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Cancer Patent Abstract
(-)-(2S,4S)-1-(2-Hydroxymethyl-1,3-dioxolan-4-yl)cytosine (also
referred to as (-)-OddC) or its derivative and its use to treat
cancer in animals, including humans.
Cancer Patent Claims
We claim:
1. A method for treating a cancer selected from the group consisting
of non-small cell lung cancer, colon cancer, breast cancer, prostate
cancer, ovarian cancer, leukemia, CNS cancer, melanoma, renal cancer,
and lymphoma cancer in a host animal comprising administering to
said animal in need thereof an effective amount of a compound of
the formula ##STR00005## wherein R.sup.1 and R.sup.2 are hydrogen,
or a pharmaceutically acceptable salt thereof.
2. The method according to claim 1 wherein said cancer is prostate
cancer.
3. The method according to claim 1 wherein said cancer is non-small
cell lung cancer.
4. The method according to claim 1 wherein said cancer is colon
cancer.
5. The method according to claim 1 wherein said cancer is breast
cancer.
6. The method according to claim 1 wherein said cancer is ovarian
cancer.
7. The method according to claim 1 wherein said cancer is lymphoma
cancer.
8. The method according to claim 1 wherein said cancer is leukemia.
9. A method for treating a cancer selected from the group consisting
of non-small cell lung cancer, colon cancer, breast cancer, prostate
cancer, ovarian cancer, leukemia, CNS cancer, melanoma, renal cancer,
and lymphoma cancer in a human comprising administering to said
human in need thereof an effective amount of a compound of the formula
##STR00006## wherein R.sup.1 and R.sup.2 are hydrogen, or a pharmaceutically
acceptable salt thereof.
10. The method according to claim 9 wherein said cancer is prostate
cancer.
11. The method according to claim 9 wherein said cancer is non-small
cell lung cancer.
12. The method according to claim 9 wherein said cancer is colon
cancer.
13. The method according to claim 9 wherein said cancer is breast
cancer.
14. The method according to claim 9 wherein said cancer is ovarian
cancer.
15. The method according to claim 9 wherein said cancer is lymphoma
cancer.
16. The method according to claim 9 wherein said leukemia is acute
lymphoblastic leukemia.
Cancer Patent Description
FIELD OF THE INVENTION
This invention is in the area of medicinal chemistry, and in particular
is (-)-(2S,4S)-1-(2-hydroxymethyl-1,3-dioxolan-4-yl)cytosine (also
referred to as (-)-OddC) or its derivative, and its use to treat
cancer in animals, including humans.
BACKGROUND OF THE INVENTION
A tumor is an unregulated, disorganized proliferation of cell growth.
A tumor is malignant, or cancerous, if it has the properties of
invasiveness and metastasis. Invasiveness refers to the tendency
of a tumor to enter surrounding tissue, breaking through the basal
laminas that define the boundaries of the tissues, thereby often
entering the body's circulatory system. Metastasis refers to the
tendency of a tumor to migrate to other areas of the body and establish
areas of proliferation away from the site of initial appearance.
Cancer is now the second leading cause of death in the United States.
Over 8,000,000 persons in the United States have been diagnosed
with cancer, with 1,208,000 new diagnoses expected in 1994. Over
500,000 people die annually from the disease in this country.
Cancer is not fully understood on the molecular level. It is known
that exposure of a cell to a carcinogen such as certain viruses,
certain chemicals, or radiation, leads to DNA alteration that inactivates
a "suppressive" gene or activates an "oncogene".
Suppressive genes are growth regulatory genes, which upon mutation,
can no longer control cell growth. Oncogenes are initially normal
genes (called prooncogenes) that by mutation or altered context
of expression become transforming genes. The products of transforming
genes cause inappropriate cell growth. More than twenty different
normal cellular genes can become oncogenes by genetic alteration.
Transformed cells differ from normal cells in many ways, including
cell morphology, cell-to-cell interactions, membrane content, cytoskeletal
structure, protein secretion, gene expression and mortality (transformed
cells can grow indefinitely).
All of the various cell types of the body can be transformed into
benign or malignant tumor cells. The most frequent tumor site is
lung, followed by colorectal, breast, prostate, bladder, pancreas,
and then ovary. Other prevalent types of cancer include leukemia,
central nervous system cancers, including brain cancer, melanoma,
lymphoma, erythroleukemia, uterine cancer, and head and neck cancer.
Cancer is now primarily treated with one or a combination of three
types of therapies: surgery, radiation, and chemotherapy. Surgery
involves the bulk removal of diseased tissue. While surgery is sometimes
effective in removing tumors located at certain sites, for example,
in the breast, colon, and skin, it cannot be used in the treatment
of tumors located in other areas, such as the backbone, nor in the
treatment of disseminated neoplastic conditions such as leukemia.
Chemotherapy involves the disruption of cell replication or cell
metabolism. It is used most often in the treatment of leukemia,
as well as breast, lung, and testicular cancer.
There are five major classes of chemotherapeutic agents currently
in use for the treatment of cancer: natural products and their derivatives;
anthracyclines; alkylating agents; antiproliferatives (also called
antimetabolites); and hormonal agents. Chemotherapeutic agents are
often referred to as antineoplastic agents.
The alkylating agents are believed to act by alkylating and cross-linking
guanine and possibly other bases in DNA, arresting cell division.
Typical alkylating agents include nitrogen mustards, ethyleneimine
compounds, alkyl sulfates, cisplatin, and various nitrosoureas.
A disadvantage with these compounds is that they not only attack
malignant cells, but also other cells which are naturally dividing,
such as those of bone marrow, skin, gastro-intestinal mucosa, and
fetal tissue.
Antimetabolites are typically reversible or irreversible enzyme
inhibitors, or compounds that otherwise interfere with the replication,
translation or transcription of nucleic acids.
Several synthetic nucleosides have been identified that exhibit
anticancer activity. A well known nucleoside derivative with strong
anticancer activity is 5-fluorouracil. 5-Fluorouracil has been used
clinically in the treatment of malignant tumors, including, for
example, carcinomas, sarcomas, skin cancer, cancer of the digestive
organs, and breast cancer. 5-Fluorouracil, however, causes serious
adverse reactions such as nausea, alopecia, diarrhea, stomatitis,
leukocytic thrombocytopenia, anorexia, pigmentation, and edema.
Derivatives of 5-fluorouracil with anti-cancer activity have been
described in U.S. Pat. No. 4,336,381, and in Japanese patent publication
Nos. 50-50383, 50-50384, 50-64281, 51-146482, and 53-84981.
U.S. Pat. No. 4,000,137 discloses that the peroxidate oxidation
product of inosine, adenosine, or cytidine with methanol or ethanol
has activity against lymphocytic leukemia.
Cytosine arabinoside (also referred to as Cytarabin, araC, and
Cytosar) is a nucleoside analog of deoxycytidine that was first
synthesized in 1950 and introduced into clinical medicine in 1963.
It is currently an important drug in the treatment of acute myeloid
leukemia. It is also active against acute lymphocytic leukemia,
and to a lesser extent, is useful in chronic myelocytic leukemia
and non-Hodgkin's lymphoma. The primary action of araC is inhibition
of nuclear DNA synthesis. Handschumacher, R. and Cheng, Y., "Purine
and Pyrimidine Antimetabolites", Cancer Medicine, Chapter XV-1,
3rd Edition, Edited by J. Holland, et al., Lea and Febigol, publishers.
5-Azacytidine is a cytidine analog that is primarily used in the
treatment of acute myelocytic leukemia and myelodysplastic syndrome.
2-Fluoroadenosine-5'-phosphate (Fludara, also referred to as FaraA))
is one of the most active agents in the treatment of chronic lymphocytic
leukemia. The compound acts by inhibiting DNA synthesis. Treatment
of cells with F-araA is associated with the accumulation of cells
at the G1/S phase boundary and in S phase; thus, it is a cell cycle
S phase-specific drug. Incorporation of the active metabolite, F-araATP,
retards DNA chain elongation. F-araA is also a potent inhibitor
of ribonucleotide reductase, the key enzyme responsible for the
formation of DATP.
2-Chlorodeoxyadenosine is useful in the treatment of low grade
B-cell neoplasms such as chronic lymphocytic leukemia, non-Hodgkins'
lymphoma, and hairy-cell leukemia. The spectrum of activity is similar
to that of Fludara. The compound inhibits DNA synthesis in growing
cells and inhibits DNA repair in resting cells.
Although a number of chemotherapeutic agents have been identified
and are currently used for the treatment of cancer, new agents are
sought that are efficacious and which exhibit low toxicity toward
healthy cells.
Therefore, it is an object of the present invention to provide
compounds that exhibit anti-tumor, and in particular, anti-cancer,
activity.
It is another object of the present invention to provide pharmaceutical
compositions for the treatment of cancer.
It is further object of the present invention to provide a method
for the treatment of cancer.
SUMMARY OF THE INVENTION
A method and composition for the treatment of cancer in humans
and other host animals is disclosed that includes administering
an effective amount of (-)-(2S,4S)-1-(2-hydroxymethyl-1,3-dioxolan-4-yl)cytosine
(also referred to as (-)-OddC), a pharmaceutically acceptable derivative
thereof, including a 5' or N.sup.4 alkylated or acylated derivative,
or a pharmaceutically acceptable salt thereof, optionally in a pharmaceutically
acceptable carrier.
In a preferred embodiment, (-)-(2S,4S)-1-(2-hydroxymethyl-1,3-dioxolan-4-yl)cytosine
is provided as the indicated enantiomer and substantially in the
absence of its corresponding enantiomer (i.e., in enantiomerically
enriched, including enantiomerically pure form).
It is believed that (-)-(2S,4S)-1-(2-hydroxymethyl-1,3-dioxolan-4-yl)cytosine
is the first example of an "L"-nucleoside that exhibits
anti-tumor activity. (-)-(2S,4S)-1-(2-Hydroxymethyl-1,3-dioxolan-4-yl)cytosine
has the structure illustrated in Formula I.
##STR00001##
It has been discovered that (-)-(2S,4S)-1-(2-hydroxymethyl-1,3-dioxolan-4-yl)cytosine
exhibits significant activity against cancer cells and exhibits
low toxicity toward healthy cells in the host. Nonlimiting examples
of cancers that can be treated with this compound include lung,
colorectal, breast, prostate, bladder, pancreas, ovarian, leukemia,
and lymphoma.
In an alternative embodiment, a method and composition for the
treatment of cancer in humans and other host animals is disclosed
that includes administering an effective amount of a compound of
the formula:
##STR00002## wherein R is F, Cl, --CH.sub.3, --C(H).dbd.CH.sub.2,
--C.dbd.CH, or --C.dbd.N and R.sup.1 is hydrogen, alkyl, acyl, monophosphate,
diphosphate, or triphosphate, or a pharmaceutically acceptable derivative
thereof, optionally in a pharmaceutically acceptable carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 indicates the ID.sub.50 of (-)-OddC and a combination of
(-)-OddC+THU (tetrahydrouridine, a cytidine deaminase inhibitor)
on colon cancer cells. The graph plots growth inhibition as a percentage
of control growth vs. concentration (.mu.M). In the graph, the data
for (-)-OddC alone is represented by (.circle-solid.) and the data
for (-)-OddC+THU is represented by (--.tangle-solidup.--).
FIG. 2 is a graph of tumor growth weight for mouse carcinoma (Colon
38) treated twice a day with (-)-OddC in a dosage amount of 25 mg/kgbid.
The graph plots tumor growth as a percentage of original tumor weight
vs. days. Treatment of the mice occurred in days 1, 2, 3, 4 and
5. In the graph, the data for the control (no administration of
(-)-OddC) is represented by ( ), the data for (-)-OddC is represented
by (--.tangle-solidup.--).
FIG. 3 indicates the survival rate of P388 leukemic mice that have
been treated with (-)-OddC. The graph plots percentage of survival
vs. days treated. Treatment of the mice occurred in days 1, 2, 3,
4 and 5. In the graph, the survival rate of the control (no administration
of (-)-OddC) is represented by (.circle-solid.), the survival rate
of those administered (-)-OddC at 25 mg/kgbid twice a day is represented
by (--.DELTA.--), and the survival rate of mice administered (-)-OddC
once a day at 50 mg/kgbid is represented by (O).
FIG. 4 is a plot of the relative sensitivity of certain cancer
cell lines to (-)-OddC on the basis of GI50. Bars extending to the
right represent sensitivity of the cell line to (-)-OddC in excess
of the average sensitivity of all tested cell lines. Since the bar
scale is logarithmic, a bar 2 units to the right implies the compound
achieved GI50 for the cell line at a concentration one-hundredth
the mean concentration required over all cell lines, and thus the
cell line is unusually sensitive to (-)-OddC. Bars extending to
the left correspondingly imply sensitivity less than the mean.
DETAILED DESCRIPTION OF THE INVENTION
The invention as disclosed herein is a method and composition for
the treatment of tumors, and in particular, cancer in humans or
other host animals, that includes administering an effective amount
of (-)-(2S,4S)-1-(2-hydroxymethyl-1,3-dioxolan-4-yl)cytosine, a
physiologically acceptable derivative of the compound, including
a 5' or N.sup.4 alkylated or acylated derivative, or a physiologically
acceptable salt thereof, optionally in a pharmaceutically acceptable
carrier.
(-)-(2S,4S)-1-(2-Hydroxymethyl-1,3-dioxolan-4-yl)cytosine is referred
to as an "L"-nucleoside. Since the 2 and 5 carbons of
the dioxolane ring are chiral, their nonhydrogen substituents (CH.sub.2OH
and the cytosine base, respectively) can be either cis (on the same
side) or trans (on opposite sides) with respect to the dioxolane
ring system. The four optical isomers therefore are represented
by the following configurations (when orienting the dioxolane moiety
in a horizontal plane such that the oxygen in the 3-position is
in front): cis (with both groups "up", which corresponds
to the configuration of naturally occurring nucleosides, referred
to as a "D"-nucleoside), cis (with both groups "down",
which is the non-naturally occurring configuration, referred to
as an "L"-nucleoside), trans (with the C2 substituent
"up" and the CS substituent "down"), and trans
(with the C2 substituent "down" and the CS substituent
"up"). It is believed that (-)-(2S,4S)-1-(2-hydroxymethyl-1,3-dioxolan-4-yl)cytosine
or its derivative is the first example of an "L"-nucleoside
that exhibits anti-tumor activity. This is surprising, in light
of the fact that this "L" nucleoside configuration does
not occur in nature.
As used herein, the term "enantiomerically enriched"
refers to a nucleoside composition that includes at least approximately
95%, and preferably approximately 97%, 98%, 99%, or 100% of a single
enantiomer of that nucleoside. In a preferred embodiment, (-)-(2S,4S)-1-(2-hydroxymethyl-1,3-dioxolan-4-yl)cytosine
is provided as the indicated enantiomer and substantially in the
absence of its corresponding enantiomer (i.e., in enantiomerically
enriched, including enantiomerically pure form).
The active compound can be administered as any derivative that
upon administration to the recipient, is capable of providing directly
or indirectly, the parent (-)-OddC compound, or that exhibits activity
itself. Nonlimiting examples are the pharmaceutically acceptable
salts (alternatively referred to as "physiologically acceptable
salts") of (-)-OddC, the 5-derivatives as illustrated above,
and the 5' and N.sup.4 acylated or alkylated derivatives of the
active compound (alternatively referred to as "physiologically
active derivatives"). In one embodiment, the acyl group is
a carboxylic acid ester (--C(O)R) in which the non-carbonyl moiety
of the ester group is selected from straight, branched, or cyclic
alkyl (typically C.sub.1 to C.sub.18, and more typically C.sub.1
to C.sub.5), alkaryl, aralkyl, alkoxyalkyl including methoxymethyl,
aralkyl including benzyl, aryloxyalkyl such as phenoxymethyl; aryl
including phenyl optionally substituted with halogen, C.sub.1 to
C.sub.4 alkyl or C.sub.1 to C.sub.4 alkoxy; sulfonate esters such
as alkyl or aralkyl sulphonyl including methanesulfonyl, the mono,
di or triphosphate ester, trityl or monomethoxytrityl, substituted
benzyl, trialkylsilyl (e.g. dimethyl-t-butylsilyl) or diphenylmethylsilyl.
Aryl groups in the esters optimally comprise a phenyl group.
Specific examples of pharmaceutically acceptable derivatives of
(-)-O-ddC include, but are not limited to:
##STR00003## wherein R is F, Cl, --CH.sub.3, --C(H).dbd.CH.sub.2,
--C.dbd.CH, or --C.dbd.N, and R.sub.1 and R.sub.2 are independently
selected from the group consisting of hydrogen, alkyl and acyl,
specifically including but not limited to methyl, ethyl, propyl,
butyl, pentyl, hexyl, isopropyl, isobutyl, sec-butyl, t-butyl, isopentyl,
amyl, t-pentyl, 3-methylbutyryl, hydrogen succinate, 3-chlorobenzoate,
cyclopentyl, cyclohexyl, benzoyl, acetyl, pivaloyl, mesylate, propionyl,
butyryl, valeryl, caproic, caprylic, capric, lauric, myristic, palmitic,
stearic, oleic, and amino acids including but not limited to alanyl,
valinyl, leucinyl, isoleucinyl, prolinyl, phenylalaninyl, tryptophanyl,
methioninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl,
asparaginyl, glutaminyl, aspartoyl, glutaoyl, lysinyl, argininyl,
and histidinyl. In a preferred embodiment, the derivative is provided
as the indicated enantiomer and substantially in the absence of
its corresponding enantiomer (i.e., in enantiomerically enriched,
including enantiomerically pure form).
(-)-OddC or its derivative can be provided in the form of pharmaceutically
acceptable salts. As used herein, the term pharmaceutically acceptable
salts or complexes refers to salts or complexes of (-)-OddC or its
derivatives that retain the desired biological activity of the parent
compound and exhibit minimal, if any, undesired toxicological effects.
Nonlimiting examples of such salts are (a) acid addition salts formed
with inorganic acids (for example, hydrochloric acid, hydrobromic
acid, sulfuric acid, phosphoric acid, nitric acid, and the like),
and salts formed with organic acids such as acetic acid, oxalic
acid, tartaric acid, succinic acid, malic acid, ascorbic acid, benzoic
acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid,
naphthalenesulfonic acids, naphthalenedisulfonic acids, and polygalacturonic
acid; (b) base addition salts formed with polyvalent metal cations
such as zinc, calcium, bismuth, barium, magnesium, aluminum, copper,
cobalt, nickel, cadmium, sodium, potassium, and the like, or with
an organic cation formed from N,N-dibenzylethylene-diamine, ammonium,
or ethylenediamine; or (c) combinations of (a) and (b); e.g., a
zinc tannate salt or the like.
Modifications of the active compound, specifically at the N.sup.4
and 5'-0 positions, can affect the solubility, bioavailability and
rate of metabolism of the active species, thus providing control
over the delivery of the active species. Further, the modifications
can affect the anticancer activity of the compound, in some cases
increasing the activity over the parent compound. This can easily
be assessed by preparing the derivative and testing its anticancer
activity according to the methods described herein, or other method
known to those skilled in the art.
II. Preparation of the Active Compounds
(-)-OddC can be prepared according to the method disclosed in detail
in PCT International Publication No. WO 92/18517, published on Oct.
29, 1992, or by the method disclosed in Scheme 1 and working examples
1 7 provided below, or by any other method known to those skilled
in the art. These methods, or other known methods, can be adapted
for the preparation of the exemplified derivatives of (-)-OddC.
##STR00004##
EXAMPLE 1
Preparation of 6-Anhydro-L-gulose
6-Anhydro-L-gulose was prepared in one step from L-gulose by the
treatment of L-gulose with an acid, e.g., 0.5N HCl, in 60% yield
(Evans, M. E., et al., Carbohydr. Res. (1973), 28, 359). Without
selective protection, as was done before (Jeong, L. S. et al. Tetrahedron
Lett. (1992), 33, 595 and Beach, J. W. et al. J. Org. Chem. (1992,
in press), (2) was directly converted to dioxolane triol (3) by
oxidation with NaIO.sub.4, followed by reduction with NaBH.sub.4,
which without isolation, was converted to isopropylidene derivative
(4). Benzoylation to (5), deprotection to (6), and oxidation of
diol (6) gave the acid (7). Oxidative decarboxylation of (7) with
Pb(OAc).sub.4 in dry THF gave the acetate (8), the key intermediate
in good yield. The acetate was condensed with the desired pyrimidines
(e.g., silylated thymine and N-acetylcytosine) in the presence of
TMSOTf to afford an .alpha.,.beta.-mixture, which was separated
on a silica gel column to obtain the individual isomers (9 and 10).
Debenzoylation with methanolic ammonia gave the desired (-)-OddC.
(11).
EXAMPLE 2
Preparation of (-)-1,6-Anhydro-.alpha.-L-gulopyranose (2)
A mixture of L-gulose (1) (33 g, 0.127 mol) and 0.5 N HCl (330
mL, 0.165 mol) was refluxed for 20 hours. The mixture was cooled
and neutralized to pH 6 by a resin (Dowex-2, HCO.sub.3-form) with
air bubbling. The resin was recycled by washing with 10% HCl, water,
methanol, water and saturated NaHCO.sub.3 solution. The reaction
mixture was filtered and the resin was washed with water (500 mL).
The combined filtrate was concentrated to dryness and dried in vacuo
overnight. The residue was purified over a column (5 cm depth, silica
gel, mesh, CHCl.sub.3--CH.sub.3OH, 10:1) to give a slightly yellow
solid, which was recrystallized from absolute alcohol to give a
colorless solid (2) [R.sub.f=0.43 (CHCl.sub.3--CH.sub.3OH, 5:1),
7.3 g, 35.52%]. The L-gulose R.sub.f=0.07, 11 g) obtained was recycled
to give (2) (5 g, total yield 60%): mp 142.5 145.degree. C.; .sup.1H
NMR (DMSO-d.sub.6) .delta. 3.22 3.68 (m, 4H, H-2, -3, -4 and -6a),
3.83 (d, J.sub.6b,6a=7.25 Hz, 1H, H.sub.b-6), 4.22 (pseudo t, J.sub.5,6a=4.61
and 4.18 Hz, H, H-5), 4.46 (d, J.sub.2-OH,2=6.59 Hz, 1H, 2-OH, exchangeable
with D.sub.2O), 4.62 (d, J.sub.3-OH,3=5.28 Hz, 1H, 3-OH, exchangeable
with D.sub.2O), 5.07 (d, J.sub.4-OH,4=4.84 Hz, 1H, 4-OH, exchangeable
with D.sub.2O), 5.20 (d, J.sub.1,2=2.19 Hz, 1H, H-1). [.alpha.].sub.D.sup.25-50.011
(c, 1.61, CH.sub.3OH).
EXAMPLE 3
Preparation of (-)-(1'S,2S,4S)-4-(1,2-Dihydroxyethyl-1,2-O-Isopropylidene)-2-hydroxymeth-
yl)-dioxolane (4)
A solution of NaIO.sub.4 (22.36 g, 0.1 mol) in water (300 mL) was
added in a dropwise manner over 10 minutes to a solution of (2)
(11.3 g, 0.07 mol) in methanol (350 mL) cooled to 0.degree. C. The
mixture was stirred mechanically for 15 minutes. NaBH.sub.4 (7.91
g, 0.21 mol) was added to this mixture and the reaction mixture
was stirred for 10 minutes at 0.degree. C. The white solid was filtered
off and the solid was washed with methanol (300 mL). The combined
filtrate was neutralized by 0.5 N HCl (.about.200 mL) and concentrated
to dryness. The residue was dried in vacuo overnight. The syrupy
residue was triturated with methanol-acetone (1:5, 1200 mL) using
a mechanical stirrer (5 hours) and the white solid (1st.) was filtered
off. The filtrate was concentrated to dryness and the residue was
dissolved in acetone (500 mL) and followed by p-toluene sulfonic
acid (6.63 g, 0.035 mol). After stirring for 6 hours, the mixture
was neutralized by triethylamine, the solid (2nd.) was filtered
off and the filtrate was concentrated to dryness. The residue was
dissolved in ethyl acetate (350 mL) and washed with water (50 mL.times.2),
dried (MgSO.sub.4), filtered, and evaporated to give crude (4) (3.6
g) as a yellowish syrup. The water layer was concentrated to dryness
and dried in vacuo. The solid obtained (1st and 2nd) was combined
with the dried water layer and recycled by stirring for 1 hour in
10% methanol-acetone (900 mL) and p-toluene sulfonic acid (16 g,
0.084 mol) to yield crude (4) (5.6 g). The crude (4) was purified
by a dry column over silica gel (CH.sub.3OH--CHCl.sub.3, 1% 5%)
to give (4) [R.sub.f=0.82(CHCl.sub.3--CH.sub.3OH, 10:1), 8.8 g,
61.84%] as a colorless oil. .sup.1H NMR(DMSO-d.sub.6) .delta. 1.26
and 1.32 (2.times.s, 2.times.3 H, isopropylidene), 3.41 (dd, J.sub.CH2OH,OH=6.04
Hz, J.sub.CH2OH,2=3.96 Hz, 2H, CH.sub.2OH), 3.56 4.16 (m, 6H, H-4,
-5, -1' and -2'), 4.82 (t, J.sub.OH,CH2=6.0 Hz, 1 H, CH.sub.2OH,
exchangeable with D.sub.2O), 4.85 (t, J.sub.2OH,CH2OH=3.96 Hz, 1H,
H-2). [.alpha.].sub.D.sup.25-12.48 (c, 1.11, CHCl.sub.3), Anal,
Calcd for C.sub.9H.sub.16O.sub.5: C, 52.93; H, 7.90. Found:C, 52.95;
H, 7.86.
EXAMPLE 4
Preparation of (+)-(1'S,2S,4S)-4-(1,2-Dihydroxymethyl-1,2-O-Isopropylidene)-2-(O-benzoyl-
oxymethyl)-dioxolane (5)
Benzoyl chloride (6.5 mL, 0.056 mol) was added in a dropwise manner
to a solution of (4) (8.5 g, 0.042 mol) in pyridine-CH.sub.2Cl.sub.2
(1:2, 120 mL) at 0.degree. C. and the temperature was raised to
room temperature. After stirring for 2 hours, the reaction was quenched
with methanol (10 mL) and the mixture was concentrated to dryness
in vacuo. The residue was dissolved in CH.sub.2Cl.sub.2 (300 mL)
and washed with water (100 mL.times.2), brine, dried (MgSO.sub.4),
filtered, evaporated to give a yellowish syrup, which was purified
by silica gel column chromatography (EtOAc-Hexane 4% -30%) to yield
(5) [R.sub.f=0.45 (Hexane-EtOAc, 3:1), 10.7 g, 83.4%] as a colorless
oil. 1H NMR (CDCl.sub.3) .delta. 1.35 and 1.44 (2.times.s, 2.times.3H,
isopropylidene) 3.3 4.35 (m 6H, H-4, -5, -1' and -2'), 4.44 (d,
J=3.96 Hz, 2H, CH.sub.2--OBz), 5.29 (t, J=3.74 Hz, 1H, H-2), 7.3
7.64, 8.02 8.18 (m, 3H, 2H, --OBz). [.alpha.].sub.D.sup.25+10.73(c,
1.75, CH.sub.3OH). Anal. Calcd for C.sub.16H.sub.20O.sub.6:C, 62.33;
H, 6.54. Found: C, 62.39; H, 6.54.
EXAMPLE 5
Preparation of (+)-(1'S,2S,4S)-4-(1,2-Dihydroxyethyl)-2-(O-benzoyloxymethyl)-dioxolane
(6)
A mixture of (5) (5.7 g. 0.018 mol) and p-toluene sulfonic acid
(1.05 g. 0.0055 mol) in methanol (70 mL) was stirred at room temperature
for 2 hours. The reaction was not completed, so the solvent was
evaporated to half of the original volume and additional methanol
(50 mL) and p-toluene sulfonic acid (0.7 g, 3.68 mmol) were added.
After stirring for one more hour, the reaction mixture was neutralized
with triethyl amine and the solvent was evaporated to dryness.
The residue was purified by silica gel column chromatography (Hexane-EtOAC,
10% 33%) to give (6) [R.sub.f=0.15(Hexane-EtOAc, 1:1), 4.92 g, 99.2%]
as a colorless syrup .sup.1H NMR (DMSO-d.sub.6)) .delta. 3.43 (m,
2H, H-2'), 3.67 4.1 (m, 4H, H-4, -5 and -1'), 4.32 (d, J=3.73 Hz,
2H, CH.sub.2--OBz), 4.60 (t, J=5.72 Hz, 2'-OH, exchangeable with
D.sub.2O), 5.23 (t, J=3.96 Hz, 1H, H-2), 7.45 7.7, 7.93 8.04 (m,
3H, 2H, --OBz), [.alpha.].sub.D.sup.25+9.16 (c,1.01, CHCl.sub.3).
Anal. Calcd for C.sub.13H.sub.16O.sub.6:C, 58.20; H, 6.01. Found:
C, 58.02; H, 6.04.
EXAMPLE 6
Preparation of (-)-(2S,4S) and (2S,4R)-4-Acetoxy-2-(O-benzoyloxymethyl)-dioxolane
(8)
A solution of NaIO.sub.4 (10.18 g, 0.048 mol) in water (120 mL)
was added to a solution of (6) (3.04 g, 0.011 mol) in CCl.sub.4:CH.sub.3CN
(1:1, 160 mL), followed by RuO.sub.2 hydrate (0.02 g). After the
reaction mixture was stirred for 5 hours, the solid was removed
by filtration over Celite and the filtrate was evaporated to 1/3
volume. The residue was dissolved in CH.sub.2Cl.sub.2 (100 mL) and
the water layer was extracted with CH.sub.2Cl.sub.2 (100 mL.times.2).
The combined organic layer was washed with brine (50 mL), dried
(MgSO.sub.4), filtered, evaporated to dryness and dried in vacuo
for 16 hours to give crude (7) (2.6 g, 91%).
To a solution of crude (7) (2.6, 0.01 mol) in dry THF (60 mL) were
added Pb(OAc).sub.4(5.48 g, 0.0124 mol) and pyridine (0.83 mL, 0.0103
mol) under N.sub.2 atmosphere. The mixture was stirred for 45 minutes
under N.sub.2 and the solid was removed by filtration. The solid
was washed with ethyl acetate (60 mL) and the combined organic layer
was evaporated to dryness. The residue was purified by silica gel
column chromatography (Hexane-EtOAc, 2:1) to yield (8) [R.sub.f=0.73
and 0.79 (Hexane-EtOAc, 2:1), 1.9 g, 69.34%] as a colorless oil.
.sup.1H NMR (CDCl.sub.3) .delta. 1.998, 2.11 (2.times.s, 3H, --OAc),
3.93 4.33 (m, 2H, H-5), 4.43, 4.48 (2.times.d, J=3.73, 3.74 Hz,
2H, CH.sub.2OBz), 5.46, 5.55 (2.times.t, J=4.18, 3.63 Hz, 1H, H-2),
6.42 (m, 1H, H-4), 7.33 759, 8.00 8.15 (m, 3H, 2H, --OBZ). [.alpha.].sub.D.sup.25-12.53
(c, 1.11, CHCl.sub.3). Anal. Calcd for C.sub.13H.sub.14O.sub.6;
C, 58.64; H, 5.30. Found C, 58.78; H, 5.34.
EXAMPLE 7
Preparation of (-)-(2S,4S)-1-[2-(benzoyl)-1,3-dioxolan-4-yl]cytosine(9)
and (+)-(2S,4R)-1-[2-(benzyloxy)-1,3-dioxolan-4-yl)cytosine (10)
A mixture of N.sup.4-acetylcytosine (1.24 g, 7.52 mmol) in dry
dichloroethane (20 mL), hexamethyldisilazane (15 mL), and ammonium
sulfate (cat. amount) was refluxed for 4 hours under a nitrogen
atmosphere. The resulting clear solution was cooled to room temperature.
To this silylated acetylcytosine was added a solution of (8) (1.0
g, 3.76 mmol) in dry dichloroethane (10 mL) and TMSOTf (1.46 mL
7.55 mmol). The mixture was stirred for 6 hours. Saturated NaHCO.sub.3
(10 mL) was added and the mixture was stirred for another 15 minutes
and filtered through a Celite pad. The filtrate was evaporated and
the solid was dissolved in EtOAc and washed with water and brine,
dried, filtered and evaporated to give the crude product. This crude
product was purified on a silica column (5% CH.sub.3OH/CHCl.sub.3)
to yield a pure .alpha.,.beta. mixture of (9) and (10) (0.40 g,
30%) and the .alpha.,.beta. mixture of (13) and (14) (0.48 g, 40%).
The mixture of (14) was reacetylated for separation, the combined
.alpha.,.beta. mixture was separated by a long silica column (3%
CH.sub.3OH/CHCl.sub.3) to yield (9) (0.414 g, 30.7%) and (10) (0.481
g, 35.6%) as foams. These foams were triturated with CH.sub.3OH
to obtain white solids. 9: UV (CH.sub.3OH) .lamda. max 298 nm; Anal.
(C.sub.17H.sub.17N.sub.3O.sub.8) C, H, N. 10: UV (CH.sub.3OH) .lamda.
max 298 nm.
EXAMPLE 8
Preparation of (-)-(2S,4S)-1-(2-Hydroxymethyl-1,3-dioxolan-4-yl)cytosine
(11)
A solution of (9) (0.29 g, 0.827) in CH.sub.3OH/NH.sub.3 (50 mL,
saturated at 0.degree. C.) was stirred at room temperature for 10
hours. The solvent was evaporated and the crude (11) was purified
on preparative silica plates (20% CH.sub.3OH/CHCl.sub.3) to give
an oil. This was crystallized from CH.sub.2Cl.sub.2/hexane to give
(11) (0.136 g, 77.7%) as a white solid. UV .lamda. max 278.0 nm
(.epsilon. 11967) (pH 2), 270.0 nm (.epsilon. 774) (pH 7), 269.0
nm (.epsilon.8379) (pH 11); Anal. (C.sub.8H.sub.11N.sub.3O.sub.4)C,H,N.
II. Pharmaceutical Compositions
Humans, equines, canines, bovines and other animals, and in particular,
mammals, suffering from cancer can be treated by administering to
the patient an effective amount of (-)-OddC or its derivative or
a pharmaceutically acceptable salt thereof optionally in a pharmaceutically
acceptable carrier or diluent, either alone, or in combination with
other known anticancer or pharmaceutical agents. This treatment
can also be administered in conjunction with other conventional
cancer therapies, such as radiation treatment or surgery.
These compounds can be administered by any appropriate route, for
example, orally, parenterally, intravenously, intradermally, subcutaneously,
or topically, in liquid, cream, gel, or solid form, or by aerosol
form.
The active compound is included in the pharmaceutically acceptable
carrier or diluent in an amount sufficient to deliver to a patient
a therapeutically effective amount for the desired indication, without
causing serious toxic effects in the patient treated. A preferred
dose of the compound for all of the herein-mentioned conditions
is in the range from about 10 ng/kg to 300 mg/kg, preferably 0.1
to 100 mg/kg per day, more generally 0.5 to about 25 mg per kilogram
body weight of the recipient per day. A typical topical dosage will
range from 0.01 3% wt/wt in a suitable carrier.
The compound is conveniently administered in any suitable unit
dosage form, including but not limited to one containing 1 to 3000
mg, preferably 5 to 500 mg of active ingredient per unit dosage
form. A oral dosage of 25 250 mg is usually convenient.
The active ingredient is preferably administered to achieve peak
plasma concentrations of the active compound of about 0.00001 30
mM, preferably about 0.1 30 .mu.M. This may be achieved, for example,
by the intravenous injection of a solution or formulation of the
active ingredient, optionally in saline, or an aqueous medium or
administered as a bolus of the active ingredient.
The concentration of active compound in the drug composition will
depend on absorption, distribution, inactivation, and excretion
rates of the drug as well as other factors known to those of skill
in the art. It is to be noted that dosage values will also vary
with the severity of the condition to be alleviated. It is to be
further understood that for any particular subject, specific dosage
regimens should be adjusted over time according to the individual
need and the professional judgment of the person administering or
supervising the administration of the compositions, and that the
concentration ranges set forth herein are exemplary only and are
not intended to limit the scope or practice of the claimed composition.
The active ingredient may be administered at once, or may be divided
into a number of smaller doses to be administered at varying intervals
of time.
Oral compositions will generally include an inert diluent or an
edible carrier. They may be enclosed in gelatin capsules or compressed
into tablets. For the purpose of oral therapeutic administration,
the active compound or its prodrug derivative can be incorporated
with excipients and used in the form of tablets, troches, or capsules.
Pharmaceutically compatible binding agents, and/or adjuvant materials
can be included as part of the composition.
The tablets, pills, capsules, troches and the like can contain
any of the following ingredients, or compounds of a similar nature:
a binder such as microcrystalline cellulose, gum tragacanth or gelatin;
an excipient such as starch or lactose, a dispersing agent such
as alginic acid, Primogel, or corn starch; a lubricant such as magnesium
stearate or Sterotes; a glidant such as colloidal silicon dioxide;
a sweetening agent such as sucrose or saccharin; or a flavoring
agent such as peppermint, methyl salicylate, or orange flavoring.
When the dosage unit form is a capsule, it can contain, in addition
to material of the above type, a liquid carrier such as a fatty
oil. In addition, dosage unit forms can contain various other materials
which modify the physical form of the dosage unit, for example,
coatings of sugar, shellac, or enteric agents.
The active compound or pharmaceutically acceptable salt thereof
can be administered as a component of an elixir, suspension, syrup,
wafer, chewing gum or the like. A syrup may contain, in addition
to the active compounds, sucrose as a sweetening agent and certain
preservatives, dyes and colorings and flavors.
The active compound or pharmaceutically acceptable salts thereof
can also be mixed with other active materials that do not impair
the desired action, or with materials that supplement the desired
action, such as other anticancer agents, antibiotics, antifungals,
antiinflammatories, or antiviral compounds.
Solutions or suspensions used for parenteral, intradermal, subcutaneous,
or topical application can include the following components: a sterile
diluent such as water for injection, saline solution, fixed oils,
polyethylene glycols, glycerine, propylene glycol or other synthetic
solvents; antibacterial agents such as benzyl alcohol or methyl
parabens; antioxidants such as ascorbic acid or sodium bisulfite;
chelating agents such as ethylenediaminetetraacetic acid; buffers
such as acetates, citrates or phosphates and agents for the adjustment
of tonicity such as sodium chloride or dextrose. The parental preparation
can be enclosed in ampoules, disposable syringes or multiple dose
vials made of glass or plastic.
If administered intravenously, preferred carriers are physiological
saline or phosphate buffered saline (PBS).
In one embodiment, the active compounds are prepared with carriers
that will protect the compound against rapid elimination from the
body, such as a controlled release formulation, including implants
and microencapsulated delivery systems. Biodegradable, biocompatible
polymers can be used, such as ethylene vinyl acetate, polyanhydrides,
polyglycolic acid, collagen, polyorthoesters, and polylactic acid.
Methods for preparation of such formulations will be apparent to
those skilled in the art.
Liposomal suspensions may also be pharmaceutically acceptable carriers.
These may be prepared according to methods known to those skilled
in the art, for example, as described in U.S. Pat. No. 4,522,811
(which is incorporated herein by reference in its entirety). For
example, liposome formulations may be prepared by dissolving appropriate
lipid(s) (such as stearoyl phosphatidyl ethanolamine, stearoyl phosphatidyl
choline, arachadoyl phosphatidyl choline, and cholesterol) in an
inorganic solvent that is then evaporated, leaving behind a thin
film of dried lipid on the surface of the container. An aqueous
solution of the active compound are then introduced into the container.
The container is then swirled by hand to free lipid material from
the sides of the container and to disperse lipid aggregates, thereby
forming the liposomal suspension.
III. Biological Activity
A wide variety of biological assays have been used and are accepted
by those skilled in the art to assess anti-cancer activity of compounds.
Any of these methods can be used to evaluate the activity of the
compounds disclosed herein.
One common method of assessing activity is through the use of the
National Cancer Institute's ("NCI") test panels of cancer
cell lines. These tests evaluate the in vitro anti-cancer activity
of particular compounds, and provide predictive data with respect
to the use of tested compounds in vivo. Other assays include in
vivo evaluations of the compound's effect on human or mouse tumor
cells implanted into or grafted onto nude mice.
A. In Vivo Activity of (-)-OddC
(-)-OddC was tested for anticancer activity in vivo against the
P388 leukemia cell line and the C38 colon cancer cell line. Examples
8 and 9 provide the experimental details and results of these tests.
EXAMPLE 9
In Vivo Treatment of Leukemia P388 Cells with (-)-O-ddC
10.sup.6 Leukemia P388 cells were implanted ip to BDF1 mice obtained
from Southern Research Institute, Alabama. (-)-OddC was administered
ip twice daily for five days starting one day after tumor cell implantation.
Using this protocol, 75 mg/kg/dose was shown to be toxic to the
mice.
FIG. 3 and Table 1 show the results of these studies. In FIG. 3,
(.circle-solid.) represents the data for the control (untreated
animals), (--.DELTA.--) represents the survival rate of those administered
(-)-OddC at 25 mg/kgbid twice a day, and (.largecircle.) represents
the survival rate of mice administered (-)-OddC once a day at 50
mg/kgbid. Of the six mice treated with 25 mg/kg/dose of (-)-OddC,
there is one long term survivor, and the life span of the remaining
five mice was increased by 103%.
TABLE-US-00001 TABLE 1 Dosage.sup.a Mean Survival ILS.sup.b Death
Cures.sup.c/ Group (mg/kg) Route Time (days) (%) Time (day) Total
Control -- -- 13.3 -- 11, 12, 13 0/6 13, 13, 18 -OddC 25 .times.
2 .times. 5 ip 27 103 18, 20, 22, 1/6 25, 33, 45 Inoculum: 10.sup.6
P388 cells were inoculated into each mouse ip on day 0 .sup.aTreatment
was given twice a day on days 1 to 5 .sup.bIncreased Life Span percent
above control .sup.cSurvivors equal or greater than 45 day life
span
EXAMPLE 10
In Vivo Treatment of Colon 38 Tumor Cells with (-)-OddC
Colon 38 tumor cells were implanted sc to BDF1 mice. (-)-OddC was
administered to the mice twice daily for five days, at a dosage
of 25 mg/kg/dose. The colon tumor cell growth was retarded as shown
in FIG. 2. In FIG. 2, (.circle-solid.) represents the data from
the control animals, and (.tangle-solidup.) represents the data
from the mice treated with (-)-OddC.
B. In Vitro Testing of (-)-OddC
(-)-OddC was evaluated in the NCI's cancer screening program. The
test measures the inhibition of various cancer cell lines at various
concentrations of (-)-OddC. The cell lines which were tested are
set forth in Table 2.
Table 2 also provides the concentration at which GI50 and TGI were
observed in the tested cell lines. GI50, TGI and LC50 are values
representing the concentrations at which the PG (percent of growth
inhibition), defined below, is +50, 0, and -50, respectively. These
values were determined by interpolation from dose response curves
established for each cell line, plotted as a function of PG v. log.sub.10
concentration of (-)-OddC.
PG is the measured effect of (-)-OddC on a cell line and was calculated
according to one of the following two expressions: If (Mean OD.sub.test-Mean
OD.sub.tzero).gtoreq.0. then PG=100.times.(Mean OD.sub.test-Mean
OD.sub.tzero)/(Mean OD.sub.ctrl-Mean OD.sub.tzero) If (Mean OD.sub.test-Mean
OD.sub.tzero)<0. then PG=100.times.(Mean OD.sub.test-Mean OD.sub.tzero)/(Mean
OD.sub.tzero) Where: Mean OD.sub.tzero=The average of optical density
measurements of SRB-derived color just before exposure of cells
to the test compound. Mean OD.sub.test=The average of optical density
measurements of SRB-derived color after 48 hours exposure of cells
to the test compound. Mean OD.sub.ctrl=The average of optical density
measurements of SRB-derived color after 48 hours with no exposure
of cells to the test compound.
In Table 2, the first two columns describe the subpanel (e.g.,
leukemia) and cell line (e.g., CCRF-CEM) which were treated with
(-)-OddC. Column 3 indicates the log.sub.10 at which GI50 occurred
and column 4 indicates the log.sub.10 at which TGI occurred. If
these response parameters could not be obtained by interpolation,
the value given for each response parameter is the highest concentration
tested and is preceded by a ">" sign. For example,
if all the PG at al concentrations of (-)-OddC given to a particular
cell line exceeds +50, then this parameter can not be obtained by
interpolation.
TABLE-US-00002 TABLE 2 Panel Cell Line Log.sub.10GI50 Log.sub.10TGI
Leukemia CCRF-CEM -6.64 >-4.00 RL-60 (TB) -6.28 >-4.00 K-562
-4.59 >-4.00 BSOLT-4 -6.66 -4.39 RPMI-2.26 -4.03 >-4.00 SR
-5.95 >-4.00 Non-Small A549/ATCC -6.01 >-4.00 Cell Lung Cancer
BKVX >-4.00 >-4.00 HOP-62 -6.23 -4.71 NCI-H23 -4.92 >-4.00
NCI-H322M >-4.00 >-4.00 NCI-H460 -4.32 >-4.00 NCI-H522
-6.06 >-4.00 Colon COLO205 -4.03 >-4.00 Cancer HCT-116 -5.23
>-4.00 HCT-15 -5.39 >-4.00 HT29 >-4.00 >-4.00 K2112
>-4.00 >-4.00 CNS Cancer SP-268 -5.18 >-4.00 SP-295 -6.24
>-4.00 SNB-19 -5.71 >-4.00 U251 -4.91 >-4.00 Melanoma LOX
D6VI -6.39 >-4.00 MALME-3M -4.51 >-4.00 M14 -6.27 -5.07 SK-MEL-28
-4.31 >-4.00 SK-MEL-5 -4.91 >-4.00 UACC-257 >-4.00 >-4.00
UACC-62 -5.53 >-4.00 Ovarian OROV1 -4.03 >-4.00 Cancer OVCAR-3
-4.44 >-4.00 OVCAR-4 >-4.00 >-4.00 OVCAR-5 -4.41 >-4.00
OVCAR-8 -5.82 >-4.00 SK-OV-3 -5.35 >-4.00 Renal 785-4 -5.36
>-4.00 Cancer ACHN -6.46 >-4.00 CAKI-1 -6.65 -4.87 RXF-393
-6.17 >-4.00 SN12C -6.27 >-4.00 TK-30 >-4.00 >-4.00
UO-31 -5.60 >-4.00 Prostate PC-3 -6.29 >-4.00 Cancer DU-145
-6.97 >-4.00 Breast MCF7 -5.95 >-4.00 Cancer MCF7/ADR-RES
-4.97 >-4.00 MDA-MB- >-4.00 >-4.00 231/ATCC HS578T >-4.00
>-4.00 MDA-MB-435 -4.62 >-4.00 MDA-N -4.33 >-4.00 BT-549
-4.59 >-4.00 T-47D >-4.00 >-4.00
FIG. 4 is a graph that displays the relative selectivity of (-)-OddC
for a particular cell line. Bars extending to the right represent
sensitivity of the cell line to (-)-OddC in excess of the average
sensitivity of all tested cell lines. Since the bar scale is logarithmic,
a bar 2 units to the right implies the compound exhibited a GISO
for the cell line at a concentration one-hundredth the mean concentration
required over all cell lines, and thus the cell line is unusually
sensitive to (-)-OddC. Bars extending to the left correspondingly
imply sensitivity less than the mean. These cell lines can be easily
determined from Table 2, as the log.sub.10 concentration will be
preceded by a ">".
It can be seen from FIG. 4 that at least one cell line of each
type of cancer cell tested exhibited sensitivity to (-)-OddC. Certain
prostate cancer cell lines, leukemia cell lines, and colon cell
lines show extreme sensitivity to (-)-OddC.
EXAMPLE 11
Comparison of (-)-OddC and AraC
As discussed in the Background of the Invention, cytosine arabinoside
(also referred to as Cytarabin, araC, and Cytosar) is a nucleoside
analog of deoxycytidine used in the treatment of acute myeloid leukemia.
It is also active against acute lymphocytic leukemia, and to a lesser
extent, is useful in chronic myelocytic leukemia and non-Hodgkin's
lymphoma. The primary action of araC is inhibition of nuclear DNA
synthesis. It was of interest to compare the toxicity to tumor cells
of (-)-OddC and AraC.
Cells in logarithmic growth were plated at a density of 5000 cells/mL/well
in 24-well plates. Drugs were added to the cells at different dosages
and cultures were maintained for a period of three generations.
At the end of this time, methylene blue assays were performed and/or
cell numbers were directly counted. Methylene blue is a die which
binds in a stoichiometric manner to proteins of viable cells and
can be used to indirectly quantitate cell number (Finlay, 1984).
IC.sub.50 values were determined by interpolation of the plotted
data. Each value shown is the mean.+-.standard deviation of five
experiments with each data point done in duplicate.
In all of the tumor cell lines tested, (-)-OddC was more cytotoxic
than AraC. (-)-OddC was significantly more effective than AraC in
the KB nasopharyngeal carcinoma cell line and in the two prostate
carcinoma lines DU-145 and PC-3. HepG2 cells originate from hepatocellular
carcinoma and the 2.2.15 line is derived from HepG2 cells which
were transfected with a copy of the hepatitis B virus genome. CEM
cells are derived from acute lymphoblastic leukemia. (-)-OddU, the
compound which would be formed by the deamination of (-)-OddC was
not toxic in any of the cell lines tested. Enzymatic studies indicate
that, unlike AraC whose clinical efficacy is greatly diminished
by its susceptibility to deamination, (-)-OddC is not a substrate
for deaminase.
It has been determined that (-)-OddC can be phosphorylated to mono-,
di- and tri-phosphate nucleotide in vivo. It appears that (-)-OddC
exhibits its cellular toxicity in a phosphorylated form because
cells that are incapable of phosphorylating the compound are much
less sensitive to the compound. The first enzyme responsible for
its phosphorylation is human deoxycytidine kinase. In vitro enzymatic
studies indicate that (-)-OddC can be phosphorylated by this enzyme.
Unlike araC, (-)-OddC is not deaminated by cytidine deaminase.
The presence of cytidine deaminase in solid tumor tissues could
be a key contributing factor responsible for the lack of activity
of araC in solid tumors. This could partly explain why (-)-OddC
is active against HepG2 cells in nude mice, whereas araC is inactive.
It also explains why (-)-OddC has different spectrums of anti-tumor
activity from that of araC. Furthermore, the presence of cytidine
deaminase in the gastrointestinal tract could also play an important
role in why araC cannot be taken orally. The lack of action of cytidine
deaminase to (-)-OddC may explain why (-)-OddC could still have
anti-tumor activity if given orally.
TABLE-US-00003 BIOCHEMICAL STUDIES OF (-)-OddC In vitro cytotoxicity
of AraC, (-)-OddC and (-)-OddU ID.sub.50 (.mu.M) Cell Line AraC
(-)-OddC (-)-OddU KB 0.152 .+-. .010 0.048 .+-. .021 >30 DU-145
0.170 .+-. .035 0.024 .+-. .020 >30 PC-3 0.200 .+-. .078 0.056
.+-. .039 >30 HepG2 0.125 .+-. .013 0.110 .+-. .050 >30 2.2.15
0.145 .+-. .007 0.110 .+-. .011 >30 CEM 0.030 .+-. .010 0.025
.+-. .030 >30
IV. Use of (-)-OddC in Oligonucleotides and in Antisense Technology
Antisense technology refers in general to the modulation of gene
expression through a process wherein a synthetic oligonucleotide
is hybridized to a complementary nucleic acid sequence to inhibit
transcription or replication (if the target sequence is DNA), inhibit
translation (if the target sequence is RNA) or to inhibit processing
(if the target sequence is pre-RNA). A wide variety of cellular
activities can be modulated using this technique. A simple example
is the inhibition of protein biosynthesis by an antisense oligonucleotide
bound to mRNA. In another embodiment, a synthetic oligonucleotide
is hybridized to a specific gene sequence in double stranded DNA,
forming a triple stranded complex (triplex) that inhibits the expression
of that gene sequence. Antisense oligonucleotides can be also used
to activate gene expression indirectly by suppressing the biosynthesis
of a natural repressor or directly by reducing termination of transcription.
Antisense Oligonucleotide Therapy (AOT) can be used to inhibit the
expression of pathogenic genes, including those which are implicated
in the uncontrolled growth of benign or malignant tumor cells or
which are involved in the replication of viruses, including HIV
and HBV.
The stability of the oligonucleotides against nucleases is an important
factor for in vivo applications. It is known that 3'-exonuclease
activity is responsible for most of the unmodified antisense oligonucleotide
degradation in serum. Vlassov, V. V., Yakubov, L. A., in Prospects
for Antisense Nucleic Acid Therapy of Cancers and AIDS, 1991, 243
266, Wiley-Liss, Inc., New York; Nucleic Acids Res., 1993, 21, 145.
The replacement of the nucleotide at the 3'-end of the oligonucleotide
with (-)-OddC or its derivative can stabilize the oligonucleotide
against 3'-exonuclease degradation. Alternatively or in addition,
an internal nucleotide can be replaced by (-)-OddC or its derivative
to resist the degradation of the oligonucleotide by endonucleases.
Given the disclosure herein, one of ordinary skill in the art will
be able to use (-)-OddC or its derivative to stabilize a wide range
of oligonucleotides against degradation by both exonucleases and
endonucleases, including nucleosides used in antisense oligonucleotide
therapy. All of these embodiments are considered to fall within
the scope of this invention. Example 11 provides one, non-limiting,
example of the use of (-)-OddC to resist the activity of a 3'-exonuclease.
EXAMPLE 11
Resistance to 3'-Exonuclease Activity by (-)-OddC
The human cytosolic exonuclease activity from human H9 (T-type
lymphocytic leukemic cells) was determined by sequencing gel assay.
Briefly, the 3'-terminated substrate was prepared from a 20 or 23
base-long DNA primer with the following sequence:
TABLE-US-00004 3'-CAATTTTGAATTTCCTTAACTGCC-5' 24 1
The primers were labelled at the 5'-end with [.tau.-.sup.32P] ATP,
annealed to complementary RNA templates and terminated at the 3'
end with dTTP (20 mer) dCTP (23 mer) or (-)-OddCTP (23 mer) in a
standing start reaction catalyzed by HIV-1 RT. Under these conditions,
the 20mer was terminated with dTMP (A) the 23mer was terminated
with dCMP (B) or (-)-O-ddCMP(C). These single stranded DNA substrates
were used to assay their susceptibility to the cytoplasmic exonuclease.
The assays were done in 10 .mu.l reactions containing 50 mM Tris-HCl
pH 8.0, 1 mM MgCl.sub.2, 1 mM dithiothreitol, 0.1 mg/ml bovine serum
albumin, 0.18 .mu.Ci/ml 3'-terminated substrate and 2 .mu.l of the
exonuclease (0.03 units). The reactions were incubated at 37.degree.
C. for the indicated times and terminated by adding 4 .mu.l 98%
formamide, 10 mM EDTA and 0.025% bromophenol blue. The samples were
denatured at 100.degree. C. for 5 minutes followed by rapid cooling
on ice. The unreacted material as well as the reaction products
were separated on 15% polyacrylamide/urea sequencing gels and visualized
by autoradiography. The oligonucleotide with (-)-OddC at the 3'-end
was at least five times more resistant to 3'-exonuclease than the
other oligonucleotides.
Modifications and variations of the present invention in the treatment
of cancer will be obvious to those skilled in the art from the foregoing
detailed description of the invention. Such modifications and variations
are intended to come within the scope of the appended claims.
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