U.S. patent number RE40,418 [Application Number 11/222,485] was granted by the patent office on 2008-07-01 for treatment of neoplasms with cci-779/ekb-569 combination.
This patent grant is currently assigned to Wyeth. Invention is credited to James J. Gibbons, Jr., Sridhar K. Rabindran.
United States Patent |
RE40,418 |
Rabindran , et al. |
July 1, 2008 |
Treatment of neoplasms with CCI-779/EKB-569 combination
Abstract
This invention provides the use of a combination of CCI-779 and
EKB-569 in the treatment of neoplasms.
Inventors: |
Rabindran; Sridhar K. (Chestnut
Ridge, NY), Gibbons, Jr.; James J. (Westwood, NJ) |
Assignee: |
Wyeth (Madison, NJ)
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Family
ID: |
23203475 |
Appl.
No.: |
11/222,485 |
Filed: |
September 8, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
|
Reissue of: |
10212889 |
Aug 6, 2002 |
06617333 |
Sep 9, 2003 |
|
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Current U.S.
Class: |
514/291; 514/312;
514/313; 514/311; 514/314; 514/922; 514/183 |
Current CPC
Class: |
A61P
35/00 (20180101); A61P 43/00 (20180101); C07C
229/30 (20130101); A61P 35/04 (20180101); C07C
237/16 (20130101); A61K 31/47 (20130101); A61K
31/395 (20130101); A61K 31/00 (20130101); A61P
35/02 (20180101); A61K 31/4706 (20130101); A61K
31/436 (20130101); A61K 31/395 (20130101); A61K
2300/00 (20130101); A61K 31/436 (20130101); A61K
2300/00 (20130101); A61K 31/47 (20130101); A61K
2300/00 (20130101); A61K 31/4706 (20130101); A61K
2300/00 (20130101); Y10S 514/922 (20130101) |
Current International
Class: |
A61K
31/00 (20060101); A61K 31/44 (20060101); A61K
31/47 (20060101) |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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0 448 078 |
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Sep 1991 |
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EP |
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0 525 960 |
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Mar 1996 |
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EP |
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0 722 720 |
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Jul 1996 |
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EP |
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0 525 960 |
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Feb 2003 |
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EP |
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WO 02/13802 |
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Feb 2002 |
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WO |
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WO 03/020266 |
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Mar 2003 |
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WO |
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WO 2004/066919 |
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Aug 2004 |
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WO |
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|
Primary Examiner: Krass; Frederick
Attorney, Agent or Firm: Milowsky; Arnold S. Howson &
Howson LLP
Claims
What is claimed is:
1. A method of treating a neoplasm .Iadd.in which uncontrolled cell
proliferation results from deregulation of epidermal growth factor
receptor (EGFR) kinase .Iaddend.in a mammal in need thereof, which
comprises providing to said mammal a synergistically effective
amount of a combination comprising .Iadd.rapamycin 42-ester with
3-hydroxy-2-(hydroxymethyl)-2-methylpropionic acid
.Iaddend.(CCI-779) and .Iadd.4-dimethylamino-but-2-enoic acid
[4-(3-chloro-4-fluoro-phenylamino)-3-cyano-7-ethoxy-quinolin-6-yl]-amide
.Iaddend.(EKB-569).
2. The method according to claim 1, wherein the neoplasm is renal
cancer.
3. The method according to claim 1, wherein the neoplasm is soft
tissue sarcoma.
4. The method according to claim 1, wherein the neoplasm is breast
cancer.
5. The method according to claim 1, wherein the neoplasm is a
neuroendocrine tumor of the lung.
6. The method according to claim 1, wherein the neoplasm is
cervical cancer.
7. The method according to claim 1, wherein the neoplasm is uterine
cancer.
8. The method according to claim 1, wherein the neoplasm is a head
and neck cancer.
9. The method according to claim 1, wherein the neoplasm is
glioma.
10. The method according to claim 1, wherein the neoplasm is
non-small cell lung cancer.
11. The method according to claim 1, wherein the neoplasm is
prostate cancer.
12. The method according to claim 1, wherein the neoplasm is
pancreatic cancer.
13. The method according to claim 1, wherein the neoplasm is
lymphoma.
14. The method according to claim 1, wherein the neoplasm is
melanoma.
15. The method according to claim 1, wherein the neoplasm is small
cell lung cancer.
16. The method according to claim 1, wherein the neoplasm is
ovarian cancer.
17. .[.The.]. .Iadd.A .Iaddend.method .[.according to claim 1,
wherein the neoplasm is.]. .Iadd.of treating .Iaddend.colon cancer
.Iadd.in a mammal in need thereof, which comprises providing to
said mammal a synergistically effective amount of a combination
comprising rapamycin 42-ester with
3-hydroxy-2(hydroxymethyl)-2-methylpropionic acid (CCI-779) and
4-dimethylamino-but-2-enoic acid
[4-(3-chloro-4-fluoro-phenylamino)-3-cyano-7-ethoxy-quinolin-6-yl]-amide
(EKB-569).Iaddend..
18. The method according to claim 1, wherein the neoplasm is
esophageal cancer.
19. The method according to claim 1, wherein the neoplasm is
gastric cancer.
20. The method according to claim 1, wherein the neoplasm is
leukemia.
21. .[.The.]. .Iadd.A .Iaddend.method .[.according to claim 1,
wherein the neoplasm is.]. .Iadd.of treating .Iaddend.colorectal
cancer .Iadd.in a mammal in need thereof, which comprises providing
to said mammal a synergistically effective amount of a combination
comprising rapamycin 42-ester with
3-hydroxy-2(hydroxymethyl)-2-methylpropionic acid (CCI-779) and
4-dimethylamino-but-2-enoic acid
[4-(3-chloro-4-fluoro-phenylamino)-3-cyano-7-ethoxy-quinolin-6-yl]-amide
(EKB-569).Iaddend..
.[.22. The method according to claim 1, wherein the neoplasm is
unknown primary cancer..].
23. A method of treating a neoplasm .Iadd.in which uncontrolled
cell proliferation results from deregulation of epidermal growth
factor receptor (EGFR) kinase .Iaddend.in a mammal in need thereof,
which comprises providing to said mammal an effective amount of a
synergistic combination comprising .Iadd.rapamycin 42-ester with
3-hydroxy-2-(hydroxymethyl)-2-methylpropionic acid
.Iaddend.(CCI-779) and .Iadd.4-dimethylamino-but-2-enoic acid
[4-(3-chloro-4-fluoro-phenylamino)-3-cyano-7-ethoxy-quinolin-6-yl]-amide
.Iaddend.(EKB-569), wherein either CCI-779, EKB-569, or both are
provided in subtherapeutically effective amounts.
24. The method according to claim 23 in which CCI-779 is provided
in a subtherapeutically effective amount.
25. The method according to claim 23 in which EKB-569 is provided
in a subtherapeutically effective amount.
26. The method according to claim 23 in which both CCI-779 and
EKB-569 are provided in subtherapeutically effective amounts.
27. An antineoplastic combination .Iadd.useful in treatment of
neoplasms in which uncontrolled cell proliferation results from by
deregulation of epidermal growth factor receptor kinase
.Iaddend.which comprises an antineoplastic effective amount of a
synergistic combination of .Iadd.rapamycin 42-ester with
3-hydroxy-2-(hydroxymethyl)-2-methylpropionic acid
.Iaddend.(CCI-779) and .Iadd.4-dimethylamino-but-2-enoic acid
[4-(3-chloro-4-fluoro-phenylamino)-3-cyano-7-ethoxy-quinolin-6-yl]-amide
.Iaddend.(EKB-569).
Description
.Iadd.CROSS-REFERENCE TO OTHER REISSUE APPLICATIONS.Iaddend.
.Iadd.More than one reissue application has been filed for the
reissue of U.S. Pat. No. 6,617,333. Reissue divisional application
No. 11/645,330, filed Dec. 22, 2006, and reissue divisional
application No. 11/645,327, filed Dec. 22, 2006, are currently
pending..Iaddend.
BACKGROUND OF THE INVENTION
This application claims priority from copending provisional
application Serial No. 60/310,646, filed Aug. 7, 2001, the entire
disclosure of which is hereby incorporated by reference.
This invention relates to the use of combinations of rapamycin
42-ester with 3-hydroxy-2-(hydroxymethyl)-2-methylpropionic acid
(CCI-779) and 4-dimethylamino-but-2-enoic acid
[4-(3-chloro-4-fluoro-phenylamino)-3-cyano-7-ethoxy-quinolin-6-yl]-amide
(EKB-569).
Rapamycin is a macrocyclic triene antibiotic produced by
Streptomyces hygroscopicus, which was found to have anti-fungal
activity, particularly against Candida albicans, both in vitro and
in vivo [C. Vezina et al., J. Antibiot. 28, 721 (1975); S. N.
Sehgal et al., J. Antibiot. 28, 727 (1975); H. A. Baker et al., J.
Antibiot. 31, 539 (1978); U.S. Pat. Nos. 3,929,992; and 3,993,749].
Additionally, rapamycin alone (U.S. Pat. No. 4,885,171) or in
combination with picibanil (U.S. Pat. No. 4,401,653) has been shown
to have antitumor activity.
The immunosuppressive effects of rapamycin have been disclosed in
FASEB 3, 3411 (1989). Cyclosporin A and FK-506, other macrocyclic
molecules, also have been shown to be effective as
immunosuppressive agents, therefore useful in preventing transplant
rejection [FASEB 3, 3411 (1989); FASEB 3, 5256 (1989); R. Y. Calne
et al., Lancet 1183 (1978); and U.S. Pat. No. 5,100,899]. R. Martel
et al. [Can. J. Physiol. Pharmacol. 55, 48 (1977)] disclosed that
rapamycin is effective in the experimental allergic
encephalomyelitis model, a model for multiple sclerosis; in the
adjuvant arthritis model, a model for rheumatoid arthritis; and
effectively inhibited the formation of IgE-like antibodies.
Rapamycin is also useful in preventing or treating systemic lupus
erythematosus [U.S. Pat. No. 5,078,999], pulmonary inflammation
[U.S. Pat. No. 5,080,899], insulin dependent diabetes mellitus
[U.S. Pat. No. 5,321,009], skin disorders, such as psoriasis [U.S.
Pat. No. 5,286,730], bowel disorders [U.S. Pat. No. 5,286,731],
smooth muscle cell proliferation and intimal thickening following
vascular injury [U.S. Pat. Nos. 5,288,711 and 5,516,781], adult
T-cell leukemia/lymphoma [European Patent Application 525,960 A1],
ocular inflammation [U.S. Pat. No. 5,387,589], malignant carcinomas
[U.S. Pat. No. 5,206,018], cardiac inflammatory disease [U.S. Pat.
No. 5,496,832], and anemia [U.S. Pat. No. 5,561,138].
Rapamycin 42-ester with
3-hydroxy-2-(hydroxymethyl)-2-methylpropionic acid (CCI-779) is
ester of rapamycin which has demonstrated significant inhibitory
effects on tumor growth in both in vitro and in vivo models. The
preparation and use of hydroxyesters of rapamycin, including
CCI-779, are disclosed in U.S. Pat. No. 5,362,718.
CCI-779 exhibits cytostatic, as opposed to cytotoxic properties,
and may delay the time to progression of tumors or time to tumor
recurrence. CCI-779 is considered to have a mechanism of action
that is similar to that of sirolimus. CCI-779 binds to and forms a
complex with the cytoplasmic protein FKBP, which inhibits an
enzyme, mTOR (mammalian target of rapamycin, also known as
FKBP12-rapamycin associated protein [FRAP]). Inhibition of mTOR's
kinase activity inhibits a variety of signal transduction pathways,
including cytokine-stimulated cell proliferation, translation of
mRNAs for several key proteins that regulate the G1 phase of the
cell cycle, and IL-2-induced transcription, leading to inhibition
of progression of the cell cycle from G1 to S. The mechanism of
action of CCI-779 that results in the G1.fwdarw.S phase block is
novel for an anticancer drug.
In vitro, CCI-779 has been shown to inhibit the growth of a number
of histologically diverse tumor cells. Central nervous system (CNS)
cancer, leukemia (T-cell), breast cancer, prostate cancer, and
melanoma lines were among the most sensitive to CCI-779. The
compound arrested cells in the G1 phase of the cell cycle.
In vivo studies in nude mice have demonstrated that CCI-779 has
activity against human tumor xenografts of diverse histological
types. Gliomas were particularly sensitive to CCI-779 and the
compound was active in an ortho-topic glioma model in nude mice.
Growth factor (platelet-derived)-induced stimulation of a human
glioblastoma cell line in vitro was markedly suppressed by CCI-779.
The growth of several human pancreatic tumors in nude mice as well
as one of two breast cancer lines studied in vivo also was
inhibited by CCI-779.
Protein tyrosine kinases are a class of enzymes that catalyze the
transfer of a phosphate group from ATP or GTP to tyrosine residue
located on protein substrates. Protein tyrosine kinases clearly
play a role in normal cell growth. Many of the growth factor
receptor proteins function as tyrosine kinases and it is by this
process that they effect signaling. The interaction of growth
factors with these receptors is a necessary event in normal
regulation of cell growth. However, under certain conditions, as a
result of either mutation or overexpression, these receptors can
become deregulated; the result of which is uncontrolled cell
proliferation which can lead to tumor growth and ultimately to the
disease known as cancer [Wilks A. F., Adv. Cancer Res., 60, 43
(1993) and Parsons, J. T.; Parsons, S. J., Important Advances in
Oncology, De Vita V. T. Ed., J. B. Lippincott Co., Phila., 3
(1993)]. Among the growth factor receptor kinases and their
proto-oncogenes that have been identified and which are targets of
the compounds of this invention are the epidermal growth factor
receptor kinase (EGF-R kinase, the protein product of the erbB
oncogene), and the product produced by the erbB-2 (also referred to
as the neu or HER2) oncogene. Since the phosphorylation event is a
necessary signal for cell division to occur and since overexpressed
or mutated kinases have been associated with cancer, an inhibitor
of this event, a protein tyrosine kinase inhibitor, will have
therapeutic value for the treatment of cancer and other diseases
characterized by uncontrolled or abnormal cell growth. For example,
overexpression of the receptor kinase product of the erbB-2
oncogene has been associated with human breast and ovarian cancers
[Slamon, D. J., et al., Science, 244, 707 (1989) and Science, 235,
1146 (1987)]. Deregulation of EGF-R kinase has been associated with
epidermoid tumors [Reiss, M., et al., Cancer Res., 51, 6254
(1991)], breast tumors [Macias, A., et al., Anticancer Res., 7, 459
(1987)], and tumors involving other major organs [Gullick, W. J.,
Brit. Med. Bull., 47, 87 (1991)]. Because of the importance of the
role played by deregulated receptor kinases in the pathogenesis of
cancer, many recent studies have dealt with the development of
specific PTK inhibitors as potential anti-cancer therapeutic agents
[some recent reviews: Burke, T. R., Drugs Future, 17, 119 (1992)
and Chang, C. J.; Geahlen, R. L., J. Nat. Prod., 55, 1529
(1992)].
4-Dimethylamino-but-2-enoic acid
[4-(3-chloro-4-fluoro-phenylamino)-3-cyano-7-ethoxy-quinolin-6-yl]-amide
(EKB-569) is an EGFR kinase inhibitor which has significant
inhibitory effects on tumor growth in both in vitro and in vivo
models. The preparation and use of EGFR kinase inhibitors, such as
EKB-569, are disclosed in U.S. Pat. No. 6,002,008.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows cytotoxicity curves of EKB-569, CCI-779, and
combinations of EKB-569+CCI-779 in HCT116 cells.
FIG. 2 shows isobolograms (at the 50% effect level) of a
EKB-569+CCI-779 combination.
FIG. 3 shows isobolograms for EKB-569+CCI-779 combinations derived
from different endpoints ranging from 50-65%.
FIG. 4 shows a 3-dimensional analysis of the synergistic
interaction of a EKB-569+CCI-779 combination.
FIG. 5 shows a contour plot of the 3-dimensional synergy plot of a
EKB-569+CCI-779 combination.
DESCRIPTION OF THE INVENTION
This invention provides the use of combinations of CCI-779 and
EKB-569 as antineoplastic combination chemotherapy. In particular,
these combinations are useful in the treatment of renal cancer,
soft tissue cancer, breast cancer, neuroendocrine tumor of the
lung, cervical cancer, uterine cancer, head and neck cancer,
glioma, non-small lung cell cancer, prostate cancer, pancreatic
cancer, lymphoma, melanoma, small cell lung cancer, ovarian cancer,
colon cancer, esophageal cancer, gastric cancer, leukemia,
colorectal cancer, and unknown primary cancer. This invention also
provides combinations of CCI-779 and EKB-569 for use as
antineoplastic combination chemotherapy, in which the dosage of
either CCI-779 or EKB-569 or both are used in subtherapeutically
effective dosages.
As used in accordance with this invention, the term "treatment"
means treating a mammal having a neoplastic disease by providing
said mammal an effective amount of a combination of CCI-779 and
EKB-569 with the purpose of inhibiting growth of the neoplasm in
such mammal, eradication of the neoplasm, or palliation of the
mammal.
As used in accordance with this invention, the term "providing,"
with respect to providing the combination, means either directly
administering the combination, or administering a prodrug,
derivative, or analog of one or both of the components of the
combination which will form an effective amount of the combination
within the body.
The preparation of CCI-779 is described in U.S. Pat. No. 5,362,718,
which is hereby incorporated by reference. An improved preparation
of CCI-779 is disclosed in U.S. patent application Ser. No.
09/670,358, now U.S. Pat. No. 6,277,983, which is hereby
incorporated by reference. When CCI-779 is used as an
antineoplastic agent, it is projected that initial i.v. infusion
dosages will be between about 0.1 and 100 mg/m.sup.2 when
administered on a daily dosage regimen (daily for 5 days, every 2-3
weeks), and between about 0.1 and 1000 mg/m.sup.2 when administered
on a once weekly dosage regimen. Oral or intravenous infusion are
the preferred routes of administration, with intravenous being more
preferred.
EKB-569 can be prepared according to the procedures described in
U.S. Pat. No. 6,002,008, which is incorporated by reference.
Preferred procedures for the preparation of EKB-569 are provided
herein. When EKB-569 is used as an antineoplastic agent it is
projected that the initial oral dosage will be between 1 and 100 mg
per day. Depending on patient tolerance, EKB-569 can be
administered daily for a treatment period, such as 14 days,
followed by a rest period (no drug administered), or can be
administered on a continuous basis for a longer treatment period
(for example, 6 months or longer).
The antineoplastic activity of the CCI-779 plus EKB-569 combination
was confirmed in in vitro standard pharmacological test procedure;
the following briefly describes the procedure used and the results
obtained.
Cell Proliferation Procedure--HCT 116 colon adenocarcinoma cells
were maintained in RPMI 1640 medium (Life Technologies, Inc.,
Gaithersburg, Md.) supplemented with 10% fetal bovine serum (FBS,
Life Technologies) and 50 .mu.g/ml gentamicin (Life Technologies)
under 7% CO.sub.2 at 37.degree. C. Cells were plated in 96-well
microtiter dishes (6000 cells/well) in 200 .mu.l RPMI 1640 medium
containing 5% FBS and 50 .mu.g/ml gentamicin and incubated
overnight at 37.degree. C. Compound dilutions were prepared in the
same medium, at 5.times. final concentration, and 50 .mu.l of the
drug dilution was added to the cell-containing wells. For studies
involving combinations of two drugs, serial dilutions of one
compound were prepared in the presence of a fixed dose of a second
compound. Alternatively, a checkerboard dilution series was
employed. Cells were cultured for three days in the presence of the
drugs. Untreated cells were included as controls. The percentage of
surviving cells was determined using sulforhodamine B (SRB,
Sigma-Aldrich, St Louis, Mo.), a protein binding dye. Cellular
protein was precipitated in each well by the addition of 50 .mu.l
of 50% cold trichloroacetic acid. After 1 hour, the plates were
washed extensively in water and dried. SRB dye reagent (0.4% SRB in
1% acetic acid, 80 .mu.l per well) was added and plates were kept
at room temperature for ten minutes. Plates were then washed
thoroughly in 1% acetic acid and dried. Cell-associated dye was
dissolved in 10 mM Tris (150 .mu.l) and the absorbance was read at
540 nm in a microtiter plate reader. The concentration of compound
that caused a fixed percentage inhibition of growth was determined
by plotting cell survival (relative to untreated cells) against the
compound dose.
Synergy Evaluation--Isobolograms were used to study the interaction
of two pharmacological agents. Here, the concentration of each drug
alone which produces a certain endpoint (e.g 50% inhibition of cell
growth, IC.sub.50), is plotted on the two graphical axes. The
straight line connecting the two points represents equally
effective concentrations of all combinations of the two drugs if
the interaction is purely additive. A shift of the isobologram to
the left of the predicted cytotoxicity (curve with concave side up)
represents a synergistic interaction. Conversely, a shift to the
right (isobologram with the convex side up) represents an
antagonistic interaction. When isobolograms for different
end-points were plotted on the same graph, the concentration of
each drug was expressed as the fraction of the concentration of
each drug alone that produced the same effect. This produces a
symmetrical isobologram with unit-less measures on each axis, and
allows a direct comparison of different endpoints.
A second model for studying drug interactions was proposed by
Prichard and Shipman [Antiviral Research 14:181-206 (1990)]. This
is a 3-dimensional model: one for each drug and the third for the
biological effect. Theoretical additive interactions are calculated
from the individual dose-response curves, based on a dissimilar
sites model of additivity (Bliss independence). The calculated
additive surface, representing predicted cytotoxicity is subtracted
from the experimental surface to reveal areas of enhanced toxicity
(synergy) or reduced toxicity (antagonism). The resulting surface
appears as a horizontal plane at 0% inhibition above the calculated
additive surface, if the interaction is additive. Peaks and valleys
deviating from this plane are indicative of synergy and antagonism,
respectively. MacSynergyll, a Microsoft Excel-based software was
used to perform all calculations automatically. This spreadsheet
calculates the theoretical additive interactions, and locates and
quantifies synergistic or antagonistic interactions that are
significant at the 95% confidence levels. The results were plotted
as a 3-dimensional plot, or as a contour plot.
Results--HCT 116 cells were chosen as they express low, but
detectable levels of EGFR, and are sensitive to inhibition by EGFR
inhibitors. The cells are somewhat resistant to CCI-779, but are
inhibited by high doses (5-10 .mu.g/ml) of this drug. HCT-116 cells
were cultured in the presence of EKB-569 alone, CCI-779 alone, or a
dilution series of EKB-569 with fixed doses of CCI-779. Following
growth for 3 days, cell survival was determined using the SRB test
procedure. Cytotoxicity curves are shown in FIG. 1. EKB-569
produced an IC.sub.50 value of 0.31 .mu.g/ml in HCT116 cells. When
this compound was combined with 2.08 .mu.g/ml CCI-779 (which caused
41% inhibition of growth when administered alone), the IC.sub.50
value is reduced to 0.03 .mu.g/ml, a 10-fold decrease. When
combined with 0.026 .mu.g/ml CCI-779 (which alone inhibits cell
proliferation by 36%), the IC.sub.50 value dropped to 0.051
.mu.g/ml, a 6-fold decrease. Similar results were observed when
dose-response curves were produced with CCI-779 in the presence of
fixed doses of EKB-569. To identify the nature of this drug
interaction, isobolograms (at 50% effect level) of the combination
of EKB-569 and CCI-779 were generated (FIG. 2). The isobologram was
deeply indented with the concave side up, indicating a substantial
synergistic interaction between the two drugs. At the most
synergistic point, 0.03 .mu.g/ml of EKB-569 combined with 0.077
.mu.g/ml CCI-779 was iso-effective with 0.31 .mu.g/ml of EKB-569
alone or 4.3 .mu.g/ml CCI-779 alone (IC.sub.50 for each drug
alone). Thus, a 10-fold reduction in the dose of EKB-569 and a
50-fold reduction in the dose of CCI-779 was required to inhibit
cell proliferation by 50% when the drugs were combined, compared to
either drug alone. Isobolograms derived from different endpoints,
ranging from 50 to 65% were also examined. As shown in FIG. 3., the
isobolograms produced were almost superimposable, indicating
synergy at all effect levels tested.
The interaction between EKB-569 and CCI-779 was also evaluated
using a 3-dimensional analysis. Here, pharmacological interactions
are presented in a 3-dimensional plot with the plane at 0%
representing additive interaction, and peaks and valleys
representing areas of synergy or antagonism, between the two drugs.
In FIG. 4, the combination of EKB-569 and CCI-779 resulted in a
broad area of synergistic interaction consistent with the results
shown in the isobologram studies. A contour plot of the
3-dimensional synergy plot facilitates the identification of the
concentration of drugs at which greatest synergistic toxicity
occurs (FIG. 5). A broad area of synergy was observed at 0.0005 to
3 .mu.g/ml CCI-779 and 0.16 to 0.4 .mu.g/ml EKB-569. Within this
area, two peaks of maximum synergy occurred at 0.0005 to 0.003
.mu.g/ml and 0.05 to 0.3 .mu.g/ml of CCI-779 and 0.25 to 0.37
.mu.g/ml EKB-569.
Based on the results of these standard pharmacological test
procedures, combinations of CCI-779 plus EKB-569 acted
synergistically together, and are useful as antineoplastic therapy.
More particularly, these combinations are useful in the treatment
of renal carcinoma, soft tissue sarcoma, breast cancer,
neuroendocrine tumor of the lung, cervical cancer, uterine cancer,
head and neck cancer, glioma, non-small cell lung cancer, prostate
cancer, pancreatic cancer, lymphoma, melanoma, small cell lung
cancer, ovarian cancer, colon cancer, esophageal cancer, gastric
cancer, leukemia, colorectal cancer, and unknown primary cancer. As
these combinations contain at least two active antineoplastic
agents, the use of such combinations also provides for the use of
combinations of each of the agents in which one or both of the
agents is used at subtherapeutically effective dosages, thereby
lessening toxicity associated with the individual chemotherapeutic
agent.
In providing chemotherapy, multiple agents having different
modalities of action are typically used as part of a chemotherapy
"cocktail." It is anticipated that the combinations of this
invention will be used as part of a chemotherapy cocktail that may
contain one or more additional antineoplastic agents depending on
the nature of the neoplasia to be treated. For example, this
invention also covers the use of the CCI-779/EKB-923 combination
used in conjunction with other chemotherapeutic agents, such as
antimetabolites (i.e., 5-fluorouracil, floxuradine, thioguanine,
cytarabine, fludarabine, 6-mercaptopurine, methotrexate,
gemcitabine, capecitabine, pentostatin, trimetrexate, or
cladribine); DNA crosslinking and alkylating agents (i.e.,
cisplatin, carboplatin, streptazoin, melphalan, chlorambucil,
carmustine, methclorethamine, lomustine, bisulfan, thiotepa,
ifofamide, or cyclophosphamide); hormonal agents (i.e., tamoxifen,
roloxifen, toremifene, anastrozole, or letrozole); antibiotics
(i.e., plicamycin, bleomycin, mitoxantrone, idarubicin,
dactinomycin, mitomycin, doxorubicin or daunorubicin);
immunomodulators (i.e., interferons, IL-2, or BCG); antimitotic
agents (i.e., estramustine, paclitaxel, docetaxel, vinblastine,
vincristine, or vinorelbine); topoisomerase inhibitors (i.e.,
topotecan, irinotecan, etoposide, or teniposide.); and other agents
(i.e., hydroxyurea, trastuzumab, altretamine, retuximab,
L-asparaginase, or gemtuzumab ozogamicin).
As used in this invention, the combination regimen can be given
simultaneously or can be given in a staggered regimen, with CCI-779
being given at a different time during the course of chemotherapy
than EKB-923. This time differential may range from several
minutes, hours, days, weeks, or longer between administration of
the two agents. Therefore, the term combination does not
necessarily mean administered at the same time or as a unitary
dose, but that each of the components are administered during a
desired treatment period. The agents may also be administered by
different routes. For example, in the combination of CCI-779 plus
EKB-569, it is anticipated that the CCI-779 will be administered
orally or parenterally, with parenterally being preferred, while
the EKB-569 may be administered parenterally, orally, or by other
acceptable means. These combination can be administered daily,
weekly, or even once monthly. As typical for chemotherapeutic
regimens, a course of chemotherapy may be repeated several weeks
later, and may follow the same timeframe for administration of the
two agents, or may be modified based on patient response.
As typical with chemotherapy, dosage regimens are closely monitored
by the treating physician, based on numerous factors including the
severity of the disease, response to the disease, any treatment
related toxicities, age, health of the patient, and other
concomitant disorders or treatments.
Based on the results obtained with the CCI-779 plus EKB-569
combinations, it is projected that the initial i.v. infusion dosage
of CCI-779 will be between about 0.1 and 100 mg/m.sup.2, with
between about 2.5 and 70 mg/m.sup.2 being preferred. It is also
preferred that the CCI-779 be administered by i.v., typically over
a 30 minute period, and administered about once per week. The
initial daily dosages of EKB-569 will be between about 1 and 100
mg, with between 5 and 75 mg being preferred. After one or more
treatment cycles, the dosages can be adjusted upwards or downwards
depending on the results obtained and the side effects
observed.
Oral formulations containing the active compounds of this invention
may comprise any conventionally used oral forms, including tablets,
capsules, buccal forms, troches, lozenges and oral liquids,
suspensions or solutions. Capsules may contain mixtures of the
active compound(s) with inert fillers and/or diluents such as the
pharmaceutically acceptable starches (e.g. corn, potato or tapioca
starch), sugars, artificial sweetening agents, powdered celluloses,
such as crystalline and microcrystalline celluloses, flours,
gelatins, gums, etc. Useful tablet formulations may be made by
conventional compression, wet granulation or dry granulation
methods and utilize pharmaceutically acceptable diluents, binding
agents, lubricants, disintegrants, surface modifying agents
(including surfactants), suspending or stabilizing agents,
including, but not limited to, magnesium stearate, stearic acid,
talc, sodium lauryl sulfate, microcrystalline cellulose,
carboxymethylcellulose calcium, polyvinylpyrrolidone, gelatin,
alginic acid, acacia gum, xanthan gum, sodium citrate, complex
silicates, calcium carbonate, glycine, dextrin, sucrose, sorbitol,
dicalcium phosphate, calcium sulfate, lactose, kaolin, mannitol,
sodium chloride, talc, dry starches and powdered sugar. Preferred
surface modifying agents include nonionic and anionic surface
modifying agents. Representative examples of surface modifying
agents include, but are not limited to, poloxamer 188, benzalkonium
chloride, calcium stearate, cetostearyl alcohol, cetomacrogol
emulsifying wax, sorbitan esters, colloidal silicon dioxide,
phosphates, sodium dodecylsulfate, magnesium aluminum silicate, and
triethanolamine. Oral formulations herein may utilize standard
delay or time release formulations to alter the absorption of the
active compound(s). The oral formulation may also consist of
administering the active ingredient in water or a fruit juice,
containing appropriate solubilizers or emulsifiers as needed.
In some cases it may be desirable to administer the compounds
directly to the airways in the form of an aerosol.
The compounds may also be administered parenterally or
intraperitoneally. Solutions or suspensions of these active
compounds as a free base or pharmacologically acceptable salt can
be prepared in water suitably mixed with a surfactant such as
hydroxy-propylcellulose. Dispersions can also be prepared in
glycerol, liquid polyethylene glycols and mixtures thereof in oils.
Under ordinary conditions of storage and use, these preparation
contain a preservative to prevent the growth of microorganisms.
The pharmaceutical forms suitable for injectable use include
sterile aqueous solutions or dispersions and sterile powders for
the extemporaneous preparation of sterile injectable solutions or
dispersions. In all cases, the form must be sterile and must be
fluid to the extent that easy syringability exists. It must be
stable under the conditions of manufacture and storage and must be
preserved against the contaminating action of microorganisms such
as bacteria and fungi. The carrier can be a solvent or dispersion
medium containing, for example, water, ethanol, polyol (e.g.,
glycerol, propylene glycol and liquid polyethylene glycol),
suitable mixtures thereof, and vegetable oils.
For the purposes of this disclosure, transdermal administrations
are understood to include all administrations across the surface of
the body and the inner linings of bodily passages including
epithelial and mucosal tissues. Such administrations may be carried
out using the present compounds, or pharmaceutically acceptable
salts thereof, in lotions, creams, foams, patches, suspensions,
solutions, and suppositories (rectal and vaginal).
Transdermal administration may be accomplished through the use of a
transdermal patch containing the active compound and a carrier that
is inert to the active compound, is non toxic to the skin, and
allows delivery of the agent for systemic absorption into the blood
stream via the skin. The carrier may take any number of forms such
as creams and ointments, pastes, gels, and occlusive devices. The
creams and ointments may be viscous liquid or semisolid emulsions
of either the oil-in-water or water-in-oil type. Pastes comprised
of absorptive powders dispersed in petroleum or hydrophilic
petroleum containing the active ingredient may also be suitable. A
variety of occlusive devices may be used to release the active
ingredient into the blood stream such as a semi-permeable membrane
covering a reservoir containing the active ingredient with or
without a carrier, or a matrix containing the active ingredient.
Other occlusive devices are known in the literature.
Suppository formulations may be made from traditional materials,
including cocoa butter, with or without the addition of waxes to
alter the suppository's melting point, and glycerin. Water soluble
suppository bases, such as polyethylene glycols of various
molecular weights, may also be used.
The following provides the preparation of EKB-569 from commercially
available starting materials or starting materials that can be made
according to available literature procedures.
Preparation of 4-dimethylaminocrotonic acid from
TMS-4-bromocrotonate
##STR00001##
211 ml dimethylamine (2M in THF, 0.422 moles) was added drop-wise
to a solution of 50 g TMS-4-bromocrotonate (0.211 moles, 75.9% by
GC-MS) in 250 ml of THF at 0-50 C. under N.sub.2. The reaction
mixture was stirred at room temperature for 30 minutes. A white
solid by-product was filtered off. 2 ml water was added to the
filtrate followed by seeding. The crystals formed were filtered and
washed with ether to give 18.3 g (from two crops) off-white solid
product. Yield was 67.2% (98% purity by GC-MS, NMR was consistent
with the structure).
Preparation of methyl 4-dimethylaminocrotonate from
methyl-4-bromocrotonate
##STR00002##
120 ml dimethylamine (2M in THF, 0.24 moles) was added drop-wise to
a solution of 20 g methyl 4-bromocrotonate (85% purity, 0.095
moles) in 150 ml of THF at 0-50 C. under N.sub.2. The reaction
mixture was stirred for 15 minutes at room temperature. TLC (9:1
CH.sub.2Cl.sub.2:MeOH with few drops of Et.sub.3N) showed residual
methyl 4-bromocrotonate. The reaction mixture was heated to 40-450
C. for 15 minutes. A white solid by-product was filtered off. The
filtrate was evaporated to give a yellow oil (14 g). The yellow oil
was dissolved in 100 ml CH.sub.2Cl.sub.2 and washed with H.sub.2O
twice. The aqueous layer was back extracted with 100 ml
CH.sub.2Cl.sub.2. The CH.sub.2Cl.sub.2 layers were combined, dried
over MgSO.sub.4 and filtered. The filtrate was evaporated to give
an oil (12 g). Yield was 88%. NMR indicated desired product with
trace methyl 4-bromocrotonate.
Preparation of Methyl 4-N,N-dimethylaminocrotonate hydrochloride on
large scale
##STR00003##
A 3 L flask was charged with tetrahydrofuran (0.71 kg, 0.80 L).
Methyl 4-bromocrotonate (0.20 kg, 0.13 L, d=1.522 g/mL) was added
and rinsed with tetrahydrofuran (0.18 kg, 0.20 L). The solution was
stirred and cooled to 0-10.degree. C. An additional funnel was
charged with a solution of dimethylamine in tetrahydrofuran and
added over (1 h 15 min) keeping the temperature at 0-10.degree. C.
The mixture was stirred for a minimum of 30 mins and checked for
reaction completion by TLC. The reaction was complete when there is
.ltoreq.2% detectable starting material (methyl 4-bromocrotonate)
present. The mixture was filtered cold on a Buchner funnel into a 3
L multi-neck flask, rinsed with pre-chilled (0-10.degree. C.)
tetrahydrofuran (2.times.0.18 kg, 2.times.0.20 L), and suction
maintained until dripping stops. The flask was equipped with an
agitator, thermometer, and a setup for vacuum distillation. The
solution was concentrated by distillation under a reduced pressure
of (125-200 mm Hg) and at a maximum pot temperature of 40.degree.
C.) to a pot volume of (200 mL). Isopropanol (0.22 kg, 0.28 L) was
added and the mixture cooled to 0-10.degree. C. The distillation
stillhead was replaced with an addition funnel charged with a
solution of HCl in isopropanol, which was added over 45 min until
pH of 2.0-3.0 was reached, while maintaining a temperature
0-10.degree. C. The mixture was held for a minimum 30 min, and
fileted cold on a Buchner funnel, rinsed with isopropanol
(2.times.0.12 kg, 2.times.0.15 L). The filter cake was dammed and
suction maintained until dripping stopped. The product was dried in
a vacuum oven at 50.degree. C. and 10 mm Hg for 18-20 h.
Preparation of 4-dimethylaminocrotonic acid hydrochloride from
methyl 4-dimethylaminocrotonate
##STR00004##
A NaOH solution (3.35 g in 25 ml H.sub.2O, 0.084 moles) was added
drop-wise to a solution of 12 g methyl 4-dimethylaminocrotonate
(0.084 moles) in 100 ml MeOH at room temperature. The reaction
mixture was heated to 40-45.degree. C. for 1 hour then cooled to
room temperature. The pH was adjusted to 1.about.2 with 5 N HCl.
The mixture was concentrated to a thick oil which was triturated
with dehydrated alcohol to form a solid. The solid by-product was
filtered off. The filtrate was evaporated to an oil which was
triturated with IPA. Seven (7.0) g of white solid product was
obtained. Yield was 50% with the purity 86.3% by GC-MS.
Preparation of 4-N,N-dimethylaminocrotonic acid hydrochloride on
large scale
##STR00005##
A 2 L multi-neck flask was equipped with agitator thermometer,
addition funnel, and nitrogen protection. The flask was charged
with ethanol (0.39 kg, 0.50 L). Methyl 4-N,N-dimethylamino
crotonate hydrochloride (0.125 kg) was added and rinsed with
ethanol (0.10 kg, 0.125 L). The suspension was stirred and cooled
to 0-10.degree. C. The addition funnel was charged with sodium
hydroxide (50%) (0.11 kg, 0.072 L, d=1.53 g/mL) and addd over 20
min keeping the temperature at 0-10.degree. C. A slight exotherm
was observed and the mixture turned yellow. The mixture was stirred
for a minimum of 15 min, and then warmed to 18-22.degree. C., and
held for a minimum of 4 h. The reaction was checked for completion
by TLC. The reaction is complete when there is .ltoreq.2%
detectable starting material (methyl 4-N,N-dimethylaminocrotonate
hydrochloride) present. The mixture was cooled to 0-10.degree. C.
An addition funnel was charged with a solution of HCl in
isopropanol and added over 40 min until pH 2.0-3.0 was attained,
while maintaining the pot temperature of 0-10.degree. C. The
mixture was sturred for a minimum of 30 min, and filtered cold on a
Buchner funnel into a 2 L multi-neck flask, rinsed with cold
ethanol (0-10.degree. C.) (2.times.0.05 kg, 2.times.0.063 L) with
suction maintained until dripping stops. The flask was equpped with
an agitator, thermometer, and setup for vacuum distillation.
Solvent was removed under a reduced pressure of 50-100 mm Hg and at
a maximum pot temperature of (40.degree. C.) to a pot volume of
160-180 mL. Isopropanol (0.049 kg, 0.063 L) was added, and the
mixture warmed to 35-40.degree. C. over 10 min. Acetone (0.10 kg,
0.13 L) was added over 20 min while maintaining the pot temperature
at 35-40.degree. C. The mixture was seeded and cooled to ambient
temperature 20-25.degree. C., and held there for a minimum of 12-18
h. The mixture was cooled to 0-10.degree. C., held there for a
minimum of 1 h. A mixture of isopropanol (0.049 kg, 0.063 L) and
acetone (0.10 kg, 0.13 L) was prepared, stirred to homogenize, and
cooled to 0-10.degree. C. The mixture was filtered cold on a
Buchner funnel, rinsed with isopropanol/acetone (2.times.0.074 kg,
2.times.0.96 L), and the filter caked dammed while maintaining
suction until dripping stopped. The product was dried in a vacuum
oven at 50.degree. C. and 10 mm Hg for 18-20 h.
Preparation of 4-dimethylaminocrotonyl anilide from
4-dimethylaminocrotonic acid hydrochloride
##STR00006##
Thionyl chloride (0.36 ml, 0.005 moles) was added dropwise to a
solution of 0.33 g 4-dimethylaminocrotonic acid hydrochloride
(0.002 moles) in 15 ml CH.sub.2Cl.sub.2 containing 2 drops of DMF
at 0.degree. C. under N2. The reaction mixture was refluxed for 30
min. Then 0.72 ml aniline (0.008 moles) was added drop-wise to the
reaction mixture at 0.degree. C. and stirred for 1 hour at room
temperature. A solid by-product was filtered. The filtrate was
evaporated to give an oil (0.6 g). GC-MS data shows that the oil is
11.7% 4-dimethylaminocrotonic acid hydrochloride and 85% of desired
product.
Preparation and isolation of 4-N,N-dimethylaminocrotonoylchloride
hydrochloride
A well stirred suspension 4-dimethylaminocrotonic acid
hydrochloride (5.0 g, 30 mmol) in cold (0.degree. C.) THF (40 mL)
and DMF (2 pipet drops) was treated with oxalyl chloride (3.15 mL,
36 mmol). The mixture was stirred at 20-25.degree. C. for 3 h then
cooled to 0.degree. C. and held for 30 min. The solids were
collected on Buchner funnel (under a blanket of nitrogen) and
washed with cold (0.degree. C.) THF (3.times.5 mL). The product was
dried under vacuum (.about.1 torr) at 40-50 2.degree. C. for 3 h to
give 4.0 g of 4-dimethylaminocrotonoyl chloride hydrochloride. This
material is characterized as its methyl ester by treatment of the
solid with methanol.
Alternatively, the title compound can be prepared in CH.sub.3CN and
used directly for the coupling step:
Preparation of EKB-569
A 3 L multi-neck flask was equipped with an agitator, thermometer,
dip tube, and nitrogen protection. The flask was charged with
N-methyl pyrrolidinone (0.77 kg, 0.75 L, d=1.033 g/mL). At ambient
temperature,
4-[3-chloro-4-fluorophenyl]amino-6-amino-3-cyano-7-ethoxy quinoline
(0.0748 kg) ]see, U.S. Pat. No. 6,002,008] was added and the
mixture stirred while heating to 40-45.degree. C. and hold for 15
min. The flask was cooled to 0-10.degree. C. The mixture containing
4-N,N-dimethylaminocrotonoyl chloride hydrochloride was transferred
via dip tube and positive nitrogen pressure to the 3 L flask over
30-45 min, while maintaining 0-10.degree. C. The mixture was kept
at 0-10.degree. C. for a minimum of 2 h. The reaction was checked
for completion by HPLC. The reaction is complete when there is
.ltoreq.2% of the starting material
(4-[3-chloro-4-fluorophenyl]amino-6-amino-3-cyano-7-ethoxy
quinoline) present. A 12 L multi-neck flask equipped with agitator,
thermometer, dip tube, and nitrogen protection was charged with
water (2.61 kg, 2.61 L). Sodium bicarbonate (0.209 kg) was added
and stirred until a solution was obtained. The solution was cooled
to 20-24.degree. C. The NMP-CH.sub.3CN mixture was transferred, via
dip tube and positive nitrogen pressure, to the 12 L flask over
45-60 min, while maintaining 20-24.degree. C. The mixture was
maintained at 20-24.degree. C. for a minimum of 1 h, and filtered
on a Buchner funnel, and rinsed with water (3.times.0.40 kg,
3.times.0.40 L) with suction being maintained until dripping stops.
The product was dried in a vacuum oven at 50.degree. C. and 10 mm
Hg for 28-30 h to give 78.5 g (86% yield) of product.
* * * * *