U.S. patent application number 16/018847 was filed with the patent office on 2019-05-23 for methods of treating various cancers using an axl/cmet inhibitor alone or in combination with other agents.
This patent application is currently assigned to IGNYTA, INC.. The applicant listed for this patent is IGNYTA, INC.. Invention is credited to Thelma S. ANGELES, Mark A. ATOR, Mangeng M. CHENG, Bruce D. DORSEY, Robert L. HUDKINS, Bruce A. RUGGERI.
Application Number | 20190151317 16/018847 |
Document ID | / |
Family ID | 52432571 |
Filed Date | 2019-05-23 |
![](/patent/app/20190151317/US20190151317A1-20190523-C00001.png)
![](/patent/app/20190151317/US20190151317A1-20190523-C00002.png)
![](/patent/app/20190151317/US20190151317A1-20190523-C00003.png)
![](/patent/app/20190151317/US20190151317A1-20190523-C00004.png)
![](/patent/app/20190151317/US20190151317A1-20190523-D00001.png)
![](/patent/app/20190151317/US20190151317A1-20190523-D00002.png)
![](/patent/app/20190151317/US20190151317A1-20190523-D00003.png)
![](/patent/app/20190151317/US20190151317A1-20190523-D00004.png)
![](/patent/app/20190151317/US20190151317A1-20190523-D00005.png)
![](/patent/app/20190151317/US20190151317A1-20190523-D00006.png)
![](/patent/app/20190151317/US20190151317A1-20190523-D00007.png)
View All Diagrams
United States Patent
Application |
20190151317 |
Kind Code |
A1 |
ANGELES; Thelma S. ; et
al. |
May 23, 2019 |
METHODS OF TREATING VARIOUS CANCERS USING AN AXL/cMET INHIBITOR
ALONE OR IN COMBINATION WITH OTHER AGENTS
Abstract
This application describes the use of the compound ##STR00001##
or a salt thereof, either alone or in combination with other
therapeutically active agents, for the treatment of particular
cancers, including any solid or hematological cancer in which AXL
or c-Met is over-expressed.
Inventors: |
ANGELES; Thelma S.; (West
Chester, PA) ; ATOR; Mark A.; (Paoli, PA) ;
CHENG; Mangeng M.; (Stoughton, PA) ; DORSEY; Bruce
D.; (Ambler, PA) ; HUDKINS; Robert L.;
(Chester Springs, PA) ; RUGGERI; Bruce A.; (West
Chester, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
IGNYTA, INC. |
San Diego |
CA |
US |
|
|
Assignee: |
IGNYTA, INC.
San Diego
CA
|
Family ID: |
52432571 |
Appl. No.: |
16/018847 |
Filed: |
June 26, 2018 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14907514 |
Jan 25, 2016 |
10028956 |
|
|
PCT/US14/49028 |
Jul 31, 2014 |
|
|
|
16018847 |
|
|
|
|
61861482 |
Aug 2, 2013 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 35/00 20180101;
A61K 31/513 20130101; A61K 31/541 20130101; A61K 31/517 20130101;
A61K 31/47 20130101; A61K 45/06 20130101; A61K 31/517 20130101;
A61K 2300/00 20130101; A61K 31/513 20130101; A61K 2300/00
20130101 |
International
Class: |
A61K 31/513 20060101
A61K031/513; A61K 31/517 20060101 A61K031/517; A61K 45/06 20060101
A61K045/06 |
Claims
1.-27. (canceled)
28. A method of treating non-small cell lung cancer in a subject,
the method comprising administering to the subject a combination of
(i) erlotinib or gefitinib, and (ii) a compound having the
structure of: ##STR00004## or a pharmaceutically acceptable salt
thereof, wherein the non-small cell lung cancer is resistant or
insensitive to treatment with one or more EGFR inhibitors.
29. The method of claim 28, wherein the one or more EGFR inhibitors
are erlotinib and/or gefitinib.
30. The method of claim 28, wherein the one or more EGFR inhibitors
is erlotinib.
31. The method of claim 28, wherein the one or more EGFR inhibitors
is gefitinib.
32. The method of claim 28, wherein the compound, or a
pharmaceutically acceptable salt thereof, is administered to the
subject from one to four times daily.
33. The method of claim 32, wherein the compound, or a
pharmaceutically acceptable salt thereof, is administered to the
subject in an amount from about 10 mg/kg to about 55 mg/kg.
Description
BACKGROUND
[0001] The present application relates to novel compounds that are
inhibitors of the receptor tyrosine kinases AXL and c-MET. The
compounds are suitable for treatment of AXL or c-MET-mediated
disorders such as cancer, and the development of resistance to
cancer therapies.
[0002] Receptor tyrosine kinases (RTKs) are transmembrane proteins
that transduce signals from the extracellular environment to the
cytoplasm and nucleus to regulate normal cellular processes,
including survival, growth, differentiation, adhesion, and
mobility. Abnormal expression or activation of RTKs has been
implicated in the pathogenesis of various human cancers, linked
with cell transformation, tumor formation and metastasis. These
observations have led to intense interest in the development of
tyrosine kinase inhibitors as cancer therapeutics (Rosti et al,
Crit. Rev. Oncol. Hematol. 2011. [Epub ahead of print]; Gorden et
al, J. Oncol. Pharm. Pract. 2011. [Epub ahead of print]; Grande et
al, Mol. Cancer Ther. 2011, 10, 569).
[0003] AXL is a member of the TAM (TYRO3, AXL, MER) receptor
tyrosine kinase (RTK) family originally identified as a
transforming gene expressed in cells from patients with chronic
myelogenous leukemia (O'Bryan et. al Mol. Cell Biol. 1991, 11,
5016) or chronic myeloproliferative disorder (Janssen et. al
Oncogene, 1991, 6, 2113). AXL activation occurs by binding of its
cognate protein ligand, growth arrest specific 6 (Gash), homotypic
dimerization through its extracellular domain or cross-talk via the
interleukin (IL)-15 receptor or HER2. AXL signaling stimulates
cellular responses, including activation of phosphoinositide
3-kinase-Akt, extracellular signal-regulated kinase (ERK) and p38
mitogen-activated protein kinase cascades, the NF-.kappa.B pathway,
and signal transducer and activator of transcription (STAT)
signaling (Hafizi et. al Cytokine Growth Factor Rev., 2006, 17,
295). Numerous biological consequences of AXL signaling, including
invasion, migration, survival signaling, angiogenesis, resistance
to chemotherapeutic and targeted drugs, cell transformation, and
proliferation, represent undesirable traits associated with cancer
(Linger et al. Adv. Cancer Res., 2008, 100, 35; Hafizi et. al
Cytokine Growth Factor Rev., 2006, 17, 295; Holland et al, Cancer
Res. 2005, 65, 9294).
[0004] AXL receptors regulate vascular smooth muscle homeostasis
(Korshunov et al, Circ. Res. 2006, 98, 1446) and are implicated in
the control of oligodendrocyte cell survival (Shankar et al, J.
Neurosci. 2003, 23, 4208). Studies in knockout mice have revealed
that TAM receptors play pivotal roles in innate immunity by
inhibiting inflammation in macrophages and dendritic cells (Sharif
et al, J. Exp. Med. 2006, 203, 1891; Rothlin et al, Cell. 2007,
131, 1124), promoting the phagocytosis of apoptotic cells (Lu et
al, Nature. 1999, 398, 723; Lu & Lemke, Science. 2001, 293,
306; Prasad et al, Mol. Cell Neurosci. 2006, 3, 96) and stimulating
the differentiation of natural killer cells (Park et al, Blood
2009, 113, 2470).
[0005] AXL has been found to be constitutively activated due to
gene amplification and/or altered protein expression (O'Bryan et
al, J. Biol. Chem. 1995, 270, 551; Linger et al, Expert Opin. Ther.
Targets. 2010, 14, 1073; Mudduluru et al, Oncogene, 2011, 30,
2888). Altered expression of AXL has been reported in a variety of
human cancers (Crosier et al, Leuk. Lymphoma. 1995, 18, 443;
Challier et al, Leukemia, 1996, 10, 781; Ito et al, Thyroid. 1999,
9, 563; Sun et al, Oncology. 2004, 66, 450; Green et al, Br. J.
Cancer. 2006, 94, 1446; Liu et al, Blood. 2010, 116, 297) and is
associated with invasiveness and metastasis in lung cancer (Shieh
et al, Neoplasia. 2005, 7, 1058), prostate cancer (Shiozawa et al,
Neoplasia. 2010, 12, 116), breast cancer (Zhang et al, Cancer Res.
2008, 68, 1905), esophageal cancer (Hector et al, Cancer Biol.
Ther. 2010, 10, 1009), ovarian cancer (Rankin et al, Cancer Res.
2010, 70, 7570), pancreatic cancer (Koorstra et al, Cancer Biol.
Ther. 2009, 8, 618; Song et al, Cancer, 2011, 117, 734), liver
cancer (He et al, Mol. Carcinog. 2010, 49, 882), gastric cancer (Wu
et al, Anticancer Res. 2002, 22, 1071; Sawabu et al, Mol Carcinog.
2007, 46, 155), thyroid cancer (Avilla et al, Cancer Res. 2011, 71,
1792), renal cell carcinoma (Chung et al, DNA Cell Biol. 2003, 22,
533; Gustafsson et al, Clin. Cancer Res. 2009, 15, 4742) and
glioblastoma (Hutterer et al, Clin. Cancer Res. 2008, 14, 130).
[0006] Indeed, AXL overexpression is associated with late stage and
poor overall survival in many of those human cancers (Rochlitz et
al, Leukemia, 1999, 13, 1352; Vajkoczy et al, Proc Natl. Acad. Sci.
2006, 103, 5799). AXL contributes to at least three of the six
fundamental mechanisms of malignancy in human, by promoting cancer
cell migration and invasion, involving in tumor angiogenesis, and
facilitating cancer cell survival and tumor growth (Holland et al,
Cancer Res. 2005, 65, 9294; Tai et al, Oncogene. 2008, 27, 4044; Li
et al, Oncogene, 2009, 28, 3442; Mudduluru et al, Mol. Cancer Res.
2010, 8, 159). AXL is strongly induced by epithelial-to-mesenchymal
transitions (EMT) in immortalized mammary epithelial cells and AXL
knockdown completely prevented the spread of highly metastatic
breast carcinoma cells from the mammary gland to lymph nodes and
several major organs and increases overall survival (Gjerdrum et
al, Proc. Natl. Acad. Sci. USA. 2010, 107, 1124; Vuoriluoto et al,
Oncogene. 2011, 30, 1436), indicating AXL represents a critical
downstream effector of tumor cell EMT requiring for cancer
metastasis.
[0007] AXL is also induced during progression of resistance to
therapies including imatinib in gastrointestinal stromal tumors
(Mahadevan et al, Oncogene. 2007, 26, 3909) and Herceptin and EGFR
inhibitor therapy (e.g. lapatinib) in breast cancer (Liu et al,
Cancer Res. 2009, 69, 6871) via a "tyrosine kinase switch", and
after chemotherapy in acute myeloid leukemia (Hong et al, Cancer
Lett. 2008, 268, 314). AXL knockdown was also reported to lead to a
significant increase in chemosensitivity of astrocytoma cells in
response to chemotherapy treatment (Keating et al, Mol. Cancer
Ther. 2010, 9, 1298). These data indicate AXL as an important
mediator for tumor resistance to conventional chemotherapy and
molecular-based cancer therapeutics.
[0008] The c-MET receptor was initially identified as the TPR-MET
oncogene in an osteosarcoma cell line treated with a chemical
carcinogen. The TPR-Met protein is able to transform and confer
invasive and metastatic properties to non-tumorigenic cells
(Sattler et. al, Current Oncology Rep., 2007, 9, 102). The
oncogenic potential is a result of spontaneous dimerization and
constitutive activation of TPR-MET. Aberrant expression of HGF and
c-MET is associated with the development and poor prognosis of a
wide range of solid tumors, including breast, prostate, thyroid,
lung, stomach, colorectal, pancreatic, kidney, ovarian, and uterine
carcinoma, malignant glioma, uveal melanoma, and osteo- and
soft-tissue sarcoma (Jaing et. al Critical Rev. Oncol/Hematol.,
2005, 53, 35). Gastric tumors with an amplification of the wt-c-MET
gene are more susceptible to MET inhibition, thereby making c-MET
an attractive target (Smolen et. al Proc. Natl. Acad. Sci. USA,
2006, 103, 2316).
[0009] In vitro and in vivo studies have shown that increased and
dysregulated c-MET activation leads to a wide range of biological
responses associated with the malignant phenotype. These responses
include increased motility/invasion, increased tumorigenicity,
enhanced angiogenesis, protection of carcinoma cells from apoptosis
induced by DNA-damaging agents such as adriamycin, ultraviolet
light, and ionizing radiation, and enhanced rate of repair of DNA
strand breaks [Comoglio et. al J. Clin. Invest., 2002, 109, 857,
Sattler et. al Current Oncology Rep., 2007, 9, 102; Fan et. al,
Mol. Cell Biol., 2001, 21, 4968). Based upon these data, HGF may
enhance mutagenicity following DNA damage, allowing tumor cells
with genetic damage to survive, and thus leading to resistance to
chemo- and radiotherapeutic treatment regimens (Fan et. al, Mol.
Cell Biol., 2001, 21, 4968; Hiscox et. al Endocrine-Related Cancer,
2004, 13, 1085).
[0010] MET amplification plays a unique critical role in mediating
resistance of non-small cell lung cancer to EGFR inhibitors (e.g.
Tarceva.TM., Iressa.TM., Tykerb.TM.) the resistance of HER2
positive breast cancer to trastuzumab (Sattler et. al, Update
Cancer Ther., 2009, 3, 109; Engleman et. al, Science, 2007, 316,
1039, Shattuck et. al Cancer Res., 2008, 68, 1471, Agarwal et. al,
Br. J. Cancer, 2009, 100, 941; Kubo et. al, Int. J. Cancer 2009,
124, 1778). Inhibition of c-MET in Tarceva.TM. or Iressa.TM.
resistant cells using shRNA or small molecules alone or in
combination with an EGFR inhibitor overcame MET-mediated resistance
to EGFR inhibitors [Agarwal et. al, Br. J. Cancer, 2009, 100, 941;
Bachleitner-Hoffman et. al, Mol. Cancer Ther., 2008, 7, 3499, Tang
et. al, Br. J. Cancer, 2008, 99, 911; Bean et. al, Proc. Natl.
Acad. Sci. USA, 2007, 104, 20932). Due to the pleiotropic,
pro-tumorigenic activities of the HGF-c-MET axis, inhibiting this
pathway would be predicted to have potent anti-tumor effects in
many common cancers through multiple complimentary mechanisms.
SUMMARY
[0011] The present application describes the use of a particular
AXL/c-Met inhibitor, CEP-40783, or a salt thereof, either alone or
in combination with other therapeutically active agents, for the
treatment of particular cancers, including any solid or
hematological cancer in which AXL or c-Met is over-expressed. The
structure of CEP-40783 is shown below:
##STR00002##
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 depicts the binding assay data for CEP-40783
inhibition of AXL and c-Met.
[0013] FIG. 2 depicts dissociation rate data of CEP-40783 from AXL
and c-Met.
[0014] FIG. 3A depicts the effects on AXL phosphorylation in female
SCID mice bearing NCI-H1299 NSCLC xenografts after oral
administration of CEP-40873.
[0015] FIG. 3B depicts the effects on c-Met phosphorylation in
nu/nu mice bearing GLT-16 gastric carcinoma subcutaneous
tumorgrafts after oral administration of CEP-40873.
[0016] FIG. 4 depicts the anti-tumor efficacy of orally
administered CEP-40783 in NIH3T3/AXL tumor xenografts in female
SCID mice.
[0017] FIG. 5 depicts the anti-tumor efficacy of orally
administered CEP-40783 in GTL-16 gastric carcinoma xenografts in
female athymic nude mice.
[0018] FIG. 6 depicts the anti-tumor efficacy of orally
administered CEP-40783 in EBC-1 Human NSCLC xenografts in female
athymic nude mice.
[0019] FIG. 7 depicts the effects of different dosing schedules of
CEP-40873 on growth of EBC-1 Human NSCLC xenografts.
[0020] FIG. 8 depicts the anti-tumor effects of orally administered
CEP-40783 in female Balc/c mice implanted (i.v.) with 4T1-Luc2
murine mammary carcinoma cells.
[0021] FIGS. 9A and 9B depict the effect of oral administration of
CEP-40783 on metastases of MDA-MB-231-Leu orthotopic breast tumor
xenografts in female nude mice.
[0022] FIG. 10 depicts the effects of oral administration of
CEP-40783 in erlotinib-insensitive primary NSCLC human
TumorGraphs.TM..
[0023] FIG. 11 depicts the activity of CEP-40783 and erlotinib in
"erlotinib-sensitive" NSCLC TumorGraft.TM. having activated AXL and
c-Met.
DESCRIPTION
[0024] As used herein, the following terms have the meanings
ascribed to them below unless specified otherwise.
[0025] The term "about" refers to .+-.10% of a given value.
[0026] "Pharmaceutical composition" refers to a composition having
a safety and/or efficacy profile suitable for administration to a
subject, including a human.
[0027] "Pharmaceutically acceptable" when used by itself or in
conjunction with another term or terms refers to materials, such
as, for example, an active ingredient, salt, excipient, carrier,
vehicle, or diluent that is generally chemically and/or physically
compatible with the other ingredients comprising a formulation,
and/or is generally physiologically compatible with the recipient
thereof.
[0028] "Subject" refers to a member of the class Mammalia. Examples
of mammals include, without limitation, humans, primates,
chimpanzees, rodents, mice, rats, rabbits, horses, livestock, dogs,
cats, sheep, and cows.
[0029] "Therapeutically effective amount" refers to an amount of a
compound sufficient to improve or inhibit the worsening or severity
of one or more symptoms associated with a particular disorder or
condition that is being treated in a particular subject or subject
population. It should be appreciated that the determination or
selection of the dosage form(s), dosage amount(s), and route(s) of
administration is within the level of ordinary skill in the
pharmaceutical and medical arts.
[0030] "Treatment" refers to the acute or prophylactic diminishment
or alleviation of at least one symptom or characteristic associated
with or caused by a disorder being treated. For example, treatment
can include diminishment of a symptom of a disorder or complete
eradication of either a symptom and/or the disorder itself. It
should be understood that the terms "preventing" and "preventative"
and "prophylactic" are not absolute but rather refer to uses and
results where the administration of a compound or composition
diminishes the likelihood or seriousness of a condition, symptom,
or disease state, and/or may delay the onset of a condition,
symptom, or disease state for a period of time. In some
embodiments, the terms "treating", "treated", and "treatment" refer
to curative uses and results as well as uses and results that
diminish or reduce the severity of a particular condition, symptom,
disorder, or disease described herein.
[0031] As used herein, the terms "therapeutically active agent" and
"therapeutic agent", whether used alone or in conjunction with
another term or terms, refers to any compound, i.e. a drug or a
salt thereof, that may be or has been found to be useful in the
treatment of a particular condition, symptom, disease or disorder
and is not CEP-40783.
[0032] The compounds (including CEP-40783 and/or any other
therapeutically active agent) described herein may be isolated and
used per se as a free base or may be isolated in the form of a
salt. It should be understood that the terms "salt(s)" and "salt
form(s)" whether used by themselves or in conjunction with another
term or terms encompasses all inorganic and organic salts,
including industrially acceptable salts, as defined herein, and
pharmaceutically acceptable salts, as defined herein, unless
otherwise specified. As used herein, industrially acceptable salts
are salts that are generally suitable for manufacturing and/or
processing (including purification) as well as for shipping and
storage, but may not be salts that are typically administered for
clinical or therapeutic use. Industrially acceptable salts may be
prepared on a laboratory scale, i.e. multi-gram or smaller, or on a
larger scale, i.e. up to and including a kilogram or more.
Pharmaceutically acceptable salts, as used herein, are salts that
are generally chemically and/or physically compatible with the
other ingredients comprising a formulation, and/or are generally
physiologically compatible with the recipient thereof.
Pharmaceutically acceptable salts may be prepared on a laboratory
scale, i.e. multi-gram or smaller, or on a larger scale, i.e. up to
and including a kilogram or more. It should be understood that
pharmaceutically acceptable salts are not limited to salts that are
typically administered or approved (by a regulatory authority such
as FDA) for clinical or therapeutic use in humans. A practitioner
of ordinary skill will readily appreciate that some salts are both
industrially acceptable as well as pharmaceutically acceptable
salts. It should be understood that all such salts, including mixed
salt forms, are within the scope of the application.
[0033] In one aspect, the present application provides a compound
that is
##STR00003##
or a salt thereof. The compound can be referred to by the chemical
name
3-(4-Fluorophenyl)-1-isopropyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-c-
arboxylic acid
[4-(6,7-dimethoxyquinolin-4-yloxy)-3-fluorophenyl]-amide and is
also known as CEP-40783.
[0034] In another aspect, the present application provides for
methods of treatment of various cancers including any solid or
hematological cancer in which AXL or c-Met is over-expressed, where
the method comprises the administration of CEP-40783 or a
pharmaceutically acceptable salt thereof to a subject in recognized
need of such treatment. In another aspect, the present application
provides for the use of CEP-40783 or a pharmaceutically acceptable
salt thereof in the preparation of a medicament for treating a
solid or hematological cancer in which AXL or c-Met is
over-expressed, wherein the medicament is subsequently administered
to a subject in recognized need thereof. Preferably, CEP-40783 or a
pharmaceutically acceptable salt thereof is administered to the
subject in a pharmaceutical composition that comprises a
pharmaceutically acceptable excipient.
[0035] In another aspect, the application describes methods of
treating particular cancers including, but not limited to, non
small cell lung cancer (NSCLC), breast cancer, gastric cancer and
pancreatic cancer using CEP-40783 or a pharmaceutically acceptable
salt thereof. In some embodiments the cancer is NSCLC that is
resistant or insensitive to treatment with EGFR inhibitors. In some
embodiments, CEP-40783 or a pharmaceutically acceptable salt
thereof, is administered as a single agent. In other embodiments
CEP-40783, or a pharmaceutically acceptable salt thereof, is
administered in combination with another therapeutic agent. In some
embodiments the other therapeutic agent is erlotinib. In still
other embodiments the other therapeutic agent is gefitinib.
[0036] In any of the aforementioned methods of treatment (or
medical uses) the solid or hematological cancer in which AXL or
c-Met is over-expressed may be treated prophylactically, acutely or
chronically using CEP-40783 or a salt thereof. In some embodiments,
CEP-40783 or a salt thereof may be used in combination with another
therapeutic agent. In some embodiments CEP-40783 or a
pharmaceutically acceptable salt thereof is administered
simultaneously with the other therapeutic agent. In other
embodiments CEP-40783 or a pharmaceutically acceptable salt thereof
is administered sequentially, i.e., administered before or after
the other therapeutic agent. In such embodiments, the CEP-40783 or
a pharmaceutically acceptable salt thereof is administered after
the subject exhibits some degree of resistance or insensitivity to
treatment with another therapeutic agent. In some embodiments, the
other therapeutic agent is erlotinib. In still other embodiments
the other therapeutic agent is gefitinib.
[0037] In the therapeutic applications described herein, CEP-40783
or a pharmaceutically acceptable salt thereof, can be administered
in a wide variety of oral and/or parenteral dosage forms. In one
embodiment, the compounds of the present invention are delivered
orally. Parenteral administration should be understood as
administration by injection, that is, intravenously,
intramuscularly, intracutaneously, subcutaneously, intraduodenally,
or intraperitoneally. In certain embodiments, the compounds of the
present invention are administered intravenously or subcutaneously.
Also, the compounds described herein can be administered by
inhalation, for example, intranasally. Additionally, the compounds
of the present invention can be administered transdermally. The
compounds can also be delivered rectally, bucally or by
insufflation.
[0038] Determination of the proper dosage for a particular
situation is within the skill of the practitioner. Generally,
treatment is initiated with smaller dosages which are less than the
optimum dose of the compound. Thereafter, the dosage is increased
by small increments until the optimum effect under the
circumstances is reached. For convenience, the total daily dosage
may be divided and administered in portions during the day, if
desired. For example in some embodiments CEP-40783 or a salt
thereof is administered from one to four times per day. A typical
dose is about 1 mg to about 1,000 mg, such as about 5 mg to about
500 mg. In certain embodiments, the typical dose is about 1 mg to
about 300 mg, such as about 5 mg to about 250 mg. In still other
embodiments, the typical dose is about 10 mg to 100 mg. In some
embodiments CEP-40783 or a salt thereof is dosed relative to body
weight. For example, in some embodiments CEP-40783 or a salt
thereof is administered in an amount of about 0.1 mg/kg to about
500 mg/kg, such as about 1 mg/kg to about 100 mg/kg, or to about 5
mg/kg to about 75 mg/kg. In some embodiments CEP-40783 or a salt
thereof is administered in an amount of about 10 mg/kg to about 55
mg/kg.
[0039] CEP-40783 can be prepared using any number of different
methods, including, for example, using the methods described in WO
2013/0074633.
Biology
[0040] CEP-40783 is an orally active, potent and selective AXL and
c-Met kinase inhibitor, with enzyme IC.sub.50 values of 7 nM and 12
nM, respectively. In AXL-transfected 293GT cells, CEP-40783 was
27-fold more active compared to recombinant enzyme with an
IC.sub.50 value of 0.26 nM. Comparably high cellular potency was
observed in NCI-H1299 human NSCL cells (IC.sub.50=0.1 nM).
CEP-40783 also demonstrated superior activity against c-Met in
GTL-16 cells (IC.sub.50=6 nM). The increased inhibitory activity of
CEP-40783 in cells may be due to its extended residence time on
both AXL and c-Met, consistent with a Type II mechanism. The
prolonged residence time of CEP-40783 at the target may provide for
improved in vivo efficacy, selectivity and therapeutic index.
Additionally, CEP-40783 showed high kinome selectivity against 298
kinases with an S90 of 0.04 (fraction of kinases showing >90%
inhibition at 1 .mu.M).
[0041] A summary of PK data across various species is presented in
Table 1 below:
TABLE-US-00001 TABLE 1 Parameters Rat.sup.a Dog.sup.b Monkey.sup.b
% F 57 100 77 C.sub.max (ng/mL) 593 1172 260 AUC.sub.0-t (ng h/mL;
p.o.) 16334 14429 3030 t.sub.1/2 i.v. (h) 21.5 27.6 14.5 CL
(mL/min/kg) 1.4 0.5 2.2 V.sub.d (L/kg) 2.6 1.3 1.2
.sup.aAdministered at 1 mg/kg i.v. and 3 mg/kg p.o.
.sup.bAdministered at 0.5 mg/kg i.v. and p.o.
CEP-40783 Demonstrates Time-Dependent Binding Kinetics for
Inhibition of AXL and c-Met
[0042] Time-dependent binding assays were performed in Greiner low
volume white 384-well plates. Assay buffer consisted of 50 mM Hepes
(pH 7.5), 10 mM MgCl.sub.2, 1 mM EGTA, and 0.01% Brij-35, while
compound dilution buffer contained 1% DMSO in assay buffer.
Compound dilution buffer (5 .mu.L) was added to the assay plate.
Serial half-log dilutions of CEP-40783 were prepared in DMSO at
150.times. final assay concentration in a 384-well polypropylene
plate and 100 .mu.L transferred robotically to the assay plate.
Kinase tracer (5 .mu.L; Invitrogen PV5592) was added to all the
wells. The final tracer concentration was 10 nM AXL and 100 nM for
c-Met. LanthaScreen Eu-anti-GST antibody (5 .mu.L of 6 nM, 2 nM
final; Invitrogen PV5594) was added to DMSO (no inhibitor) control
wells while the remaining wells received 5 .mu.L of the same
antibody plus 15 nM GST-tagged enzyme (5 nM final). Kinetic
readings were immediately initiated on an EnVision.TM. 2104 plate
reader (PerkinElmer) fitted with a laser light source (337 nm), a
Lance/DELFIA dual mirror, and APC (665 nm) and europium (615 nm)
filters. Thirty readings were taken at 4-min intervals and the 665
nm/615 nm emission ratio was calculated. The average ratio
corresponding to the no enzyme control was subtracted from all the
data. Inhibition curves for compounds were generated by plotting
percent control activity versus log 10 of the concentration of
compound. IC.sub.50 values were calculated by nonlinear regression
using the sigmoidal dose-response (variable slope) equation in
XLfit The fold-shift in IC.sub.50 was calculated by dividing the
initial value by the lowest value obtained, and the time the
maximal change was observed was also recorded (tmax).
[0043] When tested using a LanthaScreen.TM. Eu-kinase binding assay
CEP-40783 displayed time-dependent binding kinetics for AXL and
c-Met which is consistent with a Type II mechanism (See FIG. 1).
The binding data shows a 35-fold shift in the IC.sub.50 value in
CEP-40783 inhibition of AXL from time t.sub.0 to t.sub.max of 2
hrs. CEP-40783 also displayed time-dependent inhibition of c-Met in
which a 38-fold shift in the IC.sub.50 was noted.
Dissociation of CEP-40783 from AXL and c-Met
[0044] Rates of dissociation of CEP-40783 from AXL and c-Met were
also determined using the LanthaScreen.TM. Eu-kinase binding assay.
Assay buffer was prepared as for association, and used for all
dilutions. A 5-4, aliquot of 80 nM LanthaScreen Eu-anti-GST
antibody: 20 nM GST-tagged enzyme mix was added to the Greiner low
volume white 384-well plate, with antibody only added to control
wells. Compound (100 nL) in DMSO was added by pintool at 2000-fold
over the final assay concentration. DMSO was added to no inhibitor
control wells. After a one-hour incubation at ambient temperature
to allow formation of the enzyme-inhibitor complex, 2 .mu.L of the
reaction mix was transferred to a 384 Optiplate (PerkinElmer) and
78 .mu.L of kinase tracer 236 was added. The final tracer
concentration was 100 nM for AXL and 200 nM for c-Met. Readings
were immediately initiated and total of 60 time points were taken
at 2-min intervals, at 25.degree. C. or 37.degree. C. The
background subtracted ratio was normalized to the no inhibitor
control to calculate the % tracer bound, which was plotted with
respect to time. The data was fitted to the one phase association
model in GraphPad Prism (La Jolla Calif.).
[0045] As seen in FIG. 2 CEP-40783 displays very slow "off-rates"
for AXL and c-Met at 25.degree. C., which is illustrative of
pseudo-irreversible binding kinetics. The dissociation rates were
slightly enhanced by higher temperature (37.degree. C.). It is
believed that the potent AXL and c-Met cellular activities of
CEP-40783 can be explained by the slow dissociation rates, which
results in prolonged drug-receptor residence times. Type I kinase
inhibitors have been shown to exhibit rapid dissociation rates
using this technology.
CEP-40783 Inhibits AXL and c-Met Phosphorylation in Tumor
Xenografts
[0046] Female Nu/Nu mice (6-8 weeks, Charles River Laboratory,
Wilmington, Mass.) were maintained 5/cage in microisolator units on
a standard laboratory diet (Teklad Labchow, Harlan Teklad, Madison,
Wis.). Animals were housed under humidity- and
temperature-controlled conditions and the light/dark cycle was set
at 12-hour intervals. Mice were quarantined at least 1 week prior
to experimental manipulation. Experiments were approved (Protocol
03-023) by the Institutional Animal Care and Use Committee of Teva
Pharmaceuticals Inc.
[0047] Briefly, NCI-H1299 NSCL cells (for AXL studies) or GTL-16
gastric carcinoma cells (for c-Met studies) were collected and
resuspended in DMEM medium at density of 5.times.10.sup.7/mL and an
aliquot (100 .mu.L) of the cell suspension (5.times.10.sup.6 cells)
was inoculated subcutaneously to the left flank of each mouse with
a 23 g needle. When the tumor xenograft volumes reached
approximately 300-500 mm.sup.3 the mice received a single oral
administration of either PEG-400 vehicle or indicated doses of
CEP-40783 at 100 .mu.L/dose. At indicated time points post dosing,
the mice (3 mice at each time point) were sacrificed by
decapitation and blood was collected in 1.5 mL microcentrifuge
tubes containing 20 .mu.L of heparin sodium (10,000 unit/mL in
H.sub.2O, Cat.sup.#0210193191, MP Biomedical, Solon, Ohio) and left
on ice briefly. The tubes were centrifuged at 20,817.times.g
(Eppendorf Centrifuge 5417R with a FA45-30-11 rotor) for 8 minutes
at 4.degree. C. and the plasma was collected and transferred to 1.5
mL microfuge tubes, which were then stored at -80.degree. C. The
tumors were excised and weighed, cut into small pieces with a
scalpel and placed into a round-bottom 14 mL tube (Cat.sup.#352059,
Becton Dickinson, Franklin, N.J.) on ice. Two volumes of FRAK lysis
buffer without detergent [10 mM Tris, pH 7.5, 50 mM sodium
chloride, 20 mM sodium fluoride, 2 mM sodium pyrophosphate, 0.1%
BSA, plus freshly prepared 1 mM activated sodium vanadate, 4 mM
DTT, 1 mM PMSF and the protease inhibitor cocktail III (1:100
dilution, Cat.sup.#539134, Calbiochem, La Jolla, Calif.)] were
added to 1 volume of tumor (eg, 500 .mu.L FRAK lysis buffer were
added to 250 mg tissue). The tissues were then disrupted with a
hand-held tissue blender for 2-3 cycles, 10-15 seconds each cycle
with 1-2 minute interval. The lysates were then sonicated twice,
4-5 strokes each time. The tissue lysates were transferred to 1.5
mL microfuge tubes and centrifuged at 20,817.times.g (Eppendorf
Centrifuge 5417R with a FA45-30-11 rotor) for 10 minutes at
4.degree. C. The supernatants (12 .mu.L) were transferred to 1.5 mL
micro-centrifuge tubes containing 108 .mu.L FRAK lysis buffer and
40 .mu.L of 4.times.LDS sample buffer (Cat.sup.# NP0007,
Invitrogen) with freshly added 100 nM dithiotreitol (Cat# F820-02,
JT Baker, Phillipsburg, N.J.). The remaining supernatants were
stored at -80.degree. C. The compound levels in both plasma and
tumor lysates were measured by LC-MS/MS. Immunoblot analyses of
phospho-c-Met and total c-Met, and phosphor-AXL and total AXL for
tumor PD analyses were carried out according to the protocols
provided by the antibody suppliers (Cell Signaling Technology). The
rabbit phospho-c-Met (Y1234/1235) (Cat#3129) and c-Met antibodies
(Cat#3127) and rabbit phospho-AXL(Y702) (Cat#5724) and AXL
antibodies (Cat#4939) were purchased from Cell Signaling Technology
(Beverly, Mass.). The samples were heat-inactivated at 90.degree.
C. for 5 minutes; 20 .mu.L of each sample was resolved by NuPAGE 7%
Tris-acetate gels (Cat# EA03552Box, Invitrogen) at 150 V until the
dye front was out of the gels. The gels were transferred to
nitrocellulose membranes (Cat# LC2000, Invitrogen) for 2 hours at
30V constant using a wet XCell II blot module (Cat# EL9051,
Invitrogen). The membranes were blocked in Tris-buffered saline
(TBS) containing 0.2% Tween-20 (TBST) and 3% Nestle Carnation
nonfat milk (Nestle USA Inc, Solon, Ohio) at room temperature (RT)
for 1 hour. The membranes were incubated with anti-phospho-c-Met
(Tyr1234/1235) or anti-phospo AXL antibody (Tyr702; diluted 1:1000
in TBST containing 3% bovine serum albumin) for 1.5 hours at RT or
overnight at 40.degree. C. while rocking gently. After washing 3
times with TBST for 10 minutes each time, the membranes were
incubated with goat-anti-rabbit antibody conjugated with
horseradish peroxidase (HRP) (Cat# W401B, Promega, Madison, Wis.)
diluted in TBST containing 3% nonfat-milk for 1 hour at RT while
rocking gently. After washing 3 times with TBST for 10 minutes each
time and one time with TBS for 5 minutes, the membranes were
incubated with 5 mL of ECL.TM.-Western blotting detection reagents
(Cat# RPN2106, GE Healthcare UK, Buckinghamshire, UK) for 5 minutes
and exposed to Kodak chemiluminescence BioMax films (Cat#178, 8207;
Carestream Health Inc, Rochester, N.Y.) for visualization. The
membranes were then stripped by incubating with stripping buffer
(62.5 mM Tris HCl pH 6.8, 2% SDS and 100 mM 2-mercaptoethanol) for
30 minutes at 56.degree. C., and re-blotted with anti-c-Met and
anti-AXL antibody and then goat anti-rabbit-HRP secondary antibody
diluted 1:10,000. The films imaging individual bands of phospho-
and total AXL and phospho- and total c-Met were scanned (HP Scanjet
7400c, Hewlett-Packard Company, Palo Alto, Calif.) and quantified
with Gel-Pro Analyzer software (Media Cybernetics, Inc, Bethesda,
Md.). The magnitude of normalized AXL and normalized c-Met
phosphorylation of each tumor sample relative to vehicle control
tumor samples was then calculated.
[0048] In PK/PD studies, CEP-40783 showed dose- and time-dependent
inhibition of AXL phosphorylation using NCI-H1299 NSCL xenografts
with .about.80% target inhibition at 0.3 mg/kg 6 h post dose and
complete target inhibition to >90% inhibition at 1 mg/kg between
6-24 h, while a 10 mg/kg po dose resulted in complete AXL
inhibition up to 48 h post dosing. Female Scid mice bearing
NCI-H1299 NSCLC were administered CEP-40783 as indicated and plasma
and tumor samples were collected at 6 hrs post-administration (FIG.
3A). Effects on phospho-AXL (Cell Signaling #5724) and total AXL
(Cell Signaling #4977) in tumor samples were detected by
immunoblotting and the magnitude of inhibition of normalized AXL
phosphorylation was calculated.
[0049] Nu/nu mice bearing GTL-16 gastric carcinoma sc tumor
xenografts were dosed were dosed as indicated and plasma and tumor
samples were collected 6 and 24 hrs post dose (FIG. 3 B). Effects
on c-Met phosphorylation were determined using ELISA (Invitrogen
KH00281 and KH02031) and the magnitude of inhibition of normalized
c-Met phosphorylation was calculated.
CEP-40783 Inhibits the Growth of NIH3T3/AXL Xenografts
[0050] The engineered NIH3T3/AXL cell line was generated as
follows. The human AXL cDNA was subcloned into the pQCXIP vector
and the sequences were confirmed by sequencing at Children's
Hospital of Philadelphia. PT67 cells (5.times.10.sup.5) in 4 mL
DMEM+10% FBS were seeded in each well of 6-well plates (Cat#353046,
Becton Dickinson, Franklin Lakes, N.J.) and cultured at 37.degree.
C. in a humidified incubator with 5% CO.sub.2 overnight. The cells
were transfected with 2 .mu.g of pQCXIP-AXL with Lipofectamine.TM.
2000 (Cat.sup.#52887, Invitrogen, Carlsbad, Calif.) transfection
reagent per the manufacturer's protocol. In brief, the DNA in 500
.mu.L culture media was mixed with 500 .mu.L media containing 20
.mu.L Lipofectamine.TM. 2000 transfection reagent, and the mixture
was incubated at room temperature for 20 minutes. After the culture
media was aspirated and the dishes were washed with 2 mL 1.times.
Dulbecco's Phosphate Buffered Saline (PBS, Cat#21-031-CM,
Mediatech, Manassas, Va.), 1 mL of the appropriate transfection
mixture was added to each well. The wells were returned to the
37.degree. C. humidified incubator with 5% CO.sub.2 and carefully
rocked every 30 minutes and after 3 hours of incubation, the
mixture was removed and 4 mL fresh complete media medium was added
to each dish. The supernatants were collected about 48 hours and
again at 60 hours after transfection and were filtered through a
0.45 .mu.m filter. The medium were aliquoted and stored in
-80.degree. C. until use. Infection of NIH3T3 cells with AXL
retroviruses to generate stable NIH3T3/AXL cell lines: The NIH3T3
cells seeded in 10 cm culture dishes were changed to 4.5 mL of the
collected medium containing AXL retroviruses plus freshly added
polybrene (final concentration of 8 .mu.g/mL). Six to eight hours
later, 6 mL of complete DMEM medium was added into each culture
dish and the cells were incubated with the medium for 48 hours. The
cells were 1:4 split, and then selected with 0.5 .mu.g/mL puromycin
until all the uninfected NIH3T3 cells had died. The cells were then
expanded and AXL expression and tyrosine phosphorylation in cells
were confirmed by immunoblotting prior to in vivo xenograft
studies.
[0051] Female Scid/Beige mice (6-8 weeks, Taconic, Hudson, N.Y.)
were maintained 5/cage in microisolator units on a standard
laboratory diet (Teklad Labchow, Harlan Teklad, Madison, Wis.).
Animals were housed under humidity- and temperature-controlled
conditions and the light/dark cycle was set at 12-hour intervals.
Mice were quarantined at least 1 week prior to experimental
manipulation. All animal studies were conducted under protocol
#03-023 approved by the Institutional Animal Care and Use Committee
(IACUC) of Teva Pharmaceuticals Inc.
[0052] NIH3T3/AXL cells were collected and resuspended in DMEM
medium at density of 5.times.10.sup.7/mL. An aliquot (100 .mu.L) of
the cell suspension (5.times.10.sup.6 cells) was inoculated
subcutaneously to the left flank of each mouse with a 23 g needle.
The mice were monitored and the tumor sizes were measured. When the
NIH3T3/AXL tumor volumes reached 300 mm.sup.3, the tumor-bearing
mice were randomized into different treatment groups (8-10
mice/group) and were administered either vehicle (PEG-400) or
CEP-40783 formulated in PEG-400 at indicated doses, qd, with 100
.mu.L per dosing volume. The length (L) and width (W) of each tumor
were measured with a vernier caliper and the mouse body weight was
determined every two to three days. Tumor volumes were calculated
with the formula of 0.5236*L*W*(L+W)/2. Statistical analyses of
tumor volumes and mouse body weights were carried out with the
Mann-Whitney Rank Sum Test. Plasma and tumor samples were obtained
at 2 hours post final dose at each dose level, and the compound
levels in plasma and tumor lysates were measured by LC-MS/MS.
[0053] As shown in FIG. 4 female SCID mice bearing subcutaneous
NIH3T3-AXL tumor xenografts were administered either vehicle or
CEP-40783 at the following doses: 0.3 mg/kg, 1 mg/kg, or 10 mg/kg)
by mouth (PO/per os), once a day (qd). Tumor sizes and mouse body
weights were measured and recorded every two to three days and the
absolute tumor volumes were calculated. Administration of CEP-40783
resulted in complete tumor regressions at all doses tested which is
consistent with sustained and significant pharmacodynamic
inhibition of AXL activity in these AXL-dependent tumors.
CEP-40783 Inhibits the Growth of GTL-16 Gastric Carcinoma
Xenografts
[0054] The human gastric cancer cell line, GTL-16 was purchased
from ATCC (American Tissue Culture Collection, Manassas, Va.) and
cultured in DMEM medium with 10% fetal bovine serum (FBS, Cat#
SH3007003, Hyclone Laboratory Inc, Logan, Utah). Female Nu/Nu mice
(6-8 weeks, Charles River Laboratory, Wilmington, Mass.) were
maintained 5/cage in microisolator units on a standard laboratory
diet (Teklad Labchow, Harlan Teklad, Madison, Wis.). Animals were
housed under humidity- and temperature-controlled conditions and
the light/dark cycle was set at 12-hour intervals. Mice were
quarantined at least 1 week prior to experimental manipulation.
Experiments were approved (Protocol 03-023) by the Institutional
Animal Care and Use Committee of Teva Pharmaceuticals Inc. GTL-16
cells were collected and resuspended in DMEM medium at density of
5.times.10.sup.7/mL and an aliquot (100 .mu.L) of the cell
suspension (5.times.10.sup.6 cells) was inoculated subcutaneously
to the left flank of each mouse with a 23 g needle. The mice were
then monitored daily. When tumor volumes were approximately 200
mm.sup.3, mice were randomized into different treatment groups
(8-10 mice/group) and administered orally either vehicle (PEG-400)
or CEP-40783 formulated in PEG-400 at indicated doses, qd, with 100
.mu.L per dosing volume. The length (L) and width (W) of each tumor
was measured with a vernier caliper and the mouse body weight was
determined every 2-3 days. The tumor volumes were then calculated
with the formula of 0.5236*L*W*(L+W)/2. Statistical analyses of
tumor volumes and mouse body weight were carried out using the
Mann-Whitney Rank Sum Test. **p<0.01 vehicle as compared to
CEP-40783 treated groups. Plasma and tumor samples were obtained at
2 hours post final dose at each dose level, and the compound levels
in plasma and tumor lysates were measured by LC-MS/MS. The TGI
values were calculated at the end of study by comparing the tumor
volumes (TV) of each CEP-40783-treatment group with those of
vehicle-treated group with the following formula: 1-(the last day
TV of compound-treated group/the last day TV of vehicle-treated
group).
[0055] These data (FIG. 5) demonstrate that oral administration of
CEP-40783 results in significant anti-tumor efficacy (tumor stasis
and regressions) at 10 and 30 mg/kg in this cMet-dependent tumor
model.
CEP-40783 Inhibits the Growth of EBC-1 NSCLC Xenografts
[0056] The human NSCL cancer cell line, EBC-1 was purchased from
ATCC (American Tissue Culture Collection, Manassas, Va.) and
cultured in DMEM medium with 10% fetal bovine serum (FBS, Cat#
SH3007003, Hyclone Laboratory Inc, Logan, Utah). Female Nu/Nu mice
(6-8 weeks, Charles River Laboratory, Wilmington, Mass.) were
maintained 5/cage in microisolator units on a standard laboratory
diet (Teklad Labchow, Harlan Teklad, Madison, Wis.). Animals were
housed under humidity- and temperature-controlled conditions and
the light/dark cycle was set at 12-hour intervals. Mice were
quarantined at least 1 week prior to experimental manipulation.
Experiments were approved (Protocol 03-023) by the Institutional
Animal Care and Use Committee of Teva Pharmaceuticals Inc. GTL-16
cells were collected and resuspended in DMEM medium at density of
5.times.10.sup.7/mL and an aliquot (100 .mu.L) of the cell
suspension (5.times.10.sup.6 cells) was inoculated subcutaneously
to the left flank of each mouse with a 23 g needle. The mice were
then monitored daily. When tumor volumes were approximately 250
mm.sup.3, mice were randomized into different treatment groups
(8-10 mice/group) and administered orally either vehicle (PEG-400)
or CEP-40783 formulated in PEG-400 at indicated doses, qd, with 100
.mu.L per dosing volume. The length (L) and width (W) of each tumor
was measured with a vernier caliper and the mouse body weight was
determined every 2-3 days. The tumor volumes were then calculated
with the formula of 0.5236*L*W*(L+W)/2. Statistical analyses of
tumor volumes and mouse body weight were carried out using the
Mann-Whitney Rank Sum Test. **p<0.01 vehicle as compared to
CEP-40783 treated groups. Plasma and tumor samples were obtained at
2 hours post final dose at each dose level, and the compound levels
in plasma and tumor lysates were measured by LC-MS/MS. The TGI
values were calculated at the end of study by comparing the tumor
volumes (TV) of each CEP-40783-treatment group with those of
vehicle-treated group with the following formula: 1-(the last day
TV of compound-treated group/the last day TV of vehicle-treated
group).
[0057] As shown in FIG. 6, oral administration of CEP-40783 at
doses of 10 mg/kg and 30 mg/kg resulted in tumor regressions, with
tumor stasis and partial regressions observed at 3 mg/kg in this
c-Met dependent tumor model.
Discontinuous and Alternate Oral Dosing Schedules of CEP-40783
Maintain Significant Anti-Tumor Efficacy
[0058] Similar methods to those described above for FIG. 6 were
employed to evaluate the anti-tumor effects of discontinuous and
alternate oral dosing schedules of CEP-40783. Upon EBC-1 tumor
xenografts reaching approximately 200 mm.sup.3, mice were
randomized into different treatment groups (8-10 mice/group) and
administered orally either vehicle (PEG-400) or CEP-40783
formulated in PEG-400 at 10 mg/kg qd. After a period of 7 days of
continuous dosing, animals were then switched to either a 7 day
on/7 day off 10 mg/kg qd dosing regimen, or intermittent twice
weekly (q2d) or thrice weekly (q3d) oral dosing at a 10 mg/kg qd
dose for a total of 21 days.
[0059] As seen in FIG. 7 significant anti-tumor efficacy is
maintained when CEP-40783 is dosed orally for 7 days followed by
dosing holidays (qdx7, dosing every 7 days) and/or alternate dose
schedules, i.e., q3d (dosing every three days) or q2d (dosing every
two days). Specifically, partial responses were seen in groups that
were dosed orally with CEP-40783 every 7 days (40%) every 3 days
(70%) and every 2 days (40%). Complete tumor regressions were seen
in 10% and 20% of animals in the qdx7 and q2d dosing groups,
respectively, which is indicative that discontinuous and
intermittent dosing of CEP-40783 retains highly significant
anti-tumor activity consistent with its tumor pharmacodynamic
profile.
CEP-40783 Inhibits 4T1-Luc2 Systemic Dissemination
[0060] The murine breast cancer cell line 4T1-luciferase tagged
cell line generated by stable transfection of the firefly
luciferase gene expressed from the SV40 promoter was purchased from
Caliper Life Sciences. The cells were grown in DMEM medium
supplemented with 10% fetal bovine serum (FBS, Cat.sup.# SH3007003,
Hyclone, Logan, Utah). D-Luciferin firefly/potassium salt (Cat. #
L-8220) was purchased from Biosynth International, Inc., Itasca,
Ill.). Female Balb-c mice (6-8 weeks) were purchased from Charles
River laboratories. The Balb/c mice were injected with
0.5.times.10.sup.5 4 T1-Luc2 cultured tumor cells iv (tail vein)
two days after the start of treatments with either PEG-400 or
CEP-40783 formulated in PEG-400 (dosed at 1, 10 and 30 mg/kg, po,
qd). Two days later, the mouse body weights were measured twice a
week and the mice were subjected for full body in vivo imaging
thrice a week. Treatments were given for a total of 23 days, with
21 days post tumor cell seeding. Bioluminescence image analysis was
performed at the end of the study using a Caliper Life Sciences
(Xenogen) Spectrum in vivo imaging machine. Each cage of five mice
was anesthetized using isofluorane and imaged for a series of time
points from 0.5-5 minutes max. Image analysis was performed using
Living Image Software (vs. 4.0, 2010) using subjective but equally
sized gates or regions of interest (ROIs) overlaid on each animal
image. Counts were converted to average radiance or mean photon
flux; expressed as photons per second per cm.sup.2 of surface area,
(p/s/cm.sup.2/sr). Mann-Whitney non-parametric, 1- or 2-way ANOVA
were used as statistical tests, a p-value less than 0.05 were
considered significant. Statistical software used was Graph Pad
Prism (vs. 5.01, 2007), and calculations were performed using
Microsoft Office Excel (Professional, 2003).
[0061] As seen in FIG. 8 oral administration of CEP-40783 results
in highly significant reductions in systemic 4T1 mammary tumor
dissemination at the 10 mg/kg and 30 mg/kg doses, and a
significant, albeit of lesser magnitude, reduction in tumor
dissemination at the 1 mg/kg dose as well.
CEP-40783 Decreases Lymph Node Metastases of MDA-MB-2312 Leu
Orthotopic Breast Cancer Implants
[0062] The human breast cancer cell line
MDA-MB-231-Leu-D3H2LN-luciferase expressing cell line was purchased
from Caliper Life Sciences. This tumor cell line was generated by
stable transfection of the firefly luciferase gene expressed from
the SV40 promoter and further selected for its metastatic potential
upon isolation from a spontaneous lymph node metastasis of
MDA-MB-231-Luc cells in immune-compromised mice. The cells were
grown in DMEM medium supplemented with 10% fetal bovine serum (FBS,
Cat.sup.# SH3007003, Hyclone, Logan, Utah). D-Luciferin
firefly/potassium salt (Cat. # L-8220) was purchased from Biosynth
International, Inc., Itasca, Ill.). Female nu/nu mice (6 to 8 weeks
old) were purchased from Harlan Laboratories. MDA-MB-231-luc-D3H2LN
(2.times.10.sup.6) cells were implanted orthotopically into the
mammary fat pad of nu/nu mice and two days later, the mice were
grouped (10 mice per group) and orally administered either vehicle
(PEG-400) or CEP-40783 formulated in PEG-400 at 10 and 30 mg/kg qd.
Mouse body weights were measured on day 0 and then every 7 days,
and bioluminescence image analysis performed at the end of the
study using the Caliper Life Sciences (Xenogen) Spectrum imager as
detailed in FIG. 6. Images of the primary tumor and potential
metastatic tumors in lymph nodes were taken weekly, starting on day
7 post cell implantation. Prior to image acquisition, each mouse
were give a 0.2 ml intra-peritoneal injection of D-Luciferin
firefly/potassium salt and images were acquired 6-10 minutes post
substrate administration. The primary tumors were covered with
electrical tape or thick black paper to avoid interference in
imaging metastatic tumors residing in lymph nodes. The mice with
bioluminescent images disseminated into peritoneal cavity on the
first imaging were discarded. The study ended when the
bioluminescent signals from the primary tumors became too strong to
effectively assess the detection of metastatic tumors in lymph
nodes. At the end of study, the mice were sacrificed and the plasma
and primary tumors were collected for LC/MS analysis. Image
analyses was performed after all the images had been taken, using
Living Image Software (vs. 4.0, 2010). Counts were converted to
average radiance or mean photon flux; expressed as photons per
second (p/s).
[0063] FIG. 9A shows the quantitation of radiance values in primary
tumors on Day 14 post inoculation. FIG. 9B shows quantitation of
radiance values on Day 21 post-inoculation for lymph node
metastases. Values represent mean.+-.SEM of radiance values.
Statistical analyses were performed using Mann-Whitney Rank Sum
Test or 1- or 2-way ANOVA for significance differences between
treatment groups. *p<0.05-vehicle as compared to CEP-40783
treated. These data (FIGS. 8, 9A and 9B) show that oral
administration is efficacious in reducing spontaneous lymph node
and pulmonary metastatic tumor burden in these models.
AXL/c-Met Dual Inhibition: Therapeutic Utility
[0064] AXL and c-Met activation are estimated to be the underlying
mechanism(s) responsible for the acquisition of anti-EGFR
resistance in up to 50% of EGFR-mutated NSCLC patients. Other
cancer types with a high prevalence of constitutive AXL and/or
c-Met activation include: breast cancer, non-small cell lung cancer
(NSCLC), pancreatic cancer, gastric cancer, esophageal cancer, and
ovarian cancer.
[0065] Table 2 (below) summarizes the results of a number of
experiments where CEP-40783 has demonstrated efficacy in a number
of erlotinib-insensitive NSCLC models and has shown superiority to
an optimal paclitaxel dosing regimen.
TABLE-US-00002 TABLE 2 % Tumor Growth Inhibition CEP-40783
CEP-40783 Paclitaxel Study Duration 10 mg/kg 30 mg/kg 10 mg/kg
Model Name (days) po/qd po/qd iv/q4dx3 CTG-0157 14 3% 85%*.dagger.
41% CTG-0165 37 116%*.dagger. 118%*.dagger. 56%* CTG-0159 14 46%*
66%*.dagger. 25%* CTG-0170 10 104%*.dagger. 107%*.dagger. 77%*
CTG-0192 32 85%* 71%* 105%* Data from CTG-0157 and CTG-0159 is on
file but not included here. *Indicates statistical significance (P
< 0.05) compared to the control group. .dagger.Indicates
statistical significance (P < 0.05) compared to the group
treated with paclitaxel.
[0066] As shown in Table 2 oral administration of CEP-40783 results
in significant efficacy (TGI and/or tumor regression) in all human
primary NSCLC TumorGrafts. CTG-0192 is an erlotinib-sensitive model
which was found to be the least CEP-40783 sensitive tumor relative
to paclitaxel.
Efficacy in Erlotinib-Insensitive Primary NSCLC Human
TumorGrafts.TM.
[0067] FIG. 10 presents data from two erlotinib-insensitive human
TumorGraft models (CTG-0165 and CTG-0170). AXL and/or c-Met are
constitutively activated in these models and, as shown, oral
CEP-40783 demonstrates superior efficacy in both models as
evidenced by tumor regressions at the 10 mg/kg and 30 mg/kg doses
relative to standard of care (SoC) therapy, namely paclitaxel 10
mg/kg, iv, q4dx2).
Activity of CEP-40783 and Erlotinib in "Erlotinib-Sensitive" NSCLC
TumorGraft.TM. with Activated AXL and c-Met
[0068] FIG. 11 presents data from an erlotinib-sensitive NSCLC
TumorGraft.TM. model (CTG-0192) with activated AXL and c-Met. As
seen in the graph (FIG. 11), in the animals dosed with erlotinib
(35 mg/kg, po, qdx34) there was initial sensitivity followed by
acquired erlotinib-resistance at (approx.) day 29. Dosing was
discontinued at day 34. Moreover, upon discontinuation of dosing,
the rate of tumor regrowth was similar to control/vehicle.
[0069] Tumor regressions were achieved in the combination treatment
group (CEP-40783/erlotinib) and in the group treated with CEP-40783
as a single agent significant TGI/tumor stasis was maintained even
after dosing was discontinued (day 34).
[0070] Unlike erlotinib, there was no acquired resistance to
CEP-40783 during the treatment phase of the experiment. In
addition, there was a long-lasting anti-tumor effect in the
CEP-40783 (alone) group even after discontinuation of
treatment.
[0071] Methods (for the experiments shown in FIGS. 10 and 11)
[0072] Female immunocompromised nu/nu mice (Harlan) between 6-9
weeks of age were housed on irradiated papertwist-enriched 1/8''
corncob bedding (Sheperd) in individual HEPA ventilated cages
(Innocage.RTM. IVC, Innovive USA) on a 12-hour light-dark cycle at
68-74.degree. F. (20-23.degree. C.) and 30-70% humidity. Animals
were fed water ad libitum (reverse osmosis, 2 ppm Cl.sub.2) and an
irradiated Test rodent diet (Teklad 2919) consisting of 19%
protein, 9% fat, and 4% fiber. [0073] Tumor Models: Champions
TumorGraft models of NSCLC are owned by Champions Oncology. In this
study, animals were implanted unilaterally on the right flank with
tumor fragments harvested from host animals. When tumors reached
approximately 150-250 mm.sup.3, animals were matched by tumor
volume into treatment and control groups and dosing initiated (Day
0); mice were ear-tagged and followed individually throughout the
experiment. [0074] CEP-40783 was formulated using vehicle
components (PEG400) supplied by Champions. Erlotinib (manufacturer:
OSI Pharmaceuticals; National Drug Code: 50242-062-01; Lot #:
US0002) was formulated using 100% PEG-400 as the vehicle according
to manufacturer's specifications.
[0075] Efficacy measurements: Beginning Day 0, tumor dimensions
were measured twice weekly by digital caliper (Fowler Ultra-Cal IV)
and data including individual and mean estimated tumor volumes
(Mean TV.+-.SEM) were recorded for each group; tumor volume was
calculated using the formula
TV=width.sup.2.times.length.times.0.52. At study completion,
percent tumor growth inhibition (% TGI) values were calculated and
reported for each treatment group (T) versus control (C) using
initial (i) and final (f) tumor measurements by the formula: %
TGI=1-T.sub.f-T.sub.i/C.sub.f-C; single agent or combination
therapies resulting in a TGI>50 at study completion are
considered active in the tested model at the evaluated treatment
regimen according to NCI guidelines. Individual mice reporting a
tumor volume .ltoreq.50% of the Day 0 measurement for two
consecutive measurements over a seven day period were considered
partial responders (PR). If the PR persisted until study
completion, percent tumor regression (% TR) was determined using
the formula: % TR=1-T.sub.f/T.sub.i.times.100; a mean value was
calculated if multiple PR mice occurred in one group. Individual
mice lacking palpable tumors (<4.times.4 mm.sup.2 for two
consecutive measurements over a seven day period) were classified
as complete responders (CR); a CR that persisted until study
completion was considered a tumor-free survivor (TFS). TFS animals
are excluded from efficacy calculations. Statistical differences in
tumor volume were determined using a two-tailed One-Way Analysis of
Variance (ANOVA) followed by the Dunett's multiple comparisons test
comparing treated groups with control and combinations with
standard agent alone when possible. All data collected in this
study was managed electronically and stored on a redundant server
system.
[0076] The data described herein clearly demonstrates that
CEP-40783 exhibits potent AXL and c-Met pharmacodynamic and
anti-tumor efficacy in established tumor xenograft models. In all
of the above studies CEP-40783 was well tolerated with no
compound-related body weight loss. As such, CEP-40783 may have
potential therapeutic utility in multiple human tumor types in
which c-Met and AXL activity play a critical role in tumor
formation, local invasion and metastasis. The data further suggests
that CEP-40783 can be used as adjuvant/neoadjuvant therapy in
combination with standard of care chemotherapies to enhance overall
efficacy and/or to limit or prevent metastatic dissemination of the
primary tumor (resectable or non-resectable).
* * * * *