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Cancer Patent Abstract
Materials and methods for treating certain cancers are described,
preferably cancers that result from the up-regulation of the RAF-MEK-ERK
pathway, and more preferably chronic myelogenous leukemia, and which
cancer is preferably resistant to the inhibition of the Bcr-Abl
tyrosine kinase, imatinib.
Cancer Patent Claims
We claim:
1. A method for treating a patient suffering from cancer, wherein
said patients' cancer exhibits up-regulation of the RAF-MEK-ERK
pathway, comprising administering to the cancer patient an effective
dose of an inhibitor of said RAF-MEK-ERK pathway, wherein said cancer
is CML, and said inhibitor is selected from the group consisting
of Bay 43-9006 and CI-1040.
2. The method of claim 1, wherein said CML is resistant to an inhibitor
of Bcr-Abl tyrosine kinase.
3. The method of claim 2, wherein said inhibitor of Bcr-Abl tyrosine
kinase is imatinib.
4. The method of claim 2, wherein said inhibitor of the RAF-MEF-ERK
pathway is Bay 43-9006.
5. The method of claim 2, wherein said inhibitor of the RAF-MEF-ERK
pathway is CI-1040.
6. The method of claim 1, further comprising administering an inhibitor
of Bcr-Abl tyrosine kinase.
7. The method of claim 6, wherein said inhibitor of the RAF-MEF-ERK
pathway is Bay 43-9006.
8. The method of claim 6, wherein said inhibitor of the RAF-MEF-ERK
pathway is CI-1040.
Cancer Patent Description
FIELD OF THE INVENTION
The invention described herein is in the field of cancer therapy,
and preferably for the treatment of chronic myelogenous leukemia.
BACKGROUND OF THE INVENTION
A goal of modern cancer therapy is to identify molecules in signal
transduction pathways that affect cell growth, and particularly
those that cause a normal cell to become cancerous. One such pathway
is the RAF-MEK-ERK pathway, and the up-regulation of one or more
of its members is thought to be responsible for a number of cancers.
For example, patients with chronic myelogenous leukemia, herein
after referred to as CML, who are in either the chronic or blast
phase typical achieve remissions in response to the marketed drug
Gleevec.TM., also referred to as imatinib or STI571 (N. Eng. J.
Med. 244, 1031 [2001]; N. Eng. J. Med 244, 1038 [2001]). CML is
characterized by the Philadelphia chromosomal translocation (Ph+)
resulting in a Bcr-Abl fusion protein. Imatinib treats CML by blocking
Bcr-Abl kinase activity.
While the remissions achieved with imatinib during the chronic
phase of CML are durable, patients with remissions achieved during
the blast phase usually relapse within 2-6 months (N. Eng. J. Med.
244, 1038 [2001]). Resistance to Imatinib results in reactivation
of Bcr-Abl kinase activity. Recently, it has been shown that these
relapses are usually due to imatinib-resistance that occur either
by over-expression of the translocated Bcr/Abl gene, or mutation
of the imatinib target gene, namely the Abl kinase (Science 293,
876 [2001]). Resistance often correlates with mutations in the Abl
kinase domain, including T315I and E255K.
The Abl kinase was chosen as a molecular target in the treatment
against cancer since 95% of patients with CML have activation of
the Abl pathway that occurs through chromosomal translocations that
result in fusion of the Bcr and Abl genes. As mentioned above, a
key pathway that is up-regulated in CML cells that are resistant
to imatinib is the RAF-MEK-ERK pathway. Therefore, treatment with
inhibitors of the RAF-MEK-ERK pathway should lead to remissions
in patients with imatinib resistant CML.
SUMMARY OF THE INVENTION
The invention described herein presents methods and compositions
for treating cancers that involve up-regulation of one or more molecules
in the pathway: RAF-MEK-ERK.
An object of the invention is a description of inhibitors of the
RAF-MEK-ERK pathway that are beneficially applied to the treatment
of certain forms of cancer, preferably CML, and more preferably
to those forms of CML that are resistant to Bcr-Abl kinase inhibitors,
and most preferably to those forms of CML that are resistant to
the Bcr-Abl kinase inhibitor, imatinib.
Another object of the invention is a description of RAF inhibitors,
preferably Bay 43-9006, alone or in combination with Bcr-Abl kinase
inhibitors, preferably imatinib, for the treatment of CML.
Still another object of the invention is a description of MEK inhibitors,
preferably CI-1040, alone or in combination with Bcr-Abl kinase
inhibitors, preferably imatinib, for the treatment of CML.
Another object of the invention is a description of methods and
compositions for formulating and administering inhibitors of the
RAF-MEK-ERK pathway, preferably in combination with Bcr-Abl kinase
inhibitors and more preferably with the Bcr-Abl kinase inhibitor,
imatinib.
These and other objects of the present invention will become apparent
to a skilled practitioner of the art upon a full consideration of
the invention.
BRIEF DESCRIPTION OF THE FIGURES
FIG. 1 shows the RAF-MEK-ERK pathway that becomes up-regulated
in certain cancer cells, including chronic myelogenous leukemia.
Also shown are the compounds BAY 43-9006, and CI-1040, and the proteins
in the pathway that they affect, RAF and MEK, respectively.
FIG. 2 shows the structure of BAY 43-9006.
FIG. 3 shows the effects of the RAF kinase inhibitor, BAY 43-9006,
and imatinib, on imatinib-resistant CML Cells.
FIG. 4 shows the effects of the RAF kinase inhibitor, BAY 43-9006,
on imatinib-resistant CML Cells. The results are from 3 experiments.
FIG. 5 shows the effects of the RAF kinase inhibitor, BAY 43-9006,
on ERK phosphorylation.
DESCRIPTION OF THE INVENTION
All publications, including patents and patent applications, mentioned
in this specification are herein incorporated by reference to the
same extent as if each individual publication was specifically and
individually indicated to be incorporated by reference in its entirety.
Based on the pathway shown in FIG. 1, it will be appreciated that
in cancers where Raf, MEK, or ERK are up-regulated, compounds that
inhibit the activities of these molecules will have beneficial effects
for treating such cancers. An example of one such cancer, also shown
in FIG. 1, is chronic myelogenous leukemia. Thus, treating patients
with non-toxic doses of, preferably, 200-400 mg and higher of the
Raf kinase inhibitor BAY 43-9006 (Endocr. Relat. Cancer 8, 219 [2001])
will result in remissions, or minimally stabilization of the growth
of the cancer. Furthermore, treating patients with non-toxic doses
of, preferably, 200-400 mg and higher of the MEK inhibitor PD184352
(now designated CI-1040, Oncogene 19, 6594 (2000) will also lead
to remissions or cancer growth stabilization in these patients.
Definitions
Unless defined otherwise, all technical and scientific terms used
herein have the same meaning as commonly understood by one of ordinary
skill in the art to which this invention belongs. Generally, the
nomenclature used herein and the laboratory procedures described
below are those well known and commonly employed in the art.
BAY 43-9006 is described in U.S. patent application Ser. No. 09/425,228
and PCT/US00/00648, as are numerous other RAF kinase inhibitors.
The MEK inhibitor, CI-1040, is described in Oncogene 19, 6594 (2000).
Imatinib, or Gleevec,.TM. is described in U.S. Pat. No. 5,521,184.
By "up-regulated" or "up-regulation" of the
RAF-MEK-ERK pathway is meant elevated levels, by whatever molecular
mechanism, of one or more of the proteins, RAF MEK, or ERK, or increases
in their enzymatic activity, or changes in their normal substrate
affinity when compared to normal cells.
It will be appreciated that the compounds that affect RAF, MEK,
or ERK may be used alone, or in combination. They may also be used
in combination with other compounds known to affect particular cancers
where the RAF-MEK-ERK pathway is up-regulated. For example, the
drug imatinib (Gleevec.TM.) is used to treat CML patients; thus,
imatinib resistance that develops in certain advanced staged CML
patients that causes up-regulation of the RAF-MEK-ERK pathway can
be treated with the appropriate compounds that control the up-regulation
of the appropriate molecules in the RAF-MEK-ERK pathway. Such compounds
can be combined with imatinib for treatment of a CML patient before
imatinib resistance develops, or without imatinib after resistance
has developed. The pathway in question and the sites of action of
BAY 43-9006, CI-1040 and imatinib are illustrated in FIG. 1.
There are several important rationale for applying inhibitors to
the RAF-MEK-ERK pathway for the treatment of CML. First, cells from
leukemia patients (Ph+) have elevated phospho-ERK levels at least
in part due to elevated, or up-regulated, RAF kinase activity. Second,
imatinib treatment results in decreased phospho-ERK levels. Also,
cells from imatinib-resistant patients have re-elevated phospho-ERK
levels.
Pharmaceutical Compositions and Modes of Administration
The present invention also relates to pharmaceutical compositions
for administration to humans that comprise an inhibitor of the RAF-MEK-ERK
pathway alone, or in combination with imatinib. Such would include
compositions for enteral administration, such as nasal, buccal,
rectal or, especially, oral, and parenteral administration, such
as intravenous, intramuscular or subcutaneous. The compositions
comprise the inhibitor on its own or, preferably, together with
a pharmaceutically acceptable carrier. The dose of inhibitor depends
on the disease to be treated, preferably CML, and more preferably
imatinib resistant CML, and the age, weight and individual condition
of a patient, and the mode of administration.
The invention relates also to processes, and to the use of inhibitors
of the RAF-MEK-ERK pathway in the preparation of pharmaceutical
compositions alone, or in combination with imatinib. Preference
is given to a pharmaceutical composition that is suitable for administration
to a human suffering from a cancer that is responsive to inhibition
of a protein tyrosine kinase. Preferably the cancer is CML, and
more preferably it is imatinib resistant CML which composition comprises
an inhibitor, or a salt thereof where salt-forming groups are present,
in an amount that is effective in inhibiting the protein tyrosine
kinase, together with at least one pharmaceutically acceptable carrier.
Preference is also given to a pharmaceutical composition for the
prophylactic or, especially, therapeutic treatment of preferably
CML, and more preferably imatinib-resistant CML, which composition
comprises as active ingredient an inhibitor, or a pharmaceutically
acceptable salt thereof, in an amount that is prophylactically or,
especially, therapeutically effective against the mentioned diseases.
The pharmaceutical compositions comprise from approximately 1%
to approximately 95% of the appropriate inhibitor, dosage forms
that are in single dose form preferably comprising from approximately
20% to approximately 90% active ingredient, and dosage forms that
are not in single dose form preferably comprising from approximately
5% to approximately 20% active ingredient. Unit dose forms are,
for example, dragees, tablets, ampoules, vials, suppositories or
capsules. Other dosage forms are, for example, ointments, creams,
pastes, foams, drops, sprays, dispersions, etc. The pharmaceutical
compositions of the present invention are prepared in a manner known
per se, for example by means of conventional mixing, granulating,
confectioning, dissolving or lyophilising processes.
There are preferably solutions that can be used with the appropriate
inhibitor, including suspensions or dispersions, especially isotonic
aqueous solutions, dispersions or suspensions, which, for example
in the case of lyophilized compositions comprising the active ingredient
on its own or together with a carrier, e.g. mannitol, may be prepared
before use. The pharmaceutical compositions may be sterilized and/or
may comprise excipients, for example preservatives, stabilizers,
wetting agents and/or emulsifiers, solubilisers, salts for regulating
the osmotic pressure and/or buffers, and are prepared in a manner
known per se, for example by means of conventional dissolving or
lyophilising processes. The said solutions or suspensions may comprise
viscosity-increasing substances, such as sodium carboxy-methylcellulose,
carboxymethylcellulose, dextran, polyvinylpyrrolidone or gelatin,
or also solubilisers, for example .RTM.Tween 80>polyoxyethylene(20)
sorbitan monooleate; trademark of ICI Americas, Inc, USA.
Synthetic or semi-synthetic oils as are know in the art may used
for injection of the appropriate inhibitor. Particularly useful
can be liquid fatty acid esters which contain a long-chain fatty
acid having from 8 to 22, especially from 12 to 22, carbon atoms.
Pharmaceutical compositions will preferably be used in oral form,
and can be obtained, for example, by combining a RAF-MEK-ERK pathway
inhibitor, with or without a Bcr-Abl tyrosine kinase inhibitor,
with one or more solid carriers, granulating a resulting mixture,
where appropriate, and processing the mixture or granules, if desired,
where appropriate with the addition of additional excipients, to
form tablets or dragee cores. Suitable carriers are especially fillers,
such as sugars, for example lactose, saccharose, mannitol or sorbitol,
cellulose preparations and/or calcium phosphates, for example tri-calcium
phosphate or calcium hydrogen phosphate, and also binders, such
as starches, for example corn, wheat, flee or potato starch, methylcellulose,
hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose and/or
polyvinylpyrrolidone, and/or, if desired, disintegrators, such as
the above-mentioned starches and also carboxymethyl starch, cross-linked
polyvinylpyrrolidone, or alginic acid or a salt thereof, such as
sodium alginate. Additional excipients are especially flow conditioners
and lubricants, for example silicic acid, talc, stearic acid or
salts thereof, such as magnesium or calcium stearate, and/or polyethylene
glycol, or derivatives thereof.
Pharmaceutical compositions for oral administration are also hard
gelatin capsules, and soft sealed capsules consisting of gelatin
and a plasticiser, such as glycerol or sorbitol. The hard gelatin
capsules may comprise an appropriate inhibitor in the form of granules,
for example in admixture with fillers, such as corn starch, binders
and/or glidants, such as talc or magnesium stearate, and, where
appropriate, stabilizers. In soft capsules the active ingredient
is preferably dissolved or suspended in suitable liquid excipients,
such as fatty oils, paraffin oil or liquid polyethylene glycols
or fatty acid esters of ethylene glycol or propylene glycol, it
likewise being possible to add stabilizers and detergents, for example
of the polyoxyethylene sorbitan fatty acid ester type.
Other oral dosage forms are, for example, syrups prepared in customary
manner which comprise the appropriate inhibitor, for example, in
suspended form and in a concentration of approximately from 5% to
20%, preferably approximately 10% or in a similar concentration
that provides a suitable single dose when administered, for example,
in a measure of 5 or 10 ml. Also suitable are, for example, powdered
or liquid concentrates for the preparation of shakes, for example
in milk. Such concentrates may also be packed in single dose quantities.
For parenteral administration of the appropriate inhibitor there
are suitable, especially, aqueous solutions of an active ingredient
in water-soluble form, for example in the form of a water-soluble
salt, or aqueous injection suspensions that comprise viscosity-increasing
substances, for example sodium carboxymethylcellulose, sorbitol
and/or dextran, and, if desired, stabilizers. The active ingredient,
where appropriate together with excipients, can also be in the form
of a lyophilisate and be made into a solution prior to parenteral
administration by the addition of suitable solvents.
The instant invention relates to a method for the treatment of
the pathological conditions mentioned above, especially those which
are responsive to inhibition of tyrosine protein kinases, preferably
CML, and more preferably CML that is resistant to Bcr-Abl tyrosine
kinase inhibitors. Thus a RAF-MEK-ERK pathway inhibitor, with or
without a Bcr-Abl tyrosine kinase inhibitor, may be administered
prophylactically or therapeutically as such, or in the form of a
pharmaceutical composition.
The Examples which follow are illustrative of specific embodiments
of the invention, and various uses thereof. They are set forth for
explanatory purposes only, and are not to be taken as limiting the
invention
EXAMPLES
Example 1
Effect of RAF Kinase Inhibitor on Imatinib-Resistant CML Cells
To test whether CML cells, and in particular imatinib-resistant
CML cells, are sensitive to inhibition of the RAF-MEK-ERK pathway,
we analyzed the response of these cells to the Raf inhibitor BAY
43-9006, and to imatinib. Parental IL3-dependent Ba/F3 mouse hematopoietic
cells, and derivative cell lines (obtained from Dr. Charles Sawyers,
University of California at Los Angeles, and described by Tipping
A. J., et al., in Leukemia, 2002, Dec; 16(12):2349-57) that are
independent of IL3 due to exogenous expression of wild type Bcr-Abl,
or two of the most frequently reported Bcr-Abl kinase domain mutants,
E255K and T315I, were exposed to either imatinib or BAY 43-9006.
The Bcr-Abl kinase domain mutants E255K and T315I are resistant
to imatinib.
Briefly, cell culture methods were as follows. Ba/F3 mouse pre-B
cells containing empty expression vector (Ba/F3) or stable expressing
wildtype Bcr-Abl (P210 WT), or the imatinib resistant T315I or E255K
mutants (P210 T315I, P210 E255K) were cultured in RPMI medium containing
10% serum. Cell proliferation assays were performed as follows.
On day 1, 2.times.10.sup.5 cells were plated in 24 well plates with
1.2 ml of media. The appropriate compound, at the chosen concentration
was then added to the media in a final DMSO concentration of 0.2%.
On day 3, the cells were resuspended, and 0.5 ml of the cell suspension
was and diluted with PBS (1:2). Finally, cell proliferation (cell
viability) was measured by trypan blue dye exclusion using CEDEX
system (Innovatis).
The results are shown in FIG. 3. Only those Ba/F3 cells expressing
wild type Bcr-Abl were sensitive to imatinib. IC50s for inhibition
by imatinib were 0.5 .mu.M for the wild type expressing cells and
over ten-fold higher for the three other cell lines. In contrast,
as shown in FIG. 4, the proliferation of all four cell lines was
inhibited by BAY 43-9006, with IC50s ranging from 4-8 .mu.M.
Thus, these results suggest that CML patients who are or are not
resistant to imatinib would respond favorably to inhibitors of the
RAF-MEK-ERK pathway such as the Raf inhibitor, BAY 43-9006.
Example 2
Effect of Raf Inhibitor on ERK Phosphorylation
A second experiment was conducted to further establish that sensitivity
to imatinib and BAY 43-9006 correlates with inhibition of the RAF-MEK-ERK
pathway, as determined by measurement of Erk phosphorylation. The
experiment was conducted using parental IL3-dependent Ba/F3 mouse
hematopoietic cells, and derivative cell lines that are independent
of IL3 due to exogenous expression of wildtype Bcr-Abl, or two of
the most frequently reported Bcr-Abl kinase imatinib resistant domain
mutants E255K and T315I (obtained from Dr. Charles Sawyers, University
of California at Los Angeles, and described by Shah, N., et al.
(August 2002) Cancer Cell, vol. 2: pages 117-125). The experiment
was conducted as follows. On day 1, 2.times.10.sup.6 cells were
plated in a 6 well plate in medium containing 10% serum, and on
day 2 the cells were treated with the relevant compound dissolved
in DMSO. The final DMSO concentration was 0.2%. Next, the cells
were spun down, and washed once with 2 ml of ice-cold phosphate
buffered saline, and then lysed with 150 ml buffer. Next, 50 ul
of cell lysates was electrophoresed in 10% Tris-Glycine gels, followed
by treatment with a 1.degree. antibody which was a phospho-ERK polyclonal
rabbit antibody used at 1:1000 dilution. It was obtained from Cell
Signaling. The 1.degree. was followed by a 2.degree. antibody which
was goat anti-rabbit horse radish peroxidase antibody, and it was
used at a dilution of 1:1000. Lastly, Westerns were developed with
an ECL kit obtained from Amersham.
FIG. 5 shows the results. Only those Ba/F3 cells expressing wild
type Bcr-Abl were sensitive to imatinib. In contrast, phospho-ERK
levels in these cells, as well as Bcr-Abl kinase imatinib resistant
domain mutants, E255K and T315I, also showed a decrease in phospho-ERK
levels in the presence of BAY 43-9006.
These results suggest that CML patients who are or are not resistant
to imatinib would respond to inhibitors of the RAF-MEK-ERK pathway
such as the RAF inhibitor, BAY 43-9006.
The invention now being fully described, it will be apparent to
one of ordinary skill in the art that many changes and modifications
can be made thereto without departing from the spirit or scope of
the appended claims. |