isoelectric point of about 5.8; a molecular weight of about 53,800 Daltons and an isoelectric point of about 5.5; a molecular weight of about 47,900 Daltons and an isoelectric point of about 5.6; a molecular weight of about 46,000 Daltons and an isoelectric point of about 5.1, or a molecular weight of about 44,900 Daltons and an isoelectric point of about 6.6, wherein the molecular weight is determined by conventional polyacrylamide gel electrophoresis methodologies, and the isoelectric point is determined by conventional isoelectric focusing methodologies.

In another embodiment of the kit, the reference sample may comprise a negative and/or positive control. The negative control being indicative of a normal cervical cell type and the positive control being indicative of cervical cancer.

In another aspect, the invention provides a method for treating cervical cancer. The method comprises administering to a patient with cervical cancer, a therapeutically-effective amount of a compound, preferably an antibody, and most preferably a monoclonal antibody, which binds specifically to a target cervical cancer-associated protein thereby to inactivate the protein. The target protein being characterized as having a molecular weight of from about 44,900 Daltons to about 69,400 Daltons, as determined by standard polyacrylamide gel electrophoresis techniques and an isoelectric point of from about 5.1 to about 6.6, as determined by standard isoelectric focusing techniques, and wherein the target protein is further characterized as being a non-chromatin protein which is detectable at a higher level in a human cervical cancer cell than in a normal human cervical cell, as determined by two-dimensional gel electrophoresis. Similarly, it is contemplated that the compound may comprise a small molecule, for example, as small organic molecule, which inhibits or reduces the biological activity of the target cervical cancer-associated protein.

In another aspect, the invention provides another method for treating cervical cancer. The method comprises the step of administering to a patient diagnosed as having cervical cancer, a therapeutically-effective amount of a compound which reduces in vivo the expression of a target cervical cancer-associated protein thereby to reduce in vivo the expression of the target protein. In a preferred embodiment, the compound is a nucleobase containing sequence, such as, an anti-sense nucleic acid sequence or anti-sense peptide nucleic acid (PNA) molecule, complementary to a nucleic acid sequence encoding at least a portion of the target protein. After administration, the anti-sense nucleic acid sequence or anti-sense PNA molecule binds to the nucleic acid sequences encoding, at least in part, the target protein thereby to reduce in vivo expression of the target cervical cancer-associated protein.

Thus, the invention provides a wide range of methods and compositions for detecting and treating cervical cancer in an individual. Specifically, the invention provides cervical cancer-associated proteins, which permit specific and early, preferably before metastases occur, detection of cervical cancer in an individual. In addition, the invention provides kits useful in the detection of cervical cancer in an individual. In addition, the invention provides methods utilizing the cervical cancer-associated proteins as targets and indicators, for treating cervical cancers and for monitoring of the efficacy of such a treatment. These and other numerous additional aspects and advantages of the invention will become apparent upon consideration of the following figures, detailed description, and claims which follow.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1a is a high resolution two-dimensional gel electrophoresis pattern of nuclear matrix proteins isolated from a cervical cancer tissue sample. Tumor-associated proteins encircled and marked with reference numbers 1-5 correspond to proteins CvC-1 to CvC-5, listed in Table 2.

FIG. 1b is a high resolution two-dimensional gel electrophoresis pattern of nuclear matrix proteins isolated from a normal cervical tissue sample. As a reference, the relative positions corresponding to the CvC-1 to CvC-5 proteins of FIG. 1a are encircled and marked with reference numbers 1-5.

FIG. 2a is a high resolution two-dimensional gel electrophoresis pattern of nuclear matrix proteins isolated from the cervical carcinoma-derived cell line C33A. Tumor-associated proteins CvC-2 and CvC-5 are encircled and marked with reference numbers 2 and 5.

FIG. 2b is a high resolution two-dimensional gel electrophoresis pattern of nuclear matrix proteins isolated from CaSki cells. Tumor-associated proteins CvC-1 and CvC-3 are encircled and marked with reference numbers 1 and 3.

For each of the above figures, molecular weight standards are indicated on the ordinate axes (M.sub.r .times.10.sup.3) and isoelectric points are shown on the abscissae.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions for the detection and treatment of cervical cancer. The invention is based, in part, upon the discovery of cervical cancer-associated proteins which generally are present at detectably higher levels in cancerous cervical cells than in normal cervical cells, as determined by two-dimensional gel electrophoresis.

The cervical cancer-associated proteins may act as marker proteins useful in the detection of cervical cancer or as target proteins for therapy of cervical cancer. For example, it is contemplated that, the marker proteins and binding moieties, for example, antibodies that bind to the marker proteins or nucleic acid probes which hybridize to nucleic acid sequences encoding the marker proteins, may be used to detect the presence of cervical cancer in an individual. Furthermore, it is contemplated that, the skilled artisan may produce novel therapeutics for treating cervical cancer which include, for example: antibodies which can be administered to an individual that bind to and reduce or eliminate the biological activity of the target protein in vivo; nucleic acid or peptide nucleic acid sequences which hybridize with genes or gene transcripts encoding the target proteins thereby to reduce expression of the target proteins in vivo; or small molecules, for example, organic molecules which interact with the target proteins or other cellular moieties, for example, receptors for the target proteins, thereby to reduce or eliminate biological activity of the target proteins.

Set forth below are methods for isolating cervical cancer-associated proteins, methods for detecting cervical cancer using cervical cancer-associated proteins as markers, and methods for treating individuals afflicted with cervical cancer using cervical cancer-associated proteins as targets for cancer therapy.

1. Identification and Purification of Cervical Cancer-associated Proteins.

Marker proteins of the invention, as disclosed herein are identified by (i) isolating proteins from normal cervical tissue and from cervical cancer tissue using a nuclear matrix purification protocol, such as those described generally in U.S. Pat. Nos. 4,882,268 and 4,885,236, or Fey et al. (1986) supra (ii) fractionating the resulting nuclear matrix protein preparations by 2-D gel electrophoresis, (iii) visualizing the resulting protein patterns, for example, by silver staining, and (iv) identifying polypeptide spots on the resulting 2-D gel electropherograms which generally are detectable in samples isolated from cervical cancer cells but not detectable in samples isolated from normal cervical cells.

Marker proteins associated with cervical cancer tissue were isolated as described herein using a modification of the method of Fey et al. (Fey et al. (1986) supra). Briefly, cervical cancer tissue is minced into small (1 mm.sup.3) pieces and homogenized with a Teflon pestle on ice and treated with a buffered solution containing 0.5% Triton-X-100, vanadyl riboside complex plus a protease inhibitor cocktail (phenylmethyl sulfonyl fluoride, aprotinin, and leupeptin) to remove lipids and soluble proteins. Tumor cells from cell lines can be harvested by trypsinization and treated in the same way as for homogenized tumor tissue. Stromal aggregates are removed by filtering the homogenate through a 250 micron nylon screen followed by a centrifugation step.

Soluble cytoskeletal proteins are removed by incubating the pellet in an extraction buffer containing 250 mM (NH.sub.4).sub.2 SO.sub.4, 0.5% Triton-X-100, vanadyl riboside complex plus a protease inhibitor cocktail for 10 minutes on ice followed by centrifugation. Chromatin is removed by incubating the pellet in DNAase I in a buffered solution containing a protease inhibitor cocktail for 45 minutes at 25.degree. C.

The remaining pellet fraction, containing the target proteins and intermediate filaments, is solubilized in a disassembly buffer containing 8 M urea, protease inhibitor cocktail plus 1% 2-mercaptoethanol. Insoluble contaminants, primarily carbohydrates and extracellular matrix, are removed by ultracentrifugation. Intermediate filaments are allowed to reassemble upon removal of urea by dialysis in assembly buffer containing protease inhibitor cocktail and removed by ultracentrifugation, leaving the target proteins in the supernatant fraction. Protein concentration can be determined by the Coomassie Plus Protein Assay Kit (Pierce Chemicals, Rockford, Ill.) using a bovine gamma globulin standard. Proteins are immediately precipitated in 80% ethanol and stored at -80.degree. C. until use.

It is contemplated that, after identification, the resulting cervical cancer-associated proteins may be isolated by preparing a nuclear matrix protein preparation, such as the one described above, electrophoresing the resulting proteins on a 2-D gel, and after some means of visualization, isolating the protein of interest from the resulting 2-D gel. Alternatively, it is contemplated that the marker protein, once identified, can be isolated, using standard protein purification methodologies well known to those of ordinary skill in the art, such as affinity chromatography, to yield substantially pure marker proteins. As used herein, the term “substantially pure” is understood to mean at least 80% pure as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

2. Detection of Cervical Cancer.

Once cervical cancer-associated proteins have been identified, they may be used as markers to determine whether an individual has cervical cancer and/or cervical dysplasia, and if so, suitable detection methods can be used to monitor the status of the disease.

Using the marker proteins, the skilled artisan can produce a variety of detection methods for detecting cervical cancer in a human. The methods, typically comprise the steps of detecting, by some means, the presence of one or more cervical cancer-associated proteins in a tissue or body fluid sample of the human. The accuracy and/or reliability of the method for detecting cervical cancer in a human may be further enhanced by detecting the presence of a plurality of cervical cancer-associated proteins in a preselected tissue or body fluid sample. The detection step may comprise one or more of the protocols described hereinbelow.

2.A. Protein Detection Methods.

The marker protein in a sample may be reacted with a binding moiety capable of specifically binding the marker protein. The binding moiety may comprise, for example, a member of a ligand-receptor pair, i.e., a pair of molecules capable of having a specific binding interaction. The binding moiety may comprise, for example, a member of a specific binding pair, such as antibody-antigen, enzyme-substrate, nucleic acid-nucleic acid, protein-nucleic acid, protein-protein, or other specific binding pair known in the art. Binding proteins may be designed which have enhanced affinity for a target protein. Optionally, the binding moiety may be linked with a detectable label, such as an enzymatic, fluorescent, radioactive, phosphorescent or colored particle label. The labeled complex may be detected, e.g., visually or with the aid of a spectrophotometer or other detector.

The marker proteins also may be detected using gel electrophoresis techniques available in the art. In two-dimensional gel electrophoresis, the proteins are separated first in a pH gradient gel according to their isoelectric point. The resulting gel then is placed on a second polyacrylamide gel, and the proteins separated according to molecular weight (see, for example, O.degree. Farrell (1975) J. Biol. Chem. 250: 4007-4021).

One or more marker proteins may be detected by first isolating proteins from a sample obtained from an individual suspected of having cervical cancer, and then separating the proteins by two-dimensional gel electrophoresis to produce a characteristic two-dimensional gel electrophoresis pattern. The pattern then may be compared with a standard gel pattern produced by separating, under the same or similar conditions, proteins isolated from normal or cancer cells. The standard may be stored or obtained in an electronic database of electrophoresis patterns. The presence of a cervical cancer-associated protein in the two-dimensional gel provides an indication of the presence of a cervical cancer in the sample being tested. The detection of two or more proteins in the two-dimensional gel electrophoresis pattern further enhances the accuracy of the assay. The presence of a plurality, e.g., two to five, cervical cancer-associated proteins on the two-dimensional gel provides a strong indication of the presence of a cervical cancer in the sample. The assay thus permits the early detection and treatment of cervical cancer.

2B. Immunoassay.

A marker cervical cancer-associated protein may also be detected using any of a wide range of immunoassay techniques available in the art. For example, the skilled artisan may employ the sandwich immunoassay format to detect cervical cancer in a body fluid sample. Alternatively, the skilled artisan may use conventional immuno-histochemical procedures for detecting the presence of the cervical cancer-associated protein in a tissue sample, for example, in a Pap smear, using one or more labeled binding proteins (See Example 5, hereinbelow).

In a sandwich immunoassay, two antibodies capable of binding the marker protein generally are used, e.g., one immobilized onto a solid support, and one free in solution and labeled with detectable chemical compound. Examples of chemical labels that may be used for the second antibody include radioisotopes, fluorescent compounds, and enzymes or other molecules which generate colored or electrochemically active products when exposed to a reactant or enzyme substrate. When a sample containing the marker protein is placed in this system, the marker protein binds to both the immobilized antibody and the labeled antibody, to form a “sandwich” immune complex on the support’s surface. The complexed protein is detected by washing away non-bound sample components and excess labeled antibody, and measuring the amount of labeled antibody complexed to protein on the support’s surface.

Both the sandwich immunoassay and the tissue immunohistochemical procedure are highly specific and very sensitive, provided that labels with good limits of detection are used. A detailed review of immunological assay design, theory and protocols can be found in numerous texts in the art, including “Practical Immunology”, Butt, W. R., ed., (1984) Marcel Dekker, New York and “Antibodies, A Laboratory Approach” Harlow et al. eds.(1988) Cold Spring Harbor Laboratory.

In general, immunoassay design considerations include preparation of antibodies (e.g., monoclonal or polyclonal antibodies) having sufficiently high binding specificity for the target protein to form a complex that can be distinguished reliably from products of nonspecific interactions. As used herein, the term “antibody” is understood to mean binding proteins, for example, antibodies or other proteins comprising an immunoglobulin variable region-like binding domain, having the appropriate binding affinities and specificities for the target protein. The higher the antibody binding specificity, the lower the target protein concentration that can be detected. A preferred binding specificity is such that the binding protein has a binding affinity for the target protein of greater than about 10.sup.5 M.sup.-1, preferably greater than about 107 M.sup.-1.

Antibodies to an isolated target cervical cancer-associated protein which are useful in assays for detecting a cervical cancer in an individual may be generated using standard immunological procedures well known and described in the art. See, for example, Practical Immunology, Butt, N. R., ed., Marcel Dekker, NY, 1984. Briefly, an isolated target protein is used to raise antibodies in a xenogeneic host, such as a mouse, goat or other suitable mammal.

The marker protein is combined with a suitable adjuvant capable of enhancing antibody production in the host, and injected into the host, for example, by intraperitoneal administration. Any adjuvant suitable for stimulating the host’s immune response may be used. A commonly used adjuvant is Freund’s complete adjuvant (an emulsion comprising killed and dried microbial cells and available from, for example, Calbiochem Corp., San Diego, or Gibco, Grand Island, N.Y.). Where multiple antigen injections are desired, the subsequent injections comprise the antigen in combination with an incomplete adjuvant (e.g., cell-free emulsion).

Polyclonal antibodies may be isolated from the antibody-producing host by extracting serum containing antibodies to the protein of interest. Monoclonal antibodies may be produced by isolating host cells that produce the desired antibody, fusing these cells with myeloma cells using standard procedures known in the immunology art, and screening for hybrid cells (hybridomas) that react specifically with the target protein and have the desired binding affinity.

Antibody binding domains also may be produced biosynthetically and the amino acid sequence of the binding domain manipulated to enhance binding affinity with a preferred epitope on the target protein. Specific antibody methodologies are well understood and described in the literature. A more detailed description of their preparation can be found, for example, in “Practical Immunology” (1984) supra).

In addition, genetically engineered biosynthetic antibody binding sites, also known in the art as BABS or sFv’s, may be used in the practice of the instant invention. Methods for making and using BABS comprising (i) non-covalently associated or disulfide bonded synthetic V.sub.H and V.sub.L dimers, (ii) covalently linked V.sub.H -V.sub.L single chain binding sites, (iii) individual V.sub.H or V.sub.L domains, or (iv) single chain antibody binding sites are disclosed, for example, in U.S. Pat. Nos.: 5,091,513; 5,132,405; 4,704,692; and 4,946,778, the disclosures of which are incorporated herein by reference. Furthermore, BABS having requisite specificity for the cervical cancer-associated proteins can be derived by phage antibody cloning from combinatorial gene libraries (see, for example, Clackson et al. (1991) Nature 352: 624-628). Briefly, a library of phage each of which express on their coat surface, BABS having immunoglobulin variable regions encoded by variable region gene sequences derived from mice pre-immunized with isolated cervical cancer-associated proteins, or fragments thereof, are screened for binding activity against immobilized cervical cancer-associated protein. Phage which bind to the immobilized cervical cancer-associated proteins are harvested and the gene encoding the BABS sequenced. The resulting nucleic acid sequences encoding the BABS of interest then may be expressed in conventional expression systems to produce the BABS protein.

The isolated cervical cancer-associated protein also may be used for the development of diagnostic and other tissue evaluating kits and assays to monitor the level of the proteins in a tissue or fluid sample. For example, the kit may include antibodies or other specific binding proteins which bind specifically with the cervical cancer-associated proteins and which permit the presence and/or concentration of the cervical cancer-associated proteins to be detected and/or quantitated in a tissue or fluid sample.

Suitable kits for detecting cervical cancer-associated proteins are contemplated to include, e.g., a receptacle or other means for capturing a sample to be evaluated, and means for detecting the presence and/or quantity in the sample of one or more of the cervical cancer-associated proteins described herein. As used herein, “means for detecting” in one embodiment includes one or more antibodies specific for these proteins and means for detecting the binding of the antibodies to these proteins by, e.g., a standard sandwich immunoassay as described herein. Where the presence of a protein within a cell is to be detected, e.g., as from a tissue sample, the kit also may comprise means for disrupting the cell structure so as to expose intracellular proteins.

2.C Nucleic Acid-based Assays.

The presence of a cervical cancer in an individual also may be determined by detecting, in a tissue or body fluid sample, a nucleic acid molecule encoding a cervical cancer-associated protein. Using methods well known to those of ordinary skill in the art, the cervical cancer-associated proteins of the invention may be sequenced, and then, based on the determined sequence, oligonucleotide probes designed for screening a cDNA library (see, for example, Maniatis et al. (1989) supra).

A target nucleic acid molecule encoding a marker cervical cancer-associated protein may be detected using a labeled binding moiety, capable of specifically binding the target nucleic acid. The binding moiety may comprise, for example, a protein, a nucleic acid or a peptide nucleic acid. Additionally, a target nucleic acid, such as an mRNA encoding a cervical cancer-associated protein, may be detected by conducting, for example, a Northern blot analysis using labeled oligonucleotides, e.g., nucleic acid fragments complementary to and capable of hybridizing specifically with at least a portion of a target nucleic acid. While any length oligonucleotide may be utilized to hybridize an mRNA transcript, oligonucleotides typically within the range of 8-100 nucleotides, preferably within the range of 15-50 nucleotides, are envisioned to be most useful in standard hybridization assays.

The oligonucleotide selected for hybridizing to the target nucleic acid, whether synthesized chemically or by recombinant DNA methodologies, is isolated and purified using standard techniques and then preferably labeled (e.g., with .sup.35 S or .sup.32 P) using standard labeling protocols. A sample containing the target nucleic acid then is run on an electrophoresis gel, the dispersed nucleic acids transferred to a nitrocellulose filter and the labeled oligonucleotide exposed to the filter under suitable hybridizing conditions, e.g. 50% formamide, 5.times. SSPE, 2.times. Denhardt’s solution, 0.1% SDS at 42.degree. C., as described in Maniatis et al. (1989) supra. Other useful procedures known in the art include solution hybridization, and dot and slot RNA hybridization. The amount of the target nucleic acid present in a sample optionally then is quantitated by measuring the radioactivity of hybridized fragments, using standard procedures known in the art.

In addition, oligonucleotides also may be used to identify other sequences encoding members of the target protein families. The methodology also may be used to identify genetic sequences associated with the nucleic acid sequences encoding the proteins described herein, e.g., to identify non-coding sequences lying upstream or downstream of the protein coding sequence, and which may play a functional role in expression of these genes. Additionally, binding assays may be conducted to identify and detect proteins capable of a specific binding interaction with a nucleic acid encoding a cervical cancer-associated protein, which may be involved e.g., in gene regulation or gene expression of the protein. In a further embodiment, the assays described herein may be used to identify and detect nucleic acid molecules comprising a sequence capable of recognizing and being specifically bound by a cervical cancer-associated protein.

In addition, it is anticipated that using a combination of appropriate oligonucleotide primers, i.e., more than one primer, the skilled artisan may determine the level of expression of a target gene in vivo by standard polymerase chain reaction (PCR) procedures, for example, by quantitative PCR. Conventional PCR based assays are discussed, for example, in Innes et al (1990) “PCR Protocols; A guide to methods and Applications”, Academic Press and Innes et al. (1995) “PCR Strategies” Academic Press, San Diego, Calif.

3. Identification of Proteins Which Interact In Vivo With Cervical Cancer-associated Proteins.

In addition, it is contemplated that the skilled artisan, using procedures like those described hereinbelow, may identify other molecules which interact in vivo with the cervical cancer-associated proteins described herein. Such molecules also may provide possible targets for chemotherapy.

By way of example, cDNA encoding proteins or peptides capable of interacting with cervical cancer-associated proteins can be determined using a two-hybrid assay, as reported in Durfee et al. (1993) Genes & Develop. 7: 555-559, the disclosure of which is incorporated herein by reference. The principle of the two hybrid system is that noncovalent interaction of two proteins triggers a process (transcription) in which these proteins normally play no direct role, because of their covalent linkage to domains that function in this process. For example, in the two-hybrid assay, detectable expression of a reporter gene occurs when two fusion proteins, one comprising a DNA-binding domain and one comprising a transcription initiation domain, interact.

The skilled artisan can use a host cell that contains one or more reporter genes, such as yeast strain Y153, reported in Durfee et al. (1993) supra. This strain carries two chromosomally located reporter genes whose expression is regulated by Gal4. A first reporter gene, is the E. coli lacZ gene under the control of the Gal4 promoter. A second reporter gene is the selectable HIS3 gene. Other useful reporter genes may include, for example, the luciferase gene, the LEU2 gene, and the GFP (Green Fluorescent Protein) gene.

Two sets of plasmids are used in the two hybrid system. One set of plasmids contain DNA encoding a Gal4 DNA-binding domain fused in frame to DNA encoding a cervical cancer-associated protein. The other set of plasmids contain DNA encoding a Gal4 activation domain fused to portions of a human cDNA library constructed from human lymphocytes. Expression from the first set of plasmids result in a fusion protein comprising a Gal4 DNA-binding domain and a cervical cancer-associated protein. Expression from the second set of plasmids produce a transcription activation protein fused to an expression product from the lymphocyte cDNA library. When the two plasmids are transformed into a gal-deficient host cell, such as the yeast Y153 cells described above, interaction of the Gal DNA binding domain and transcription activation domain occurs only if the cervical cancer-associated protein fused to the DNA binding domain binds to a protein expressed from the lymphocyte cDNA library fused to the transcription activating domain. As a result of the protein-protein interaction between the cervical cancer-associated protein and its in vivo binding partner detectable levels of reporter gene expression occur.

In addition to identifying molecules which interact in vivo with the cervical cancer-associated proteins, the skilled artisan may also screen for molecules, for example, small molecules which alter or inhibit specific interaction between a cervical cancer-associated protein and its in vivo binding partner.

For example, host cell can be transfected with DNA encoding a suitable DNA binding domain/cervical cancer-associated protein hybrid and a translation activation domain/putative cervical cancer-associated protein binding partner, as disclosed above. The host cell also contains a suitable reporter gene in operative association with a cis-acting transcription activation element that is recognized by the transcription factor DNA binding domain. The level of reporter gene expressed in the system is assayed. Then, the host cell is exposed to a candidate molecule and the level of reporter gene expression is detected. A reduction in reporter gene expression is indicative of the candidate’s ability to interfere with complex formation or stability with respect to the cervical cancer-associated protein and its in vivo binding partner. As a control, the candidate molecule’s ability to interfere with other, unrelated protein-protein complexes is also tested. Molecules capable of specifically interfering with a cervical cancer-associated protein/binding partner interaction, but not other protein-protein interactions, are identified as candidates for production and further analysis. Once a potential candidate has been identified, its efficacy in modulating cell cycling and cell replication can be assayed in a standard cell cycle model system.

Candidate molecules can be produced as described hereinbelow. For example, DNA encoding the candidate molecules can be inserted, using conventional techniques well described in the art (see, for example, Maniatis (1989) supra) into any of a variety of expression vectors and transfected into an appropriate host cell to produce recombinant proteins, including both full length and truncated forms. Useful host cells include E. coli, Saccharomyces cerevisiae, Pichia pastoris, the insect/baculovirus cell system, myeloma cells, and various other mammalian cells. The full length forms of such proteins are preferably expressed in mammalian cells, as disclosed herein. The nucleotide sequences also preferably include a sequence for targeting the translated sequence to the nucleus, using, for example, a sequence encoding the eight amino acid nucleus targeting sequence of the large T antigen, which is well characterized in the art. The vector can additionally include various sequences to promote correct expression of the recombinant protein, including transcription promoter and termination sequences, enhancer sequences, preferred ribosome binding site sequences, preferred mRNA leader sequences, preferred protein processing sequences, preferred signal sequences for protein secretion, and the like. The DNA sequence encoding the gene of interest can also be manipulated to remove potentially inhibiting sequences or to minimize unwanted secondary structure formation. As will be appreciated by the practitioner in the art, the recombinant protein can also be expressed as a fusion protein.

After translation, the protein can be purified from the cells themselves or recovered from the culture medium. The DNA can also include sequences which aid in expression and/or purification of the recombinant protein. The DNA can be expressed directly or can be expressed as part of a fusion protein having a readily cleavable fusion junction.

The DNA may also be expressed in a suitable mammalian host. Useful hosts include fibroblast 3T3 cells, (e.g., NIH 3T3, from CRL 1658) COS (simian kidney ATCC, CRL-1650) or CH0 (Chinese hamster ovary) cells (e.g., CHO-DXB11, from Chasin (1980) Proc. Nat’l . Acad. Sci. USA 77 :4216-4222), mink-lung epithelial cells (MV1Lu), human foreskin fibroblast cells, human glioblastoma cells, and teratocarcinoma cells. Other useful eukaryotic cell systems include yeast cells, the insect/baculovirus system or myeloma cells.

In order to express a candidate molecule, the DNA is subcloned into an insertion site of a suitable, commercially available vector along with suitable promoter/enhancer sequences and 3′ termination sequences. Useful promoter/enhancer sequence combinations include the CMV promoter (human cytomegalovirus (MIE) promoter) present, for example, on pCDM8, as well as the mammary tumor virus promoter (MMTV) boosted by the Rous sarcoma virus LTR enhancer sequence (e.g., from Clontech, Inc., Palo Alto). A useful inducable promoter includes, for example, A Zn.sup.2+ induceable promoter, such as the Zn.sup.2+ metallothionein promoter (Wrana et al. (1992) Cell 71: 1003-1014) Other induceable promoters are well known in the art and can be used with similar success. Expression also can be further enhanced using trans-activating enhancer sequences. The plasmid also preferably contains an amplifiable marker, such as DHFR under suitable promoter control, e.g., SV40 early promoter (ATCC #37148). Transfection, cell culturing, gene amplification and protein expression conditions are standard conditions, well known in the art, such as are described, for example in Ausubel et al., ed.,(1989) “Current Protocols in Molecular Biology”, John Wiley & Sons, NY. Briefly, transfected cells are cultured in medium containing 5-10% dialyzed fetal calf serum (dFCS), and stably transfected high expression cell lines obtained by amplification and subcloning and evaluated by standard Western and Northern blot analysis. Southern blots also can be used to assess the state of integrated sequences and the extent of their copy number amplification.

The expressed candidate protein is then purified using standard procedures. A currently preferred methodology uses an affinity column, such as a ligand affinity column or an antibody affinity column. The column is then washed, and the candidate molecules selectively eluted in a gradient of increasing ionic strength, changes in pH, or addition of mild detergent. It is appreciated that in addition to the candidate molecules which bind to the cervical cancer-associated proteins, the cervical cancer associated proteins themselves may likewise be produced using such recombinant DNA technologies.

4. Cervical Cancer Therapy and Methods for Monitoring Therapy.

The skilled artisan, after identification of cervical cancer-associated proteins and proteins which interact with the cervical cancer-associated proteins, can develop a variety of therapies for treating cervical cancer. Because the marker proteins described herein are present at detectably higher levels in cervical cancer cells relative to normal cervical cells, the skilled artisan may employ, for example, the marker proteins and/or nucleic acids encoding the marker proteins as target molecules for a cancer chemotherapy.

4.A. Anti-sense-based Therapeutics.

A particularly useful cancer therapeutic envisioned is an oligonucleotide or peptide nucleic acid sequence complementary and capable of hybridizing under physiological conditions to part, or all, of the gene encoding the marker protein or to part, or all, of the transcript encoding the marker protein thereby to reduce or inhibit transcription and/or translation of the marker protein gene. Alternatively, the same technologies may be applied to reduce or inhibit transcription and/or translation of the proteins which interact with the cervical cancer-associated proteins.

Anti-sense oligonucleotides have been used extensively to inhibit gene expression in normal and abnormal cells. See, for example, Stein et al. (1988) Cancer Res. 48: 2659-2668, for a pertinent review of anti-sense theory and established protocols. In addition, the synthesis and use of peptide nucleic acids as anti-sense-based therapeutics are described in PCT publications PCT/EP92/01219 published Nov. 26, 1992, PCT/US92/10921 published Jun. 24, 1993, and PCTIUS94/013523 published Jun. 1, 1995, the disclosures of which are incorporated herein by reference. Accordingly, the anti-sense-based therapeutics may be used as part of chemotherapy, either alone or in combination with other therapies.

Anti-sense oligonucleotide and peptide nucleic acid sequences are capable of hybridizing to a gene and/or mRNA transcript and, therefore, may be used to inhibit transcription and/or translation of the protein described herein. It is appreciated, however, that oligoribonucleotide sequences generally are more susceptible to enzymatic attack by ribonucleases than are deoxyribonucleotide sequences. Hence, oligodeoxyribonucleotides are preferred over oligoribonucleotides for in vivo therapeutic use. It is appreciated that the peptide nucleic acid sequences, unlike regular nucleic acid sequences, are not susceptible to nuclease degradation and, therefore, are likely to have greater longevity in vivo. Furthermore, it is appreciated that peptide nucleic acid sequences bind complementary single stranded DNA and RNA strands more strongly than corresponding DNA sequences (see, for example, PCT/EP92/20702 published Nov. 26, 1992). Accordingly, peptide nucleic acid sequences are preferred for in vivo therapeutic use.

Therapeutically useful anti-sense oligonucleotides or peptide nucleic acid sequences may be synthesized by any of the known chemical oligonucleotide and peptide nucleic acid synthesis methodologies well known and thoroughly described in the art. Alternatively, a complementary sequence to part or all of the natural mRNA sequence may be generated using standard recombinant DNA technologies.

Since the complete nucleotide sequence encoding the entire marker protein as well as additional 5′ and 3′ untranslated sequences are known for each of the marker proteins and/or can be determined readily using techniques well known in the art, anti-sense oligonucleotides or peptide nucleic acids which hybridize with any portion of the mRNA transcript or non-coding sequences may be prepared using conventional oligonucleotide and peptide nucleic acid synthesis methodologies.

Oligonucleotides complementary to, and which hybridizable with any portion of the mRNA transcripts encoding the marker proteins are, in principle, effective for inhibiting translation of the target proteins as described herein. For example, as described in U.S. Pat. No. 5,098,890, issued Mar. 24, 1992, the disclosure of which is incorporated herein by reference, oligonucleotides complementary to mRNA at or near the translation initiation codon site may be used to inhibit translation. Moreover, it has been suggested that sequences that are too distant in the 3′ direction from the translation initiation site may be less effective in hybridizing the mRNA transcripts because of potential ribosomal “read-through”, a phenomenon whereby the ribosome is postulated to unravel the anti-sense/sense duplex to permit translation of the message.

A variety of sequence lengths of oligonucleotide or peptide nucleic acid may be used to hybridize to mRNA transcripts. However, very short sequences (e.g., sequences containing less than 8-15 nucleobases) may bind with less specificity. Moreover, for in vivo use, short oligonucleotide sequences may be particularly susceptible to enzymatic degradation. Peptide nucleic acids, as mentioned above, likely are resistant to nuclease degradation. Where oligonucleotide and peptide nucleic acid sequences are to be provided directly to the cells, very long sequences may be less effective at inhibition because of decreased uptake by the target cell. Accordingly, where the oligonucleotide or peptide nucleic acid is to be provided directly to target cells, oligonucleotide and/or peptide nucleic acid sequences containing about 8-50 nucleobases, and more preferably 15-30 nucleobases, are envisioned to be most advantageous.

An alternative means for providing anti-sense oligonucleotide sequences to a target cell is gene therapy where, for example, a DNA sequence, preferably as part of a vector and associated with a promoter, is expressed constitutively inside the target cell. Recently, Oeller et al. (Oeller et al. (1992) Science 254: 437-539, the disclosure of which is incorporated herein by reference) described the in vivo inhibition of the ACC synthase enzyme using a constitutively expressible DNA sequence encoding an anti-sense sequence to the full length ACC synthase transcript. Accordingly, where the anti-sense oligonucleotide sequences are provided to a target cell indirectly, for example, as part of an expressible gene sequence to be expressed within the cell, longer oligonucleotide sequences, including sequences complementary to substantially all the protein coding sequence, may be used to advantage.

Finally, therapeutically useful oligonucleotide sequences envisioned also include not only native oligomers composed of naturally occurring nucleotides, but also those comprising modified nucleotides to, for example, improve stability and lipid solubility and thereby enhance cellular uptake. For example, it is known that enhanced lipid solubility and/or resistance to nuclease digestion results by substituting a methyl group or sulfur atom for a phosphate oxygen in the internucleotide phosphodiester linkage. Phosphorothioates (”S-oligonucleotides” wherein a phosphate oxygen is replaced by a sulfur atom), in particular, are stable to nuclease cleavage, are soluble in lipids, and are preferred, particularly for direct oligonucleotide administration. S-oligonucleotides may be synthesized chemically using conventional synthesis methodologies well known and thoroughly described in the art.

Preferred synthetic internucleoside linkages include phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidate, and carboxymethyl esters. Furthermore, one or more of the 5′-3′ phosphate group may be covalently joined to a low molecular weight (e.g., 15-500 Da) organic group, including, for example, lower alkyl chains or aliphatic groups (e.g., methyl, ethyl, propyl, butyl), substituted alkyl and aliphatic groups (e.g., aminoethyl, aminopropyl, aminohydroxyethyl, aminohydroxypropyl), small saccharides or glycosyl groups. Other low molecular weight organic modifications include additions to the internucleoside phosphate linkages such as cholesteryl or diamine compounds with varying numbers of carbon residues between the amino groups and terminal ribose. Oligonucleotides with these linkages or with other modifications can be prepared using methods well known in the art (see, for example, U.S. Pat. No. 5,149,798).

Suitable oligonucleotide and or peptide nucleic acid sequences which inhibit transcription and/or translation of the marker proteins can be identified using standard in vivo assays well characterized in the art. Preferably, a range of doses is used to determine effective concentrations for inhibition as well as specificity of hybridization. For example, in the cases of an oligonucleotide, a dose range of 0-100.mu.g oligonucleotide/ml may be assayed. Further, the oligonucleotides may be provided to the cells in a single transfection, or as part of a series of transfections. Anti-sense efficacy may be determined by assaying a change in cell proliferation over time following transfection, using standard cell counting methodology and/or by assaying for reduced expression of marker protein, e.g., by immunofluorescense. Alternatively, the ability of cells to take up and use thymidine is another standard means of assaying for cell division and may be used here, e.g., using .sup.3 H thymidine. Effective anti-sense inhibition should inhibit cell division sufficiently to reduce thymidine uptake, inhibit cell proliferation, and/or reduce detectable levels of marker proteins.

It is anticipated that therapeutically effective oligonucleotide or peptide nucleic acid concentrations may vary according to the nature and extent of the neoplasm, the particular nucleobase sequence used, the relative sensitivity of the neoplasm to the oligonucleotide or peptide nucleic acid sequence, and other factors. Useful ranges for a given cell type and oligonucleotide and/or peptide nucleic acid may be determined by performing standard dose range experiments. Dose range experiments also may be performed to assess toxicity levels for normal and malignant cells. It is contemplated that useful concentrations may range from about 1 to 100 .mu.g/ml per 10.sup.5 cells.

For in vivo use, the anti-sense oligonucleotide or peptide nucleic acid sequences may be combined with a pharmaceutical carrier, such as a suitable liquid vehicle or excipient, and optionally an auxiliary additive or additives. Liquid vehicles and excipients are conventional and are available commercially. Illustrative thereof are distilled water, physiological saline, aqueous solutions of dextrose, and the like. For in vivo cancer therapies, the anti-sense sequences preferably can be provided directly to malignant cells, for example, by injection directly into the tumor. Alternatively, the oligonucleotide or peptide nucleic acid may be administered systemically, provided that the anti-sense sequence is associated with means for directing the sequences to the target malignant cells.

In addition to administration with conventional carriers, the anti-sense oligonucleotide or peptide nucleic acid sequences may be administered by a variety of specialized oligonucleotide delivery techniques. For example, oligonucleotides may be encapsulated in liposomes, as described in Mannino et al. (1988) Bio Technology 6: 682, and Felgner et al. (1989) Bethesda Res. Lab. Focus 11:21. Lipids useful in producing liposomal formulations include, without limitation, monoglycerides, diglycerides, sulfatides, lysolecithin, phospholipids, saponin, bile acids, and the like. Preparation of such liposomal formulations is within the level of skill in the art (see, for example, in U.S. Pat. No. 4,235,871; U.S. Pat. No. 4,501,728; U.S. Pat. No. 4,837,028; and U.S. Pat. No. 4,737,323). The pharmaceutical composition of the invention may further include compounds such as cyclodextrins and the like which enhance delivery of oligonucleotides into cells. When the composition is not administered systemically but, rather, is injected at the site of the target cells, cationic detergents (e.g. Lipofectin) may be added to enhance uptake. In addition, reconstituted virus envelopes have been successfully used to deliver RNA and DNA to cells (see, for example, Arad et al. (1986) Biochem. Biophy. Acta. 859: 88-94).

For therapeutic use in vivo, the anti-sense oligonucleotide and/or peptide nucleic acid sequences are administered to the individual in a therapeutically effective amount, for example, an amount sufficient to reduce or inhibit target protein expression in malignant cells. The actual dosage administered may take into account whether the nature of the treatment is prophylactic or therapeutic in nature, the age, weight, health of the patient, the route of administration, the size and nature of the malignancy, as well as other factors. The daily dosage may range from about 0.01 to 1,000 mg per day. Greater or lesser amounts of oligonucleotide or peptide nucleic acid sequences may be administered, as required. As will be appreciated by those skilled in the medical art, particularly the chemotherapeutic art, appropriate dose ranges for in vivo administration would be routine experimentation for a clinician. As a preliminary guideline, effective concentrations for in vitro inhibition of the target molecule may be determined first.

4.B. Binding Protein-based Therapeutics.

As mentioned above, a cancer marker protein or a protein that interacts with the cancer marker protein may be used as a target for chemotherapy. For example, a binding protein designed to bind the marker protein essentially irreversibly can be provided to the malignant cells, for example, by association with a ligand specific for the cell and known to be absorbed by the cell. Means for targeting molecules to particular cells and cell types are well described in the chemotherapeutic art.

Binding proteins maybe obtained and tested using technologies well known in the art. For example, the binding portions of antibodies maybe used to advantage. It is contemplated, however, that intact antibodies or BABS, which preferably, have been humanized may be used in the practice of the invention. As used herein, the term “humanized” is understood to mean a process whereby the framework region sequences of a non-human immunoglobulin variable region are replaced by human variable region sequences. Accordingly, it is contemplated that such humanized binding proteins will elicit a weaker immune response than their unhumanized counterparts. Particularly useful are binding proteins identified with high affinity for the target protein, e.g., greater than about 10.sup.9 M.sup.-1. Alternatively, DNA encoding the binding protein may be provided to the target cell as part of an expressible gene to be expressed within the cell following the procedures used for gene therapy protocols well described in the art. See, for example, U.S. Pat. No. 4,497,796, and “Gene Transfer”, Vijay R. Baichwal, ed., (1986). It is anticipated that, once bound by binding protein, the target protein the will be inactivated or its biological activity reduced thereby inhibiting or retarding cell division.

As described above, suitable binding proteins for in vivo use, may be combined with a suitable pharmaceutical carrier, such as physiological saline or other useful carriers well characterized in the medical art. The pharmaceutical compositions may be provided directly to malignant cells, for example, by direct injection, or may be provided systemically, provided the binding protein is associated with means for targeting the protein to target cells. Finally, suitable dose ranges and cell toxicity levels may be assessed using standard dose range experiments. Therapeutically effective concentrations may range from about 0.01 to about 1,000 mg per day. As described above, actual dosages administered may vary depending, for example, on the nature of the malignancy, the age, weight and health of the individual, as well as other factors.

4. C. Small Molecule-based Therapeutics.

After having isolating cervical cancer-associated nuclear matrix proteins, the skilled artisan can, using methodologies well known in the art, can screen small molecule libraries (either peptide or non-peptide based libraries) to identify candidate molecules that reduce or inhibit the biological function of the cervical cancer-associated proteins. The small molecules preferably accomplish this function by reducing the in vivo expression of the target molecule, or by interacting with the target molecule thereby to inhibit either the biological activity of the target molecule or an interaction between the target molecule and its in vivo binding partner.

It is contemplated that, once the candidate small molecules have been elucidated, skilled artisan may enhance the efficacy of the small molecule using rational drug design methodologies well known in the art. Alternatively, the skilled artisan may use a variety of computer programs which assist the skilled artisan to develop quantitative structure activity relationships (QSAR) which further to assist the design of additional candidate molecules de novo. Once identified, the small molecules may be produced in commercial quantities and subjected to the appropriate safety and efficacy studies.

It is contemplated that the screening assays may be automated thereby facilitating the screening of a large number of small molecules at the same time. Such automation procedures are within the level of skill in the art of drug screening and, therefore, are not discussed herein. Candidate peptide based small molecules may be produced by expression of an appropriate nucleic acid sequence in a host cell or using synthetic organic chemistries. Similarly, non-peptidyl-based small molecules may be produced using conventional synthetic organic chemistries well known in the art.

As described above, for in vivo use, the identified small molecules may be combined with a suitable pharmaceutical carrier, such as physiological saline or other useful carriers well characterized in the medical art. The pharmaceutical compositions may be provided directly to malignant cells, for example, by direct injection, or may be provided systemically, provided the binding protein is associated with means for targeting the protein to target cells. Finally, suitable dose ranges and cell toxicity levels may be assessed using standard dose range experiments. As described above, actual dosages administered may vary depending, for example, on the nature of the malignancy, the age, weight and health of the individual, as well as other factors.

4.D. Methods for Monitoring the Status of Cervical Cancer in an Individual.

The progression of the cervical cancer or the therapeutic efficacy of chemotherapy may be measured using procedures well known in the art. For example, the efficacy of a particular chemotherapeutic agent can be determined by measuring the amount of a cervical cancer-associated protein released from cervical cancer cells undergoing cell death. As reported in PCT publication PCT/US92/09220, published May 13, 1993, incorporated by reference herein, soluble nuclear matrix proteins and fragments thereof are released by cells upon cell death. Such soluble nuclear matrix proteins can be quantitated in a body fluid and used to monitor the degree or rate of cell death in a tissue.

For example, the concentration of a body fluid-soluble nuclear matrix proteins or a fragment thereof released from cells is compared to standards from healthy, untreated tissue. Fluid samples are collected at discrete intervals during treatment and compared to the standard. It is contemplated that changes in the level of a body fluid soluble cervical cancer-associated protein, will be indicative of the efficacy of treatment (that is, the rate of cancer cell death). It is contemplated that the release of body fluid soluble interior nuclear matrix proteins can be measured in blood, plasma, urine, sputum, vaginal secretion, and breast exudate.

Where the assay is used to monitor tissue viability or progression of cervical cancer, the step of detecting the presence and abundance of the marker protein or its transcript in samples of interest is repeated at intervals and these values then are compared, the changes in the detected concentrations reflecting changes in the status of the tissue. For example, an increase in the level of cervical cancer-associated proteins may correlate with progression of the cervical cancer. Where the assay is used to evaluate the efficacy of a therapy, the monitoring steps occur following administration of the therapeutic agent or procedure (e.g., following administration of a chemotherapeutic agent or following radiation treatment). Similarly, a decrease in the level of cervical cancer-associated proteins may correlate a regression of the cervical cancer.

Thus, cervical cancer may be identified by the presence of cervical cancer-associated proteins as taught herein. Once identified, the cervical cancer may be treated using compounds which reduce in vivo the expression and/or biological activity of the cervical cancer-associated proteins. Furthermore, the methods provided herein can be used to monitor the progression of the disease and/or treatment of the disease. The following non limiting examples provide details of the isolation and characterization of cervical cancer-associated proteins and methods for their use in the detection of cervical cancer.

EXAMPLE 1

Isolation of Cervical Cancer-Associated Nuclear Matrix Proteins From Cervical Cancer Tissue Samples and Cell Lines.

Cervical cancer-associated proteins were identified by comparing silver stained 2-D gel patterns of proteins isolated from normal and cancerous cervical cells.

Fresh cervical carcinoma tissue was obtained from patients undergoing hysterectomy for clinically localized (stage IB, II or III, International Federation of Gynecology and Obstetrics or FIGO classification) carcinomas of the cervix from the Instituto Nacional de Cancerologia in Mexico City, Mexico, in accordance with Scientific and Ethics Committee Review Board approval. A small number of tumor tissues were obtained under Institutional Review Board approval from the Pittsburgh Cancer Center (Pittsburgh, Pa.). Normal cervical tissue was obtained under Institutional Review Board approval from patients undergoing hysterectomy for causes unrelated to abnormal cervical histopathology, via the Cooperative Human Tissue Network (Columbus, Ohio). Clinical staging and tumor histopathology for twenty patients who provided tissue samples for use in these experiments are shown in Table 1. With the exception of one case of adenosquamous carcinoma, all of the tumors were squamous cell carcinomas. A majority of these were of the large cell non-keratinizing type. All the patients had localized disease with clinical stages ranging from IB to IIIB (Table 1).

TABLE 1 ______________________________________ Patient Age, Clinical Staging and Histopathology ______________________________________ Case Number Patient Age FIGO Stage Histopathological Diagnosis ______________________________________ 1 37 IB .sup. LCNKS.sup..dagger. 2 49 IB .sup. LCKS.sup..dagger-dbl. 3 32 IB Squamous, mod. well diff.* 4 60 IIA LCNKS 5 63 III Adenosquamous 6 35 IB LCKS 7 44 IIIB LCNKS 8 31 IB Squamous, poorly diff..sup..sctn. 9 31 IB LCNKS 10 38 IIB LCKS 11 65 IIB LCNKS 12 35 IB LCNKS 13 43 IB LCNKS 14 65 III LCNKS 15 52 IIB LCKS 16 47 III LCNKS 17 33 IB LCNKS 18 51 IIIB LCNKS 19 45 IIB LCNKS 20 39 IIB LCNKS ______________________________________ FIGO Stage IB IIA IIB III IIIB ______________________________________ n 9 1 5 3 2 ______________________________________ .sup..dagger.,Large cell nonkeratinizing squamous cell carcinoma .sup..dagger-dbl.,Large cell keratinizing squamous cell carcinoma *,Squamous cell carcinoma, moderately well differentiated .sup..sctn.,Squamous cell carcinoma, poorly differentiated

Fresh tissue was obtained during surgery, placed into transport medium (RPMI 1640 supplemented with gentamicin and 10% fetal calf serum (GIBCO)), packed in ice, and shipped to Matritech, Inc. by overnight carrier. In a small number of cases where immediate shipment could not be arranged, tissues specimens were snap-frozen in liquid nitrogen and sent on dry ice to Matritech, Inc. by overnight carrier. Minimum size of tissue specimens was 0.2 gram. Diagnosis was obtained from pathology reports that accompanied each specimen.

Nuclear matrix proteins were isolated from cervical cancer tissue using a modification of the method of Fey et al. (1986) supra. Fresh cervical cancer tissue specimens, ranging in size from about 0.2 g to about 1.0 g, were obtained from 20 different patients. Tissue specimens were minced into small (1 mm.sup.3) pieces and homogenized with a Teflon pestle on ice and treated with a buffered solution containing 0.5% Triton-X-100, vanadyl riboside complex (RNAase inhibitor, Five Prime-Three Prime, Inc.) plus a protease inhibitor cocktail containing phenylmethyl sulfonyl fluoride (Sigma Chemical Co.), aprotinin and leupeptin (Boehringer Mannheim), to remove lipids and soluble proteins.

Stromal aggregates were removed by filtering the homogenate through 250 micron Nitex nylon screen (Tetko, Inc.) followed by a centrifugation step (600.times.g, 4.degree. C., 5min). Soluble cytoskeletal proteins were removed by incubating the pellet in an extraction buffer containing 250 mM (NH.sub.4).sub.2 SO.sub.4, 0.5% Triton X-100, vanadyl riboside complex and protease inhibitor cocktail on ice for 10 minutes followed by centrifugation (600.times.g, 4.degree. C., 5 min).

Chromatin was removed by incubating the pellet in DNAase (100 mg/mL, Boehringer-Mannheim) in a buffered solution containing protease inhibitor cocktail for 45 minutes at 25.degree. C. The remaining pellet fraction, which contained nuclear matrix proteins and intermediate filaments, was solubilized in disassembly buffer containing 8 M urea, protease inhibitor cocktail and 1% (vol/vol) 2-mercaptoethanol. Insoluble contaminants, primarily carbohydrates and extracellular matrix were removed by ultracentrifugation (163,000.times.g, 20.degree. C., 1 hr). Intermediate filaments were allowed to reassemble upon removal of urea by dialysis in an assembly buffer containing 150 mM KCl, 24 mM imidazole HCl, 5 mM MgCl.sub.2, 0.125 mM EGTA and 2 mM dithiothreitol (DTT) with protease inhibitors and were removed by ultracentrifugation (109,000.times.g, 15.degree. C., 1.5 hr), leaving the nuclear matrix proteins in the supernatant fraction.

In addition, cervical cancer-associated proteins were isolated from CaSki, ME-180, C33A, HeLa (S3 subline), SiHa, C4-1, C4-11, and HT-3 cervical tumor cell lines. Each cell line was obtained from the American Type Culture Collection (ATCC) and maintained at 37.degree. C. in 5% CO.sub.2 in Dulbecco’s Modified Eagles Medium supplemented with 10% fetal calf serum, gentamicin, fungizone and 0.12% SeraExtend (Irvine Scientific). For nuclear matrix extraction studies, cells were grown to approximately 80% confluence in 10 stage cell culture factories (Nunc), harvested by trypsinization, counted and extracted in the same manner as homogenized tumor tissue. Protein concentration of nuclear matrix proteins was determined by the Coomassie Plus Protein Assay Kit (Pierce Chemical) using a bovine gamma globulin standard. Proteins were immediately precipitated in 80% ethanol and stored at -80.degree. C. until use.

The resulting nuclear matrix proteins were next characterized by high-resolution two-dimensional gel electrophoresis according to the procedure of O’Farrell (1975) J. Biol. Chem. 250: 4007-4021(1975), on an Investigator 2-D system (Oxford Glycosystems, Bedford, Mass.). Nuclear matrix proteins were solubilized for isoelectric focusing (IEF) analysis in sample buffer containing 9 M urea, 65 mM 3-[(cholamidopropyl)dimethylamino]-1-propanesulfate (CHAPS), 2.2% ampholytes, and 140 mM dithiothreitol (DTT). Two hundred micrograms of nuclear matrix proteins were loaded per gel.

One-dimensional isoelectric focusing was carried out for 18,000 volt-hours using 1 mm.times.18 mm gel tubes. Following first dimension electrophoresis, gels were extruded from gel tubes, equilibrated for 2 minutes in a buffer containing 0.3 M Tris base, 0.075 M Tris-HCl, 3.0% SDS, 50 mM DTT, and 0.01% bromophenol blue and placed on top of 1 mm 10% Tris-glycine-SDS Duracryl (Oxford Glycosystems) high tensile strength polyacrylamide electrophoresis slab gels. Second dimension slab gels were electrophoresed at 16 Watts per gel and 12.degree. C. constant temperature for approximately 5 hours. Molecular weight standards consisted of bovine albumin (M.sub.r 66,000), ovalbumin (M.sub.r 45,000), glyceraldehyde-3-phosphate dehydrogenase (M.sub.r 36,000), carbonic anhydrase (M.sub.r 29,000), bovine pancreatic trypsinogen (M.sub.r 24,000), and soybean trypsin inhibitor (M.sub.r 20,100) (Sigma Chemical Co.). Isoelectric points were determined using internal control proteins with well-characterized isoelectric points. Following electrophoresis, gels were fixed in a solution containing 40% ethanol/10% acetic acid followed by treatment with a solution containing 0.5% glutaraldehyde. Gels were washed extensively and silver stained according to the method of Rabillioud et al. (Rabillioud et al. (1992) Electrophoresis 13: 429-439) and dried between sheets of cellophane paper.

Silver-stained gels were imaged using a MasterScan Biological Imaging System (CSP, Inc., Billerica, Mass.) according to the manufacturer’s instructions. Digital filtering algorithms were used to remove both uniform and non-uniform background without removing critical image data. Two-D scan (TM) two-dimensional gel analysis and database software (version 3.1) using multiple Gaussian least-squares fitting algorithms were used to compute spot patterns into optimal-fit models of the data as reported by Olson et al. (1980) Anal. Biochem. 169: 49-70. Triangulation from the internal standards was used to precisely determine the molecular weight and isoelectric point of each target protein of interest. Interpretive densitometry was performed using specific software application modules to integrate the data into numeric and graphical reports for each gel being analyzed.

EXAMPLE 2

Identification of Cervical Cancer-associated Nuclear Matrix Proteins Having Differential Appearance on 2-D Gels.

As described in the previous Example, 2-D gel electrophoresis patterns were obtained by fractionating proteins isolated from either normal or cancerous cervical cells. FIG. 1a shows a typical cervical cancer-associated nuclear matrix protein pattern obtained from cervical cancer tissue. FIG. 1b shows a typical gel pattern produced by nuclear matrix proteins obtained from a normal cervical tissue sample. Approximately 600 proteins were resolved per gel. Most of the proteins observed were always present, irrespective of the type of cervical tissue under investigation.

Comparison of FIGS. 1 and 2 reveals that, while most proteins in the cancer and non-cancer samples are identical, there are five proteins that are unique to the cervical cancer sample (labeled in FIG. 1). The proteins, designated CvC-1 through CvC-5, were detected in 20 tissue samples obtained from patients diagnosed with cervical carcinoma but were not detected in cervical tissue isolated from a group of 10 normal individuals. Table 2 identifies proteins, designated CvC-1 through CvC-5, by their approximate molecular weight and isoelectric point. Both the molecular weight and isoelectric point values listed in Table 1 are approximate and accurate to within 2,000 Daltons for molecular weight and to within 0.2 pI units for isoelectric point. A detailed analysis to identify proteins common to normal cervical tissue but absent from cervical cancer tissue did not reveal any proteins that were specifically associated with normal cervical tissue.

TABLE 2 ______________________________________ Cervical Cancer-associated Proteins Molecular Isoelectric Cervical Normal Peptide Weight Point Cancer Cervical ______________________________________ CvC-1 69,408 5.78 + – CvC-2 53,752 5.54 + – CvC-3 47,887 5.60 + – CvC-4 46,006 5.07 + – CvC-5 44,864 6.61 + – ______________________________________

In addition, the expression of nuclear matrix proteins isolated from cervical cancer cell lines was investigated, the results of which are summarized in Table 3, below. It is known that tumors of epithelial cell origin are characterized by the presence of stroma and other elements, such as those resulting from infiltrating inflammatory cells. Detection of nuclear matrix or matrix-associated proteins in tumor cell lines derived from cervical epithelial cell tumors reduces the possibility that the proteins are the result of stromal or other types of contamination of the nuclear matrix preparation.

2-D gel electrophoresis patterns were obtained from samples containing cervical cancer cells derived from cervical cancer cell lines. FIG. 2a shows a cervical cancer-associated nuclear matrix protein pattern obtained from the cervical cancer cell line C33A. In FIG. 2a, tumor-associated proteins CvC-2 and CvC-5 are encircled and identified with numbers 2 and 5. FIG. 2b shows a gel pattern produced by nuclear matrix proteins obtained from the cervical cancer cell line CaSki a normal cervical tissue sample. In FIG. 2b, tumor associated proteins CvC-1 and CvC-3 are encircled and identified with numbers 1 and 3.

Four of the five tumor-associated proteins (CvC1 to CvC-3 and CvC-5) were reproducibly detected in one or more cervical tumor cell lines (FIG. 2, Table 3), confirming the epithelial origin of the proteins. Expression of the fifth protein, CvC-4, was variable but could be detected in the C33A tumor cell line (Table 3).

TABLE 3 ______________________________________ Cervical Carcinoma-associated Protein Expression in Cervical Tumor Cell Lines Histopathol- Tumor ogy of tumor Nuclear matrix proteins expressed* cell line or origin CvC-1 CvC-2 CvC-3 CvC-4 CvC-5 ______________________________________ CaSKI.sup..dagger. Epidermoid + .sup. tr.sup..dagger-dbl. + – + SiHa Squamous – tr +++ – + cell HeLa Adeno- tr – +++ – + carcinoma ME-180.sup..dagger. Epidermoid – tr + – + C33A Squamous + ++ – var.sup..sctn. + cell C4-I Squamous tr – +++ – + cell C4-II Squamous – - – - tr cell HT-3.sup..dagger. Epidermoid tr – + – tr ______________________________________ *Nuclear matrix proteins were extracted from tumor cell lines obtained from the American Type Culture Collection using Fey and Penman extraction methodology. .sup..dagger. Tumor cell lines arising from metastatic epidermoid carcinoma originating from cervix. .sup..dagger-dbl. Indicates low level expression, detected by silver stain. .sup..sctn. Indicates variable expression, detected by silver stain.

Two of the cervical cancer-associated proteins specific to cervical cancer cells were isolated and processed for microsequence analysis.

EXAMPLE 3

Characterization of Cervical Cancer-Associated Nuclear Matrix Protein Markers.

Two protein staining spots detectable on a 2-D gel corresponding to CvC-3 and CvC-5 were isolated, the protein harvested and subjected to microsequence analysis, as described hereinbelow.

For sequencing of the cervical cancer-associated polypeptides CvC-3 and CvC-5, the nuclear matrix fraction from HeLa cells were electrophoresed on two-dimensional gels as described above. Each gel was loaded with 300 micrograms of protein isolated by the nuclear matrix protein isolation procedure, as described above. Following the second-dimension of electrophoresis, proteins were visualized by reverse staining. Briefly, gels were soaked in 200 mM imidazole for 10 minutes, rinsed for 1 minute in water, followed by 1-2 minutes in 300 mM zinc chloride (Fernandez-Patron et al. (1992) Bio Techniques 12: 564-573). After the protein-containing spots began to appear, the gels were placed in water, and the relevant gels spots excised. The isolated gel spots representing individual cervical cancer-associated polypeptides were pooled and destained by a 5 minute wash in 2% citric acid, followed by several washes in 100 mM Tris hydrochloride at pH 7.0 to raise the pH within the gel pieces.

Each set of pooled gel fragments was then diluted with an equal volume of 2.times. sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) sample buffer (250 mM Tris-Cl, 2% SDS, 20% glycerol, 0.01% bromophenol blue and 10% .beta.-mercaptoethanol, pH 6.8) and incubated at 75.degree. C. for 3 minutes. The gel fragment-containing samples were then cooled on ice and loaded onto a 4% polyacrylamide stacking/11% polyacrylamide separating SDS-PAGE gel, and electrophoresed in 1.times. Tank Buffer (24 mM Tris-HCl, 192 mM glycine, 1% SDS, pH 8.3) to focus the gel spots into bands. Molecular weight markers (BioRad #161-0304) were used on each gel to relate the observed molecular weights on the one-and two-dimensional gels. Following electrophoresis, these gels were electroblotted onto Immobilon PVDF membranes (Oxford Glycosystems, Inc.) (Towbin et al. (1979) Proc. Nat’l. Acad. Sci. USA 76: 4350-4354) as modified by Matsudaira (Matsudaira et al. (1987) J. Biol. Chem. 262: 10035) for the mini-gel format. The membranes were then stained for 1 minute with Buffalo Black (0.1% in 1% acetic acid, 40% methanol) and rinsed with water. Regions of membrane containing polypeptide bands were excised with a clean scalpel.

The PVDF-bound polypeptides were then subjected to tryptic peptide mapping and microsequencing (Fernandez et al. (1994) Analytical Biochem. 218: 112-117) at the Microchemistry Facility at the Worcester Foundation for Biomedical Research using a Hewlett Packard Model 1090M HPLC. Sequence determinations were made on an Applied Biosystems ProCise Sequenator, and most were confirmed by MALDI-TOF mass spectrometry of individual peptides. Other peptides were identified by mass analysis alone, or mass analysis of carboxypeptidase-digested material.

Microseguence Analysis of CvC-3 Peptides.

Using the methodology described above, CvC-3 was isolated from approximately 120 two-dimensional gels of HeLa nuclear matrix and refocused on Immobilon-PVDF membrane for microsequence analysis. Although only one protein was observed by silver staining the 2-D gel location identified as CvC-3, refocusing of the protein on a one dimensional 11% minigel resulted in the resolution of two clearly separable protein bands. These proteins were labeled as CvC-3H and CvC-3L and submitted separately for microsequence analysis. Analysis of the tryptic maps indicates that two different proteins were contained in the two bands seen on the refocusing minigel, since little overlap was observed in the peak retention times of the two peptides.

Ten masses were detected by mass spectrometry from seven of the CvC-3H peaks. Amino acid sequence was obtained for three peptides, two by Edman degradation and one by carboxypeptidase-MALDI-TOF analysis. The sequences obtained for these peptides, shown in Table 4 match a protein known as IEF SSP 9502 or “novel human nuclear phosphoprotein”. (Honore et. al. (1994) supra; GenBank Accession #LO7758). The complete amino acid sequence for this protein, as derived from a gene sequence, is shown in SEQ. ID No.: 10. Seven other masses from peak fractions separated on the CvC-3H tryptic map also matched those of predicted tryptic fragments from this protein. Mass correlation data of tryptic peptides from CvC-3H are summarized in Table 4. The predicted molecular weight of the nuclear phosphoprotein, based upon its nucleotide sequence is 55 kDa, whereas its observed molecular weight by 2-D gel analysis is 79 kDa (Honore et al. (1994) supra).

TABLE 4 __________________________________________________________________________ Mass Correlation of CvC-3H-derived Tryptic Peptides Observed Predicted SEQ. Peak Mass (Da) Mass (Da) Delta Sequence ID. No. Protein __________________________________________________________________________ 4 1110.64 1109.25 0.13% PAASLAVHTDK 1 IEF SSP 9502 5 834.62 835.92 0.16% FSGQIER 2 IEF SSP 9502 7 1056.57 1057.26 0.07% RLIAEAKEK 3 IEF SSP 9502 8 1187.45 1185.37 0.18% PSLVHSRDM 4 IEF SSP 9502 10 1774.73 1766.93 0.44% VWDISTVSSVNEAFGR* 5 IEF SSP 9502 10 1802.22 1805.02 0.16% LVLGSARNSSISGPFGSR 6 IEF SSP 9502 11 2746.27 2743.02 0.12% SDKPIFTLNAHNDEISGLDLSSQIK** 7 IEF SSP 9502 12 2412.23 2409.68 0.11% VQTLQFHPFE AQTLISGSYDK* 8 IEF SSP 9502 12 2475.13 2483.98 0.36% MGVLFCSSCC PDLPFIYAFGGQK 9 IEF SSP 9502 __________________________________________________________________________ *Underlining reflects sequences confirmed by Edman degradation. **Bolded underlining reflects sequence confirmed by carboxypeptidase digestion.

In addition, seven masses were detected by mass spectrometry from four peaks derived from tryptic digestion of CvC-3L. One of these was directly sequenced and was found to be identical to cytokeratin 17 (Troyanovsky et al. (1992), supra; GenBank Accession # Q04695). Six other masses from fractions separated on the CvC-3L tryptic map also matched those of predicted tryptic fragments of human cytokeratin 17. The amino acid sequence for this protein, from Troyanovsky et al. (1992), supra, is shown in SEQ. ID No.: 18. Mass correlation data of tryptic peptides from CvC-3L are summarized in Table 5. The apparent molecular weight of CvC-3L (47.9 kDa) is consistent with the detection of a full length molecule of cytokeratin 17 (Predicted molecular weight, 48 kDa) in cervical tumors.

TABLE 5 __________________________________________________________________________ Mass Correlation of CvC-3L-derived Tryptic Peptides Observed Predicted SEQ. Peak Mass (Da) Mass (Da) Delta Sequence ID No. Protein __________________________________________________________________________ 4 995.46 994.03 0.14% DYSQYYR 11 Cytokeratin 17 4 1244.97 1242.34 0.21% NHEEEMNALR 12 Cytokeratin 17 9 1518.03 1516.67 0.09% LLEGEDAHLTQYK* 13 Cytokeratin 17 10 791.19 790.94 0.03% ILNEMR 14 Cytokeratin 17 10 835.16 832.91 0.27% SEISELR 15 Cytokeratin 17 12 1144.21 1144.21 0.00% DAEDWFFSK 16 Cytokeratin 17 12 1187.57 1186.33 0.10% LSVEADINGLR 17 Cytokeratin 17 __________________________________________________________________________ *Underlining reflects sequences confirmed by Edman degradation.

Microsequence Analysis of CvC-5 Peptides.

The gel spot identified as CvC-5 was collected from HeLa nuclear matrix from the same preparative two-dimensional gels that were used for the collection of CvC-3. Approximately 100 gel spots were collected as described and refocused on Immobilon-PVDF membrane for microsequence analysis. During the initial identification of tumor associated proteins it was noted that in some cervical tumors, two proteins appeared to migrate very closing together in the location identified as CvC-5. Only one protein was clearly apparent. However, when the expression of this protein was examined in cervical tumor cell lines, 3 of 8 cell lines showed the presence of at least two proteins in the area defined by CvC-5 (Table 3). Without wishing to be bound by theory, one explanation for the apparent detection of only one protein in the CvC-5 gel spot in many tumors is that one of the proteins may be more abundant, thereby masking the presence of other closely migrating proteins. When CvC-5 gel spots were pooled and refocused onto a one dimensional minigel, only one diffusely stained protein band was detected.

The tryptic map of the diffuse band containing the polypeptide components of the CvC-5 gel spot contained approximately 30 resolved peaks. Mass analysis was performed on 12 of these peaks and 30 masses were obtained. Six amino acid sequences were obtained by automated Edman degradation, revealing the presence of three distinct polypeptides. The first of these is a protein known as TDP-43 or TAR DNA binding protein (Out et. al. (1995) supra; GenBank Accession # U23731). The complete amino acid sequence, as derived from the gene sequence for this protein, is shown in SEQ. ID. No. 26. The apparent molecular weight of 43 kDa suggests identification of the intact protein in cervical tumors. Six other masses from fractions separated on the CvC-5 tryptic map also matched those of predicted tryptic fragments from this protein. Mass correlation data and peptide sequence data of tryptic peptides matching TDP-43 are shown in Table 6.

TABLE 6 __________________________________________________________________________ Mass Correlation of CvC-5 Derived Tryptic Peptides. Observed Predicted SEQ. Peak Mass (Da) Mass (Da) Delta Sequence ID. No. Protein __________________________________________________________________________ 12 1729.01 1726.79 0.13% FGGNPGGFGNQGGFGNSR 19 TDP43 13 655.72 653.78 0.30% WCDCK 20 TDP43 13 834.24 833.89 0.04% TTEQDLK 21 TDP43 14 682.63 681.79 0.12% GFGFVR 22 TDP43 16 1511.88 1511.66 0.01% LPNSKQSQDEPLR 23 TDP43 21 1280.01 1281.41 0.11% KMDETDASSAVK 24 TDP43 25 1342.84 1341.61 0.09% TSDLIVLGLPWK* 25 TDP43 __________________________________________________________________________ *Underlining reflects sequences confirmed by Edman degradation.

Sequence information obtained for three peptides matched a nuclear pore protein known as nucleoporin or Nup358 (Wu et. al. (1995) supra, Gen Bank Accession #L41840). The complete amino acid sequence, as derived from the gene sequence, is shown in SEQ. ID. No.:34. Mass correlation data for five additional masses identified from the CvC-5 tryptic map which matched predicted tryptic fragments of Nup358 are shown in Table 7. The location of the sequences matching Nup358 suggests our isolation of a C-terminal fragment of the intact protein (M.sub.r 358 kDa) from cervical tumors.

TABLE 7 __________________________________________________________________________ Mass Correlation of CvC-5 Derived Tryptic Peptides. Observed Predicted SEQ. Peak Mass (Da) Mass (Da) Delta Sequence ID No. Protein __________________________________________________________________________ 9 613.14 614.66 0.25% NYYR* 27 nup358 10 613.20 614.66 0.24% NYYR* 28 nup358 11 702.22 701.78 0.06% VQEAQK 29 nup358 16 938.37 939.10 0.08% EVADCFK 30 nup358 17 2459.64 2458.54 0.04% HDGTGGQSIYGDKFEDENFDVK** 31 nup358 21 1419.00 1419.71 0.05% ITMELFXNIVPR** 32 nup358 21 2773.58 2771.11 0.09% HTGPGLLSMANQGQNTNNXXFVIXLK** 33 nup358 __________________________________________________________________________ *Denotes a peptide that appeared in two adjacent HPLC fractions **Underlining reflects sequences confirmed by Edman degradation

The third polypeptide identified in the CvC-5 gel spot is a fragment of lamin A (Fisher et. al. (1986), supra; GenBank Accession #M13452). Two sequences matching lamin A were obtained by Edman degradation (Table 8). Nine additional masses from fragments of the CvC-5 tryptic map match predicted masses of tryptic fragments from lamin A. Mass correlation data for these additional masses were shown in Table 8. The amino acid sequence for this protein, (Fisher et. al. (1986) supra), is shown in SEQ. ID No.: 46.

TABLE 8 __________________________________________________________________________ Mass Correlation of CvC-5 derived Tryptic Peptides. Observed Predicted Seq. Peak Mass (Da) Mass (Da) Delta Sequence ID No. Protein __________________________________________________________________________ 7 667.10 666.69 0.06% EFESR 35 lamin A 8 569.50 568.63 0.15% TYSAK* 36 lamin A 8 585.78 587.63 0.31% LDNAR 37 Iamin A 11 569.10 568.63 0.08% TYSAK* 38 lamin A 11 1025.18 1023.11 0.20% NIYSEELR 39 lamin A 12 805.83 803.91 0.24% TALSEKR 40 lamin A 17 1349.52 1347.56 0.15% LALDMEIHAYR** 41 lamin A 17 1009.78 1009.18 0.06% EMAEMRAR 42 lamin A 21 1912.74 1913.07 0.02% EELDFQKNIYSEELR* 43 lamin A 22 1896.58 1894.13 0.13% MQQQLDEYQELLDIK** 44 lamin A 22 1913.03 1913.07 0.00% EELDFQKNIYSEELR* 45 lamin A __________________________________________________________________________ *Denotes a peptide that appeared in two adjacent HPLC fractions **Underlining reflects sequences confirmed by Edman degradation

Cervical cancer-associated proteins may be identified using well-known techniques based upon the partial amino acid sequences provided above. Thus, the cervical cancer-associated proteins detected according to methods of the invention may be referred to as comprising a continuous sequence shown in the above-noted sequence fragments. It is appreciated that the skilled artisan, in view of the foregoing disclosure, would be able to produce an antibody directed against any cervical cancer-associated protein identified by the methods described herein. Moreover, the skilled artisan, in view of the foregoing disclosure, would be able to produce nucleic acid sequences which encode the fragments described above, as well as nucleic acid sequences complementary thereto. In addition, the skilled artisan using conventional recombinant DNA methodologies, for example, by screening a cDNA library with such a nucleic acid sequence, would be able to isolate full length nucleic acid sequences encoding target cervical cancer-associated proteins. Such full length nucleic acid sequences, or fragments thereof, may be used to generate nucleic acid-based detection systems or therapeutics.

EXAMPLE 4

Production of Antibodies Which Bind Specifically to Cervical Cancer-associated Proteins.

Once identified, a cervical cancer-associated protein, such as a CvC-1 through CvC-5, may be detected in a tissue or body fluid sample using numerous binding assays that are well known to those of ordinary skill in the art. For example, as discussed above, a cervical cancer-associated protein may be detected in either a tissue or body fluid sample using an antibody, for example, a monoclonal antibody, which bind specifically to an epitope disposed upon the cervical cancer-associated protein. In such detection systems, the antibody preferably is labeled with a detectable moiety.

Provided below is an exemplary protocol for the production of an anti-cervical cancer-associated monoclonal antibody. Other protocols also are envisioned. Accordingly, the particular method of producing antibodies to target proteins is not envisioned to be an aspect of the invention.

Balb/c by J mice (Jackson Laboratory, Bar Harbor, Me.) are injected intraperitoneally with the target protein, e.g., CvC-3 protein isolated from HeLa cell nuclear matrix, every 2 weeks until the immunized mice obtain the appropriate serum titer. Thereafter, the mice are injected with 3 consecutive intravenous boosts. Freund’s complete adjuvant (Gibco, Grand Island) is used in the first injection, incomplete Freund’s in the second injection; and saline is used for subsequent intravenous injections. The animal is then sacrificed and its spleen removed. Spleen cells (or lymph node cells) then are fused with a mouse myeloma line, e.g., using the method of Kohler et al. (1975) Nature 256: 495, the disclosure of which is incorporated herein by reference. Hybridomas producing antibodies that react with the target proteins then are cloned and grown as ascites. Hybridomas are screened by nuclear reactivity against the cell line that is the source of the immunogen, and by tissue immunohistochemistry using standard procedures known in the immunology art. Detailed descriptions of screening protocols, ascites production and immunoassays also are disclosed in PCT/US92/09220 published May 13, 1993, the disclosure of which is incorporated herein by reference.

EXAMPLE 5

Antibody-based Assay for Detecting Cervical Cancer in an Individual

The following assay has been developed for tissue samples, however, it is contemplated that similar assays for testing fluid samples may be developed without undue experimentation. A typical assay may employ a commercial immunodetection kit, for example, the ABC Elite Kit from Vector Laboratories, Inc.

A biopsy sample, for example, a Pap smear is removed from the patient under investigation in accordance with the appropriate medical guidelines. The sample then is applied to a glass microscope slide and the sample fixed in cold acetone for 10 minutes. Then, the slide is rinsed in distilled water and pretreated with a hydrogen peroxide containing solution (2 mL 30% H.sub.2 O.sub.2 and 30 mL cold methanol). The slide is then rinsed in a Buffer A comprising Tris Buffered Saline (TBS) with 0.1% Tween and 0.1% Brij. A mouse anti-cervical cancer-associated protein monoclonal antibody in Buffer A is added to the slide and the slide then incubated for one hour at room temperature. The slide is then washed with Buffer A, and a secondary antibody (ABC Elite Kit, Vector Labs, Inc) in Buffer A is added to the slide. The slide is then incubated for 15 minutes at 37.degree. C. in a humidity chamber. The slides are washed again with Buffer A, and the ABC reagent (ABC Elite Kit, Vector Labs, Inc.) is then added to the slide for amplification of the signal. The slide is then incubated for a further 15 minutes at 37.degree. C. in the humidity chamber.

The slide then is washed in distilled water, and a diamino benzenedine (DAB) substrate added to the slide for 4-5 minutes. The slide is then rinsed with distilled water, counterstained with hematoxylin, rinsed with 95% ethanol, rinsed with 100% ethanol, and then rinsed with xylene. A cover slip is then applied to the slide and the result observed by light microscopy.

Equivalents

The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Abstract
The present invention relates to methods for preventing the development of epithelial ovarian cancer by administering progestin products, either alone or in combination with other agents such as estrogen products.

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Inventors: Rodriguez; Gustavo C. (Durham, NC), Hughes, Jr.; Claude L. (Mebane, NC)
Assignee: New Life Pharmaceuticals Inc. (Chicago, IL)

Appl. No.: 08/713,834
Filed: September 13, 1996

Claims

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What is claimed are:

1. A method of preventing ovarian cancer in a post-menopausal female subject in need thereof who is no longer ovulating comprising administering to the post-menopausal female subject a progestin product sensitive to preventing ovarian cancer in an amount effective to increase apoptosis in ovarian epithelial cells of the female subject.

2. A method of preventing ovarian cancer in a post-menopausal female subject in need thereof who is no longer ovulating comprising administering to the post-menopausal female subject a composition consisting essentially of a progestin product sensitive to preventing ovarian cancer.

3. A method of preventing ovarian cancer in a post-menopausal female subject in need thereof who is no longer ovulating comprising administering a progestin product sensitive to preventing ovarian cancer to the post-menopausal female subject, in an amount effective to increase apoptosis in ovarian epithelial cells of the female subject, according to a regimen that is not effective for contraception.

4. The method of claim 3 wherein the progestin product is administered or delivered in an amount less than sufficient for contraceptive effect.

5. The method of claim 3 wherein the progestin product is administered for a duration of less than one menstrual cycle.

6. The method of claim 3 wherein the progestin product is administered for nonconsecutive menstrual cycles.

7. The method of claim 3 wherein the progestin product is administered for one or more menstrual cycles for fewer than 21 consecutive days in each cycle.

8. The method of claim 3 wherein the progestin product is administered or delivered with a less than daily frequency.

9. The method of claim 3 wherein the progestin product is administered for one or more menstrual cycles according to a regimen that fails to maintain a contraceptive blood level of the drug or its active metabolite for 21 consecutive days in each cycle.

10. The method of claim 1 wherein a progestin product is administered or delivered to a female subject at a dose higher than equivalent to 10 mg of norethindrone orally per day.

11. The method of claim 1 wherein a progestin product is administered or delivered to said post-menopausal female subject who is no longer ovulating at a dose higher than equivalent to 5 mg of norethindrone orally per day.

12. The method of claim 1 wherein a progestin product is administered or delivered to said post-menopausal female subject who is no longer ovulating at a dose less than equivalent to 1.25 mg of norethindrone orally per day.

13. The method of claim 1 wherein the female subject is at high risk of developing ovarian cancer.

14. The method of claim 1 wherein the female subject is at high risk of developing ovarian cancer.

15. A method of increasing apoptosis in ovarian epithelial cells of a post-menopausal female subject in need thereof comprising administering to the post-menopausal female subject who is no longer ovulating a composition consisting essentially of a progestin product sensitive to inducing apoptosis in an amount effective to increase apoptosis in ovarian epithelial cells of the female subject.

16. A method of increasing apoptosis in ovarian epithelial cells of a post-menopausal female subject in need thereof comprising administering to the post-menopausal female subject who is no longer ovulating a progestin product sensitive to inducing apoptosis in an amount effective to increase apoptosis in ovarian epithelial cells of the female subject.

17. The method of claim 16 wherein the progestin product is administered or delivered in an amount less than sufficient for contraceptive effect.

18. The method of claim 16 wherein the progestin product is administered for a duration of less than one menstrual cycle.

19. The method of claim 16 wherein the progestin product is administered for nonconsecutive menstrual cycles.

20. The method of claim 16 wherein the progestin product is administered for one or more menstrual cycles for fewer than 21 consecutive days in each cycle.

21. The method of claim 16 wherein the progestin product is administered or delivered with a less than daily frequency.

22. The method of claim 16 wherein the progestin product is administered for one or more menstrual cycles according to a regimen that fails to maintain a contraceptive blood level of the drug or its active metabolite for 21 consecutive days in each cycle.

23. A method of increasing apoptosis in ovarian epithelial cells of a female subject in need thereof comprising administering to a female subject an amount of progestin product effective to increase apoptosis in ovarian epithelial cells of the female subject wherein a progestin product is administered or delivered to a female subject at a dose higher than equivalent to 10 mg of norethindrone orally per day.

24. A method of increasing apoptosis in ovarian epithelial cells of a female subject in need thereof comprising administering to a post-menopausal female subject an amount of progestin product effective to increase apoptosis in ovarian epithelial cells of the female subject wherein a progestin product is administered or delivered to a post-menopausal female subject who is no longer ovulating at a dose higher than equivalent to 5 mg of norethindrone orally per day.

25. A method of increasing apoptosis in ovarian epithelial cells of a female subject in need thereof comprising administering to a female subject an amount of progestin product effective to increase apoptosis in ovarian epithelial cells of the female subject wherein a progestin product is administered or delivered to a post-menopausal female subject who is no longer ovulating at a dose less than equivalent to 1.25 mg of norethindrone orally per day.

26. The method of claim 24 wherein the progestin product is administered or delivered at a dose higher than equivalent to 10 mg of norethindrone orally per day.

27. The method of claim 24 wherein the progestin product is administered or delivered with a less than daily frequency.

28. The method of claim 25 wherein a progestin product is administered or delivered to a female subject at a dose less than equivalent to 1 mg of norethindrone orally per day.

29. The method of claim 25 wherein a progestin product is administered or delivered to a female subject at a dose less than equivalent to 0.5 mg of norethindrone orally per day.

30. The method of claim 25 wherein a progestin product is administered or delivered to a female subject at a dose less than equivalent to 0.2 mg of norethindrone orally per day.

31. The method of claim 25 wherein the progestin product is administered or delivered with a less than daily frequency.

32. A method of increasing apoptosis in ovarian epithelial cells of a post-menopausal female subject who is no longer ovulating in need thereof comprising administering to a female subject an amount of progestin product effective to increase apoptosis in ovarian epithelial cells of the female subject wherein a progestin product is administered or delivered to said post-menopausal female subject at a dose greater than equivalent to 1.0 mg of norethindrone orally per day.

33. The method of claim 32 wherein the progestin product is administered or delivered at a dose greater than equivalent to at least 2.5 mg of norethindrone orally per day.

34. A method of increasing apoptosis in ovarian epithelial cells of a post-menopausal female subject who is no longer ovulating in need thereof comprising administering to said post-menopausal female subject an amount of progestin product effective to increase apoptosis in ovarian epithelial cells of the female subject wherein said progestin product is administered or delivered to said post-menopausal female subject at a dose of greater than 1.0 mg of progestin orally per day.

35. The method of claim 34 wherein the progestin product is administered or delivered at a dose greater than 2.5 mg of progestin orally per day.

36. The method of claim 35 wherein the progestin product is administered or delivered at a dose greater than 5.0 mg of progestin orally per day.

37. A method of increasing apoptosis in ovarian epithelial cells of a post-menopausal female subject who is no longer ovulating in need thereof comprising administering to said post-menopausal female subject an amount of progestin product effective to increase apoptosis in ovarian epithelial cells of the female subject wherein a progestin product is administered or delivered to said post-menopausal female subject at a dose less than 2.5 mg of progestin orally per day.

38. The method of claim 37 wherein the progestin product is administered or delivered at a dose less than 1.0 mg of progestin orally per day.
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Description

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FIELD OF THE INVENTION

The present invention relates generally to methods of preventing the development of ovarian cancer by administering progestin products, alone or in association with other hormones such as estrogen products.

BACKGROUND OF THE INVENTION

Ovarian cancer is the fourth leading cause of cancer deaths among women in the United States and causes more deaths than all other gynecological malignancies combined. In the United States, a woman’s lifetime risk of developing ovarian cancer is 1 in 70. In 1992, about 21,000 cases of ovarian cancer were reported, and about 13,000 women died from the disease. [Chapter 321, Ovarian Cancer, Harrison's Principles of Internal Medicine, 13th ed., Isselbacher et al., eds., McGraw-Hill, New York (1994), pages 1853-1858; American Cancer Society Statistics, Cancer J. Clinicians, 45:30 (1995). Epithelial ovarian cancer, the most common ovarian cancer, has a distinctive pattern of spread: in addition to metastasis through the lymphatic and blood vessels to areas such as the liver, lung and brain, cancer cells may also migrate through the peritoneum to produce multiple metastatic nodules in the visceral and parietal peritoneum and the hemidiaphragms. Early stage ovarian cancer is often asymptomatic and is detected coincidentally by palpating an ovarian mass on pelvic examination. In premenopausal patients, about 95% of these masses are benign. Even after menopause, 70% of masses are benign but detection of any enlargement requires exploratory surgery. In postmenopausal women with a pelvic mass, a markedly elevated serum CA-125 level of greater than 95 U/ml indicates malignancy with a 96% positive predictive value. [Chapter 321, Ovarian Cancer, Harrison's Princples of Internal Medicine, supra.]

Epithelial ovarian cancer is seldom encountered in women less than 35 years of age. Its incidence increases sharply with advancing age and peaks at ages 75 to 80, with the median age being 60 years. The single most important risk factor for this cancer is a strong family history of breast or ovarian cancer. In families in which ovarian, breast, endometrial or colon cancer can be tracked as an apparent autosomal dominant trait, the risk of this cancer can be as high as 50%. Having a single first-degree relative with ovarian cancer increases a woman’s risk by at least three-fold, and having a personal history of breast or colorectal cancer increases the risk of subsequently developing ovarian cancer by two-fold. [Chapter 321, Ovarian Cancer, Harrison's Principles of Internal Medicine, supra.] In addition, those with identifiable genetic mutations in genes such as BRCA1 also have an increased risk. Baker et al., Etiology, Biology, and Epidemiology of Ovarian Cancer, Seminars in Surgical Oncology 10: 242-248, 1994; Amus et al., Genetic Epidemiology of Epithelial Ovarian Cancer, Cancer 71: 566-72, 1993; Whitmore, Characteristics Relating To Ovarian Cancer Risk: Implications for Preventing and Detection, Gynecologie Oncology 55, 515-19, 1994. Oncogenes associated with ovarian cancers include the HER-2/neu (c-erbB-2) gene, which is overexpressed in a third of ovarian cancers, the fms oncogene, and abnormalities in the p53 gene, which are seen in about half of ovarian cancers. A number of environmental factors have also been associated with a higher risk of epithelial ovarian cancer, including a high fat diet and intake of lactose in subjects with relatively low tissue levels of galactose-1-phosphate uridyl transferase.

In epidemiological studies, behavior associated with decreased ovulation, such as pregnancy, breastfeeding and use of estrogen-progestin combination oral contraceptive medications, decrease the risk of ovarian cancer; use of estrogen-progestin combination oral contraceptives for as long as 5 years can reduce the risk of ovarian cancer by 50%. Greene et al., The Epidemiology of Ovarian Cancer, Seminars Oncology, 11: 209-225, 1984; Whitmore et al., Characteristics Relating To Ovarian Cancer Risk: Collaborative Analysis of 12 US Case-Control Studies, American J. Epidemiology 136: 1212-20, 1992. Conversely, early menarche, late menopause and nulliparity (no pregnancies) have been shown to increase the risk of ovarian cancer. The risk has been shown to positively correlate with the number of ovulatory cycles in a woman’s lifetime. Wu et al., Personal and Environmental Characteristics Related To Epithelial Ovarian Cancer, American J. Epidemiology, Vol. 108(6) 1216-1227. The long-term use of ovulation-inducing ovarian hyperstimulants such as clomiphene has been shown to be associated with an increased risk of ovarian cancer in some women. Rossary et al., Ovarian Tumors in a Cohort Of Infertile Women, New Engl. J. Med., 331: 771-6, 1994. Thus, some factors that favor prolonged and persistent ovulation have been thought to increase ovarian cancer risk, whereas some factors that suppress ovulation have been thought to decrease risk. [Chapter 321, Ovarian Cancer, Harrison's Principles of Internal Medicine, supra.] These data have led to the “incessant ovulation” hypothesis for the development of ovarian cancer. Casagrande et al., “Incessant Ovulation” and Ovarian Cancer, Lancet at 170-73 (Jul. 28, 1979). This hypothesis is that repeated ovulation cycles, each of which involves the disruption and repair of the ovarian surface epithelium, may cause neoplastic transformation of the ovarian epithelium in susceptible individuals and that the risk of ovarian cancer is associated with the number of ovulation cycles in a woman’s lifetime.

There is no established pharmaceutical approach to the prevention of ovarian cancer. For all women, especially those at high risk of developing this disease, the only option available at this time is surgical removal of the ovaries, with all of the attendant risks and subsequent adverse health consequences due to resulting estrogen deficiency.

Although epidemiological evidence suggests that the use of combination oral contraceptives, which contain both an estrogen and a progestin, is associated with a subsequent reduced risk of developing epithelial ovarian cancer, the mechanism for this protective effect is unknown, and oral contraceptive preparations are not currently approved for this purpose. The reduction in risk of ovarian cancer in women who have used estrogen-progestin combination oral contraceptives for at least three years is approximately 40 percent. Moreover, this protective effect increases with the duration of use and persists for up to two decades after discontinuation of use. Rosenberg et al., A Case Control Study of Oral Contraceptive Use and Invasive Epithelial Ovarian Cancer, The WHO Collaborative Study of Neoplasia and Steroid Contraceptives; Epithelial Ovarian Cancer and Combined Oral Contraceptives, Int’l J. Epidemiology 18: 538-45, 1989; Lee et al., The Reduction in Risk of Ovarian Cancer Associated with Oral Contraceptive Use, New Engl. J. Med. 316: 650-51, 1987; Thomas P. Gross, James J. Schlesselman, The Estimated Effect of Oral Contraceptive Use on the Cumulative Risk of Epithelial Ovarian Cancer, Obstetrics Gynecology 83: 419-24, 1994; Franceschi et al., Pooled Analysis of 3 European Case-Control Studies of Epithelial Ovarian Cancer: III Oral Contraceptive Use, Int’l J. Cancer 49: 61-65, 1991.

It is commonly believed that the protective effect of oral contraceptives is related to the ability of these drugs to inhibit ovulation. Estrogen-progestin combination oral contraceptives act primarily by suppressing the pituitary gland’s production of gonadotropins, thereby inhibiting the hormonal stimulus for ovulation. These combination drugs also have direct inhibitory effects on the reproductive tract, including inducing changes in the cervical mucus that decrease the ability of sperm to enter the uterus, as well as changes in the endometrium that reduce the likelihood of implantation, and reducing fallopian tube motility and uterine secretions.

The epidemiological studies showing the protective effect of combination oral contraceptives evaluated older combination preparations which typically contained higher doses of drug than most contraceptive regimens used today. Common older regimens contained 50 micrograms or more of ethinyl estradiol (an estrogen) or 100 micrograms or more of mestranol (an estrogen) and greater than 1 mg of norethindrone, norethindrone acetate or norethynodrel (a progestin). Table 1 infra lists the progestin and estrogen content of some older regimens. All of the currently used low-dose combination oral contraceptives contain lower doses of both progestin and estrogen, as well as a lower ratio of progestin to estrogen. Consequently, it has not been definitively established that the newer low-dose combination oral contraceptives are associated with the same protective effect as the older high-dose combination contraceptives. Rosenblatt et al., High Dose and Low Dose Combined Oral Contraceptives: Protective Against Epithelial Ovarian Cancer and The Length of the Protective Effect, Eur. J. Cancer, 28: 1870-76, 1992.

Despite the overall safety of combination oral contraceptives, their use is not recommended for women smokers older than age 35, for women of all ages who are at increased risk for myocardial infarction, for women with liver disease, and for women older than age 40. Serious and potentially fatal side effects include deep vein thrombosis, pulmonary emboli, myocardial infarction, thromboembolic stroke, hemorrhagic stroke, and high blood pressure. In the 35-39 year old age group, the use of oral contraceptives among women smokers doubles their risk of death. After age 40, the mortality rate even in non-smoker women using oral contraceptives (32.0 per 100,000) is greater than women using no contraception (28.2 per 100,000), while the mortality rate for smoker women is quadrupled (117.6 vs. 28.2 per 100,000). [Chapter 340, Disorders of the Ovary and Female Reproductive Tract, Harrison's Principles of Internal Medicine, supra, pages 2017-2036.]

Progestin-only contraceptives do not reliably inhibit ovulation, but are nevertheless contraceptively effective, presumably due to direct effects on the reproductive tract. The actual contraceptive mechanism of action is unclear. Prior epidemiological studies have exhibited no consistent pattern of either increasing or decreasing risk of ovarian cancer according to duration of use. The WHO Collaborative Study of Neoplasia and Steroid Contraceptives Depot-Medroxyprogesterone Acetate(DMPA) and Risk of Epithelial Ovarian Cancer, Int’l J. Cancer. 49:191-195 (1991); Liam et al., Risk of Breast, Uterine, Corpus, and Ovarian Cancer in Women Receiving/Medroxyprogesterone Injections, J. Am. Med. Ass’n 249:2909-2912 (1983). Thus, unlike the data available for progestin-estrogen combination contraceptives, the prior art relating to progestin-only contraceptives does not suggest that the use of a progestin reduces the risk of epithelial ovarian cancer.

Estrogen, alone or with low doses of progestin, is also used as hormonal replacement therapy in menopausal women. For long term use, Premarin.RTM. (conjugated equine estrogen) is generally given at a dose of 0.625 mg orally daily (equivalent to 10 to 20 .mu.g ethinyl estradiol orally per day) or an equivalent dose transdermally. Other regimens add cyclic progestins or continuous low-dose progestins, typically 2.5 to 10 mg per day of Provera.RTM. (medroxyprogesterone acetate). One epidemiologic study has suggested that hormone replacement therapy with estrogen alone may be associated with an increased risk of developing ovarian cancer. Rodriguez et al., Estrogen Replacement Therapy and Fatal Ovarian Cancer, Am. J. Epidemiology, 141:828-835 (1995).

SUMMARY OF THE INVENTION

The present invention provides a method for preventing the development of epithelial ovarian cancer by administering progestin products, either alone or in combination with other agents, such as estrogen products. A method is provided of preventing ovarian cancer comprising administering to a female subject an amount of progestin product effective to increase apoptosis in ovarian epithelial cells of the female subject.

It is further the object of this invention to expand the clinical usage of progestin drugs beyond the current use of these drugs as oral contraceptive agents in young women or as part of estrogen-progestin hormone replacement regimens in postmenopausal women. One aspect of the invention provides a method for preventing the development of ovarian cancer comprising administering to a female subject a composition consisting essentially of a progestin product (i.e., a progestin product alone without an estrogen product).

The invention also provides a method for preventing the development of ovarian cancer comprising administering a progestin product to a female subject according to a regimen that is not effective for contraception. This can be accomplished in a number of ways, including altering the dosage of progestin product, the type of progestin product, the ratio of progestin product to estrogen product, or the timing of administration.

With regard to infertile female subjects, the present invention further provides a method for preventing the development of ovarian cancer comprising administering a progestin product according to a regimen that is different from that currently used for hormone replacement therapy. Again, this can be accomplished in a number of ways, including altering the dosage, timing, ratio of progestin product to estrogen product, or the type of progestin product.

It is contemplated that the progestin product may be concurrently administered in combination with additional agent(s), such as an estrogen product, a second progestin product, an androgenic agent, an androgen agonist, a progestin agonist, an estrogen antagonist, or another hormone product, or with other agents that induce apoptosis of ovarian epithelial cells. Such additional agent(s) may be selected to improve the activity of the progestin agent for preventing ovarian cancer or to reduce any side effects of the progestin agent. Preferably if estrogen is used as the second agent, it is used in doses lower than those currently used in combination oral contraceptive regimens or in doses selected to provide a progestin/estrogen product ratio that is higher than the ratio currently used in combination oral contraceptives.

The present invention is based on the discovery that administration of progestin alone induced an accelerated rate of apoptosis in vivo in ovarian epithelial cells of monkeys. Apoptosis is one of the most important mechanisms used for the elimination of cells that have sustained DNA damage and which are thus prone to transformation into malignant neoplasms. This novel explanation for the association between estrogen-progestin combination oral contraceptive use and a reduced risk of ovarian cancer is a complete departure from the widely accepted theory that suppression of “incessant ovulation” is responsible for this reduced risk. This finding thus relates to the discovery that progestin alone or estrogen-progestin combinations may be administered in ways that do not effectively inhibit ovulation or otherwise inhibit contraception, yet which still prevent ovarian cancer.

The invention further relates to the discovery that progestin alone induced a greater rate of apoptosis than a combination of estrogen and progestin, which in turn induced a greater rate of apoptosis than estrogen alone. The invention thus contemplates that administration of progestin alone be effective for preventing the development of ovarian cancer, contrary to the suggestions of the prior art that progestin has no effect on risk of ovarian cancer.

DETAILED DESCRIPTION OF THE INVENTION

The present invention generally relates to methods for preventing the development of epithelial ovarian cancer by administering a progestin product, either alone or in combination with other agents, such as an estrogen product. The invention provides a method of preventing ovarian cancer comprising administering to a female subject an amount of progestin product effective to increase apoptosis in ovarian epithelial cells of the female subject. The invention also provides a method of increasing apoptosis in ovarian epithelial cells of a female subject comprising administering to a female subject an amount of progestin product effective to increase apoptosis in ovarian epithelial cells of the female subject. In particular, the methods of the present invention will be particularly advantageous when applied to females at high risk of developing ovarian cancer.

In a second aspect of the invention, a method is provided for preventing the development of ovarian cancer comprising administering to a female subject a composition consisting essentially of a progestin product (i.e., a progestin product alone without an estrogen product). The female subject may be a fertile female or an infertile female, including perimenopausal and postmenopausal women. The most preferred product for administration would be an agent that provides the greatest rate of apoptosis of ovarian epithelial cells with the least side effects. Use of a progestin product for longer durations, or at higher doses, at appropriate intervals, and/or use of an agent that maximizes apoptosis, without creating unacceptable side effects, in fertile or infertile women may reduce the risk of ovarian cancer further than that previously achieved by combination oral contraceptive use, potentially by as much as 60% to 80%.

The invention further contemplates expanding the clinical usage of progestin drugs beyond the current use of these drugs as oral contraceptive agents in young women or as part of estrogen-progestin hormone replacement regimens in postmenopausal women. Specifically, a third aspect of the present invention provides a method for preventing the development of ovarian cancer comprising administering a progestin product to a fertile female subject according to a regimen that is not effective for contraception. This can be accomplished in a number of ways, including altering the dosage of progestin product, the type of progestin product, the ratio of progestin product to estrogen product, or the timing of administration. Also specifically contemplated is administration of a progestin product in doses higher than those currently used for contraception.

Oral contraceptive administration regimens are selected to simulate the normal menstrual cycle, which averages 28 days in women of reproductive age. The menstrual cycle begins at the onset of a menstrual bleeding episode and lasts until the onset of the next. Thus, day 1 of a cycle would be the first day of menstruation, and day 28 would be the day before the onset of the next menstrual bleeding episode. Oral contraceptives are typically taken daily, at the same time each day, for 21 days, followed by a placebo for the next 7 days. The female generally experiences a menstrual bleeding episode during the seven-day placebo period. Thus, a woman first starting on oral contraceptives is generally instructed to begin taking them at some time between day 1 and 7.

The oral contraceptives must be taken according to the daily regimen for a full menstrual cycle before they are effective for contraception. A woman beginning an oral contraceptive regimen is not effectively protected against conception if the oral contraceptives are taken for less than the full menstrual cycle, if they are not taken daily, and if they are not taken for 21 consecutive days. A minimum blood level of the exogenously administered estrogen or progestin hormones must be maintained daily in order to suppress ovulation. If the blood level drops too low, ovulation may occur and the other inhibitory mechanisms on the reproductive tract may fail to prevent conception.

Thus, according to this third aspect of present invention, a regimen of progestin product administration that is not effective for contraception would include, for example, administering or delivering (regardless of whether the route of administration is oral or via injection or implant) progestin product in doses lower than those effective for contraceptive use and/or lower than those previously used in contraceptives; administering progestin product with estrogen product at a progestin/estrogen ratio that is higher than that previously used in contraceptives; administering the drug for less than one menstrual cycle; administering the drug for nonconsecutive menstrual cycles, e.g., every other cycle; administering the drug for one or more menstrual cycles for fewer than 21 consecutive days in each cycle; delivering the drug (regardless of whether the route of administration is oral or via injection or implant) with a less than daily frequency; or administering the drug for one or more menstrual cycles according to a regimen that fails to maintain a contraceptive blood level of the drug or its active metabolite for 21 consecutive days in each cycle. A regimen of progestin product administration that is different from that currently used for contraception would also include administering the progestin product at a daily dose higher than that currently used for contraception.

Exemplary regimens according to this third aspect of the invention include administering progestin product at a dose less than a dose equivalent to 1 mg daily of norethindrone, more preferably less than 0.2 mg daily, or less than 0.05 mg daily, and possibly as low as 0.025 mg daily of a norethindrone equivalent dose. Another exemplary regimen includes administering progestin product at a dose higher than 10 mg daily of a norethindrone equivalent dose. A further exemplary regimen includes administering a progestin product with an estrogen product at a ratio of greater than 239:1 by weight in norethindrone/ethinyl estradiol equivalent doses. Additional exemplary regimens include administering any dose of progestin product with a less than daily frequency; or administering any dose of progestin product for a brief time, e.g., one week only, during the menstrual cycle. It is contemplated that the most desirable mode of administration may be administering the progestin product for a brief period sufficient to produce apoptotic turnover of damaged ovarian cells, followed by repeated dosing periods at intervals, for example 1, 3, 5 or 10 years, selected to provide apoptotic turnover adequate to prevent malignant transformations. The most preferable progestin product for administration would be a product that maximizes the apoptotic turnover of ovarian epithelial cells and minimizes any side effects.

The fourth aspect of the present invention provides a method for preventing the development of ovarian cancer in infertile female subjects, comprising administering a progestin product according to a regimen that is different from that currently used for hormone replacement therapy. Again, this can be accomplished in a number of ways, including altering the dosage, timing, ratio of progestin product to estrogen product, or the type of progestin product. Other contemplated regimens would include, for example, administering or delivering progestin product in doses lower or higher than those previously used in hormone replacement therapy; or administering progestin product with estrogen product at a progestin/estrogen ratio that is higher than that previously used in hormone replacement therapy.

Estrogen is the primary agent in hormone replacement therapy. Postmenopausal women are generally given estrogen alone, or with low doses of progestins. The hormones may be administered continuously or cyclically. Continuous administration is typically 0.625 mg Premarin.RTM. (a conjugated equine estrogen) daily or its equivalent, with 2.5 mg Provera.RTM. (medroxyprogesterone acetate) daily. Cyclical administration is typically 25 consecutive days of 0.625 mg Premarin.RTM. daily, with 10 mg Provera.RTM. daily on days 16 through 25, followed by 5 days of no hormone treatment (during which time these women will menstruate).

Exemplary regimens according to the fourth aspect of the present invention include doses of progestin product less than a dose equivalent to 2.5 mg of medroxyprogesterone acetate daily (equivalent to about 1.25 mg of norethindrone), or less than 0.5 mg daily of a norethindrone equivalent dose. Another exemplary regimen includes a dose of progestin product greater than a dose equivalent to 10 mg of medroxyprogesterone acetate daily (equivalent to about 5 mg of norethindrone) for 10 days every month. A further exemplary regimen includes doses of progestin product with estrogen product at a ratio of greater than 1:1 by weight in norethindrone/ethinyl estradiol equivalent doses, or a ratio of greater than 50:1 or 100:1. It is also contemplated that the most desirable mode of administration may be administering the progestin product for a brief period sufficient to produce apoptotic turnover followed by repeated dosing periods at selected intervals adequate to prevent malignant transformations. A presently preferred progestin product is levonorgestrel or other 19-nortestosterone derivatives. The most preferable progestin product for administration would be a product that maximizes the apoptotic turnover of ovarian epithelial cells and minimizes any side effects.

The present invention yet further provides a novel use of progestin product in preparation of a non-contraceptive medicament for prevention of ovarian cancer in female subjects, as well as a novel use of progestin product in preparation of a medicament for prevention of ovarian cancer in infertile female subjects.

All doses given herein are appropriate for a female subject of about 60 kg weight; the dosages naturally will vary more or less depending on the weight of the subject. The doses may be increased or decreased, and the duration of treatment may be shortened or lengthened as determined by the treating physician. The frequency of dosing will depend on the pharmacokinetic parameters of the agents and the route of administration. The optimal pharmaceutical formulation will be determined by one skilled in the art depending upon the route of administration and desired dosage. See for example, Remington’s Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, Pa. 18042) pages 1435-1712, the disclosure of which is hereby incorporated by reference. Such formulations may influence the physical state, stability, rate of in vivo release, and rate of in vivo clearance of the administered agents.

Those of ordinary skill in the art will readily optimize effective dosages and concurrent administration regimens as determined by good medical practice and the clinical condition of the individual patient. Regardless of the manner of administration, the specific dose may be calculated according to body weight, body surface area or organ size. Further refinement of the calculations necessary to determine the appropriate dosage for treatment involving each of the above mentioned formulations is routinely made by those of ordinary skill in the art and is within the ambit of tasks routinely performed by them without undue experimentation, especially in light of the dosage information and assays disclosed herein. Appropriate dosages may be ascertained through use of established assays for determining dosages in conjunction with appropriate dose-response data. The final dosage regimen will be determined by the attending physician, considering various factors which modify the action of drugs, e.g. the drug’s specific activity, the severity of the damage and the responsiveness of the patient, the age, condition, body weight, sex and diet of the patient, the severity of any infection, time of administration and other clinical factors. As studies are conducted, further information will emerge regarding the appropriate dosage levels for the treatment of various diseases and conditions.

It is contemplated that the routes of delivery of progestin products (either alone or in combination with other pharmaceuticals) could include oral, sublingual, injectable (including short-acting, depot, implant and pellet forms injected subcutaneously or intramuscularly), vaginal creams, suppositories, pessaries, rings, rectal suppositories, intrauterine devices, and transdermal forms such as patches and creams.

The present invention is related to the discovery that administration of progestin alone induced an accelerated rate of apoptosis in vivo in ovarian epithelial cells of monkeys. Apoptosis is a process whereby a genetic program within the cell is activated to trigger a specific series of events within the cell eventually leading to the death and efficient disposal of the cell. Richard Lockshin, Zahra Zakeri, The Biology of Cell Death and Its Relationship to Aging in Cellular Aging and Cell Death, pp. 167-180, 1996. Wiley-Liss Inc., Editors: N.J. Holbrook, G. Martin, R. Lockshin. C. Miligan, L. Schwartz, Programmed Cell Death During Development of Animals in Cellular Aging and Cell Death, pp. 181-208, 1996. Wiley-Liss Inc. P53-Dependent, Apoptosis in Tumor Progression and in Cancer Therapy, Scott W. Lowe, H. Earl Ruley in Cellular Aging and Cell Death, pp. 209-234, 1996. Wiley-Liss, Inc.

For cells that have sustained DNA damage, apoptosis is one of the most important mechanisms used for the elimination of these cells, the preservation of which could otherwise lead to the development of malignant neoplasms. Canman et al., DNA Damage Responses: P-53 Induction, Cell Cycle Pertubations, and Apoptosis, Cold Spring Harbor Symp. Ouant. Biol., 59:277-286 (1994). Thus, the apoptosis pathway is a virtually universal safeguard to prevent the persistence and proliferation of damaged cells that can be lethal to the organism. For normal tissues, the processes of cell proliferation and cell death are usually in a steady-state balance, and the apoptosis mechanism not only serves to prevent overgrowth of tissue, but also to eliminate those cells that are aberrant and therefore prone to resist normal growth regulatory controls.

An accelerated rate of apoptosis would facilitate the destruction and thereby removal of ovarian surface epithelial cells which have defective DNA and which have the potential to transform into malignant neoplasms. Given the importance of the apoptotic pathway for removal of abnormal cells from tissues, and thus the protection of normal tissues from neoplastic transformation, it is likely that the induction of apoptosis by progestins is one of the major (if not the major) mechanism underlying the effect of combination oral contraceptives in reducing the risk of ovarian cancer.

This novel explanation for the association between estrogen-progestin combination oral contraceptive use and a reduced risk of ovarian cancer is a complete departure from the widely accepted theory that suppression of “incessant ovulation” is responsible for this reduced risk. This finding thus leads to the discovery that progestin alone or estrogen-progestin combinations may be administered in ways that do not effectively inhibit ovulation or otherwise inhibit contraception, yet which still prevent ovarian cancer. Since the protective mechanism for progestin containing compounds is related to a direct biological effect on the ovarian epithelium, it is likely that the use of progestin drugs in postmenopausal women who are not ovulating will also be protective against the development of epithelial ovarian carcinoma.

The invention is further based on the discovery that use of progestin alone induces a more accelerated rate of apoptosis in vivo in ovarian epithelial cells of monkeys compared to the combination of estrogen and progestin, which in turn induced a greater rate of apoptosis than estrogen alone. The implications of this discovery are that the progestin component of the oral contraceptive is responsible for this effect, and that administration of progestin alone may be effective for preventing the development of ovarian cancer, contrary to established reports that it has no effect on risk of ovarian cancer. Since the human-equivalent dose of the progestin only dose given the monkeys is insufficient to reliably block ovulation in women, yet showed the greatest degree of apoptosis (and thus protection), this indicates that ovulatory blockade per se is not essential for the protective effect, and that progestin product only (or with estrogen product) in doses less than sufficient to prevent ovulation is effective in preventing ovarian cancer.

The term “progestin product” or “progestogenic agent” as used herein includes any drug which binds to the progestin receptor and induces a progestational effect. This definition thus includes all of the known progestins, derivatives of progesterone or testosterone that have progestin activity, progestin agonists, and any other agent that increases the rate of apoptosis in ovarian epithelial cells. It is contemplated that not only presently available progestins but also progestins introduced in the future will be useful according to the present invention. The known synthetic progestins are mainly derivatives of 17-alpha-hydroxy-progesterone or 19-nortestosterone. These progestins can be classified into three groups: the pregnane, estrane, and gonane derivatives. The pregnane progestins, derived from 17 alphahydroxy-progesterone, include, for example, medroxyprogesterone acetate, chlormadinone acetate, megestrol acetate, and cyproterone acetate. All of these are roughly 20% to 50% of the potency of norethindrone. The estranes, derived from 19-nortestosterone include norethindrone, norethynodrel, lynestrenol, norethindrone acetate, ethynodiol acetate, and norethindrone enanthate. All of these are metabolized to norethindrone and are roughly equivalent to the same dosage of norethindrone. The gonanes are derived from the basic estrane structure, with the addition of an ethyl group of position 13 of the molecule. This additional ethyl group confers augmented progestogenic activity, and also significant androgenic effects. Drugs in this group include, for example, norgestrel (-d and -1), norgestimate, desogestrel, and gestodene. All of these are roughly equivalent to four times the dose of norethindrone. The oral preparations currently on the market are: norgestrel 0.075 mg, medroxyprogesterone acetate 2.5 mg, 5.0 mg, and 10.0 mg, norethindrone 0.35 mg, and norethindrone acetate 0.50 mg.

Progestogenic agents have a variety of biological effects including antifertility, inhibition of midcycle luteinizing hormone surge, inhibition of ovulation, inhibition of corpus lutetium function and development, and production of a secretory endometrium. In addition, the progestins have important effects on carbohydrate metabolism, lipid and lipoprotein metabolism and have cardiovascular effects.

Progestogenic potency can be measured by other biological outcomes, including the ability of these agents to bind to the progesterone receptor. The progestogenic activity of the various progestin derivatives can vary. In a review of the literature, Dorflinger has noted that the progestogenic potency of all these estrane drugs is equivalent, and exhibit only 5-10 percent of the progestogenic activity of levonorgestrel.

In addition to their progestogenic effects, the synthetic progestins have the ability to bind to both estrogen and androgen receptors, to a varying degree. These drugs can therefore have estrogenic, androgenic, antiestrogenic or antiandrogenic effects. For example, the estrane progestins are weak estrogen agonists, and therefor have slight estrogen activity. In contrast, the gonane levonorgestrel has no estrogenic activity, but does have androgenic activity. The 19-nortestosterone derivatives have androgenic activity mediated by variable binding to the androgen receptor.

Given the diverse binding patterns of the different synthetic progestins to various receptors (progestin, androgen and estrogen receptors), the estrogenic, progestogenic and androgenic activity can vary among the different synthetic progestin formulations, thus leading to varying degrees of progestational activity and androgenic side effects. For example, the progestational binding activity of norethindrone is less than 20% that of levonorgestrel and less than 10% that of 3-ketodesogestrel, the active metabolite of the progestin desogestrel, while the binding affinity of norethindrone to the androgen receptor is similar to that of 3-ketodesogestrel, and yet both compounds have less than 50% of the nuclear cell androgenic activity of levonorgestrel.

It is contemplated that the progestins with more androgenic activity and less estrogenic activity, such as levonorgestrel, may be preferred as more potent for preventing the development of ovarian cancer. Such agents would include the 19-nortestosterone derivatives, such as norethindrone, norethynodrel, lynestrenol, norethindrone acetate, ethynodiol acetate, and norethindrone enanthate.

The term “estrogen product” as used herein includes ethinyl estradiol, mestranol (a 50 mg dosage of which is equivalent to 35 mg of ethinyl estradiol), conjugated equine estrogen, estrone, estradiol, esterified estrogens, estropipate, and other estrogen equivalents and estrogen agonists.

“Concurrent administration” or “co-administration” as used herein includes administration of the agents together, or before or after each other. The agents may be administered by different routes. For example, one agent may be administered intravenously while the second agent is administered intramuscularly, intravenously or orally. They may be administered simultaneously or sequentially, as long as they are given in a manner sufficient to allow both agents to achieve effective concentrations in the body.

The term “infertile female” as used herein includes perimenopausal and postmenopausal females past the age of reproduction and younger women not capable of conception, including ovulation, fertilization and implantation.

The tenn “effective for contraception” as used herein includes sufficient inhibition of fertility, including ovulation or implantation.

The term “contraceptive blood level” as used herein includes a blood level sufficient to inhibit fertility, including ovulation or implantation.

The term “females at high risk of developing ovarian cancer” includes females with a family history of breast or ovarian cancer, females with a prior history of breast or ovarian cancer, or females with a mutation in the BRCA1 gene or any other mutation shown to be associated with a high risk of developing ovarian cancer.

Various combinations of progestin and estrogen that have been used in oral contraceptives are shown in Table 1.

TABLE 1 ______________________________________ Previously Used Combinations of Progestin and Estrogen Noreth- EE Equi- indrone valent Dose Equivalent Dose Dose Progestin (mg) Dose Estrogen (mg) (mg) P/E Ratio ______________________________________ Noreth- 9.85 9.85 Mestranol 0.150 0.105 93.810 yndrel 5.00 5.00 0.075 0.053 95.238 2.50 2.50 0.036 0.025 99.206 2.50 2.50 0.100 0.070 35.714 Noreth- 10.00 10.00 Mestranol 0.060 0.042 238.095 indrone 2.00 2.00 0.100 0.070 28.571 1.00 1.00 0.050 0.035 28.571 1.00 1.00 0.080 0.056 17.857 Noreth- 1.00 1.00 Ethinyl 0.050 0.050 20.000 indrone 0.50 0.50 estradiol 0.035 0.035 14.286 0.40 0.40 0.035 0.035 11.429 Noreth- 2.50 2.50 EE 0.050 0.050 50.000 indrone 1.00 1.00 0.050 0.050 20.000 acetate 0.60 0.60 0.030 0.030 20.000 1.50 1.50 0.030 0.030 50.000 1.00 1.00 0.020 0.020 50.000 Ethyno- 1.00 1.00 Mestranol 0.100 0.070 14.286 diol diacetate Ethyno- 1.00 1.00 EE 0.050 0.050 20.000 diol diacetate dl- 0.50 2.00 EE 0.050 0.050 10.000 Norgestrel 0.30 1.20 0.030 0.030 10.000 ______________________________________ Equivalencies 50 mg Mestranol = 35 mg Ethinyl estradiol (EE) 0.5 mg diNorgestrel = 2 mg Norethindrone

Each block describes a specific combination of progestin and estrogen, e.g., norethynodrel and mestranol, and within each block older combinations are listed first, with successively newer combinations following, Two trends are evident. First, over time the size and ratio of the dosages has decreased, i.e., the downward trend of the progestin component is steeper than the downward trend of the estrogen component. On a relative scale, therefore, estrogen has become more important over time. Second, with this downward trend in dosage, it is apparent that the relative ratio of progestin to estrogen is also trending downward. By contrast, the present invention emphasizes the greater importance of progestin in combination with estrogen, and thus emphasizes combination ratios even higher than those ratios, e.g., 100-1, that have long since been abandoned.

Other aspects and advantages of the present invention will be understood upon consideration of the following illustrative examples. Example 1 addresses the effect of administration of progestin or estrogen products, alone or in combination, on the ovarian epithelial cells of monkeys. Example 2 addresses the effect of progestin and estrogen products, alone or in combination, on the ovaries of humans. Example 3 addresses the effect of hormonally active agents, alone or in combination, in vitro on human ovarian tissue. Example 4 addresses the effect of gonadal hypertrophy on rodent ovaries. Example 5 addresses the effect of various hormonally active products, alone or in combination, on monkey ovaries. Example 6 addresses the effect of various hormonally active agents on the ovarian tissue of transgenic mice that have been altered to have altered expression of receptors, growth factors, integrins or protooncogenes.

EXAMPLE 1

EFFECT OF ESTROGEN AND PROGESTIN IN VIVO ON MONKEY OVARIES

Young female adult cynomolgus monkeys were fed a diet for two years that contained either no hormones, the oral combination contraceptive “Triphasil.RTM.,” the estrogenic component of “Triphasil.RTM.” (ethinyl estradiol) alone, or the progestin component of “Triphasil.RTM.” (levonorgestrel) alone, each administered in the same pattern that occurs in a “Triphasil.RTM.” regimen. Doses were scaled on the basis of caloric intake, which is the accepted way to achieve human-equivalent doses. The human-equivalent doses were thus: six days of 0.030 mg ethinyl estradiol +0.050 mg levonorgestrel, followed by 5 days of 0.040 mg ethinyl estradiol +0.075 mg levonorgestrel, followed by 10 days of 0.030 mg ethinyl estradiol +0.125 mg levonorgestrel, followed by 7 days of no treatment. This cyclic regimen was repeated every 28 days continuously for 2 years.

At the completion of the two years of the study, the animals were sacrificed, and their ovaries were removed and both formalin fixed and paraffin embedded as well as flash frozen and stored at minus 70 degrees Celsius. Five-micron ovarian sections were mounted on coated slides, and stained with the Apoptag-plus kit (Oncor, Gaithersburg, Md.), which specifically labels the 3′ end of free DNA fragments in cells undergoing DNA fragmentation, a characteristic of apoptosis. After staining, cells undergoing apoptosis were easily identified by their dark brown nuclear discoloration. The ovarian surface epithelium was examined histologically to assess ovarian epithelial morphology and to determine the percentage of ovarian cells undergoing apoptosis. To calculate the percentage of ovarian epithelial cells undergoing apoptosis, both the total number of ovarian epithelial cells and the number undergoing apoptosis were counted on each five-micron ovarian section. At each step, the investigators were completely blinded with regard to which treatment group was associated with each ovary.

The ovarian surface epithelium is comprised of a single layer of epithelial cells that rests on a basement membrane overlying the ovarian cortex. In the control and non-progestin treated monkeys, the ovarian surface epithelium typically had a lush appearance with the epithelial cells containing abundant cytoplasm and visible microvilli at the surface with apoptotic cells rarely seen. In the progestin treated monkeys, the ovarian surface epithelium was observed to contain numerous brown-staining apoptotic cells.

The median percentage of ovarian epithelial cells undergoing apoptosis for each of the treatment groups is shown below ill Table 2.

TABLE 2 ______________________________________ Apoptotic Effect of Four Treatments On Monkey Epithelia Median Percent of Range of Percent of Apoptotic Apoptotic Treatment Number Cell Counts Cell Counts ______________________________________ Control 20 3.8% 0.1-33.0% Ethinyl-estriadol- 20 1.8% 0.1-28.6% only Combination Pill 17 14.5% 3.0-61.0% Levonorgestrel 18 24.9% 3.5-61.8% ______________________________________ Multiple Comparisons: Control Levonorgestrel (p < 0.001) Combination Pill Ethinylestradiol (p < 0.001) Ethinylestradiol Levonorgestrel (p < 0.001) Control Combination Pill (p < 0.05)

From Table 2, the median percentage of apoptosis in the control group of monkeys not receiving any hormonal therapy was approximately 3.8%. Statistically, this was not significantly different from the rate of apoptosis seen in the ovarian epithelium in monkeys receiving only the estrogen component of “Triphasil.RTM.,” ethinyl estradiol, in which the median percentage of apoptosis was 1.8%.

A marked and significantly greater level of apoptosis was noted in the other two groups of monkeys-those that received the combination pill (containing both ethinyl estradiol and levonorgestrel) and those that received levonorgestrel (the progestin) alone. In this latter group (progestin alone), the observed median percentage of cells undergoing apoptosis was over six times greater than the level of apoptosis observed in the control, untreated monkeys. Because the only difference between the combination pill group and estrogen-alone group is the presence of the levonorgestrel component of the combination pill, and because the degree of apoptosis of the ovarian epithelium in the estrogen-alone group was no different than that of the control group, these data demonstrate that the accelerated rate of apoptosis in the ovarian epithelium in combination pill treated monkeys is due to the effects of the progestational component (levonorgestrel) of the combination pill. Moreover, the higher rate of apoptosis among the monkeys that received a progestational agent alone than in the monkeys that received the combination pin, although not statistically significant, indicates that progestin-only treatment is more effective at inducing apoptosis of the ovarian surface epithelium than a progestin/estrogen combination treatment.

EXAMPLE 2

EFFECT OF PROGESTIN AND ESTROGEN IN VIVO ON HUMAN OVARIES

Various progestins alone, including pregnanes, estranes and gonanes, various estrogens alone, or various progestin-estrogen combinations at varying doses are administered to women for at least one month prior to a scheduled surgery for removal of the ovaries and uterus. In particular, regimens of estrogen alone, estrogen with medroxyprogesterone acetate (or another 17-hydroxy-progesterone derivative), and estrogen with levonorgestrel (or another 19-nortestosterone derivative) are evaluated. To evaluate the effects of the different dosage regimens, the ovaries are examined for various markers, including apoptosis, proliferation, expression of growth factors, expression of steroid hormone receptors, and expression of other enzymes or genes.

EXAMPLE 3

EFFECT OF HORMONALLY ACTIVE AGENTS IN VITRO ON HUMA OVARIAN TISSUE

Ovarian epithelia cultured from ovaries removed from normal women or women with epithelial ovarian cancer are treated with various progestins alone, including pregnanes, estranes and gonanes, various estrogens alone, various progestin-estrogen combinations, progesterone receptor agonists, progesterone receptor antagonists, estrogen receptor agonists, or estrogen receptor antagonists, each at varying doses and varying durations, from e.g., 24 hours to 7 days. The ovarian tissue is then examined for various markers, including apoptosis, proliferation, expression of growth factors, expression of steroid hormone receptors, and expression of other enzymes or genes. The most potent agent for inducing apoptosis is determined.

EXAMPLE 4

EFFECT OF GONADAL HYPERTROPHY ON RODENT OVARIES

The ovaries of mice or rats are modestly “hyperstimulated” by compensatory gonadal hypertrophy after unilateral oophorectomy. The ovaries of the control animals, which received no treatment, are removed and examined at age 4, 4.5, 5 and 6 months. One ovary of the test animals is removed and examined at age 4 months, and the remaining ovary of each test animal is removed and examined at either age 4.5, 5 or 6 months. The ovarian tissue is examined for various markers, including apoptosis, proliferation, expression of growth factors, expression of steroid hormone receptors, and expression of other enzymes or genes.

EXAMPLE 5

EFFECT OF HORMONALLY ACTIVE AGENTS IN VIVO ON MONKEY OVARIES

Mature young female monkeys are treated with one of the following: control, leuprolide acetate (a gonadotropin releasing hormone [GnRH or LHRH] agonist), various oral contraceptives, levonorgestrel, norethindrone, medroxyprogesterone acetate, ethinyl estradiol, testosterone, testosterone derivatives, RU-486, progestin agonists, progestin antagonists, estrogen agonists and estrogen antagonists, each at varying doses. The ovarian tissue is removed and examined for various markers, including apoptosis, proliferation, expression of growth factors, expression of steroid hormone receptors, and expression of other enzymes or genes.

EXAMPLE 6

EFFECT OF HORMONALLY ACTIVE AGENTS IN VIVO ON OVARIES OF TRANSGENIC MICE

The effect of various progestins, estrogens or androgens, each at varying doses, is evaluated on the ovarian tissue of transgenic mice that have been altered to “knockout” their progestin receptor, to have an altered expression of the estrogen receptor, to express BRCA1, or to have altered expression of growth factors, integrins or protooncogenes.

Numerous modifications and variations in the practice of the invention are expected to occur to those skilled in the art upon consideration of the foregoing description on the presently preferred embodiments thereof. Consequently the only limitations which should be placed upon the scope of the present invention are those that appear in the appended claims.

Abstract
This invention provides a method for treating cancer in an individual, wherein the cancer is a tumor in which endothelin is upregulated (e.g. tumors of the prostate, lung, liver, breast, brain, stomach, colon, endometrium, testicle, thyroid, pituatary, bladder, kidney, pancreas and meninges) by administering to the individual an effective amount of a compound of Formula I or Formula Ia, as describe herein.

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Inventors: Romerdahl; Cynthia A. (Wayland, MA)
Assignee: BASF Aktiengesellschaft (DE)

Appl. No.: 08/818,622
Filed: March 14, 1997
Claims

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What is claimed is:

1. A method of treating a solid tumor sensitive to the compounds below and in which endothelin is upregulated in a human in need of treatment, comprising administering to the human a compound of Formula Ia in sufficient quantity to inhibit growth of the solid tumor, wherein Formula Ia is: ##STR21## where R is formyl, tetrazolyl, cyano, a COOH group or a radical which can be hydrolyzed to COOH;

R.sup.2 is hydrogen, hydroxyl, NH.sub.2, NH(C.sub.1 -C.sub.4 -alkyl), N(C.sub.1 -C.sub.4 -alkyl).sub.2, halogen, C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy or C.sub.1 -C.sub.4 -alkylthio;

X is CR.sup.14 where R.sup.14 is hydrogen or C.sub.1 -C.sub.5 -alkyl;

R.sup.3 is hydrogen, hydroxyl, NH.sub.2, NH(C.sub.1 -C.sub.4 -alkyl), N(C.sub.1 -C.sub.4 -alkyl).sub.2, halogen, C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy, –NH–O–C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -alkylthio or CR.sup.3 is linked to CR.sup.14 as indicated above to give a 5- or 6-membered ring;

R.sup.4 and R.sup.5, which can be identical or different, are

phenyl or naphthyl, which can be substituted by one or more of the following radicals: halogen, nitro, cyano, hydroxyl, C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy, phenoxy, C.sub.1 -C.sub.4 -alkylthio, amino, C.sub.1 -C.sub.4 -alkylamino or C.sub.1 -C.sub.4 -dialkylamino; or

phenyl or naphthyl, which are connected together in the ortho positions via a direct linkage, a methylene, ethylene or ethenylene group, an oxygen or sulfur atom or an SO.sub.2, NH or N-alkyl group or C.sub.3 -C.sub.7 -cycloalkyl;

R.sup.6 is hydrogen; C.sub.1 -C.sub.8 -alkyl, C.sub.3 -C.sub.6 -alkenyl, C.sub.3 -C.sub.6 -alkynyl or C.sub.3 -C.sub.8 -cycloalkyl, where each of these radicals can be substituted by one or more of the following radicals: a halogen, nitro, cyano, C.sub.1 -C.sub.4 -alkoxy, C.sub.3 -C.sub.6 -alkenyloxy, C.sub.3 -C.sub.6 -alkynyloxy, C.sub.1 -C.sub.4 -alkyl thio, C.sub.1 -C.sub.4 -haloalkoxy, C.sub.1 -C.sub.4 -alkylcarbonyl, C.sub.1 -C.sub.4 -alkoxy-carbonyl, C.sub.3-8 -alkylcarbonylalkyl, C.sub.1 -C.sub.4 -alkylamino, di-C.sub.1 -C.sub.4 -alkylamino, phenyl or phenyl or phenoxy which is substituted one or more times by halogen, nitro, cyano, C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy or C.sub.1 -C.sub.4 -alkylthio;

phenyl or naphthyl, each of which can be substituted by one or more of the following radicals: halogen, nitro, cyano, hydroxyl, amino, C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy, phenoxy, C.sub.1 -C.sub.4 -alkylthio, C.sub.1 -C.sub.4 -alkylamino, C.sub.1 -C.sub.4 -dialkylamino, methylenedioxy or ethylenedioxy;

a five- or six-membered heteroaromatic moiety containing one to three nitrogen atoms and/or one sulfur or oxygen atom, which can have one to four halogen atoms and/or one or two of the following radicals: C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy, C.sub.1 -C.sub.4 -alkylthio, phenyl, phenoxy or phenylcarbonyl, wherein the phenyl radicals can have one to five halogen atoms and/or one to three of the following radicals: C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy and/or C.sub.1 -C.sub.4 -alkylthio;

Y sulfur or oxygen or a single bond;

Z sulfur, oxygen, –SO–, –SO.sub.2 — or a single bond with the proviso that R.sup.6 can be hydrogen only when Z is not a single bond.

2. The method of claim 1 wherein for the compound of Formula Ia R is a COOH group, R.sup.2 is –OMe, R.sup.3 is –OMe, R.sup.4 is phenyl, R.sup.5 is phenyl, R.sup.6 is methyl, R.sup.14 is H, Y is oxygen, and Z is oxygen.

3. The method of claim 1 wherein for the compound of Formula Ia R is a COOH group, R.sup.2 is methyl, R.sup.3 is methyl, R.sup.4 is phenyl, R.sup.5 is phenyl, R.sup.6 is methyl, R.sup.14 is H, Y is oxygen, and Z is oxygen.

4. The method of claim 1 wherein for the compound of Formula Ia R is a COOH group, R.sup.2 is methyl, R.sup.3 is methyl, R.sup.4 is phenyl, R.sup.5 is phenyl, R.sup.6 is methyl, R.sup.14 is H, Y is oxygen, and Z is a single bond.

5. The method of claim 1 wherein the solid tumor is a tumor of the prostate.

6. The method of claim 2 wherein the solid tumor is a tumor of the prostate.

7. The method of claim 3 wherein the solid tumor is a tumor of the prostate.

8. The method of claim 4 wherein the solid tumor is a tumor of the prostate.

9. A method of treating a solid tumor selected from the group consisting of prostate, lung, liver, breast, brain, stomach, colon, endometrium, testicle, thyroid, pituitary, bladder, kidney, pancreas and meninges in a human in need of treatment, comprising administering to the human a compound of Formula Ia in sufficient quantity to inhibit growth of said tumor, wherein Formula Ia is: ##STR22## where R is formyl, tetrazolyl, cyano, a COOH group or a radical which can be hydrolyzed to COOH;

R.sup.2 is hydrogen, hydroxyl, NH.sub.2, NH(C.sub.1 -C.sub.4 -alkyl), N(C.sub.1 -C.sub.4 -alkyl).sub.2, halogen, C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy or C.sub.1 -C.sub.4 -alkylthio;

X is CR.sup.14 where R.sup.14 is hydrogen or C.sub.1 -C.sub.5 -alkyl;

R.sup.3 is hydrogen, hydroxyl, NH.sub.2, NH(C.sub.1 -C.sub.4 -alkyl), N(C.sub.1 -C.sub.4 -alkyl).sub.2, halogen, C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy, –NH–O–C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -alkylthio;

R.sup.4 and R.sup.5 , which can be identical or different are

phenyl or naphthyl, which can be substituted by one or more of the following radicals: halogen, nitro, cyano, hydroxyl, C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy, phenoxy, C.sub.1 -C.sub.4 -alkylthio, amino, C.sub.1 -C.sub.4 -alkylamino or C.sub.1 -C.sub.4 -dialkylamino; or

phenyl or naphthyl, which are connected together in the ortho positions via a direct linkage, a methylene, ethylene or ethenylene group, an oxygen or sulfur atom or an SO.sub.2, NH or N-alkyl group or C.sub.3 -C.sub.7 -cycloalkyl;

R.sup.6 is hydrogen; C.sub.1 -C.sub.8 -alkyl, C.sub.3 -C.sub.6 -alkenyl, C.sub.3 -C.sub.6 -alkynyl or C.sub.3 -C.sub.8 -cycloalkyl, where each of these radicals can be substituted by one or more of the following radicals: a halogen, nitro, cyano, C.sub.1 -C.sub.4 -alkoxy, C.sub.3 -C.sub.6 -alkenyloxy, C.sub.3 -C.sub.6 -alkynyloxy, C.sub.1 -C.sub.4 -alkylthio, C.sub.1 -C.sub.4 -haloalkoxy, C.sub.1 -C.sub.4 -alkylcarbonyl, C.sub.1 -C.sub.4 -alkoxy-carbonyl, C.sub.3-8 -alkylcarbonylalkyl, C.sub.1 -C.sub.4 -alkylamino, di-C.sub.1 -C.sub.4 -alkylamino, phenyl or phenyl or phenoxy which is substituted one or more times by halogen, nitro, cyano, C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy or C.sub.1 -C.sub.4 -alkylthio;

phenyl or naphthyl, each of which can be substituted by one or more of the following radicals: halogen, nitro, cyano, hydroxyl, amino, C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy, phenoxy, C.sub.1 -C.sub.4 -alkylthio, C.sub.1 -C.sub.4 -alkylamino, C.sub.1 -C.sub.4 -dialkylamino, methylenedioxy or ethylenedioxy;

a five- or six-membered heteroaromatic moiety containing one to three nitrogen atoms and/or one sulfur or oxygen atom, which can have one to four halogen atoms and/or one or two of the following radicals: C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy, C.sub.1 -C.sub.4 -alkylthio, phenyl, phenoxy or phenylcarbonyl, wherein the phenyl radicals can have one to five halogen atoms and/or one to three of the following radicals: C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy and/or C.sub.1 -C.sub.4 -alkylthio;

Y sulfur or oxygen or a single bond;

Z sulfur, oxygen, –SO–, –SO.sub.2 — or a single bond with the proviso that R.sup.6 can be hydrogen only when Z is not a single bond.

10. The method of claim 9 wherein for the compound of Formula Ia R is a COOH group, R.sup.2 is –OMe, R.sup.3 is –OMe, R.sup.4 is phenyl, R.sup.5 is phenyl, R.sup.6 is methyl, R.sup.14 is H, Y is oxygen, and Z is oxygen.

11. The method of claim 9 wherein for the compound of Formula Ia R is a COOH group, R.sup.2 is methyl, R.sup.3 is methyl, R.sup.4 is phenyl, R.sup.5 is phenyl, R.sup.6 is methyl, R.sup.14 is H, Y is oxygen, and Z is oxygen.

12. The method of claim 9 wherein for the compound of Formula Ia R is a COOH group, R.sup.2 is methyl, R.sup.3 is methyl, R.sup.4 is phenyl, R.sup.5 is phenyl, R.sup.6 is methyl, R.sup.14 is H, Y is oxygen, and Z is a single bond.

13. A method of treating a solid tumor of the prostate in a mammal in need of treatment, comprising administering to the mammal a compound of Formula Ia in sufficient quantity to inhibit growth of said tumor, wherein Formula Ia is: ##STR23## where R is formyl, tetrazolyl, cyano, a COOH group or a radical which can be hydrolyzed to COOH;

R.sup.2 is hydrogen, hydroxyl, NH.sub.2, NH(C.sub.1 -C.sub.4 -alkyl), N(C.sub.1 -C.sub.4 -alkyl).sub.2, halogen, C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy or C.sub.1 -C.sub.4 -alkylthio;

X is CR.sup.14 where R.sup.14 is hydrogen or C.sub.1 -C.sub.5 -alkyl;

R.sup.3 is hydrogen, hydroxyl, NH.sub.2, NH(C.sub.1 -C.sub.4 -alkyl), N(C.sub.1 -C.sub.4 -alkyl).sub.2, halogen, C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy, –NH–O–C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -alkylthio;

R.sup.4 and R.sup.5, which can be identical or different are

phenyl or naphthyl, which can be substituted by one or more of the following radicals: halogen, nitro, cyano, hydroxyl, C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy, phenoxy, C.sub.1 -C.sub.4 -alkylthio, amino, C.sub.1 -C.sub.4 -alkylamino or C.sub.1 -C.sub.4 -dialkylamino; or

phenyl or naphthyl, which are connected together in the ortho positions via a direct linkage, a methylene, ethylene or ethenylene group, an oxygen or sulfur atom or an SO.sub.2, NH or N-alkyl group or C.sub.3 -C.sub.7 -cycloalkyl;

R.sup.6 is hydrogen; C.sub.1 -C.sub.8 -alkyl, C.sub.3 -C.sub.6 -alkenyl, C.sub.3 -C.sub.6 -alkynyl or C.sub.3 -C.sub.8 -cycloalkyl, where each of these radicals can be substituted by one or more of the following radicals: a halogen, nitro, cyano, C.sub.1 -C.sub.4 -alkoxy, C.sub.3 -C.sub.6 -alkenyloxy, C.sub.3 -C.sub.6 -alkynyloxy, C.sub.1 -C.sub.4 -alkylthio, C.sub.1 -C.sub.4 -haloalkoxy, C.sub.1 -C.sub.4 -alkylcarbonyl, C.sub.1 -C.sub.4 -alkoxy-carbonyl, C.sub.3-8 -alkylcarbonylalkyl, C.sub.1 -C.sub.4 -alkylamino, di-C.sub.1 -C.sub.4 -alkylamino, phenyl or phenyl or phenoxy which is substituted one or more times by halogen, nitro, cyano, C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy or C.sub.1 -C.sub.4 -alkylthio;

phenyl or naphthyl, each of which can be substituted by one or more of the following radicals: halogen, nitro, cyano, hydroxyl, amino, C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy, phenoxy, C.sub.1 -C.sub.4 -alkylthio, C.sub.1 -C.sub.4 -alkylamino, C.sub.1 -C.sub.4 -dialkylamino, methylenedioxy or ethylenedioxy;

a five- or six-membered heteroaromatic moiety containing one to three nitrogen atoms and/or one sulfur or oxygen atom, which can have one to four halogen atoms and/or one or two of the following radicals: C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy, C.sub.1 -C.sub.4 -alkylthio, phenyl, phenoxy or phenylcarbonyl, wherein the phenyl radicals can have one to five halogen atoms and/or one to three of the following radicals: C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy and/or C.sub.1 -C.sub.4 -alkylthio;

Y sulfur or oxygen or a single bond;

Z sulfur, oxygen, –SO–, –SO.sub.2 — or a single bond with the proviso that R.sup.6 can be hydrogen only when Z is not a single bond.

14. The method of claim 13 wherein for the compound of Formula Ia R is a COOH group, R.sup.2 is –OMe, R.sup.3 is –OMe, R.sup.4 is phenyl, R.sup.5 is phenyl, R.sup.6 is methyl, R.sup.14 is H, Y is oxygen, and Z is oxygen.

15. The method of claim 13 wherein for the compound of Formula Ia R is a COOH group, R.sup.2 is methyl, R.sup.3 is methyl, R.sup.4 is phenyl, R.sup.5 is phenyl, R.sup.6 is methyl, R.sup.14 is H, Y is oxygen, and Z is oxygen.

16. The method of claim 13 wherein for the compound of Formula Ia R is a COOH group, R.sup.2 is methyl, R.sup.3 is methyl, R.sup.4 is phenyl, R.sup.5 is phenyl, R.sup.6 is methyl, R.sup.14 is H, Y is oxygen, and Z is a single bond.
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Description

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BACKGROUND

Cancer is a disease for which many potentially effective treatments are available. However, due to the prevalence of cancers of various types and the serious effects cancer can have, more effective treatments, especially those with fewer adverse side effects than currently available forms of treatment, are needed.

SUMMARY OF THE INVENTION

This invention relates to novel carboxylic acid derivatives, their preparation and use in treating cancer in which endothelin is upregulated, in a mammal, for example, a human.

Endothelin is a peptide which is composed of 21 amino acids and is synthesized and released by the vascular endothelium. Endothelin exists in three isoforms, ET-1, ET-2 and ET-3. As used herein the term “endothelin” or “ET” refers to one or all isoforms of endothelin. Endothelin is a potent vasoconstrictor and has a potent effect on vessel tone. It is known that this vasoconstriction is caused by binding of endothelin to its receptor (Nature, 332, (1988) 411-415; FEBS Letters, 231, (1988) 440-444 and Biochem. Biophys. Res. Commun., 154, (1988) 868-875).

Increased or abnormal release of endothelin causes persistent vasoconstriction in the peripheral, renal and cerebral blood vessels, which may lead to illnesses. It has been reported in the literature that elevated levels of endothelin were found in the plasma of patients with hypertension, acute myocardial infarct, pulmonary hypertension, Raynaud’s syndrome, atherosclerosis and in the airways of asthmatics (Japan J. Hypertension, 12, (1989) 79, J. Vascular Med. Biology 2, (1990) 207, J. Am. Med. Association 264, (1990) 2868).

Accordingly, substances which specifically inhibit the binding of endothelin to the receptor should also antagonize the various above-mentioned physiological effects of endothelin and therefore be valuable drugs. For example, the compounds of the present invention can be used for the treatment of hypertensions, pulmonary hypertension, myocardial infarct, angina pectoris, acute kidney failure, renal insufficiency, cerebral vasospasms, cerebral ischemia, subarachnoid hemorrhages, migraine, asthma, atherosclerosis, endotoxic shock, endotoxin-induced organ failure, intravascular coagulation, restenosis after angioplasty, benign prostate hyperplasia, or hypertension or kidney failure caused by ischemia or intoxication as described in WO96/11914 and WO95/26716, the teaching of both of which are incorporated herein by reference in their entirety.

We have found that certain carboxylic acid derivatives of Formula I or Ia which are inhibitors of endothelin receptors are also useful in treating cancer, such as prostate cancer. These carboxylic acid derivatives are described herein and also in WO96/11914 and WO95/26716, the teachings of both of which are incorporated herein by reference in their entirety.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to a method for treating cancer in an individual, for example, a human, wherein the cancer is a tumor in which endothelin is upregulated (e.g. tumors of the prostate, lung, liver, breast, brain, stomach, colon, endometrium, testicle, thyroid, pituitary, bladder, kidney, pancreas and meninges). By treating is meant inhibiting (partially or totally) formation of a solid tumor in which endothelin is upregulated, reversing development of a solid tumor in which endothelin is upregulated or reducing its further progression, by administering to the individual an effective amount of one or more compound(s) of Formula I and/or Formula Ia as described below. As used herein the term an effective amount is a quantity sufficient to inhibit (partially or totally) growth of a solid tumor in which endothelin is upregulated, reverse development of a solid tumor in which endothelin is unregulated or reduce its further progression. In the present invention, Formula Ia is a subgroup of the Formula I.

One or more compounds of Formula I and Ia may be administered alone or with pharmaceutically accepted carrier or diluent appropriate for the desired route of administration. Administration can be by any of the means which are conventional for pharmaceutical, preferably oncological, agents, including oral and parenteral means such as subcutaneously, intravenously, intramuscularly, intraperitoneally, nasally or rectally.

The dosage administered to the mammal, such as a human, includes an effective amount of a compound of Formula I or Formula Ia. For a particular condition or method of treatment, the dosage can be determined empirically, using known methods, and will depend upon factors such as the biological activity; toxicity profile; the means of administration; the age, sex, health and body weight of the recipient; the nature and extent of the symptoms; the frequency of treatment; the administration of other therapies; and the effect desired.

A typical daily dose of the compound of Formula I or Ia will be from about 0.5 to about 5000 milligram per kilogram of body weight by oral administration and from about 0.1 to about 1000 milligrams per kilogram of body weight by parenteral administration. In one embodiment, wherein administration is parenteral, a daily dose will be from about 50 to about 500 milligrams per kilogram of body weight. In a particular embodiment, wherein the administration is parenterally, a daily dose will be from about 100 to about 300 milligrams per kilogram of body weight (e.g. 100, 150 or 220 milligrams per kilogram of body weight).

The novel compounds can be used in conventional solid or liquid pharmaceutical forms, e.g., as uncoated or (film) coated tablets, capsules, powders, granules, suppositories, solutions, ointments, creams or sprays. These are produced in a conventional way. The active substances can for this purpose be processed with conventional pharmaceutical aids such as tablet binders, fillers, preservatives, tablet disintegrants, flow regulators, plasticizers, wetting agents, dispersants, emulsifiers, solvents, release-slowing agents, antioxidants and/or propellent gases (cf. H. Sucker et al.: Pharmazeutische Technologie, Thieme-Verlag, Stuttgart, 1991). The administration forms obtained in this way normally contain from 0.1 to 90% by weight of the active substance.

The carboxylic acid derivatives useful in the method of the invention are compounds of Formula I: ##STR1## where R is formyl, tetrazolyl, cyano, a COOH group or a radical which can be hydrolyzed to COOH, and the other substituents have the following meanings:

R.sup.2 hydrogen, hydroxyl, NH.sub.2, NH(C.sub.1 -C.sub.4 -alkyl), N(C.sub.1 -C.sub.4 -alkyl).sub.2, halogen, C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy or C.sub.1 -C.sub.4 -alkylthio;

X nitrogen or CR.sup.14 where R.sup.14 is hydrogen or C.sub.1-5 -alkyl, or CR.sup.14 forms together with CR.sup.3 a 5- or 6-membered alkylene or alkenylene ring which can be substituted by one or two C.sub.1-4 -alkyl groups and in which in each case a methylene group can be replaced by oxygen, sulfur, –NH or –NC.sub.1-4 -alkyl;

R.sup.3 hydrogen, hydroxyl, NH.sub.2, NH(C.sub.1 -C.sub.4 -alkyl), N(C.sub.1 -C.sub.4 -alkyl).sub.2, halogen, C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy, –NH–O–C.sub.1-4 -alkyl, C.sub.1 -C.sub.4 -alkylthio or CR.sup.3 is linked to CR.sup.14 as indicated above to give a 5- or 6-membered ring;

R.sup.4 and R.sup.5 (which can be identical or different):

phenyl or naphthyl, which can be substituted by one or more of the following radicals: halogen, nitro, cyano, hydroxyl, C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy, phenoxy, C.sub.1 -C.sub.4 -alkylthio, amino, C.sub.1 -C.sub.4 -alkylamino or C.sub.1 -C.sub.4 -dialkylamino; or

phenyl or naphthyl, which are connected together in the ortho positions via a direct linkage, a methylene, ethylene or ethenylene group, an oxygen or sulfur atom or an SO.sub.2 -, NH- or N-alkyl group, or C.sub.3 -C.sub.7 -cycloalkyl; or

R.sup.4 is C.sub.1 -C.sub.10 -alkyl which can carry from one to five halogen atoms and/or one of the following radicals: C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -alkylthio, cyano, C.sub.1 -C.sub.8 -alkylcarbonyl, C.sub.1 -C.sub.8 -alkoxy-carbonyl, phenyl, phenoxy or phenylcarbonyl, where the phenyl radicals in turn can carry from one to five halogen atoms and/or from one to three of the following radicals: C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy and/or C.sub.1 -C.sub.4 -alkylthio;

C.sub.1 -C.sub.10 -aklyl which can carry from one to five halogen atoms and carries one of the following radicals: a five-membered heteroaromatic ring which contains from one to three nitrogen atoms and/or one sulfur or oxygen atom and which can carry from one to four halogen atoms and/or one or two of the following radicals: C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy, C.sub.1 -C.sub.4 -alkylthio and/or phenyl;

C.sub.3 -C.sub.12 -cycloalkyl or C.sub.3 -C.sub.12 -cycloalkenyl, each of which can contain one oxygen or sulfur atom and can carry from one to five halogen atoms and/or one of the following radicals: C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -alkylthio, cyano, C.sub.1 -C.sub.8 -alkylcarbonyl, C.sub.1 -C.sub.8 -alkoxycarbonyl, phenyl, phenoxy or phenyl-carbonyl, where the phenyl radicals in turn can carry from one to five halogen atoms and/or from one to three of the following radicals: C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy and/or C.sub.1 -C.sub.4 -alkylthio;

C.sub.3 -C.sub.6 -alkenyl or C.sub.3 -C.sub.6 -alkynyl, each of which can carry from one to five halogen atoms and/or one of the following radicals: C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -alkylthio, cyano, C.sub.1 -C.sub.8 -alkylcarbonyl, C.sub.1 -C.sub.8 -alkoxycarbonyl, phenyl, phenoxy or phenylcarbonyl, where the phenyl radicals in turn can carry from one to five halogen atoms and/or from one to three of the following radicals: C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy and/or C.sub.1 -C.sub.4 -alkylthio;

a five- or six-membered heteroaromatic ring which contains from one to three nitrogen atoms and/or one sulfur or oxygen atom and which can carry from one to four halogen atoms and/or one or two of the following radicals: C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy, C.sub.1 -C.sub.4 -alkylthio, phenyl, phenoxy or phenylcarbonyl, where the phenyl radicals in turn can carry from one to five halogen atoms and/or from one to three of the following radicals: C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy and/or C.sub.1 -C.sub.4 -alkylthio;

R.sup.4 and R.sup.5 form, together with the adjacent carbon atom, a 3- to 8-membered ring which can contain one oxygen or sulfur atom and can carry from one to three of the following radicals: C.sub.1 -C.sub.4 -alkyl, halogen, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy and/or C.sub.1 -C.sub.4 -alkylthio [sic];

R.sup.5 is hydrogen, C.sub.1 -C.sub.4 -alkyl, C.sub.3 -C.sub.6 -alkenyl, C.sub.3 -C.sub.6 -alkynyl, C.sub.3 -C.sub.8 -cycloalkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxyalkyl, C.sub.1 -C.sub.4 -alkylthioalkyl, phenyl or R.sup.5 is linked to R.sup.4 as indicated above to form a 3- to 8-membered ring;

R.sup.6 hydrogen, C.sub.1 -C.sub.8 -alkyl, C.sub.3 -C.sub.6 -alkenyl, C.sub.3 -C.sub.6 -alkynyl or C.sub.3 -C.sub.8 -cycloalkyl, where each of these radicals can be substituted one or more times by: halogen, nitro, cyano, C.sub.1 -C.sub.4 -alkoxy, C.sub.3 -C.sub.6 -alkenyloxy, C.sub.3 -C.sub.6 -alkynyloxy, C.sub.1 -C.sub.4 -alkylthio, C.sub.1 -C.sub.4 -haloalkoxy, C.sub.1 -C.sub.4 -alkylcarbonyl, C.sub.1 -C.sub.4 -alkoxy-carbonyl, C.sub.3-8 -alkylcarbonylalkyl, C.sub.1 -C.sub.4 -alkylamino, di-C.sub.1 -C.sub.4 -alkylamino, phenyl or phenyl or phenoxy which is substituted one or more times, for example one to three times, by halogen, nitro, cyano, C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy or C.sub.1 -C.sub.4 -alkylthio;

phenyl or naphthyl, each of which can be substituted by one or more of the following radicals: halogen, nitro, cyano, hydroxyl, amino, C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy, phenoxy, C.sub.1 -C.sub.4 -alkylthio, C.sub.1 -C.sub.4 -alkylamino, C.sub.1 -C.sub.4 -dialkylamino, methylenedioxy or ethylenedioxy;

a five- or six-membered heteroaromatic moiety containing one to three nitrogen atoms and/or one sulfur or oxygen atom, which can carry one to four halogen atoms and/or one or two of the following radicals: C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy, C.sub.1 -C.sub.4 -alkylthio, phenyl, phenoxy or phenylcarbonyl, it being possible for the phenyl radicals in turn to carry one to five halogen atoms and/or one to three of the following radicals: C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy and/or C.sub.1 -C.sub.4 -alkylthio;

with the proviso that R.sup.6 can be hydrogen only when Z is not a single bond;

Y sulfur or oxygen or a single bond;

Z sulfur or oxygen or a single bond.

In particular embodiments, carboxylic acid derivatives useful in the method of the invention are compounds of Formula Ia which are a subgroup of Formula I: ##STR2## where R is formyl, tetrazolyl, cyano, a COOH group or a radical which can be hydrolyzed to COOH, and the other substituents have the following meanings:

R.sup.2 hydrogen, hydroxyl, NH.sub.2, NH(C.sub.1 -C.sub.4 -alkyl), N(C.sub.1 -C.sub.4 -alkyl).sub.2, halogen, C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy or C.sub.1 -C.sub.4 -alkylthio;

X nitrogen or CR.sup.14 where R.sup.14 is hydrogen or C.sub.1-5 -alkyl, or CR.sup.14 forms together with CR.sup.3 a 5- or 6-membered alkylene or alkenylene ring which can be substituted by one or two C.sub.1-4 -alkyl groups and in which in each case a methylene group can be replaced by oxygen, sulfur, –NH or –NC.sub.1-4 -alkyl;

R.sup.3 hydrogen, hydroxyl, NH.sub.2, NH(C.sub.1 -C.sub.4 -Alkyl), N(C.sub.1 -C.sub.4 -alkyl).sub.2, halogen, C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy, –NH–O–C.sub.1-4 -alkyl, C.sub.1 -C.sub.4 -alkylthio or CR.sup.3 is linked to CR.sup.14 as indicated above to give a 5- or 6-membered ring;

R.sup.4 and R.sup.5 (which can be identical or different):

phenyl or naphthyl, which can be substituted by one or more of the following radicals: halogen, nitro, cyano, hydroxyl, C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy, phenoxy, C.sub.1 -C.sub.4 -alkylthio, amino, C.sub.1 -C.sub.4 -alkylamino or C.sub.1 -C.sub.4 -dialkylamino; or

phenyl or naphthyl, which are connected together in the ortho positions via a direct linkage, a methylene, ethylene or ethenylene group, an oxygen or sulfur atom or an SO2, NH or N-alkyl group or C.sub.3 -C.sub.7 -cycloalkyl;

R.sup.6 hydrogen, C.sub.1 -C.sub.8 -alkyl, C.sub.3 -C.sub.6 -alkenyl, C.sub.3 -C.sub.6 -alkynyl or C.sub.3 -C.sub.8 -cycloalkyl, where each of these radicals can be substituted one or more times by: halogen, nitro, cyano, C.sub.1 -C.sub.4 -alkoxy, C.sub.3 -C.sub.6 -alkenyloxy, C.sub.3 -C.sub.6 -alkynyloxy, C.sub.1 -C.sub.4 -alkyl-thio, C.sub.1 -C.sub.4 -haloalkoxy, C.sub.1 -C.sub.4 -alkylcarbonyl, C.sub.1 -C.sub.4 -alkoxy-carbonyl, C.sub.3-8 -alkylcarbonylalkyl, C.sub.1 -C.sub.4 -alkylamino, di-C.sub.1 -C.sub.4 -alkylamino, phenyl or phenyl or phenoxy which is substituted one or more times, eg. one to three times, by halogen, nitro, cyano, C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy or C.sub.1 -C.sub.4 -alkylthio;

phenyl or naphthyl, each of which can be substituted by one or more of the following radicals: halogen, nitro, cyano, hydroxyl, amino, C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy, phenoxy, C.sub.1 -C.sub.4 -alkylthio, C.sub.1 -C.sub.4 -alkylamino, C.sub.1 -C.sub.4 -dialkylamino, methylenedioxy or ethylenedioxy;

a five- or six-membered heteroaromatic moiety containing one to three nitrogen atoms and/or one sulfur or oxygen atom, which can carry one to four halogen atoms and/or one or two of the following radicals: C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy, C.sub.1 -C.sub.4 -alkylthio, phenyl, phenoxy or phenylcarbonyl, it being possible for the phenyl radicals in turn to carry one to five halogen atoms and/or one to three of the following radicals: C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy and/or C.sub.1 -C.sub.4 -alkylthio;

with the proviso that R6 can be hydrogen only when Z is not a single bond;

Y sulfur or oxygen or a single bond;

Z sulfur, oxygen, –SO–, –SO.sub.2 — or a single bond.

The compounds, and the intermediates for preparing the compounds of Formula I and Ia, such as IV and VI, may have one or more asymmetrically substituted carbon atoms. Such compounds may be in the form of the pure enantiomers or pure diastereomers or a mixture thereof. The use of an enantiomerically pure compound as active substance is preferred.

The invention furthermore relates to the use of the above-mentioned carboxylic acid derivatives for producing drugs, in particular for producing endothelin receptor inhibitors.

The invention furthermore relates to the preparation of the compounds of the Formula IV in enantiomerically pure form. Enantioselective epoxidation of an olefin with two phenyl substituents is known (J. Org. Chem. 59, 1994, 4378-4380).

The preparation of the compounds according to the invention where Z is sulfur or oxygen starts from the epoxides IV, which are obtained in a conventional manner, for example as described in J. March, Advanced Organic Chemistry, 2nd ed., 1983, page 862 and page 750, from the ketones II or the olefins III: ##STR3##

Carboxylic acid derivatives of the general Formula VI can be prepared by reacting the epoxides of the general Formula IV (eg. with R=COOR.sup.10) with alcohols or thiols of the general formula V where R.sup.6 and Z have the meanings stated above. ##STR4##

To do this, compounds of the general formula IV are heated with compounds of the formula V, in the molar ratio of about 1:1 to 1:7, preferably 1 to 3 mole equivalents, to 50-200.degree. C., preferably 80-150.degree. C.

The reaction can also take place in the presence of a diluent. All solvents which are inert toward the reagents used can be used for this purpose.

Examples of such solvents or diluents are water, aliphatic, alicyclic and aromatic hydrocarbons, which may in each case be chlorinated, such as hexane, cyclohexane, petroleum ether, naphtha, benzene, toluene, xylene, methylene chloride, chloroform, carbon tetrachloride, ethyl chloride and trichloroethylene, ethers such as diisopropyl ether, dibutyl ether, methyl tert-butyl ether, propylene oxide, dioxane and tetrahydrofuran, ketones such as acetone, methyl ethyl ketone, methyl isopropyl ketone and methyl isobutyl ketone, nitrites such as acetonitrile and propionitrile, alcohols, such as methanol, ethanol, isopropanol, butanol and ethylene glycol, esters such as ethyl acetate and amyl acetate, amides such as dimethylformamide, dimethylacetamide and N-methylpyrrolidone, sulfoxides and sulfones, such as dimethyl sulfoxide and sulfolane, bases such as pyridine, cyclic ureas such as 1,3-dimethylimidazolidin-2-one and 1,3-dimethyl-3,4,5,6-tetra-hydro-2(1H)-pyrimidinone.

The reaction is preferably carried out at a temperature in the range from 0.degree. C. to the boiling point of the solvent or mixture of solvents.

The presence of a catalyst may be advantages. Suitable catalysts are strong organic and inorganic acids, and Lewis acids. Examples thereof are, inter alia, sulfuric acid, hydrochloric acid trifluoroacetic acid, p-toluenesulfonic acid, boron trifluoride etherate and titanium (IV) alcoholates.

Compounds of the Formula VI where R.sup.4 and R.sup.5 are cycloalkyl can also be prepared by subjecting compounds of the Formula VI where R.sup.4 and R.sup.5 are phenyl, naphthyl, or phenyl or naphthyl substituted as described above, to a nuclear hydrogenation.

Compounds of the Formula VI can be obtained in enantiomerically pure form by starting from enantiomerically pure compounds of the Formula IV and reacting them in the manner described with compounds of the Formula V.

It is furthermore possible to obtain enantiomerically pure compounds of the Formula VI by carrying out a classical racemate resolution on racemic or diastereomeric compounds of the Formula VI using suitable enantiomerically pure bases such as brucine, strychnine, quinine, quinidine, cinchonidine, cinchonine, yohimbine, morphine, dehydroabietylamine, ephedrine (-), (+), deoxyephedrine (+), (-), threo-2-amino-1-(p-nitrophenyl)-1,3-propanediol (+), (-), threo-2-(N,N-dimethylamino)-1-(p-nitrophenyl)-1,3-propanediol (+), (-), .alpha.-(2-naphthyl)ethylamine (+), (-), aminomethylpinane, N,N-dimethyl-1-phenylethylamine, N-methyl-1-phenylethylamine, 4-nitrophenylethylamine, pseudoephedrine, norephedrine, norpseudoephedrine, amino acid derivatives, peptide derivatives.

The compounds according to the invention where Y is oxygen, and the remaining substituents have the meanings stated under the general Formulas I and Ia, can be prepared, for example, by reacting the carboxylic acid derivatives of the general formula VI where the substituents have the stated meanings with compounds of the general Formula VII ##STR5## where R.sup.15 is a halogen or R.sup.16 –SO.sub.2 –, where R.sup.16 can be C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl or phenyl. The reaction preferably takes place in one of the above-mentioned inert diluents with the addition of a suitable base, that is of a base which deprotonates the intermediate VI, in a temperature range from room temperature to the boiling point of the solvent.

Compounds of the Formula VII are known, some of them can be bought, or they can be prepared in a generally known manner.

It is possible to use as base an alkali metal or alkaline earth metal hydride such as sodium hydride, potassium hydride, or calcium hydride, a carbonate such as an alkali metal carbonate, for example, sodium or potassium carbonate, an alkali metal or alkaline earth metal hydroxide such as sodium or potassium hydroxide, an organometallic compound such as butyllithium, or an alkali metal amide such as lithium diisopropylamide.

The compounds according to the invention where Y is sulfur, and the remaining substituents have the meanings stated under the general Formulas I and Ia, can be prepared, for example, by reacting carboxylic acid derivatives of the general Formula VIII, which can be obtained in a known manner from compounds of the general formula VI and in which the substituents have the above-mentioned meanings, with compounds of the general formula IX, where R.sup.2, and R.sup.3 and X have the meanings stated under general Formulas I and Ia. ##STR6##

The reaction preferably takes place in one of the above-mentioned inert diluents with the addition of a suitable base, that is a base which deprotonates the intermediate IX, in a temperature range from room temperature to the boiling point of the solvent.

It is possible to use as base, besides those mentioned above, organic bases such as triethylamine, pyridine, imidazole or diazabicycloundecene.

Carboxylic acid derivatives of the Formula VIa (Z in formula VI=direct linkage) can be prepared by reacting epoxides of the Formula IV with cuprates of the Formula XI: ##STR7##

The cuprates can be prepared as described in Tetrahedron Letters 23, (1982) 3755.

Compounds of the Formulas I and Ia can also be prepared by starting from the corresponding carboxylic acid, that is compounds of the Formulas I and Ia where R is COOH, and initially converting these in a conventional manner into an activated form, such as a halide, an anhydride or imidazolide, and then reacting the latter with an appropriate hydroxy compound HOR.sup.10. This reaction can be carried out in the usual solvents and often requires addition of a base, in which case those mentioned above are suitable. These two steps can also be simplified, for example, by allowing the carboxylic acid to act on the hydroxy compound in the presence of a dehydrating agent such as a carbodiimide.

In addition, it is also possible for compounds of the Formula I to be prepared by starting from the salts of the corresponding carboxylic acids, that is from compounds of the Formulas I and Ia where R is COR.sup.1 and R.sup.1 is OM, where M can be an alkali metal cation or the equivalent of an alkaline earth metal cation. These salts can be reacted with many compounds of the Formula R.sup.1 –A where A is a conventional nucleofugic leaving group, for example halogen such as chlorine, bromine, iodine, or aryl- or alkylsulfonyl which is unsubstituted or substituted by halogen, alkyl or haloalkyl, such as toluenesulfonyl and methylsulfonyl, or another equivalent leaving group. Compounds of the formula R.sup.1 –A with a reactive substituent A are known or can be easily obtained with general expert knowledge. This reaction can be carried out in conventional solvents and advantageously takes place with the addition of a base, in which case those mentioned above are suitable.

The radical R in Formula I and Ia may vary widely. For example, R is a group ##STR8## where R.sup.1 has the following meanings: a) hydrogen;

b) succinimidyloxy;

c) a five-membered heteroaromatic moiety linked by a nitrogen atom, such as pyrrolyl, pyrazolyl, imidazolyl and triazolyl, which may carry one or two halogen atoms, in particular fluorine and chlorine and/or one or two of the following radicals:

C.sub.1 -C.sub.4 -alkyl such as methyl, ethyl, 1-propyl, 2-propyl, 2-methyl-2-propyl, 2-methyl-1-propyl, 1-butyl, 2-butyl;

C.sub.1 -C.sub.4 -haloalkyl, in particular C.sub.1 -C.sub.2 -haloalkyl such as fluoromethyl, difluoromethyl, trifluoromethyl, chlorodifluoromethyl, dichlorofluoromethyl, trichloromethyl, 1-fluoroethyl, 2-fluoroethyl, 2,2-difluoroethyl, 2,2,2-trifluoroethyl, 2-chloro-2,2-difluoroethyl, 2,2-dichloro 2-fluoroethyl, 2,2,2-trichloroethyl and pentafluoroethyl;

C.sub.1 -C.sub.4 -haloalkoxy, in particular C.sub.1 -C.sub.2 -haloalkoxy such as difluoromethoxy, trifluoromethoxy, chlorodifluoromethoxy, 1-fluoroethoxy, 2-fluoroethoxy, 2,2-difluoroethoxy, 1,1,2,2-tetrafluoroethoxy, 2,2,2-trifluoroethoxy, 2-chloro-1,1,2-trifluoroethoxy and pentafluoroethoxy, in particular trifluoromethoxy;

C.sub.1 -C.sub.4 -alkoxy such as methoxy, ethoxy, propoxy, 1-methylethoxy, butoxy, 1-methylpropoxy, 2-methylpropoxy, 1,1-dimethylethoxy, in particular methoxy, ethoxy, 1-methylethoxy;

C.sub.1 -C.sub.4 -alkylthio such as methylthio, ethylthio, propylthio, 1-methylethylthio, butylthio, 1-methylpropylthio, 2-methylpropylthio, 1,1-dimethylethylthio, in particular methylthio and ethylthio;

d) R.sup.1 furthermore is a radical ##STR9## where m is 0 or 1 and R.sup.7 and R.sup.8, which can be identical or different, have the following meanings: hydrogen, C.sub.1 -C.sub.8 -alkyl, in particular C.sub.1 -C.sub.4 -alkyl as mentioned above; C.sub.3 -C.sub.6 -alkenyl such as 2-propenyl, 2-butenyl, 3-butenyl, 1-methyl-2-propenyl, 2-methyl-2-propenyl, 2-pentenyl, 3-pentenyl, 4-pentenyl, 1-methyl-2-butenyl, 2-methyl-2-butenyl, 3-methyl-2-butenyl, 1-methyl-3-butenyl, 2-methyl-3-butenyl, 3-methyl-3-butenyl, 1,1-dimethyl-2-propenyl, 1,2-dimethyl-2-propenyl, 1-ethyl-2-propenyl, 2-hexenyl, 3-hexenyl, 4-hexenyl, 5-hexenyl, 1-methyl-2-pentenyl, 2-methyl-2-pentenyl, 3-methyl-2-pentenyl, 4-methyl-2-pentenyl, 3-methyl-3-pentenyl, 4-methyl-3-pentenyl, 1-methyl-4-pentenyl, 2-methyl-4-pentenyl, 3-methyl-4-pentenyl, 4-methyl-4-pentenyl, 1,1-dimethyl-2-butenyl, 1,1-dimethyl-3-butenyl, 1,2-dimethyl-2-butenyl, 1,2-dimethyl-3-butenyl, 1,3-dimethyl-2-butenyl, 1,3-dimethyl-3-butenyl, 2,2-dimethyl-3-butenyl, 2,3-dimethyl-2-butenyl, 2,3-dimethyl-3-butenyl, 1-ethyl-2-butenyl, 1-ethyl-3-butenyl, 2-ethyl-2-butenyl, 2-ethyl-3-butenyl, 1,1,2-trimethyl-2-propenyl, 1-ethyl-1-methyl-2-propenyl and 1-ethyl-2-methyl-2-propenyl, in particular 2-propenyl, 2-butenyl, 3-methyl-2-butenyl and 3-methyl-2-pentenyl;

C.sub.3 -C.sub.6 -alkynyl such as 2-propynyl, 2-butynyl, 3-butynyl, 1-methyl-2-propynyl, 2-pentynyl, 3-pentynyl, 4-pentynyl, 1-methyl-3-butynyl, 2-methyl-3-butynyl, 1-methyl-2-butynyl, 1,1-dimethyl-2-propynyl, 1-ethyl-2-propynyl, 2-hexynyl, 3-hexynyl, 4-hexynyl, 5-hexynyl, 1-methyl-2-pentynyl, 1-methyl-2-pentynyl, 1-methyl-3-pentynyl, 1-methyl-4-pentynyl, 2-methyl-3-pentynyl, 2-methyl-4-pentynyl, 3-methyl-4-pentynyl, 4-methyl-2-pentynyl, 1,1-dimethyl-2-butynyl, 1,1-dimethyl-3-butynyl, 1,2-dimethyl-3-butynyl, 2,2-dimethyl-3-butynyl, 1-ethyl-2-butynyl, 1-ethyl-3-butynyl, 2-ethyl-3-butynyl and 1-ethyl-1-methyl-2-propynyl, preferably 2-propynyl, 2-butynyl, 1-methyl-2-propynyl and 1-methyl-2-butynyl, in particular 2-propynyl

C.sub.3 -C.sub.8 -cycloalkyl such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl and cycloheptyl, cyclooctyl, where these alkyl, cycloalkyl, alkenyl and alkynyl groups can each carry one to five halogen atoms, in particular fluorine or chlorine and/or one or two of the following groups:

C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -alkylthio, C.sub.1 -C.sub.4 -haloalkoxy as mentioned above, C.sub.3 -C.sub.6 -alkenyloxy, C.sub.3 -C.sub.6 -alkenylthio, C.sub.3 -C.sub.6 -alkynyloxy, C.sub.3 -C.sub.6 -alkynylthio, where the alkenyl and alkynyl constituents present in these radicals preferably have the above-mentioned meanings;

C.sub.1 -C.sub.4 -alkylcarbonyl such as, in particular, methylcarbonyl, ethylcarbonyl, propylcarbonyl, 1-methylethylcarbonyl, butylcarbonyl, 1-methylpropylcarbonyl, 2-methylpropylcarbonyl, 1,1-dimethylethylcarbonyl;

C.sub.1 -C.sub.4 -alkoxycarbonyl such as methoxycarbonyl, ethoxycarbonyl, propyloxycarbonyl, 1-methylethoxycarbonyl, butyloxycarbonyl, 1-methylpropyloxycarbonyl, 2-methylpropyloxycarbonyl, 1,1-dimethylethoxycarbonyl;

C.sub.3 -C.sub.6 -alkenylcarbonyl, C.sub.3 -C.sub.6 -alkynylcarbonyl, C.sub.3 -C.sub.6 -alkenyloxycarbonyl and C.sub.3 -C.sub.6 -alkynyloxycarbonyl, where the alkenyl and alkynyl radicals are preferably defined as detailed above;

phenyl, unsubstituted or substituted one or more times, for example, one to three times, by halogen, nitro, cyano, C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy or C.sub.1 -C.sub.4 -alkylthio, such as 2-fluorophenyl, 3-chlorophenyl, 4-bromophenyl, 2-methylphenyl, 3-nitrophenyl, 4-cyanophenyl, 2-trifluoromethylphenyl, 3-methoxyphenyl, 4-trifluoroethoxyphenyl, 2-methylthiophenyl, 2,4-dichlorophenyl, 2-methoxy-3-methyl-phenyl, 2,4-dimethoxyphenyl, 2-nitro-5-cyanophenyl, 2,6-difluorophenyl;

di-C.sub.1 -C.sub.4 -alkylamino such as, in particular, dimethylamino, dipropylamino, N-propyl-N-methylamino, N-propyl-N-ethylamino, diisopropylamino, N-isopropyl-N-methylamino, N-isopropyl-N-ethylamino, N-isopropyl-N-propylamino;

R.sup.7 and R.sup.8 furthermore phenyl which can be substituted by one or more, for example, one to three, of the following radicals: halogen, nitro, cyano, C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy or C.sub.1 -C.sub.4 -alkylthio, as mentioned above in particular;

or R.sup.7 and R.sup.8 together form a C.sub.4 -C.sub.7 -alkylene chain which is closed to form a ring, is unsubstituted or substituted, for example, substituted by C.sub.1 -C.sub.4 -alkyl, and may contain a heteroatom selected from the group consisting of oxygen, sulfur, or nitrogen, such as –(CH.sub.2).sub.4 –, –(CH.sub.2).sub.5 –, –(CH.sub.2).sub.6 –, –(CH.sub.2).sub.7 –, –(CH.sub.2).sub.2 –O–(CH.sub.2).sub.2 –, –CH.sub.2 –S–(CH.sub.2).sub.3 –, –(CH.sub.2).sub.2 –O–(CH.sub.2).sub.3 –, –NH–(CH.sub.2).sub.3 –, –CH.sub.2 –NH–(CH.sub.2).sub.2 –, –CH.sub.2 –CH.dbd.CH–CH.sub.2 –, –CH.dbd.CH–(CH.sub.2).sub.3 –;

e) R.sup.1 furthermore is a group ##STR10## where k is 0, 1 and 2, p is 1, 2, 3 and 4 and R.sup.9 is C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.3 -C.sub.6 -alkenyl, C.sub.3 -C.sub.6 -alkynyl or unsubstituted or substituted phenyl, as mentioned above in particular.

f) R.sup.1 furthermore a radical OR.sup.10, where R.sup.10 is:

hydrogen, the cation of an alkali metal such as lithium, sodium, potassium or the cation of an alkaline earth metal such as calcium, magnesium, and barium or an environmentally compatible organic ammonium ion such as tertiary C.sub.1 -C.sub.4 -alkyl-ammonium or the ammonium ion;

C.sub.3 -C.sub.8 -cycloalkyl as mentioned above, which may carry one to three C.sub.1 -C.sub.4 -alkyl groups;

C.sub.1 -C.sub.8 -alkyl such as, in particular, methyl, ethyl, propyl, 1-methylethyl, butyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1,2-dimethylpropyl, 1,1-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, hexyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 1,1-dimethylbutyl, 2,2-dimethylbutyl, 3,3-dimethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethylbutyl, 2-ethylbutyl, 1-ethyl-2-methylpropyl, which can carry one to five halogen atoms, in particular fluorine and chlorine and/or one of the following radicals:

C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -alkylthio, cyano, C.sub.1 -C.sub.4 -alkylcarbonyl, C.sub.3 -C.sub.8 -cycloalkyl, C.sub.1 -C.sub.4 -alkoxycarbonyl, phenyl, phenoxy or phenylcarbonyl, where the aromatic radicals in turn can carry in each case one to five halogen atoms and/or one to three of the following radicals: nitro, cyano, C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy and/or C.sub.1 -C.sub.4 -alkylthio, as mentioned above in particular;

a C.sub.1 -C.sub.8 -alkyl as mentioned above, which can carry one to five halogen atoms, in particular fluorine and/or chlorine, and carries one of the following radicals: a 5-membered heteroaromatic moiety containing one to three nitrogen atoms, or a 5-membered heteroaromatic moiety containing a nitrogen atom and an oxygen or sulfur atom, which can carry one to four halogen atoms and/or one or two of the following radicals:

nitro, cyano, C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, phenyl, C.sub.1 -C.sub.4 -haloalkoxy and/or C.sub.1 -C.sub.4 -alkylthio. Particular mention may be made of: 1-pyrazolyl, 3-methyl-1-pyrazolyl, 4-methyl-1-pyrazolyl, 3,5-dimethyl-1-pyrazolyl, 3-phenyl-1-pyrazolyl, 4-phenyl-1-pyrazolyl, 4-chloro-1-pyrazolyl, 4-bromo-1-pyrazolyl, 1-imidazolyl, 1-benzimidazolyl, 1,2,4-triazol-1-yl, 3-methyl-1,2,4-triazol-1-yl, 5-methyl-1,2,4-triazol-1-yl, 1-benzotriazolyl, 3-isopropyl-5-isoxazolyl, 3-methyl-5-isoxazolyl, 2-oxazolyl, 2-thiazolyl, 2-imidazolyl, 3-ethyl-5-isoxazolyl, 3-phenyl-5-isoxazolyl, 3-tert-butyl-5-isoxazolyl;

a C.sub.2 -C.sub.6 -alkyl group which carries one of the following radicals in position 2: C.sub.1 -C.sub.4 -alkoxyimino, C.sub.3 -C.sub.6 -alkynyloxyimino, C.sub.3 -C.sub.6 -haloalkenyloxyimino or benzyloxyimino;

a C.sub.3 -C.sub.6 -alkenyl or C.sub.3 -C.sub.6 -alkynyl group, it being possible for these groups in turn to carry one to five halogen atoms;

R.sup.10 furthermore a phenyl radical which can carry one to five halogen atoms and/or one to three of the following radicals: nitro, cyano, C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy,

C.sub.1 -C.sub.4 -haloalkoxy and/or C.sub.1 -C.sub.4 -alkylthio, as mentioned above in particular;

a 5-membered heteroaromatic moiety which is linked via a nitrogen atom, contains one to three nitrogen atoms and can carry one or two halogen atoms and/or one or two of the following radicals: C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, phenyl, C.sub.1 -C.sub.4 -haloalkoxy and/or C.sub.1 -C.sub.4 -alkylthio. Particular mention may be made of: 1-pyrazolyl, 3-methyl-1-pyrazolyl, 4-methyl-1-pyrazolyl, 3,5-dimethyl-1-pyrazolyl, 3-phenyl-1-pyrazolyl, 4-phenyl-1-pyrazolyl, 4-chloro-1-pyrazolyl, 4-bromo-1-pyrazolyl, 1-imidazolyl, 1-benzimidazolyl, 1,2,4-triazol-1-yl, 3-methyl-1,2,4-tri-azol-1-yl, 5-methyl-1,2,4-triazol-1-yl, 1-benzotriazolyl, 3,4-dichloro-1-imidazolyl;

R.sup.10 furthermore is a group ##STR11## where R.sup.11 and R.sup.12, which can be identical or different, are: C.sub.1 -C.sub.8 -alkyl, C.sub.3 -C.sub.6 -alkenyl, C.sub.3 -C.sub.6 -alkynyl, C.sub.3 -C.sub.8 -cycloalkyl, it being possible for these radicals to carry a C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -alkylthio and/or an unsubstituted or substituted phenyl radical, as mentioned above in particular;

phenyl which can be substituted by one or more, for example one to three, of the following radicals: halogen, nitro, cyano, C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy or C.sub.1 -C.sub.4 -alkylthio, where these radicals are, in particular, those mentioned above;

or R.sup.11 and R.sup.12 together form a C.sub.3 -C.sub.12 -alkylene chain which can carry one to three C.sub.1 -C.sub.4 -alkyl groups and contain a heteroatom from the group consisting of oxygen, sulfur and nitrogen, as mentioned in particular for R.sup.7 and R.sup.8.

g) R.sup.1 furthermore is a radical ##STR12## where R.sup.13 is: C.sub.1 -C.sub.4 -alkyl, C.sub.3 -C.sub.6 -alkenyl, C.sub.3 -C.sub.6 -alkynyl, C.sub.3 -C.sub.8 -cycloalkyl as mentioned above in particular, it being possible for these radicals to carry a C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -alkylthio and/or a phenyl radical as mentioned above;

phenyl, unsubstituted or substituted, in particular as mentioned above.

h) R.sup.1 is a radical ##STR13## where R.sup.13 has the above-mentioned meaning. R can furthermore be:

tetrazolyl or cyano.

In a specific embodiment the carboxylic acid derivatives of the general Formula I, both as pure enantiomers and pure diastereomers or as mixture thereof, are those where the substituents have the following meanings:

R.sup.2 hydrogen, hydroxyl, N(C.sub.1 -C.sub.4 -alkyl).sub.2, the C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy, C.sub.1 -C.sub.4 -alkylthio groups and halogen atoms mentioned in detail for R.sup.1, especially chlorine, methyl, methoxy, ethoxy, difluoromethoxy, trifluoromethoxy;

X nitrogen or CR.sup.14 where

R.sup.14 is hydrogen or alkyl, or CR.sup.14 forms together with CR.sup.3 a 4- to 5-membered alkylene or alkenylene ring in which, in each case, a methylene group can be replaced by oxygen or sulfur, such as –CH.sub.2 –CH.sub.2 –O–, –CH.dbd.CH–O–, –CH.sub.2 –CH.sub.2 –CH.sub.2 –O–, –CH.dbd.CH–CH.sub.2 O, in particular hydrogen, –CH.sub.2 –CH.sub.2 –O–, –CH(CH.sub.3)–CH(CH.sub.3)–O–, –C(CH.sub.3).dbd.C(CH.sub.3)–O–, –CH.dbd.C(CH.sub.3)–O– or –C(CH.sub.3).dbd.C(CH.sub.3)–S;

R.sup.3 is hydrogen, hydroxyl, N(C.sub.1 -C.sub.4 -alkyl).sub.2, C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy, C.sub.1 -C.sub.4 -alkylthio groups and halogen atoms mentioned for R.sup.1, especially chlorine, methyl, methoxy, ethoxy, difluoromethoxy, trifluoromethoxy or is linked to R.sup.14 as mentioned above to give a 5- or 6-membered ring;

R.sup.4 and R.sup.5 phenyl or naphthyl, which can be substituted by one or more, for example, one to three, of the following radicals: halogen, nitro, cyano, hydroxyl, mercapto, amino, C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy, C.sub.1 -C.sub.4 -alkylthio, C.sub.1 -C.sub.4 -alkylamino, di-C.sub.1 -C.sub.4 -alkylamino, C.sub.1 -C.sub.4 -alkylcarbonyl, C.sub.1 -C.sub.4 -alkoxycarbonyl; in particular as mentioned for R.sup.7 and R.sup.8, and, or example, 3-hydroxyphenyl, 4-dimethylamino-phenyl, 2-mercaptophenyl, 3-methoxycarbonylphenyl, 4-acetyl-phenyl, 1-naphthyl, 2-naphthyl, 3-bromo-2-naphthyl, 4-methyl-1-naphthyl, 5-methoxy-1-naphthyl, 6-trifluoromethyl-1-naphthyl, 7-chlor-1-naphthyl, 8-hydroxy-1-naphthyl; or R.sup.4 and R.sup.5 form together with the adjacent carbon atom a 3- to 6-membered ring which can contain an oxygen or sulfur atom and is unsubstituted or carries from one to three, depending on the ring size, of the following radicals:

C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -haloalkoxy, C.sub.1 -C.sub.4 -alkylthio as mentioned above in general and in particular; and

phenyl or naphthyl, which are connected together in the ortho positions by a direct linkage, a methylene, ethylene or ethenylene group, an oxygen or sulfur atom or an SO.sub.2, NH or N-alkyl group, or C.sub.3 -C.sub.7 -cycloalkyl;

R.sup.4 C.sub.1 -C.sub.10 -alkyl as specifically mentioned for R.sup.1, which can carry from one to five halogen atoms such as fluorine, chlorine, bromine, iodine, in particular fluorine and chlorine, and/or one of the following radicals: alkoxy, alkylthio, cyano, alkylcarbonyl, alkoxycarbonyl, phenyl, phenoxy, phenyl-carbonyl as mentioned in general and in particular for R.sup.1 ;

C.sub.1 -C.sub.10 -alkyl as mentioned above, which can carry from one to five halogen atoms as mentioned above, in particular fluorine and chlorine, and carries a 5-membered heteroaromatic ring which is unsubstituted or substituted, as mentioned above for R.sup.1 ;

C.sub.3 -C.sub.12 -cycloalkyl, in particular C.sub.3 -C.sub.7 -cycloalkyl, or C.sub.3 -C.sub.12 -cycloalkenyl, in particular C.sub.4 -C.sub.7 -cycloalkenyl, it being possible for one methylene group in the saturated or unsaturated ring to be replaced by an oxygen or sulfur atom, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, tetrahydrofuranyl, tetrahydrothienyl, tetrahydropyranyl, tetrahydrothiopyranyl, cyclopropenyl, dihydrofuranyl, dihydrothienyl, dihydropyranyl, dihydrothiopyranyl, where the cycloalkyl and cycloalkenyl radicals can be substituted by from one to five halogen atoms as mentioned above, especially fluorine or chlorine, and/or one of the following radicals: C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -alkylthio, cyano, C.sub.1 -C.sub.8 -alkylcarbonyl, C.sub.1 -C.sub.8 -alkoxycarbonyl, phenyl, phenoxy, phenylcarbonyl as mentioned above in general and in particular;

C.sub.3 -C.sub.6 -alkenyl or C.sub.3 -C.sub.6 -alkynyl as mentioned for R.sup.1, which can carry from one to five halogen atoms as mentioned above, in particular fluorine and chlorine, and/or one of the following radicals:

C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -alkylthio, cyano, C.sub.1 -C.sub.8 -alkylcarbonyl, C.sub.1 -C.sub.8 -alkoxycarbonyl, phenyl, phenoxy, phenylcarbonyl as mentioned above in general and in particular;

5- or 6-membered hetaryl such as furyl, thienyl, pyrryl, pyrazolyl, imidazolyl, triazolyl, isoxazolyl, oxazolyl, isothiazolyl, thiazolyl, thiadiazolyl, pyridyl, pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, for example 2-furanyl, 3-furanyl, 2-thienyl, 3-thienyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 3-isothiazolyl, 4-isothiazolyl, 5-isothiazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2-imidazolyl, 4-imidazolyl, 5-imidazolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 4-pyrazolyl, 5-pyrazolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, oxa-2,4-diazolyl, oxa-3,4-diazolyl, thia-2,4-diazolyl, thia-3,4-diazolyl [sic] and triazolyl, where the heteroaromatic rings can carry from one to five halogen atoms as mentioned above, in particular fluorine and chlorine, and/or from one to three of the following radicals:

C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -alkylthio, cyano, nitro, C.sub.1 -C.sub.8 -alkylcarbonyl, C.sub.1 -C.sub.8 -alkoxycarbonyl, phenyl, phenoxy, phenylcarbonyl as mentioned above in general and in particular;

R.sup.5 hydrogen, C.sub.1 -C.sub.4 -alkyl, C.sub.3 -C.sub.6 -alkenyl, C.sub.3 -C.sub.6 -alkynyl, C.sub.3 -C.sub.8 -cycloalkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxyalkyl, C.sub.1 -C.sub.4 -alkylthioalkyl or phenyl as mentioned above for R.sup.4 in particular;

R.sup.6 is C.sub.1 -C.sub.8 -alkyl, C.sub.3 -C.sub.6 -alkenyl, C.sub.3 -C.sub.6 -alkynyl or C.sub.3 -C.sub.8 -cycloalkyl as mentioned above in particular, it being possible for these radicals in each case to be substituted one or more times by: halogen, hydroxyl, nitro, cyano, C.sub.1 -C.sub.4 -alkoxy, C.sub.3 -C.sub.6 -alkenyloxy, C.sub.3 -C.sub.6 -alkynyloxy, C.sub.1 -C.sub.4 -alkylthio, C.sub.1 -C.sub.6 -haloalkoxy, C.sub.1 -C.sub.4 -alkylcarbonyl, hydroxycarbonyl, C.sub.1 -C.sub.4 -haloalkoxy, C.sub.1 -C.sub.4 -alkylcarbonyl, hydroxycarbonyl, C.sub.1 -C.sub.4 -alkoxycarbonyl, C.sub.1 -C.sub.4 -alkylamino, di-C.sub.1 -C.sub.4 -alkylamino or unsubstituted or substituted phenyl or phenoxy, as mentioned above in particular;

phenyl or naphthyl, which can be substituted by one or more of the following radicals: halogen, nitro, cyano, hydroxyl, amino, C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy, phenoxy, C.sub.1 -C.sub.4 -alkylthio, C.sub.1 -C.sub.4 -alkylamino or C-C.sub.4 -dialkylamino, as mentioned in particular for R.sup.7 and R.sup.4 ;

a five- or six-membered heteraromatic moiety which contains one to three nitrogen atoms and/or one sulfur or oxygen atom and which can carry one to four halogen atoms and/or one or two of the following radicals: C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy, C.sub.1 -C.sub.4 -alkylthio, phenyl, phenoxy or phenylcarbonyl, it being possible for the phenyl radicals in turn to carry one to five halogen atoms and/or one to three of the following radicals: C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy and/or C.sub.1 -C.sub.4 -alkylthio, as mentioned for R.sup.4 in particular;

Y sulfur, oxygen or a single bond;

Z sulfur, oxygen –SO–, –SO.sub.2 — or a single bond.

In a further embodiment, compounds of the Formula I and Ia, both as pure enantiomers and pure diastereomers or as mixture thereof, are those in which the substituents have the following meanings:

R.sup.2 C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -alkoxy

X nitrogen or CR.sup.14, where

R.sup.14 is hydrogen or alkyl, or CR.sup.14 forms together with CR.sup.3 a 4- or 5-membered alkylene or alkenylene ring such as –CH.sub.2 –CH.sub.2 –O–, –CH.dbd.CH–O–, –CH.sub.2 –CH.sub.2 –CH.sub.2 –O–, –CH.dbd.CH–CH.sub.2 O–, in particular hydrogen, –CH.sub.2 –CH.sub.2 –O–, –CH(CH.sub.3)–CH(CH.sub.3)–O–, –c(CH.sub.3).dbd.C(CH.sub.3)–O–, –CH.dbd.C(CH.sub.3)–O– or –C(CH.sub.3).dbd.C(CH.sub.3)–S;

R.sup.3 the C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -alkylthio groups mentioned for R.sup.1, or is linked to R.sup.14 as mentioned above to give a 5- or 6-membered ring;

R.sup.4 and R.sup.5 phenyl (identical or different) which can be substituted by one or more, for example, one to three, of the following radicals: halogen, nitro, hydroxyl, C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -alkylthio or

R.sup.4 and R.sup.5 are phenyl groups which are connected together in the ortho positions by a direct linkage, a methylene, ethylene or ethenylene group, an oxygen or sulfur atom or an SO.sub.2, NH or N-alkyl group; or

R.sup.4 and R.sup.5 are C.sub.3 -C.sub.7 -cycloalkyl;

R.sup.6 C.sub.1 -C.sub.8 -alkyl, C.sub.3 -C.sub.6 -alkenyl or C.sub.3 -C.sub.8 -cycloalkyl, it being possible for these radicals in each case to be substituted one or more times by: halogen, hydroxyl, nitro, cyano, C.sub.1 -C.sub.4 -alkoxy, C.sub.3 -C.sub.6 -alkenyloxy, C.sub.1 -C.sub.4 -alkylthio;

phenyl or naphthyl, which can be substituted by one or more of the following radicals: halogen, nitro, cyano, hydroxyl, amino, C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -haloalkoxy, phenoxy, C.sub.1 -C.sub.4 -alkylthio, C.sub.1 -C.sub.4 -alkylamino or C.sub.1 -C.sub.4 -dialkylamino;

a five- or six-membered heteroaromatic moiety which contains a nitrogen atom and/or a sulfur or oxygen atom and which can carry one to four halogen atoms and/or one or two of the following radicals: C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy, C.sub.1 -C.sub.4 -alkylthio, phenyl, phenoxy or phenylcarbonyl, it being possible for the phenyl radicals in turn to carry one to five halogen atoms and/or one to three of the following radicals: C.sub.1 -C.sub.4 -alkyl, C.sub.1 -C.sub.4 -haloalkyl, C.sub.1 -C.sub.4 -alkoxy and/or C.sub.1 -C.sub.4 -alkylthio;

Y sulfur, oxygen or a single bond;

Z sulfur, oxygen, –SO–, –SO.sub.2 — or a single bond.

The beneficial effect of the compounds can be shown in the following tests:

Evaluation of Biological Activity in vivo

Compounds of the invention may be further tested in any of the various preclinical assays for in vivo anticancer activity.

For example, human tumors which have been grown in nude mice can be transplanted into new recipient mice using tumor fragments which are about 50 mg in size. The day of transplantation is designated as day 0. Five to fifteen days later, the mice are treated with the test compounds given as an intravenous or intraperitoneal injection, in groups of five to ten mice at each dose. Compounds are given daily for either 5, 10 or 15 days, at dosed from 0.1-1000 mg/kg of body weight.

Tumor diameters and body weights are measured periodically. Tumor mass is calculated using the diameters measured with Vernier calipers, and the formula:

Mean tumor weights are calculated for each treatment group relative to the untreated control tumors. The DU-145 Model is a specific example of this type of assay and is described below.

DU-145 Model

Human tumor fragments from prostate (HTB 81), which had been grown in nude mice were implanted subcutaneously via trochar, in the dorsal side of new recipient nude mice, as is well known in the art. The day of implantation is designated as day 0.

Treatment was commenced on day 11 post implantation using Compound I-1 of Table I. There were 6 mice in each treatment group with the mode of administration and amounts administered (mg/kg of body weight) described in Table XI. Compound I-1 was administered once a day for 10 days commencing, as earlier stated, at day 11 post implantation (Q1D.times.10:11).

Table IX shows that all treatment groups responded to administration of Compound I-1, as evidenced by a reduction in the %T/C Mean Tumor Weight (MTW) value.

TABLE IX ______________________________________ USE OF COMPOUND I-1 IN THE DU-145 PROSTATE TUMOR MODEL Mean Tumor Weight (MTW) Days for Tumor on Day 33 MTW Dose Weight = 1000 mg (mg) % T/C ______________________________________ Control 32.6 1232 100.00 100 mg/kg IV, Q1Dx10:11 39.3 561 45.54 150 mg/kg IV, Q1DX10:11 37.4 793 64.37 150 mg/kg IP, Q1DX10:11 59.3 407 33.04 220 mg/kg IP, Q1DX10:11 50.9 470 38.15 ______________________________________

Receptor Binding Studies

Cloned human ET.sub.A receptor-expressing CHO cells and guinea pig cerebellar membranes with >60% ET.sub.B compared with ET.sub.A receptors were used for binding studies.

The ET.sub.A receptor-expressing CHO cells were grown in F.sub.12 medium containing 10% fetal calf serum, 1% glutamine, 100 U/ml penicillin and 0.2% streptomycin (Gibco BRL, Gaithersburg, Md., USA). After 48 h, the cells were washed with PBS and incubated with 0.05% trypsin-containing PBS for 5 min. Neutralization was then carried out with F.sub.12 medium, and the cells were collected by centrifugation at 300.times.g. To lyze the cells, the pellet was briefly washed with lysis buffer (5 mM Tris-HCl, pH 7.4 with 10% glycerol) and then incubated at a concentration of 10.sup.7 cells/ml of lysis buffer at 4.degree. C. for 30 min. The membranes were centrifuged at 20,000.times.g for 10 min., and the pellet was stored in liquid nitrogen.

Guinea pig cerebella were homogenized in a Potter-Elvejhem homogenizer and membranes were obtained by differential centrifugation at 1000.times.g for 10 min. and repeated centrifugation of the supernatant at 20,000.times.g for 10 min.

Binding Assays

For the ET.sub.A and ET.sub.B receptor binding assay, the membranes were suspended in incubation buffer (50 mM Tris-HCl, pH 7.4 with 5 mM MnCl.sub.2, 40 .mu.g/ml; bacitracin and 0.2% BSA) at a concentration of 50 .mu.g of protein per assay mixture and incubated with 25 pM [.sup.125 I]-ET.sub.1 (ET.sub.A receptor assay) or 25 pM [.sup.125 I]-RZ.sub.3 (ET.sub.B receptor assay) in the presence and absence of test substance at 25.degree. C. The nonspecific binding was determined using 10.sup.-7 M ET.sub.1. After 30 min., the free and bound radioligand were separated by filtration through GF/B glass fiber filters (Whatman, England) on a Skatron cell collector (Skatron, Lier, Norway) and the filters were washed with ice-cold Tris-HCl buffer, pH 7.4 with 0.2% BSA. The radioactivity collected on the filters was quantified using a Packard 2200 CA liquid scintillation counter.

The K.sub.i values shown in Table A were determined by non-linear regression analysis using the LIGAND program.

Table A shows the effect of compounds of the Formula I as the K.sub.i [mol/l] determined in the experiments.

TABLE A ______________________________________ K.sub.i [mol/l] Compound ET-A ET-B ______________________________________ 4.42 2.5 .times. 10.sup.-7 3.0 .times. 10.sup.-6 4.58 1.6 .times. 10.sup.-7 4.7 .times. 10.sup.-6 ______________________________________

Functional in vitro Assay System to Look for Endothelin Receptor (Subtype A) antagonists

This assay system is a functional, cell-based assay for endothelin receptors. When certain cells are stimulated with endothelin 1 (ET1) they show an increase in the intracellular calcium concentration. This increase can be measured in intact cells loaded with calcium-sensitive dyes.

1-Fibroblasts which had been isolated from rats and in which an endogenous endothelin receptor of the A subtype had been detected were loaded with the fluorescent dye Fura 2-an as follows: after trypsinization, the cells were resuspended in buffer A (120 mM NaCl, 5 mM KCl, 1.5 mM MgCl.sub.2, 1 mM CaCl.sub.2, 25 mM HEPES, 10 mM glucose, pH 7.4) to a density of 2′10.sup.6 /ml and incubated with Fura 2-am (2 .mu.M), Pluronics F-127 (0.04%) and DMSO (0.2%) at 37.degree. C. in the dark for 30 min. The cells were then washed twice with buffer A and resuspended at 2.times.10.sup.6 /ml.

The fluorescence signal from 2.times.10.sup.5 cells per ml with Ex/Em 380/510 was recorded continuously at 30.degree. C. The test substances and, after an incubation time of 3 min., ET1 were added to the cells, and the maximum change in the fluorescence was determined. The response of the cells to ET1 without previous addition of a test substance was used as control and was set equal to 100%.

Table B indicates the effect of some compounds of the Formula I as the IC.sub.50 [mol/l] determined in the experiments.

TABLE B ______________________________________ Compound IC.sub.50 [mol/l] ______________________________________ 4.42 7.4 .times. 10.sup.-7 4.58 1.0 .times. 10.sup.-6 ______________________________________

Testing of ET Antagonists in vivo

Male SD rats weighing 250-300 g were anesthetized with amobarbital, artificially ventilated, vagotomized and pithed. The carotid artery and jugular vein were catheterized.

In control animals, intravenous administration of 1 .mu.g/kg ET1 led to a distinct rise in blood pressure which persisted for a lengthy period.

The test animals received an i.v. injection of the test compounds (1 ml/kg) 5 min. before the administration of ET1. To determine the ET-antagonistic properties, the rise in blood pressure in the test animals was compared with that in the control animals.

Endothelin-1-Induced Sudden Death in Mice

The principle of the test is the inhibition of the sudden heart death caused in mice by endothelin, which is probably induced by constriction of the coronary vessels, by pretreatment with endothelin receptor antagonists. Intravenous injection of 10 nmol/kg endothelin in a volume of 5 ml/kg of body weight results in death of the animals within a few minutes.

The lethal endothelin-1 dose is checked in each case on a small group of animals. If the test substance is administrated intravenously, the endothelin-1 injection which was lethal in the reference group usually takes place 5 min. thereafter. With other modes of administration, the times before administration are extended, where appropriate up to several hours.

The survival rate is recorded, and effective doses which protect 50% of the animals (ED 50) from endothelin-induced heart death for 24 h. or longer are determined.

Functional Test on Vessels for Endothelin Receptor Antagonists

Segments of rabbit aorta are, after an initial tension of 2 g and a relaxation time of 1 h. in Krebs-Henseleit solution at 37.degree. C. and pH 7.3-7.4, first induced to contract with K.sup.+. After washing out, an endothelin dose-effect plot up to the maximum is constructed.

Potential endothelin antagonists are administered to other preparations of the same vessel 15 min. before starting the endothelin dose-effect plot. The effects of the endothelin are calibrated as a % of the K.sup.+ -induced contraction. Effective endothelin antagonists result in a shift to the right in the endothelin dose-effect plot.

Synthesis Examples of Compound of Formula Ia

EXAMPLE 1

Methyl 2-hydroxy-3-methoxy-3,3-diphenylpropionate

5 g (19.6 mmol) of methyl 3,3-diphenyl-2,3-epoxypropionate were dissolved in 50 ml of absolute methanol and, at 0.degree. C., 0.1 ml of boron trifluoride etherate was added. The mixture was stirred at 0.degree. C. for 2 h. and at room temperature for a further 12 h. The solution vent was distilled out, the residue was taken up in ethyl acetate, washed with sodium bicarbonate solution and water and dried over magnesium sulfate. After removal of the solvent by distillation there remained 5.5 g (88%) of a pale yellow oil.

EXAMPLE 2

Methyl 2-hydroxy-3-phenoxy-3,3-diphenylpropionate

5 g (19.6 mmol) of methyl 3,3-diphenyl-2,3-epoxypropionate and 5.6 g (60 mmol) of phenol were heated together at 100.degree. C. for 6 h. Removal of the excess phenol by distillation under high vacuum and purification of the residue by chromatography on silica gel with hexane/ethyl acetate mixtures resulted in 4.9 g (77%) of a pale yellow oil.

EXAMPLE 3

Methyl 2-(4,6-dimethoxy-pyrimidin-2-yloxy)-3-methoxy-3,3-diphenylpropionate

2.86 g (10 mmol) of methyl 2-hydroxy-3-methoxy-3,3-diphenylpropionate were dissolved in 40 ml of dimethylformamide, and 0.3 g (12 mmol) of sodium hydride was added. The mixture was stirred for 1 h. and then 2.2 g (10 mmol) of 4,6-dimethoxy-2-methylsulfonylpyrimidine were added. After stirring at room temperature for 24 h., cautious hydrolysis was carried out with 10 ml of water, the pH was adjusted to 5 with acetic acid, and the solvent was removed by distillation under high vacuum. The residue was taken up in 100 ml of ethyl acetate, washed with water and dried over magnesium sulfate, and the solvent was distilled out. The residue was mixed with 10 ml of ether, and the resulting precipitate was filtered off with suction. After drying, 3.48 g (82%) of a white powder remained.

EXAMPLE 4

2-(4,6-Dimethoxy-pyrimidin-2-yloxy)-3-methoxy-3,3-diphenylpropionic acid

2.12 g (5 mmol) of methyl 2-(4,6-dimethoxy-pyrimidin-2-yloxy)-3-methoxy-3,3-diphenylpropionate were dissolved in 50 ml of dioxane, 10 ml of 1 N KOH solution were added, and the mixture was stirred at 100.degree. C. for 3 h. The solution was diluted with 300 ml of water and extracted with ethyl acetate to remove unreacted ester. The aqueous phase was then adjusted to pH 1-2 with dilute hydrochloric acid and extracted with ethyl acetate. After drying over magnesium sulfate and removal of the solvent by distillation, the residue was mixed with an ester/hexane mixture, and the precipitate which formed was filtered off with suction. After drying, 1.85 g (90%) of a white powder remained.

Melting point 167.degree. C.

EXAMPLE 5

Sodium 2-(4,6-dimethoxy-2-pyrimidinyloxy)-3-methoxy-3,3-diphenyl-propionate

1.68 g (4 mmol) or 2-(4,6-dimethoxy-2-pyrimidinyloxy)-3-methoxy-3,3-diphenylpropionic acid are dissolved in 4 ml of 1N NaOH+100 ml of water. The solution is freeze-dried, and the sodium salt of the carboxylic acid used is obtained quantitatively.

10 g (34.9 mmol) of methyl 2-hydroxy-3-methoxy-3,3-diphenylpropionate were dissolved in 50 ml each of methanol and glacial acetic acid, 1 ml of RuO(OH).sub.2 in dioxane was added, and hydrogenation was carried out with H.sub.2 in an autoclave at 100.degree. C. under 100 bar for 30 h. The catalyst was filtered off, the mixture was concentrated, mixed with ether and washed with NaCl solution, and the organic phase was dried and concentrated. 10.1 g of methyl 3,3-dicyclohexyl-2-hydroxy-3-methoxypropionate were obtained was an oil.

EXAMPLE 7

Methyl 2-(4,6-dimethoxy-pyrimidin-2-ylthio)-3-methoxy-3,3-diphenylpropionate

7.16 g (25 mmol) of methyl 2-hydroxy-3-methoxy-3,3-diphenylpropionate were dissolved in 50 ml of dichloromethane, 3 g (30 mmol) of triethylamine were added, and 3.2 g (28 mmol) of methane-sulfonyl chloride were added dropwise while stirring. The mixture was stirred at room temperature for 2 h., washed with water, dried over magnesium sulfate and concentrated under reduced pressure. The residue was taken up in DMF and added dropwise at 0.degree. C. to a suspension of 12.9 g (75 mmol) of 4,6-dimethoxypyrimidine-2-thiol and 8.4 g (100 mmol) of sodium bicarbonate in 100 ml of DMF. After stirring at room temperature for 2 h. and at 60.degree. C. for a further 2 h., the mixture was poured into 1 liter of ice-water, and the resulting precipitate was filtered off with suction. After drying, 3.19 g (29%) of a white powder remained.

EXAMPLE 8

Methyl 2-hydroxy-3,3-diphenylbutyrate

1.5 g (5.9 mmol) of methyl 3,3-diphenyl-2,3-epoxypropionate dissolved in 10 ml of absolute ether were added dropwise to a cuprate solution which had been prepared from 635 mg (7 mmol) of copper(I) cyanide dissolved in 10 ml of absolute ether and 8.14 ml (13 mmol) of a 1.6 normal methyllithium solution and had been cooled to -78.degree. C. The solution was stirred at -78.degree. C. for 1 h. and then allowed to warm to room temperature. It was subsequently diluted with 100 ml of ether and 100 ml of water, and the ether phase was washed with dilute citric acid and with sodium bicarbonate solution and dried over magnesium sulfate. The crude product was purified by chromatography on silica gel with cyclohexane/ethyl acetate mixtures to result in 250 mg (16%) of a pale yellow oil.

EXAMPLE 9

2-Hydroxy-3-methoxy-3,3-diphenylpropionic acid

91.11 g (0.5 mol) of benzophenone and 45.92 g (0.85 mol) of sodium methoxide were suspended in 150 ml of methyl tert-butyl ether (MTB) at room temperature. After cooling to -10.degree. C., 92.24 g (0.85 mol) of methyl chloroacetate were added in such a way that the internal temperature rose to 40.degree. C. while continuing to cool in a bath at -10.degree. C. The mixture was then stirred without cooling at the autogenous temperature for one hour. After addition of 250 ml of water and brief stirring, the aqueous phase was separated off. The MTB phase was washed with 250 ml of dilute sodium chloride solution. After the solvent had been changed to methanol (250 ml), a solution of 1 g of p-toluenesulfonic acid in 10 ml of methanol was added at room temperature. The mixture was stirred at autogenous temperature for one hour and then heated to reflux. While distilling out the methanol, 400 g of a 10% strength sodium hydroxide solution was added dropwise, and finally 60 ml of water were added. The methanol was distilled out until the bottom temperature reached 97.degree. C. After cooling to 55.degree. C., 190 ml of MTB were added and the mixture was acidified to pH 2 with about 77 ml of concentrated HCl. After cooling to room temperature, the aqueous phase was separated off and the organic phase was concentrated by distilling out 60 ml of MTB. The product was crystallized by adding 500 ml of heptane and slowly cooling to room temperature. The coarsely crystalline solid was filtered off with suction, washed with heptane and dried to constant weight in a vacuum oven at 40.degree. C.

Yield: 108.9 g (80%), HPLC >99.5% area.

EXAMPLE 10

S-2-Hydroxy-3-methoxy-3,3-diphenylpropionic acid (racemate resolution with L-proline methyl ester)

148.8 g of a 30% strength methanolic sodium methanolate solution (0.826 mol) were added dropwise to 240 g of a 57% strength methanolic L-proline methyl ester hydrochloride solution (0.826 mol) at room temperature, and 2.4 l of MTB and 225 g (0.826 mol) of 2-hydroxy-3-methoxy-3,3-diphenylpropionic acid were added. After 2680 mol of MTB/methanol mixture had been distilled out with simultaneous dropwise addition of 2.4 l of MTB, the mixture was slowly cooled to room temperature, the crystals (R-2-hydroxy-3-methoxy-3,3-diphenylpropionic acid.times.L-proline methyl ester) were filtered off with suction, and the solid was washed with 150 ml of MTB. The filtrate was concentrated by distilling out 1.5 l of MTB, and 1.0 l of water was added. The pH was adjusted to 1.2 with concentrated hydrochloric acid at room temperature and, after stirring and phase separation, the aqueous phase was separated off and extracted with 0.4 l of MTB. The combined organic phases were extracted with 0.4 l of water. The residue after the MTB had been stripped off was dissolved in 650 ml of toluene under reflux, and the product was crystallized by seeding and slow cooling. Filtration with suction, washing with toluene and drying in a vacuum oven resulted in 78.7 g of S-2-hydroxy-3-methoxy-3,3-diphenylpropionic acid (yield 35% based on the racemate).

Chiral HPLC: 100% pure

HPLC: 99.8%

EXAMPLE 11

S-2-Hydroxy-3-methoxy-3,3-diphenylpropionic acid (racemate resolution with (S)-1-(4-nitrophenyl)ethylamine)

30.5 g (0.184 mol) of (S)-1-(4-nitrophenyl)ethylamine were added to 100 g (0.368 mol) of 2-hydroxy-3-methoxy-3,3-diphenylpropionic acid in 750 ml of acetone and 750 ml of MTB under reflux, the mixture was seeded, boiled under reflux for one hour and slowly cooled to room temperature for crystallization. The crystals (S-2-hydroxy-3-methoxy-3,3-diphenylpropionic acid.times.(S)-1(4-nitrophenyl)ethylamine) were filtered off with suction and washed with MTB. The residue was suspended in 500 ml of water and 350 ml of MTB and then the pH was adjusted to 1.2 with concentrated hydrochloric acid at room temperature, and, after stirring and phase separation, the aqueous phase was separated off and extracted with 150 ml of MTB. The combined organic phases were extracted with 100 ml of water. 370 ml of MTB were distilled out and then 390 ml of n-heptane were added under reflux, and the mixture was slowly cooled to room temperature while the product crystallized. Filtration with suction, washing with n-heptane and drying in a vacuum oven resulted in 35.0 g of S-2-hydroxy-3-methoxy-3,3-diphenylpropionic acid (yield 35% based on the racemate).

Chiral HPLC: 100% pure

HPLC: 99.8%

EXAMPLE 12

Benzyl 3-methoxy-2-(4-methoxy-6,7-dihydro-5H-cyclopentapyrimidin-2-yloxy)-3,3-dip henylpropionate

24.48 g (90 mmol) of 3-methoxy-3,3-diphenyl-2-hydroxypropionic acid were dissolved in 150 ml of DMF, and 13.7 g (99 mmol) of potassium carbonate were added. The suspension was stirred at room temperature for 30 min. Then 10.7 ml (90 mmol) of benzyl bromide were added dropwise over the course of 5 min., and the mixture was stirred for 1 h., during which the temperature rose to 32.degree. C.

To this mixture were successively added 24.84 g (180 mmol) of K.sub.2 CO.sub.3 and 20.52 g (90 mmol) of 2-methanesulfonyl-4-methoxy-6,7-dihydro-5H-Ocyclopentapyrimidine, and the mixture was stirred at 80.degree. C. for 3 h.

For workup, the contents of the flask were diluted with about 600 ml of H.sub.2 O and cautiously acidified with concentrated HCl, and 250 ml of ethyl acetate were added. 31.4 g of pure product precipitated and were filtered off.

The ethyl acetate phase was separated from the mother liquor, the aqueous phase was extracted again with ethyl acetate, and the combined organic phases were concentrated. The oily residue (19 g) was purified by chromatography (cyclohexane/ethyl acetate=9/1) to result in a further 10.5 g of pure product.

Total yield: 41.9 g (82.2 mmol)=91%

Melting point 143-147.degree. C.

MS: MH+=511

EXAMPLE 13

3-Methoxy-2-(4-methoxy-6,7-dihydro-5H-cyclopentapyrimidin-2-yl-oxy)-3,3-dip henylpropionic acid

40 g (78.4 mmol) of benzyl 3-methoxy-2-(4-methoxy-6,7-dihydro-5H-cyclopentapyrimidin-2-yloxy)-3,3-dip henylpropionate were dissolved in 400 ml of ethyl acetate/methanol (4:1), about 500 mg of palladium on active carbon (10%) were added, and the mixture was exposed to a hydrogen atmosphere until no further gas was taken up. The catalyst was filtered off, the solution was evaporated, and the residue was crystallized from ether.

EXAMPLE 14

Ethyl 2S-3,3-diphenyloxirane-2-carboxylate

2.57 g (10.2 mmol) of ethyl 3,3-diphenylacrylate and 464 mg of 4-phenylpyridine N-oxide were dissolved in 24 ml of methylene chloride, and 432 mg (6.5 mol %) of (S,S)-(+)-N,N’-bis(3,5-ditert-butylsalicylidene)-1,2-cyclohexanediaminoman ganese(III) chloride were added. While cooling in ice, 6.4 ml of a 12% strength sodium hypochlorite solution were added, and the mixture was stirred while cooling in ice for 30 min. and at room temperature overnight. The solution was diluted to 200 ml with water, extracted with ether, dried and evaporated. 2.85 g of a colorless oil were obtained. Purification by MPLC (cyclohexane:ethyl acetate=9:1) resulted in 1.12 g of oil with an enantiomer ratio of about 8:1 in favor of the S configuration.

.sup.1 H-=NMR [CDCl.sub.3 ], .delta.=1.0 (t, 3H); 3.9 (m, 3H); 7.3 (m, 10H)

EXAMPLE 15

2-Methylthio-6,7-dihydro-5H-cyclopentapyrimidin-4-ol

46.9 g (330 mmol) of methyl cyclopentanone-2-carboxylate and 53.5 g (192 mmol) of S-methylisothiourea sulfate were successively added to 29.6 g (528 mmol) of KOH in 396 ml of methanol, and the mixture was stirred at room temperature overnight, acidified with 1N hydrochloric acid and diluted with water. The crystals which separated out were filtered off with suction and dried. 20 g of crystals were obtained.

EXAMPLE 16

4-Chloro-2-methylthio-6,7-dihydro-5H-cyclopentapyrimidine

255 ml of phosphorus oxychloride were added to 20 g (110 mmol) of Example 15, and the mixture was stirred at 80.degree. C. for 3 hours. Phosphorus oxychloride was evaporated off, ice was added to the residue, and the crystals which separated out were filtered off with suction. 18.5 g of a brownish solid were obtained.

EXAMPLE 17

4-Methoxy-2-methylthio-6,7-dihydro-5H-cyclopentapyrimidine

18.05 g (90 mmol) of 4-chloro-2-methylthio-6,7-dihydro-5H-cyclopentapyrimidine were dissolved in 200 ml of methanol. At 45.degree. C., 16.7 g of sodium methoxide (as 30% strength solution in methanol) were added dropwise, and the mixture was stirred for 2 hours. The solution was evaporated, taken up in ethyl acetate and acidified with dilute hydrochloric acid, and the ethyl acetate extract was evaporated. 15.5 g of an oil remained.

.sup.1 H-NMR [DMSO], .delta.=2.1 (quintet, 2H); 2.5 (s, 3H); 2.8 (dt, 4H); 3.9 (s, 3H) ppm

EXAMPLE 18

2-Methylthio-4-methoxy-6,7-dihydro-5H-cyclopentapyrimidine

15 g (76.2 mmol) of 4-methoxy-2-methylthio-6,7-dihydro-5H-cyclo-pentapyrimidine were dissolved in 160 ml of glacial acetic acid/methylene chloride (1:1), and 1.3 g of sodium tungstate were added. At 35.degree. C., 17.5 ml (170 mmol) of a 30% strength H.sub.2 O.sub.2 solution were added dropwise. The mixture was then diluted with 500 ml of water and 100 ml of methylene chloride, and the organic phase was separated off, dried and evaporated. 14 g of oil remained and were crystallized from ether.

.sup.1 H=NMR [CDCl.sub.3 ], .delta.=2.2 (quintet, 2H); 3.0 (dt., 4H); 3.3 (s, 3H); 4.1 (s, 3H) ppm

EXAMPLE 19

1-Benzenesulfonyl-3-(4,6-dimethoxy-2-pyrimidinyloxy)-4-methoxy-4,4-diphenyl -2-butanone

0.37 g (2.4 mmol) of phenyl methyl sulfone were dissolved in 10 ml of dry THF and then, at -70.degree. C., 2 eq. of butyllithium (2.94 ml; 1.6 molar solution in hexane) were added dropwise. After 1 h. at -70.degree. C., 1 g (2.4 mmol) of methyl 2-(4,6-dimethoxy-2-pyrimidinyloxy)-3-methoxy-3,3-diphenylpropionate dissolved in 5 ml of THF was added dropwise. The reaction mixture was then stirred at -70.degree. C. for 1 h. and at -10.degree. C. for 1 h. and then warmed to room temperature.

For workup, about 10 ml of saturated NH.sub.4 Cl solution were added dropwise, thorough extraction with ethyl acetate was carried out, and the combined organic phases were washed with saturated NaCl solution and dried over NA.sub.2 SO.sub.4. The residue obtained after drying and concentration was purified by chromatography on silica gel (n-heptane/ethyl acetate 15%.fwdarw.30%) and subsequently MPLC on RP silica gel (acetonitrile/H.sub.2 O+TFA); 0.3 g of a white amorphous powder was obtained as product.

EXAMPLE 20

3,3-Diphenyloxirane-2-carbonitrile

3.1 g (54.9 mmol) of sodium methoxide were suspended in 20 ml of dry THF and then, at -10.degree. C., a mixture of 5 g (27.4 mmol) of benzophenone and 4.2 g (54.9 mmol) of chloroacetonitrile was added dropwise.

The reaction mixture was stirred at -10.degree. C. for about 2 h., then poured into water and extracted several times with ethyl acetate. The combined organic phases were dried over Na.sub.2 SO.sub.4 and concentrated, and the residue was purified by chromatography on silica gel (n-heptane/ethyl acetate).

Yield: 1.2 g (20%)

.sup.1 H-NMR [CDCl.sub.3 ], .delta.=3.9 (s, 1H); 7.4-7.5 (m, 10 H) ppm

EXAMPLE 21

2-Hydroxy-3-methoxy-3,3-diphenylpropionitrile

6.5 g (29.4 mmol) of 3,3-diphenyloxirane-2-carbonitrile were dissolved in 60 ml of methanol and, at 0.degree. C., about 2 ml of boron trifluoride etherate solution were added. The mixture was stirred further at 0.degree. C. for 1 h. and then at room temperature overnight. For workup it was diluted with diethyl ether and washed with saturated NaCl solution, and the organic phase was dried over Na.sub.2 SO.sub.4 and concentrated.

The residue comprised 7.3 g of a white amorphous powder which was used directly in the subsequent reactions.

.sup.1 H-NMR [CDCl.sub.3 ], .delta.=2.95 (broad s, OH), 3.15 (s, 3H), 5.3 (s, 1H), 7.3-7.5 (m, 10) ppm

EXAMPLE 22

2-(4,6-Dimethoxy-2-pyrimidinyloxy)-3-methoxy-3,3-diphenylpropionitrile

7.3 g (28.8 mmol) of 2-hydroxy-3-methoxy-3,3-diphenylpropionitrile were dissolved in 90 ml of DMF, and 4 g (28.8 mmol) of K.sub.2 CO.sub.3 and 6.3 g (28 mmol) of 2-methanesulfonyl-4,6-dimethoxypyrimidine were added. The mixture was stirred at room temperature for about 12 h., then poured into water and extracted with ethyl acetate. The combined organic phases were washed again with H.sub.2 O, dried and concentrated. The residue obtained in this way was then purified by chromatography on silica gel (n-heptane/ethyl acetate).

Yield: 6.9 g of white amorphous powder

FAB-MS: 392 (M+H.sup.+)

.sup.1 ZHZ-NMR [CDCl.sub.3 ], .delta.=3.3 (s, 3H); 4.95 (s, 6H), 5.85 (s, 1H); 6.3(s, 1H); 7.3-7.5 (m, 10H) ppm

EXAMPLE 23

5-[1-(4,6-Dimethoxy-2-pyrimidinyloxy)-2-methoxy-2,2-diphenyl-ethyl]-1H-tetr azole

0.5 g (1.3 mmol) of nitrile was dissolved in 10 ml of toluene, and 85 mg (1.3 mmol) of NaN.sub.3 and 460 mg (1.4 mmol) of Bu.sub.3 SnCl were successively added, and then the mixture was refluxed for about 40 h. Cooling was followed by dilution with ethyl acetate and washing with 10% aqueous KF solution and with NaCl solution. After drying over MgSO.sub.4 and concentration there remained 1.0 g of a yellow oil, which was purified by chromatography on silica gel (n-heptane/ethyl acetate).

Concentration of the fractions resulted in 60 mg of the 1H-tetrazole and 110 mg of the 1-methyltetrazole, each as amorphous white solids.

5-[1-(4,6-Dimethoxy-2-pyrmidinyloxy)-2-methoxy-2,2-diphenylethyl]-1H-tetraz ole

Electrospray-MS: 435 (M+H.sup.+)

.sup.1 H-NMR (CDCl.sub.3): .delta. (ppm) 3.28 (s, 3H), 3.85 (s, 6H), 5.75 (s, 1H), 7.25-7.40 (m, 10H), 7.50 (s, 1H).

5-[1-(4,6-Dimethoxy-2-pyrimidinyloxy)-2-methoxy-2,2-diphenylethyl]-1-methyl tetrazole

Electrospray-MS; 471 (M+H.sup.+)

.sup.1 H-NMR (CDCl.sub.3): .delta. (ppm) 3.0 (s, 3H), 3.35 (s, 3H), 3.80 (s, 6H), 5.75 (s, 1H) 7.30-7.40 (m, 11H).

EXAMPLE 24

2-(4,6-Dimethoxy-2-pyrimidinyloxy)-3-methylsulfinyl-3,3-diphenylpropionic acid

1.2 g (2.9 mmol) of 2-(4,6-dimethoxy-2-pyrimidinyloxy)-3-methylthio-3,3-diphenylpropionic acid were introduced into 15 ml of glacial acetic acid at 0.degree. C. and 294 .mu.l of 30% strength H.sub.2 O.sub.2 were added dropwise. The mixture was stirred at room temperature overnight, poured into water, extracted with CH.sub.2 Cl.sub.2 and washed with sodium thiosulfate solution and brine. After drying, 1 g of substance was isolated as a white foam.

EXAMPLE 25

2-(4,6-Dimethoxy-2-pyrimidinyloxy)-3-methylsulfonyl-3,3-diphenylpropionic acid

0.6 g (1.45 mmol) of 2-(4,6-dimethoxy-2-pyrimidinyloxy)-3-methyl-sulfinyl-3,3-diphenylpropionic acid was introduced into 15 ml of glacial acetic acid at room temperature, and 294 .mu.l of 30% strength H.sub.2 O.sub.2 were added dropwise. The mixture was stirred at room temperature overnight, heated at 50.degree. C. for a further 3 h., poured into water and washed with sodium thiosulfate solution and brine. After drying, 400 mg were isolated as a white solid.

The compounds listed in Table I can be prepared in a similar way.

TABLE I __________________________________________________________________________ ##STR14## m.p. No. R.sup.1 R.sup.4, R.sup.5 R.sup.6 R.sup.2 R.sup.3 X Y Z [.degree. __________________________________________________________________________ C.] I-1 OMe Phenyl Methyl OMe OMe CH O O 81 I-2 OH Phenyl Methyl OMe OMe CH O O 167 I-3 OH Phenyl CH.sub.2 –CH.sub.2 –S–CH.sub.3 OMe OMe CH O O I-4 OH Phenyl Ethyl OMe OMe CH O O 81 (decomp.) I-5 OH Phenyl iso-Propyl OMe OMe CH O O 182 I-6 OH Phenyl Methyl OMe OMe CH O S 168 I-7 OH Phenyl CH.sub.2 –CH.sub.2 –SO.sub.2 –CH(CH.sub.3).sub.2 OMe OMe CH O O I-8 OH Phenyl CH.sub.2 –CH.sub.2 –SO.sub.2 –CH(CH.sub.3).sub.2 OMe OMe CH S O I-9 OH Phenyl CH.sub.2 –CH.sub.2 –SO.sub.2 –CH(CH.sub.3).sub.2 OMe OMe C–CH(CH.sub.3).sub.2 O O I-10 OH Phenyl CH.sub.2 –CH.sub.2 –SO.sub.2 –CH(CH.sub.3).sub.2 OMe OMe C–CH(CH.sub.3).sub.3 O O I-11 OH Phenyl CH.sub.2 –CH.sub.2 –SO.sub.2 –CH(CH.sub.3).sub.2 OMe NH–OCH.sub.3 CH O O I-12 OH Phenyl n-Propyl OMe OMe CH O O 174 I-13 OMe Phenyl n-Propyl OMe OMe CH O O I-14 OH Phenyl n-Propyl OEt OEt CH O O I-15 OH Phenyl n-Butyl OMe OMe CH O O I-16 OH Phenyl iso-Butyl OMe OMe CH O O I-17 OH Phenyl iso-Butyl OMe O–CH.sub.2 –CH.sub.2 –C O O I-18 OH Phenyl tert.-Butyl OMe OMe CH O O I-19 OH Phenyl Cyclopropyl OMe OMe CH O O I-20 OH Phenyl Cyclopentyl OMe OMe CH O O I-21 OH Phenyl Cyclohexyl OMe OMe CH O O I-22 OH Phenyl (CH.sub.3).sub.3 C–CH.sub.2 –CH.sub.2 OEt OEt CH O O I-23 OH Phenyl (CH.sub.3).sub.2 CH–CH.sub.2 –CH.sub.2 –CH.sub.2 OMe OMe CH O O 173 I-24 OH Phenyl HO–CH.sub.2 –CH.sub.2 OMe OMe CH O O I-25 OH Phenyl HO.sub.2 C–(CH.sub.2).sub.2 — OMe OMe CH O O I-26 OH Phenyl Cyclopropylmethyl OMe OMe CH O O 115 I-27 OH Phenyl H OMe OMe CH O O I-28 OH Phenyl Methyl OMe OMe CH O — I-29 OH Phenyl Phenyl OMe OMe CH O O 136 I-30 OH Phenyl Phenyl OMe O–CH(CH.sub.3)–CH.sub.2 O-C O I-31 OMe Phenyl Phenyl OMe OMe CH O O I-32 OH Phenyl 4-Isopropyl-Phenyl OMe OMe CH O O I-33 OH Phenyl 4-Me-S-Phenyl OMe OMe CH O O I-34 OH Phenyl 4-Me-O-Phenyl OMe OMe CH O O I-35 OH Phenyl 3-Et-Phenyl OMe OMe CH O O I-36 OH Phenyl 2-Me-Phenyl OMe OMe CH O O I-37 OH Phenyl 2-Cl-Phenyl OMe OMe CH O O I-38 OH Phenyl 3-Br-Phenyl OMe OMe CH O O I-39 OH Phenyl 4-F-Phenyl OMe OMe CH O O I-40 OH Phenyl 4-F-Phenyl OMe OMe CH S O I-41 OH Phenyl 4-CH.sub.3 -Phenyl OMe OMe CH O O I-42 OH Phenyl 3-NO.sub.2 -Phenyl OMe OMe CH O O I-43 OH Phenyl 2-HO-Phenyl OMe OMe CH O O I-44 OH Phenyl 3,4-Dimethoxyphenyl OMe OMe CH O O I-45 OH Phenyl 3,4-Methylenedioxyphenyl OMe OMe CH O O I-46 OH Phenyl 3,4,5-Trimethoxyphenyl OMe OMe CH O O I-47 OH Phenyl Benzyl OMe OMe CH O O I-48 OH Phenyl 2-Cl-Benzyl OMe OMe CH O O I-49 OH Phenyl 3-Br-Benzyl OMe OMe CH O O I-50 OH Phenyl 4-F-Benzyl OMe OMe CH O O I-51 OH Phenyl 2-Me-Benzyl OMe OMe CH O O I-52 OH Phenyl 2-Me-Benzyl OMe O–CH.dbd.CH–C O O I-53 OH Phenyl 3-Et-Benzyl OMe OMe CH O O I-54 OH Phenyl 4-iso-Propyl-Benzyl OMe OMe CH O O I-55 OH Phenyl 4-NO.sub.2 -Propyl-Benzyl OMe OMe CH O O I-56 OH Phenyl 2-Me-5-Propyl-Benzyl OMe OMe CH O O I-57 OH Phenyl 2-Me-5-Propyl-Benzyl OEt OEt CH O O I-58 OH Phenyl 4-Me-2-Propyl-Benzyl OMe OMe CH O O I-59 OH Phenyl 3,4-Methylenedioxybenzyl OMe OMe CH O O I-60 OH 4-F-Phenyl Methyl OMe OMe CH O O 163-165 (decomp.) I-61 OMe 4-F-Phenyl Methyl OEt OEt CH O O I-62 OH 4-Cl-Phenyl Methyl OMe OMe CH O O I-63 OH 4-Me-O-Phenyl Methyl OMe OMe CH O O I-64 OH 4-Me-O-Phenyl Ethyl OMe OMe CH O O I-65 OH 4-Me-Phenyl Methyl OMe OMe CH O O I-66 OH 4-Me-Phenyl Methyl OMe O–CH.sub.2 –CH.sub.2 –C O O I-67 OH 3-CF.sub.3 -Phenyl n-Propyl OMe OMe CH O O I-68 OH 3-CF.sub.3 -Phenyl n-Propyl OMe O–CH(CH.sub.3)–CH.sub.2 O-C O I-69 OH 4-NO.sub.2 -Phenyl Methyl OMe OMe CH O O I-70 OH 4-NO.sub.2 -Phenyl Methyl OMe O–CH.dbd.CH-C O O I-71 OH 3-Cl-Phenyl Ethyl OMe OMe CH O O I-72 OH 2-F-Phenyl Methyl OMe OMe CH O O 193-194 (decomp.) I-73 OH 2-F-Phenyl Methyl OMe OMe CH S O I-74 OH 2-Me-O-Phenyl Methyl OMe OMe CH O O I-75 OH 2-Me-O-Phenyl Methyl OMe OMe CH O S I-76 OH 3,4-Dimethoxy- Methyl OMe OMe CH O O phenyl I-77 OH 3,4-Methylenedi- Methyl OMe OMe CH O O oxyphenyl I-78 OH p-CF.sub.3 -Phenyl Methyl OMe OMe CH O O I-79 OH Phenyl Methyl OMe OEt CH O O I-80 OMe Phenyl Methyl OMe OEt CH S O I-81 OH Phenyl Ethyl OMe NH–OMe CH O O I-82 OH p-Me-O-Phenyl n-Propyl OMe OCF.sub.3 CH O O I-83 OH Phenyl Methyl OMe CF.sub.3 CH O O I-84 OH Phenyl Methyl OMe CF.sub.3 N O O I-85 OH 3,4-Dimethoxy- Benzyl Me Me O O phenyl I-86 OH 3,4-Dimethoxy- Methyl OMe O–CH.sub.2 –CH.sub.2 –C O O phenyl

I-87 OH Phenyl Methyl OMe O–CH.sub.2 –CH.sub.2 –C O O 126 (decomp.) I-88 OH Phenyl Methyl OMe O–CH(CH.sub.3)–CH.sub.2 O-C O I-89 OH Phenyl Methyl OMe N(CH.sub.3)–CH.dbd.CH–C O O 118 I-90 OH Phenyl Methyl OMe S–C(CH.sub.3).dbd.C(CH.sub.3)–C O O I-91 OH Phenyl Methyl OMe O–C(CH.sub.3).dbd.CH–C O O I-92 OH Phenyl Methyl Me O–C(CH.sub.3).dbd.CH–C O O I-93 OH Phenyl Methyl Me O–CH.dbd.CH–C O O I-94 OH 4-F-phenyl Methyl Me S–CH.dbd.CH–C O O I-95 OH 4-F-phenyl H OMe OMe CH O O I-96 OH Phenyl Methyl OMe CH.sub.2 –CH.sub.2 –CH.sub.2 –C O O 149-151 (decomp.) I-97 OH Phenyl Methyl Methyl CH.sub.2 –CH.sub.2 –CH.sub.2 –C O O 157 (decomp.) I-98 OH Phenyl Methyl Ethyl CH.sub.2 –CH.sub.2 –CH.sub.2 –CH.sub.2 –C O O I-99 OH Phenyl Methyl OMe CH.sub.2 –CH.sub.2 –CH.sub.2 –CH.sub.2 –C O O I-100 OH Phenyl Methyl Me Me CH O O I-101 OH Phenyl Methyl Et Et CH O O I-102 OH Phenyl Methyl Me Me C–CH.sub.3 O O I-103 OH Phenyl Methyl OMe Me CH O O I-104 OH Cyclohexyl Methyl OMe OMe CH O O I-105 OH Cyclohexyl Methyl OMe CH.sub.2 –CH.sub.2 –CH.sub.2 –C O O I-106 OH Phenyl Methyl OCH.sub.3 OCH.sub.3 CH S S I-107 OH Phenyl Methyl OCH.sub.3 OCH.sub.3 CH O S 134 I-108 OCH.sub.3 Phenyl Methyl OCH.sub.3 OCH.sub.3 CH S S I-109 OH Phenyl Methyl OCH.sub.3 OCH.sub.3 CH O O I-110 OCH.sub.3 2-Fluorophenyl Methyl OCH.sub.3 OCH.sub.3 CH O O I-111 OC.sub.2 H.sub.5 3-Chlorophenyl Methyl OCH.sub.3 OCH.sub.3 N O O I-112 ON(CH.sub.3).sub.2 4-Bromophenyl Methyl CF.sub.3 CF.sub.3 CH S O I-113 O–CH.sub.2 –C.dbd.CH Phenyl Ethyl OCH.sub.3 CF.sub.3 CH O O I-114 OH Phenyl Propyl OCH.sub.3 OCF.sub.3 CH O S I-115 OCH.sub.3 Phenyl i-Propyl OCH.sub.3 CH.sub.3 CH O O I-116 OC.sub.2 H.sub.5 Phenyl s-Butyl OCH.sub.3 Cl CH S O I-117 ON(CH.sub.3).sub.2 2-Methylphenyl Methyl OCH.sub.3 OCH.sub.3 CH O O I-118 ON(CH.sub.3).sub.2 3-Methoxyphenyl Methyl OCH.sub.3 OCH.sub.3 CH O O I-119 ON.dbd.C(CH.sub.3).sub.2 4-Nitrophenyl Methyl OCH.sub.3 OCH.sub.3 CH O O I-120 ON(CH.sub.3).sub.2 Phenyl 1-Phenylpropyn-3-yl OCH.sub.3 OCF.sub.3 N O S I-121 ON.dbd.C(CH.sub.3).sub.2 2-Hydroxyphenyl Methyl OCH.sub.3 CH.sub.3 N O O I-122 ONSO.sub.2 C.sub.6 H.sub.5 3-Trifluoromethyl- Methyl OCH.sub.3 Cl N O O phenyl I-123 NHPhenyl 4-Dimethylamino- Methyl OCH.sub.3 OCH.sub.3 CH S O phenyl I-124 OC.sub.2 H.sub.5 Phenyl Trifluoroethyl CH.sub.3 CH.sub.3 CH O O I-125 ON(CH.sub.3).sub.2 Phenyl Benzyl Cl Cl CH O O I-126 ON(CH.sub.3).sub.2 Phenyl 2-Methoxyethyl OCH.sub.3 –O–CH.sub.2 –CH.sub.2 — S O I-127 OH Phenyl Phenyl OCH.sub.3 OCH.sub.3 CH O O I-128 OH Phenyl Phenyl OCH.sub.3 –O–CH.sub.2 –CH.sub.2 — O O I-129 OH Phenyl Phenyl OCH.sub.3 OCH.sub.3 N O O I-130 OH Phenyl Phenyl OCH.sub.3 OCH.sub.3 CH S O I-131 OH Phenyl Phenyl OCH.sub.3 OCH.sub.3 CH S S I-132 OH Phenyl Phenyl OCH.sub.3 OCH.sub.3 CH O S I-133 OH Phenyl Phenyl OCH.sub.3 OCH.sub.3 CH O O I-134 OH Phenyl Phenyl OCH.sub.3 OCH.sub.3 CH O O I-135 OH –(CH.sub.2).sub.5 — Phenyl Phenyl OCH.sub.3 CH O O I-136 OH Phenyl 2-Thiazolyl OCH.sub.3 OCH.sub.3 CH O O I-137 OCH.sub.3 2-Fluorophenyl Phenyl OCH.sub.3 OCH.sub.3 CH O O I-138 OC.sub.2 H.sub.5 3-Chlorophenyl Phenyl OCH.sub.3 OCH.sub.3 N O O I-139 ON(CH.sub.3).sub.2 4-Bromophenyl Phenyl CF.sub.3 CF.sub.3 CH O O I-140 O–CH.sub.2 .tbd.CH Phenyl 2-Fluorophenyl OCH.sub.3 CF.sub.3 CH O O I-141 OH Phenyl 3-Chlorophenyl OCH.sub.3

OCF.sub.3 CH O S I-142 OCH.sub.3 Phenyl 4-Bromophenyl OCH.sub.3 CH.sub.3 CH O O I-143 OC.sub.2 H.sub.5 Phenyl 4-Thiazolyl OCH.sub.3 Cl CH S O I-144 ON(CH.sub.3).sub.2 2-Methylphenyl Phenyl OCH.sub.3 OCH.sub.3 CH O O I-145 ON.dbd.C(CH.sub.3).sub.2 3-Methoxyphenyl Phenyl OCH.sub.3 OCH.sub.3 CH O O I-146 OH Phenyl Methyl OCH.sub.3 –CH.sub.2 –CH.sub.2 –CH.sub.2 –C O O I-147 OH 4-Fluorophenyl Methyl OCH.sub.3 OCH.sub.3 CH O O 168 (decomp.) I-148 OH 4-Fluorophenyl Methyl OCH.sub.3 –CH.sub.2 –CH.sub.2 –CH.sub.2 –C O O I-149 NH–SO–C.sub.6 H.sub.5 4-Nitrophenyl Phenyl OCH.sub.3 OCH.sub.3 CH O O I-150 OCH.sub.3 Phenyl 3-Imidazolyl OCH.sub.3 –O–CH.sub.2 –CH.sub.2 O O I-151 OC.sub.2 H.sub.5 Phenyl 4-Imidazolyl OCH.sub.3 CF.sub.3 N S O I-152 ON(CH.sub.3).sub.2 Phenyl 2-Pyrazolyl OCH.sub.3 OCF.sub.3 N O S I-153 ON.dbd.C(CH.sub.3).sub.2 2-Hydroxyphenyl Phenyl OCH.sub.3 CH.sub.3 N O O I-154 NH–SO.sub.2 –C.sub.6 H.sub.5 3-Trifluoromethyl- Phenyl OCH.sub.3 Cl N O O phenyl I-155 NHPhenyl 4-Dimethylamino- Phenyl OCH.sub.3 OCH.sub.3 CH S O phenyl I-156 ONa Phenyl Phenyl OCH.sub.3 OCH.sub.3 CH S S I-157 O–CH.sub.2 –C.tbd.C Phenyl Phenyl OCH.sub.3 OCH.sub.3 N S S I-158 OH Phenyl Phenyl CF.sub.3 CF.sub.3 CH O S I-159 OCH.sub.3 Phenyl Phenyl OCF.sub.3 OCF.sub.3 CH O O I-160 OC.sub.2 H.sub.5 Phenyl 2-Dimethylaminophenyl CH.sub.3 CH.sub.3 CH O O I-161 ON(CH.sub.3).sub.2 Phenyl 3-Hydroxyphenyl Cl Cl CH O O I-162 ON.dbd.C(CH.sub.3).sub.2 Phenyl 4-Trifluoromethylphenyl OCH.sub.3 –O–CH.sub.2 –CH.sub.2 — S O I-163 NH–SO.sub.2 –C.sub.6 H.sub.5 Phenyl 2-Oxazolyl OCH.sub.3 CF.sub.3 N S S I-164 OH Phenyl Methyl CH.sub.3 CH.sub.3 CH O O I-165 OH Cyclohexyl Methyl OCH.sub.3 OCH.sub.3 CH O O I-166 OH Cyclohexyl Methyl OCH.sub.3 CH.sub.2 –CH.sub.2 –CH–C O O I-167 OH Phenyl Methyl N(CH.sub.3).sub.2 N(CH.sub.3).sub.2 CH O O I-168 OH Phenyl Methyl OCH.sub.3 OCH.sub.3 CH O SO.sub.2 I-169 OH Phenyl Methyl OCH.sub.3 OCH.sub.3 CH O SO.sub.2 I-170 OH 3-F-Phenyl Me OMe OMe CH O O I-171 OH 3-F-Phenyl Me OMe CH.sub.2 –CH.sub.2 –CH.sub.2 –C O O I-172 OH 4-F-Phenyl Me OMe CH.sub.2 –CH.sub.2 –CH.sub.2 –C O O 142-143 191.degree. C. I-173 OH 3-MeO-Phenyl Me OMe CH.sub.2 –CH.sub.2 –CH.sub.2 –C O O 158-161 (decomp.) I-174 OH 3-MeO-Phenyl Me OMe OMe CH O O I-175 OH 3-MeO-Phenyl Et OMe CH.sub.2 –CH.sub.2 –CH.sub.2 –C O O I-176 OH Phenyl HO–CH.sub.2 –CH.sub.2 OMe CH.sub.2 –CH.sub.2 –CH.sub.2 –C O O I-177 OH Phenyl Me NMe.sub.2 NMe.sub.2 N O O 181 I-178 OH Phenyl Me OMe OMe N O O I-179 OH I-180 NH–SO.sub.2 -Phenyl Phenyl Me OMe OMe CH O O I-181 NH–SO.sub.2 -Me Phenyl Me OMe OMe CH O O I-182 CH.sub.2 –SO.sub.2 -Phenyl Phenyl Me OMe OMe CH O O I-183 CH.sub.2 –SO.sub.2 -Me Phenyl Me OMe OMe CH O O I-184 –CN Phenyl Me OMe OMe CH O O I-185 Tetrazolyl Phenyl Me OMe OMe CH O O I-186 NH–SO.sub.2 -Phenyl Phenyl Me OMe OMe CH O O 167 I-187 N-Methyltetrazolyl Phenyl Me OMe OMe CH O O I-188 ONa Phenyl Me OMe –O–CH.sub.2 –CH.sub.2 –C– O O 122-139 (zers.) I-189 OH o-F-Phenyl Me OMe –O–CH.sub.2 –CH.sub.2 –C– O O 140-144 (decomp.) I-190 OH m-Me-Phenyl Me OMe OMe CH O O 169-177 I-191 OH m-Me-Phenyl Me OMe –O–CH.sub.2 –CH.sub.2 –C– O O 119-135 (decomp.) I-192 OH p-F-Phenyl Me OMe Me CH O O 137-140 (decomp.) I-193 OH m-F-Phenyl Me Me –O–CH.sub.2 –CH.sub.2 –C– O O 150-152 I-194 OH p-F-Phenyl Me Me –O–CH.sub.2 –CH.sub.2 –C– O O 169-170 __________________________________________________________________________

TABLE II __________________________________________________________________________ ##STR15## No. R.sup.1 A R.sup.6 R.sup.2 R.sup.3 X Y Z m.p. [.degree. C.] __________________________________________________________________________ II-1 OH Bond Methyl OMe OMe CH O O 96-98 II-2 OH CH.sub.2 Methyl OMe OMe CH O O II-3 OH CH.sub.2 –CH.sub.2 Methyl OMe OMe CH O O II-4 OH CH.dbd.CH Methyl OMe OMe CH O O II-5 OH O Methyl OMe OMe CH O O II-6 OH S Methyl OMe OMe CH O O II-7 OH NH(CH.sub.3) Methyl OMe OMe CH O O II-8 OH Bond Isopropyl OMe OMe CH O O 137-139 II-9 OH Bond p-Isopropylphenyl OMe OMe CH O O II-10 OH Bond Benzyl OMe OMe CH O O II-11 OH CH.dbd.CH Ethyl OMe OMe CH O O II-12 OH CH.dbd.CH (CH.sub.3).sub.2 –CH.sub.2 –CH.sub.2 OMe OMe CH O O II-13 OH CH.dbd.CH Cyclopropylmethyl OMe OMe CH O O II-14 OH CH.dbd.CH Methyl OMe O–CH.sub.2 –CH.sub.2 –C O O II-15 OH CH.sub.2 –CH.sub.2 Ethyl OMe O–CH.dbd.CH–C O O II-16 OH CH.sub.2 .dbd.CH.sub.2 Methyl OMe CH.sub.2 –CH.sub.2 –CH.sub.2 –C O O II-17 OH Bond Methyl OMe CH.sub.2 –CH.sub.2 –CH.sub.2 –C O O 147 __________________________________________________________________________

TABLE IV __________________________________________________________________________ ##STR16## R.sup.1 R.sup.4 R.sup.5 R.sup.6 R.sup.2 R.sup.3 X Y Z __________________________________________________________________________ OH Phenyl Methyl Methyl OCH.sub.3 OCH.sub.3 CH S S OH Phenyl Methyl Methyl OCH.sub.3 OCH.sub.3 CH O S OCH.sub.3 Phenyl Methyl Methyl OCH.sub.3 OCH.sub.3 CH S S OH Phenyl i-Propyl Methyl OCH.sub.3 OCH.sub.3 CH O O OCH.sub.3 2-Fluorophenyl Ethyl Methyl OCH.sub.3 OCH.sub.3 CH O O OC.sub.2 H.sub.5 3-Chlorophenyl Propyl Methyl OCH.sub.3 OCH.sub.3 N O O ON(CH.sub.3).sub.2 4-Bromophenyl i-Propyl Methyl CF.sub.3 CF.sub.3 CH S O ON.dbd.C(CH.sub.3).sub.2 2-Thienyl Methyl Methyl OCF.sub.3 OCF.sub.3 CH O S HNSO.sub.2 C.sub.6 H.sub.5 3-Thienyl Methyl Methyl CH.sub.3 CH.sub.3 CH O O NHPhenyl 2-Furyl Methyl Methyl Cl Cl CH O O ONa 3-Furyl Methyl Methyl OCH.sub.3 –OCH.sub.2 –CH.sub.2 — S O O–CH.sub.2 –C.dbd.CH Phenyl Ethyl Ethyl OCH.sub.3 CF.sub.3 CH O O OH Phenyl Propyl Propyl OCH.sub.3 OCF.sub.3 CH O S OCH.sub.3 Phenyl i-Propyl i-Propyl OCH.sub.3 CH.sub.3 CH O O OC.sub.2 H.sub.5 Phenyl Methyl s-Butyl OCH.sub.3 Cl CH S O ON(CH.sub.3).sub.2 2-Methylphenyl Methyl Methyl OCH.sub.3 OCH.sub.3 CH O O ON(CH.sub.3).sub.2 3-Methoxyphenyl Methyl Methyl OCH.sub.3 OCH.sub.3 CH O O ON.dbd.C(CH.sub.3).sub.2 4-Nitrophenyl Methyl Methyl OCH.sub.3 OCH.sub.3 CH O O NHPhenyl 2-Oxazolyl Methyl Methyl CF.sub.3 CF.sub.3 N S O ONa 4-Oxazolyl Methyl 3-Propenyl [sic] OCF.sub.3 OCF.sub.3 N O S O–CH.sub.2 –C.tbd.CH 5-Oxazolyl Methyl 3-Propynyl [sic] CH.sub.3 CH.sub.3 N O O OH 3-Isoxazolyl Methyl Cyclopentyl Cl Cl N O O OCH.sub.3 4-Isoxazolyl Methyl Cyclohexyl OCH.sub.3 –O–CH.sub.2 –CH.sub.2 — O O OC.sub.2 H.sub.5 5-Isoxazolyl Methyl Cyclopropylmethyl OCH.sub.3 CF.sub.3 N S O ON(CH.sub.3).sub.2 Phenyl Methyl 1-Phenyl-3-propynyl OCH.sub.3 OCF.sub.3 N O S [sic] ON.dbd.C(CH.sub.3).sub.2 2-Hydroxyphenyl Methyl Methyl OCH.sub.3 CH.sub.3 N O O ONSO.sub.2 C.sub.6 H.sub.5 3-Trifluoromethylphenyl Methyl Methyl OCH.sub.3 Cl N O O NHPhenyl 4-Dimethylaminophenyl Methyl Methyl OCH.sub.3 OCH.sub.3 CH S O ONa 2-Imidazolyl Ethyl Methyl OCH.sub.3 OCH.sub.3 CH S S O–CH.sub.2 –C.tbd.CH 4-Imidazolyl Propyl Methyl OCH.sub.3 OCH.sub.3 N S S OH 3-Pyrazolyl i-Propyl Methyl CF.sub.3 CF.sub.3 CH O S OCH.sub.3 4-Pyrazolyl Methyl Methyl OCF.sub.3 OCF.sub.3 CH O O OC.sub.2 H.sub.5 Phenyl Methyl Trifluoroethyl CH.sub.3 CH.sub.3 CH O O ON(CH.sub.3).sub.2 Phenyl Methyl Benzyl Cl Cl CH O O ON(CH.sub.3).sub.2 Phenyl Methyl 2-Methoxyethyl OCH.sub.3 –O–CH.sub.2 –CH.sub.2 — S O ON.dbd.C(CH.sub.3).sub.2 Phenylpropyl Methyl 3-Methoxycarbonyl- OCH.sub.3 CF.sub.3 N S S [sic] NH-Phenyl 2-Pyridyl Methyl 2-Chloroethyl OCH.sub.3 OCF.sub.3 N S S ONa 3-Pyridyl Methyl Methyl OCH.sub.3 CH.sub.3 N O O O–CH.sub.2 –C.tbd.CH 4-Pyridyl Methyl Methyl OCH.sub.3 Cl N O O OCH.sub.3 Phenyl CH.sub.3 Phenyl OCH.sub.3 OCH.sub.3 CH O O OH Phenyl CH.sub.3 Phenyl OCH.sub.3 OCH.sub.3 CH O O OH Phenyl CH.sub.3 Phenyl OCH.sub.3 –O–CH.sub.2 –CH.sub.2 — O O OH Phenyl CH.sub.3 Phenyl OCH.sub.3 OCH.sub.3 N O O OH Phenyl CH.sub.3 Phenyl OCH.sub.3 OCH.sub.3 CH S O OH Phenyl CH.sub.3 Phenyl OCH.sub.3 OCH.sub.3 CH S S OH Phenyl CH.sub.3 Phenyl OCH.sub.3 OCH.sub.3 CH O S OH Phenyl H Phenyl OCH.sub.3 OCH.sub.3 CH O O OH Phenyl i-Propyl Phenyl OCH.sub.3 OCH.sub.3 CH O O OH CH.sub.3 CH.sub.3 Phenyl OCH.sub.3 OCH.sub.3 CH O O OH –(CH.sub.2).sub.5 — Phenyl Phenyl

OCH.sub.3 CH O O OH Phenyl CH.sub.3 2-Thiazolyl OCH.sub.3 OCH.sub.3 CH O O OH 2-Thienyl CH.sub.3 Phenyl OCH.sub.3 OCH.sub.3 CH O O OCH.sub.3 2-Fluorophenyl Ethyl Phenyl OCH.sub.3 OCH.sub.3 CH O O OC.sub.2 H.sub.5 3-Chlorophenyl Propyl Phenyl OCH.sub.3 OCH.sub.3 N O O ON(CH.sub.3).sub.2 4-Bromophenyl i-Propyl Phenyl CF.sub.3 CF.sub.3 CH S O ON.dbd.C(CH.sub.3).sub.2 2-Thienyl Methyl Phenyl OCF.sub.3 OCF.sub.3 CH O S NH–SO.sub.2 –C.sub.6 H.sub.5 3-Thienyl Methyl Phenyl CH.sub.3 CH.sub.3 CH O O NHPhenyl 2-Furyl Methyl Phenyl Cl Cl CH O O ONa 3-Furyl Methyl Phenyl OCH.sub.3 –O–CH.sub.2 –CH.sub.2 — S O O–CH.sub.2 .tbd.CH Phenyl Ethyl 2-Fluorophenyl OCH.sub.3 CF.sub.3 CH O O OH Phenyl Propyl 3-Chlorophenyl OCH.sub.3 OCF.sub.3 CH O S OCH.sub.3 Phenyl i-Propyl 4-Bromophenyl OCH.sub.3 CH.sub.3 CH O O OC.sub.2 H.sub.5 Phenyl Methyl 4-Thiazolyl OCH.sub.3 Cl CH S O ON(CH.sub.3).sub.2 2-Methylphenyl Methyl Phenyl OCH.sub.3 OCH.sub.3 CH O O ON.dbd.C(CH.sub.3).sub.2 3-Methoxyphenyl Methyl Phenyl OCH.sub.3 OCH.sub.3 CH O O NH–SO–C.sub.6 H.sub.5 4-Nitrophenyl Methyl Phenyl OCH.sub.3 OCH.sub.3 CH O O NHPhenyl Methyl Methyl Phenyl CF.sub.3 CF.sub.3 N S O ONa Methyl Methyl 2-Methylphenyl OCF.sub.3 OCF.sub.3 N O S O–CH.sub.2 –C.tbd.CH Methyl Methyl 3-Methoxyphenyl CH.sub.3 CH.sub.3 N O O OH Methyl Methyl 4-Nitrophenyl Cl Cl N O O OCH.sub.3 Phenyl Methyl 3-Imidazolyl OCH.sub.3 –O–CH.sub.2 –CH.sub.2 — O O OC.sub.2 H.sub.5 Phenyl Methyl 4-Imidazolyl OCH.sub.3 CF.sub.3 N S O ON(CH.sub.3).sub.2 Phenyl Methyl 2-Pyrazolyl OCH.sub.3 OCF.sub.3 N O S ON.dbd.C(CH.sub.3).sub.2 2-Hydroxyphenyl Methyl Phenyl OCH.sub.3 CH.sub.3 N O O NH–SO.sub.2 –C.sub.6 H.sub.5 3-Trifluoromethylphenyl Methyl Phenyl OCH.sub.3 Cl N O O NHPhenyl 4-Dimethylaminophenyl Methyl Phenyl OCH.sub.3 OCH.sub.3 CH S O ONa 3-Imidazolyl Ethyl Phenyl OCH.sub.3 OCH.sub.3 CH S S O–CH.sub.2 –C.tbd.CH 4-Imidazolyl Propyl Phenyl OCH.sub.3 OCH.sub.3 N S S OH 3-Pyrazolyl i-Propyl Phenyl CF.sub.3 CF.sub.3 CH O S OCH.sub.3 4-Pyrazolyl Methyl Phenyl OCF.sub.3 OCF.sub.3 CH O O OC.sub.2 H.sub.5 Phenyl Methyl 2-Dimethylaminophenyl CH.sub.3 CH.sub.3 CH O O ON(CH.sub.3).sub.2 Phenyl Methyl 3-Hydroxyphenyl Cl Cl CH O O ON.dbd.C(CH.sub.3).sub.2 Phenyl Methyl 4-Trifluoromethyl- OCH.sub.3 –O–CH.sub.2 –CH.sub.2 — S O phenyl NH–SO.sub.2 –C.sub.6 H.sub.5 Phenyl Methyl 2-Oxazolyl OCH.sub.3 CF.sub.3 N S S NH-Phenyl 2-Pyridyl Methyl 4-Isoxazolyl OCH.sub.3 OCF.sub.3 N S S ONa 3-Pyridyl Methyl Phenyl OCH.sub.3 CH.sub.3 N O O O–CH.sub.2 –C.tbd.CH 4-Pyridyl Methyl Phenyl OCH.sub.3 Cl N O O __________________________________________________________________________

Synthesis of Compounds of the Formula VI

EXAMPLE 26

Methyl 3-methoxy-3-(3-methoxyphenyl-2-hydroxybutyrate

19.5 g (88 mmol) of methyl 3-(3-methoxyphenyl)-2,3-epoxybutyrate are dissolved in 200 ml of absolute methanol, and 0.1 ml of boron trifluoride etherate is added. The mixture is stirred at room temperature for 12 hours and the solvent is removed by distillation. The residue is taken up in ethyl acetate, washed with sodium bicarbonate solution and water and dried over sodium sulfate. After removal of the solvent by distillation, 21.1 g of a pale yellow remain.

Yield: 94% (1:1 mixture of diastereomers)

EXAMPLE 27

Methyl 3-benzyloxy-3-phenyl-2-hydroxybutyrate

9.6 g (50 mmol) of methyl 3-phenyl-2,3-epoxybutyrate are dissolved in 150 ml of benzyl alcohol, and 0.5 ml of concentrated sulfuric acid is added. The mixture is stirred at 50.degree. C. for 6 hours and allowed to cool to room temperature. After neutralization with sodium bicarbonate solution, the excess benzyl alcohol is removed by distillation under high vacuum, and the residue is purified by flash chromatography on silica gel with 9:1 n-hexane/ethyl acetate. After removal of the solvent by distillation, 6.5 g of a colorless oil remain.

Yield: 43% (3:2 mixture of diastereomers)

All the compounds mentioned in Table V were prepared in a similar way.

TABLE V ______________________________________ Intermediates of the Formula VI with R.sup.1 = OCH.sub.3 ##STR17## No. R.sup.6 R.sup.4 R.sup.5 DR* M.p. [.degree. C.] ______________________________________ 1.1 Methyl 3-Methoxyphenyl Methyl 1:1 Oil 1.2 Benzyl Phenyl Methyl 3:2 Oil 1.3 Methyl 2-Fluorophenyl Methyl 1:1 Oil 1.4 Methyl 4-i-Propylphenyl Methyl 1.5 Methyl 2-Methylphenyl Methyl 2:1 Oil 1.6 Methyl 3-Methylphenyl Methyl 1.7 Methyl 4-Methylphenyl Methyl 3:2 Oil 1.8 Methyl 3-Nitrophenyl Methyl 1.9 Methyl 4-Bromophenyl Methyl 3:1 Oil 1.10 Methyl 2-Furyl Methyl 1.11 Methyl 3-Furyl Methyl 1.12 Methyl 2-Thienyl Methyl 1.13 Methyl 3-Thienyl Methyl 1.14 Methyl 2-Pyridyl Methyl 1.15 Methyl 3-Pyridyl Methyl 1.16 Methyl 4-Pyridyl Methyl 1.17 Methyl 2-Thiazolyl Methyl 1.18 Methyl 3-Isoxazolyl Methyl 1.19 Methyl 4-Imidazolyl Methyl 1.20 Methyl 2-Pyrazolyl Methyl 1.21 Methyl 4-Chlorophenyl Methyl 2:1 Oil 1.22 Benzyl 3-Methylphenyl Methyl 1:1 Oil 1.23 Methyl 4-Fluorophenyl Methyl 1:1 Oil 1.24 Benzyl 4-Bromophenyl Methyl 1:1 Oil 1.25 Benzyl 4-Chlorophenyl Methyl 3:2 Oil 1.26 Benzyl 4-Fluorophenyl Methyl 1:1 Oil 1.27 Methyl Phenyl Ethyl 1:1 Oil 1.28 Methyl 3-Nitrophenyl Methyl 2:1 Oil 1.29 Ethyl 4-Methylphenyl Methyl 1:1 Oil 1.30 Benzyl 4-Methylphenyl Methyl 1:1 Oil 1.31 Benzyl Phenyl Ethyl 1:0 Oil 1.32 4-Fluorobenzyl Phenyl Methyl 1:1 Oil ______________________________________ *Diastereomer ratio

Synthesis of Compounds of the General Formula I

EXAMPLE 28

Methyl 3-benzyloxy-3-phenyl-2-(4,6-dimethoxy-2-pyrimidinyl)oxybutyrate

3 g (10 mmol) of methyl 3-benxyloxy-3-phenyl-2-hydroxybutyrate (Compound 1.1) are dissolved in 40 me of dimethylformamide, and 0.3 g (12 mmol) of sodium hydride is added. The mixture is stirred for 1 hour and then 2.2 g (10 mmol) of 4,6-dimethoxy-2-methylsulfonylpyrimidine are added. The mixture is stirred at room temperature for 24 hours and then cautiously hydrolyzed with 10 ml of water, the pH is adjusted to 5 with acetic acid, and the solvent is removed by distillation under high vacuum. The residue is taken up in 100 ml of ethyl acetate, washed with water, dried over sodium sulfate and distilled to remove solvents. 10 mol of methyl t-butyl either are added to the residue,