COMPOSITIONS COMPRISING A PI3K INHIBITOR
AND A MEK INHIBITOR AND THEIR USE FOR TREATING CANCER
CROSS-REFERENCE TO RELATED APPLICATIONS
|0001 ] This application claims the benefit of priority of U.S Provisional Application No.
61/421,465 filed December 9, 2010, U.S Provisional Application No. 61/436,258 filed
January 26, 201 1, and U.S Provisional Application No. 61/467,485 filed March 25, 201 1, all
of which are incorporated herein by reference.
BACKGROUND
[0002] There is an ongoing need in the art for more efficacious methods and compositions
in the treatment of cancer. The instant application is directed, generally, to compositions and
methods for the treatment of cancer, and more particularly, to compositions and methods
comprising inhibitors of the mitogen activated protein kinase (MEK) and/or phosphoinositide
3-kinase (PI3K) pathways.
[0003] Tumor cells treated with inhibitors of MEK kinases typically respond via inhibition
of phosphorylation of ERK, down-regulation of Cyclin D, induction of Gl arrest, and finally
undergoing apoptosis. Pharmacologically, MEK inhibition completely abrogates tumor
growth in BRaf xenograft tumors whereas Ras mutant tumors exhibit only partial inhibition in
most cases (D. B. Solit et al., Nature 2006; 439: 358-362). Thus, MEKs have been targets of
great interest for the development of cancer therapeutics.
[0004] N-((S)-2,3-dihydroxypropyl)-3-(2-fluoro-4-iodo-phenylamino)isonicotinamide (also
referred to as MSC1936369 or AS703026) is a novel, allosteric inhibitor of MEK. It
possesses relatively high potency and selectivity, having no activity against 217 kinases or 90
non-kinase targets when tested at 0 . The in vivo PK profile of AS703026 is acceptable
in mice and rats, with relatively high oral bioavailability (52 - 57%), medium or high
clearance (0.9 - 2.6 L/h/kg) and medium or long half-life (2.2 - 4.7 h). The compound is
relatively well-tolerated in mice, with a two-week maximum tolerated dose of 60 mg/kg BID.
[0005] N-(3-{[(3-{[2-chloro-5-(methoxy)phenyl]amino}quinoxalin-2-
yl)amino]sulfonyl}phenyl)-2-methylalaninamide (also known as XL147 or SAR245408) and
2-amino-8-ethyl-4-methyl-6-(l H-pyrazol-5-yl)pyrido[2,3-d]pyrimidin-7(8 H)-one (also known
I
as XL765 or SAR245409) are selective inhibitors of class I PI3 lipid kinases. XL147
inhibits the phosphorylation of downstream effectors Akt and S6 ribosomal protein (S6RP)
and targets only PI3 isoforms (inhibitor concentration, i.e., IC50 values in nanomolar (nM):
PI3Ka 39, 3383, PI3 36, 3 23). XL765 targets both PI3 isoforms (IC50 values
in nM: PI3Ka 39, 3 113, 3 43, 3 9) and mTOR (157 nM).
[0006] Oral administration of XL147 or XL765 alone inhibits tumor growth in mice bearing
xenografts in which PI3 signaling is activated, such as the PTEN-deficient PC-3 prostate
adenocarcinoma, U87-MG gliobastoma, A2058 melanoma and WM-266-4 melanoma, or the
PIK3CA mutated MCF7 mammary carcinoma. XL147 is currently undergoing several Phase
I trials for patients with solid tumors and/or lymphoma and Phase II trials for patients with
endometrial or hormone receptor-positive breast cancer. XL765 is currently undergoing
testing in Phase I clinical trials for patients with solid tumor, lymphoma or glioblastoma and
in a Phase I I trial for patients with hormone receptor-positive breast cancer.
[0007J There remains a need, however, for a cancer therapy that is more effective in
inhibiting cell proliferation and tumor growth while minimizing patient toxicity. There is a
particular need for an ME or PI3 inhibitor therapy is made more efficacious without
substantially increasing, or even maintaining or decreasing, the dosages of MEK or PI3K
inhibitor traditionally employed in the art.
SUMMARY
[0008J In one aspect, there is provided compositions and uses thereof in the treatment of a
variety of cancers.
[0009] In particular embodiments, there is provided a composition that includes a compound
having the following structural formula:
(MSCI 936369. or AS703026 or MSC6369)
a compound selected from the group consisting of
(XL 147 or SAR245408)
and
(XL765, SAR245409 or MSC0765)
|0010] In another aspect, methods of treating a patient with cancer are provided that
comprise administering to the patient a therapeutically effective amount of a compound of
Formula (1), or a pharmaceutically acceptable salt thereof, in combination with the compound
of Formula (2a) or Formula (2b), or a pharmaceutically acceptable salt thereof.
[001 1] In one embodiment, a method of treating a patient with cancer comprises
administering to the patient a first dosage of a MEK inhibitor and a second dosage of a PI3K
inhibitor, wherein said MEK inhibitor has the following structural formula:
and said PI3 inhibitor is selected from the group consisting of
and
[0012] In some embodiments, the methods involve treating cancer selected from the group
consisting of non-small cell lung cancer, breast cancer, pancreatic cancer, liver cancer,
prostate cancer, bladder cancer, cervical cancer, thyroid cancer, colorectal cancer, liver cancer,
muscle cancer, hematological malignancies, melanoma, endometrial cancer and pancreatic
cancer. In others, the cancer is selected from the group consisting of colorectal cancer,
endometrial cancer, hematological malignancies, thryoid cancer, breast cancer, melanoma,
pancreatic cancer and prostate cancer.
[0013] In some embodiments, the compositions and methods of use described herein are in
amounts (i.e., either in the composition are in an administered dosage) that synergistically
reduce tumor volume in a patient. n further embodiments, the synergistic combination
achieves tumor stasis or tumor regression.
[0014] In another aspect, a combination for use in treating cancer is provided, the
combination comprising a therapeutically effective amount of (A) the compound of Formula
( 1), or a pharmaceutically acceptable salt thereof, and (B) the compound of Formula (2a) or
Formula (2b), or a pharmaceutically acceptable salt thereof.
|0015] In one embodiment, uses of a combination comprising a therapeutically effective
amount of (A) the compound of Formula ( 1), or a pharmaceutically acceptable salt thereof,
and (B) the compound of Formula (2a) or Formula (2b), or a pharmaceutically acceptable salt
thereof, are provided for the preparation of a medicament for use in treatment of cancer.
[0016] In another aspect, kits are provided comprising: (A) the compound of Formula ( 1), or
a pharmaceutically acceptable salt thereof; (B) the compound of Formula (2a) or Formula
(2b), or a pharmaceutically acceptable salt thereof; and (C) instructions for use.
[0017] Other objects, features and advantages will become apparent from the following
detailed description. The detailed description and specific examples are given for illustration
only since various changes and modifications within the spirit and scope of the invention will
become apparent to those skilled in the art from this detailed description. Further, the
examples demonstrate the principle of the invention and cannot be expected to specifically
illustrate the application of this invention to all the examples where it will be obviously useful
to those skilled in the prior art.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1 provides a plot showing body weight change during the evaluation of the
antitumor activity of Compound ( 1) (5 mg/kg) in combination with Compound (2b) (30
mg/kg) and Compound (2a) (50 and 75 mg/kg) against human HCT 1 6 bearing SCID female
mice.
[0019] Figure 2 provides a plot showing antitumor activity of Compound (1) (5 mg/kg) in
combination with Compound (2b) (30 mg/kg) against human HCT 16 bearing SCID female
mice.
[0020] Figure 3 provides a plot showing antitumor activity of Compound (1) (5 mg/kg) in
combination with Compound (2a) (50 and 75 mg/kg) against human HCT 16 bearing SCID
female mice. The box indicates combinations achieving therapeutic synergy.
[0021] Figure 4 provides a plot showing body weight change during the evaluation of the
antitumor activity of Compound (1) (10 and 20 mg/kg) in combination with Compound (2b)
(20 mg/kg) and Compound (2a) (50 and 75 mg/kg) against human HCT 116 bearing SCID
female mice.
[0022] Figure 5 provides a plot showing antitumor activity of Compound (1) (10 and 20
mg/kg) in combination with Compound (2b) (20 mg/kg) against human HCT 116 bearing
SCID female mice.
[0023] Figure 6 provides a plot showing antitumor activity of Compound (1) (10 mg/kg) in
combination with Compound (2a) (50 and 75 mg/kg) against human HCT 116 bearing SCID
female mice.
[0024J Figure 7 provides a plot showing body weight change during the evaluation of the
antitumor activity of Compound (1) (10 and 20 mg/kg) in combination with Compound (2a)
(50 and 75 mg/kg) against human HCT 116 bearing SCID female mice.
[0025] Figures 8 provides a plot showing antitumor activity of Compound (1) (10 and 20
mg/kg) in combination with Compound (2a) (50 and 75 mg/kg) against human HCT 116
bearing SCID female mice. The box indicates combinations achieving therapeutic synergy.
[00261 Figure 9 provides a plot showing body weight change during the evaluation of the
antitumor activity of Compound (1) (10 and 20 mg/kg) in combination with Compound (2b)
(20 mg/kg) against human HCT 116 bearing SCID female mice.
[0027] Figure 10 provides a plot showing antitumor activity of Compound ( 1) ( 10 and 20
mg/kg) in combination with Compound (2b) (20 mg/kg) against human HCT 116 bearing
SCID female mice.
[0028] Figure 11 provides a plot showing percent body weight of MiaPaCa-2 tumor-bearing
mice treated with Compound (1) (5 mg/kg) and Compound (2a) (50 mg/kg) alone or in
combination.
[0029] Figure 12 provides a plot showing percent body weight of MiaPaCa-2 tumor-bearing
mice treated with Compound (1) (5 mg/kg) and Compound (2b) (30 mg kg) alone or in
combination.
[0030| Figure 3 provides a plot showing mean tumor volumes of MiaPaCa-2 tumorbearing
mice treated with Compound (1) (5 mg/kg) and Compound (2a) (50 mg/kg) alone or
in combination.
[0031] Figure 14 provides a plot showing mean tumor volumes of MiaPaCa-2 tumorbearing
mice treated with Compound (1) (5 mg/kg) and Compound (2b) (30 mg/kg) alone or
in combination.
[0032] Figures 15A and 15B provide charts showing Z-score values of Compound ( 1) for
various tumor cell lines identifying specific therapeutic applications. Selection of specific
therapeutic applications for Compound (1). Individual z-score values for each cell line are
plotted within one group corresponding to the tumor origin. An average value for all values
within one group is shown as a green triangle, and can serve as an indicator for Compound (1)
activity within one group. As for individual z-scores, z-scores below mean strong efficacy,
whereas z-scores >0 approximate resistance.
[0033] Figures 16A and 16B provide charts showing Z-score values of Compound (2b) for
various tumor cell lines identifying specific therapeutic applications. Selection of specific
therapeutic applications for Compound (2b). Individual z-score values for each cell line are
plotted within one group corresponding to the tumor origin. An average value for all values
within one group is shown as a green triangle and can serve as an indicator for Compound
(2b) activity within one group. As for individual z-scores, z-scores below zero mean strong
efficacy, whereas a z-score >0 approximate resistance.
[0034] Figure 17 provides a chart showing Z-score values of Compound (1) in combination
with Compound (2b) for various tumor cell lines.
[0035] • Figures 18A, 18B, 18C, 18D, 18E and 18F provide plots and graphs showing
combination results of Compound (1) with Compound (2b) in CRC tumor cell lines (synergy
plot & mutation analysis).
[0036] Figures 19A and 19B provide plots and graphs showing combination results of
Compound (1) with Compound (2b) in pancreatic tumor cell lines (synergy plot & mutation
analysis).
[0037] Figures 20A and 20B provide plots and graphs showing combination results of
Compound (1) with Compound (2b) in NSCLC tumor cell lines (synergy plot & mutation
analysis).
[0038] Figure 2 1 provides a plot showing body weight change during the evaluation of the
antitumor activity of Compound (1) (20 mg/kg) in combination with Compound (2b) (20
mg/kg) and Compound (2a) (75 mg/kg) against human primary colon tumors CR-LRB-009C
bearing SCID female mice.
[0039] Figure 22 provides a plot showing antitumor activity of Compound (1) (20 mg/kg) in
combination with Compound (2b) (20 mg/kg) and Compound (2a) (75 mg/kg) against human
primary colon tumors CR-LRB-009C bearing SCID female mice.
[0040] Figure 23 provides a plot showing body weight change during the evaluation of the
antitumor activity of Compound (1) (20 mg/kg) in combination with Compound (2b) (20
mg/kg) and Compound (2a) (75 mg/kg) against human primary colon tumors CR-LRB-013P
bearing SCID female mice.
[0041 Figure 24 provides a plot showing antitumor activity of Compound (1) (20 mg/kg) in
combination with Compound (2b) (20 mg/kg) and Compound (2a) (75 mg/kg) against human
primary colon tumors CR-LRB-013P bearing SCID female mice.
[0042] Figure 25 graphically depicts the results of Icyte ex vivo imaging of Evans Blue
tumor extravasation performed after treatment with either Compound (2a) or Compound (2b)
as single agents or in combination with Compound (1) in HCT1 16 xenografts.
[0043] Figures 26A and 26B graphically depict results of FMT imaging after three days of
therapy, three hours after AnnexinV-750 administration, four hours post-treatment with
Compound (1), Compound (2a) or Compound (2b) as single agents or combinations in
HCT1 16 xenografts. Tumor fluorescence was quantified in pmol of fluorophore and
standardized to the tumor volume. Statistics: Newman-Keuls after 2way Anova on Ranked
data, NS: P<0.05).
[0044] Figures 27A and 27B graphically show protein levels of cleaved-PARP and caspase-
3 in tumor extracts following treatment with Compound (1), Compound (2a) or Compound
(2b) alone or in selected combination. Statistics: Dunnett's test for one factor after one way
Anova, NS: P<0.05.
[0045] Figure 28 provides a plot showing tumor volumes of HCT1 16 tumor-bearing mice
treated with Compound (1) (10 mg/kg), Compound (2a) (50 mg/kg) or Compound (2b)(20
mg/kg) alone or in combination. To quantify apoptosis, fluorescent Annexin-Vivd'-750 was
injected iv on day 3 and day 7 after start of treatment, 1 hour post daily treatment. Animals
were imaged by F T 3 hours post probe injection.
DETAILED DESCRIPTION
[0046] In one aspect, methods for treating patients with cancer are provided. In one
embodiment, the methods comprise administering to the patient a therapeutically effective
amount of a ME inhibitor and a therapeutically effective amount of a PI3 inhibitor, as
further described below.
[0047] In one embodiment, the inventive methods and compositions comprise a MEK
inhibitor having the following structural formula:
[0048] The MEK inhibitor according to formula (1), is referred to herein as "Compound
(1)" and is known also as MSC 1936369, AS703026 or MSC6369. The preparation,
properties, and MEK-inhibiting abilities of Compound (1) are provided in, for example,
International Patent Publication No. WO 06/045514, particularly Example 115 and Table 1
therein. The entire contents of WO 06/0455 are incorporated herein by reference. Neutral
and salt forms of the compound of Formula ( 1) are all considered herein.
[0049] In other embodiments, the inventive methods and compositions comprise a P13
inhibitor having one of the following structures:
or
[0050] The PI3K inhibitor according to formula (2a), is referred to herein as "Compound
(2a)" and is known also as XL 47 or SAR245408. The PI3 inhibitor according to formula
(2b), is referred to herein as "Compound (2b)" and is known also as XL765, SAR245409 or
MSC0765. The preparation and properties of Compound (2a) are provided in, for example,
International Patent Publication No. WO 07/044729, particularly Example 357 therein. The
entire contents of WO 07/044729 are incorporated herein by reference. The preparation and
properties of Compound (2b) are provided in, for example, International Patent Publication
No. WO 07/0448 13, particularly Example 56 therein. The entire contents of WO 07/0448 13
are incorporated herein by reference.
[0051 ] In some embodiments, the compounds described above are unsolvated. In other
embodiments, one or both of the compounds used in the method are in solvated form. As
known in the art, the solvate can be any of pharmaceutically acceptable solvent, such as water,
ethanol, and the like. In general, the presence of a solvate or lack thereof does not have a
substantial effect on the efficacy of the ME or PI3 inhibitor described above.
[0052] Although the compounds in Formula ( 1), Formula (2a) and Formula (2b) are
depicted in their neutral forms, in some embodiments, these compounds are used in a
pharmaceutically acceptable salt form. The salt can be obtained by any of the methods well
known in the art, such as any of the methods and salt forms elaborated upon in WO
07/044729, as incorporated by reference herein. A "pharmaceutically acceptable salt" of the
compound refers to a salt that is pharmaceutically acceptable and that retains pharmacological
activity. It is understood that the pharmaceutically acceptable salts are non-toxic. Additional
information on suitable pharmaceutically acceptable salts can be found in Remington 's
Pharmaceutical Sciences, 1 th ed., Mack Publishing Company, Easton, PA, 1985, or S. M.
Berge, et al., "Pharmaceutical Salts," J . Pharm. Sci., 1977;66: 1- 19, both of which are
incorporated herein by reference.
[0053] Examples of pharmaceutically acceptable acid addition salts include those formed
with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,
phosphoric acid, as well as those salts formed with organic acids, such as acetic acid,
trifluoroacetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid,
pyruvic acid, lactic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid,
tartaric acid, citric acid, benzoic acid, cinnamic acid, 3-(4-hydroxybenzoyl)benzoic acid,
mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-
hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, 2-
naphthalenesulfonic acid, 4-toluenesulfonic acid, camphorsulfonic acid, glucoheptonic acid,
4,4'-methylenebis-(3-hydroxy-2-ene-l-carboxylic acid), 3-phenylpropionic acid,
trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic
acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, p-toluenesulfonic acid,
and salicylic acid.
[0054] In a first set of embodiments, the MEK inhibitor of formula ( 1) is administered
simultaneously with the PI3 inhibitor of either formula (2a) or (2b). Simultaneous
administration typically means that both compounds enter the patient at precisely the same
time. However, simultaneous administration also includes the possibility that the MEK
inhibitor and PI3K inhibitor enter the patient at different times, but the difference in time is
sufficiently miniscule that the first administered compound is not provided the time to take
effect on the patient before entry of the second administered compound. Such delayed times
typically correspond to less than 1 minute, and more typically, less than 30 seconds.
|0055] In one example, wherein the compounds are in solution, simultaneous administration
can be achieved by administering a solution containing the combination of compounds. In
another example, simultaneous administration of separate solutions, one of which contains the
MEK inhibitor and the other of which contains the PI3 inhibitor, can be employed. In one
example wherein the compounds are in solid form, simultaneous administration^can be
achieved by administering a composition containing the combination of compounds.
[0056] In other embodiments, the MEK and PI3K inhibitors are not simultaneously
administered. In this regard, the first administered compound is provided time to take effect
on the patient before the second administered compound is administered. Generally, the
difference in time does not extend beyond the time for the first administered compound to
complete its effect in the patient, or beyond the time the first administered compound is
completely or substantially eliminated or deactivated in the patient. In one set of
embodiments, the MEK inhibitor is administered before the PI3K inhibitor. In another set of
embodiments, the PI3K inhibitor is administered before the MEK inhibitor. The time
difference in non-simultaneous administrations is typically greater than 1 minute, and can be,
for example, precisely, at least, up to, or less than 5 minutes, 10 minutes, 15 minutes, 30
minutes, 45 minutes, 60 minutes, two hours, three hours, six hours, nine hours, 1 hours, 24
hours, 36 hours, or 48 hours.
[0057] In one set of embodiments, one or both of the MEK and PI3 inhibitors are
administered in a therapeutically effective (i.e., therapeutic) amount or dosage. A
"therapeutically effective amount" is an amount of the MEK or PI3K inhibitor that, when
administered to a patient by itself, effectively treats the cancer (for example, inhibits tumor
growth, stops tumor growth, or causes tumor regression). An amount that proves
"therapeutically effective amount" in a given instance, for a particular subject, may not be
effective for 100% of subjects similarly treated for the disease or condition under
consideration, even though such dosage is deemed a "therapeutically effective amount" by
skilled practitioners. The amount of the compound that corresponds to a therapeutically
effective amount is strongly dependent on the type of cancer, stage of the cancer, the age of
the patient being treated, and other facts. In general, therapeutically effective amounts of
these compounds are well-known in the art, such as provided in the supporting references
cited above.
[0058] In another set of embodiments, one or both of the MEK and PI3 inhibitors are
administered in a sub-therapeutically effective amount or dosage. A sub-therapeutically
effective amount is an amount of the MEK or PI3K inhibitor that, when administered to a
patient by itself, does not completely inhibit over time the biological activity of the intended
target.
[0059] Whether administered in therapeutic or sub-therapeutic amounts, the combination of
MEK inhibitor and PI3K inhibitor should be effective in treating the cancer. A sub
therapeutic amount of MEK inhibitor can be an effective amount if, when combined with the
PI3K inhibitor, the combination is effective in the treatment of a cancer.
[0060] In some embodiments, the combination of compounds exhibits a synergistic effect
(i.e., greater than additive effect) in treating the cancer, particularly in reducing a tumor
volume in the patient. In different embodiments, depending on the combination and the
effective amounts used, the combination of compounds can either inhibit tumor growth,
achieve tumor stasis, or even achieve substantial or complete tumor regression.
[0061] In some embodiments, Compound (1) is administered at a dosage of about 7-120 mg
po qd. Compound (2a), meanwhile, can be administered at a dosage of about 12-600 mg po
qd. Compound (2b) can be administered at a dosage of about 15-90 mg po qd.
[0062] As used herein, the term "about" generally indicates a possible variation of no more
than 10%, 5%, or 1% of a value. For example, "about 25 mg kg" will generally indicate, in its
broadest sense, a value of 22.5-27.5 mg/kg, i.e., 25 ± 10 mg/kg.
[0063] While the amounts of MEK and PI3K inhibitors should result in the effective
treatment of a cancer, the amounts, when combined, are preferably not excessively toxic to the
patient (i.e., the amounts are preferably within toxicity limits as established by medical
guidelines). In some embodiments, either to prevent excessive toxicity and/or provide a more
efficacious treatment of the cancer, a limitation on the total administered dosage is provided.
Typically, the amounts considered herein are per day; however, half-day and two-day or threeday
cycles also are considered herein.
[0064] Different dosage regimens may be used to treat the cancer. In some embodiments, a
daily dosage, such as any of the exemplary dosages described above, is administered once,
twice, three times, or four times a day for three, four, five, six, seven, eight, nine, or ten days.
Depending on the stage and severity of the cancer, a shorter treatment time (e.g., up to five
days) may be employed along with a high dosage, or a longer treatment time (e.g., ten or more
days, or weeks, or a month, or longer) may be employed along with a low dosage. In some
embodiments, a once- or twice-daily dosage is administered every other day. In some
embodiments, each dosage contains both the ME and PI3 inhibitors, while, in other
embodiments, each dosage contains either the MEK or P13K inhibitors. In yet other
embodiments, some of the dosages contain both the MEK and PI3K inhibitors, while other
dosages contain only the MEK or the PI3K inhibitor.
[0065] Examples of types of cancers to be treated with the present invention include, but are
not limited to, lymphomas, sarcomas and carcinomas, e.g., fibrosarcoma, myxosarcoma,
liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,
endotheliosarcoma, lymphangiosarcoma, synovioma, mesothelioma,
lymphangioendotheliosarcoma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon
carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, gastric cancer,
esophageal cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat
gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary
adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal
cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal
carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, non-small cell
lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma,
astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma,
hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma,
neuroblastoma, retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acute
myelocytic leukemia (myeloblasts, promyelocytic, myelomonocytic, monocytic and
erythroleukemia); chronic leukemia (chronic myelocytic (granulocytic) leukemia and chronic
lymphocytic leukemia); and polycythemia vera, lymphoma (Hodgkin's disease and nonHodgkin's
disease), multiple myeloma, Waldenstrom's macroglobulinemia and heavy chain
disease.
[0066] In some embodiments, the cancer being treated is selected from the group consisting
of non-small cell lung cancer, breast cancer, pancreatic cancer, liver cancer, prostate cancer,
bladder cancer, cervical cancer, thyroid cancer, colorectal cancer, liver cancer, and muscle
cancer. n other embodiments, the cancer is selected from colorectal cancer, endometrial
cancer, hematology cancer, thryoid cancer, triple negative breast cancer or melanoma.
[0067] The patient considered herein is typically a human. However, the patient can be any
mammal for which cancer treatment is desired. Thus, the methods described herein can be
applied to both human and veterinary applications.
[0068] The term "treating" or "treatment", as used herein, indicates that the method has, at
the least, mitigated abnormal cellular proliferation. For example, the method can reduce the
rate of tumor growth in a patient, or prevent the continued growth of a tumor, or even reduce
the size of a tumor.
[0069] In another aspect, methods for preventing cancer in an animal are provided. In this
regard, prevention denotes causing the clinical symptoms of the disease not to develop in an
animal that may be exposed to or predisposed to the disease but does not yet experience or
display symptoms of the disease. The methods comprise administering to the patient a ME
inhibitor and a PI3K inhibitor, as described herein. In one example, a method of preventing
cancer in an animal comprises administering to the animal a compound of Formula (1), or a
pharmaceutically acceptable salt thereof, in combination with a compound selected from the
group consisting of Formula (2a) and Formula (2b), or a pharmaceutically acceptable salt
thereof.
[0070] The MEK and PI3 inhibiting compounds, or their pharmaceutically acceptable salts
or solvate forms, in pure form or in an appropriate pharmaceutical composition, can be
administered via any of the accepted modes of administration or agents known in the art. The
compounds can be administered, for example, orally, nasally, parenterally (intravenous,
intramuscular, or subcutaneous), topically, transdermally, intravaginally, intravesically,
tntracistemally, or rectally. The dosage form can be, for example, a solid, semi-solid,
lyophilized powder, or liquid dosage forms, such as for example, tablets, pills, soft elastic or
hard gelatin capsules, powders, solutions, suspensions, suppositories, aerosols, or the like,
preferably in unit dosage forms suitable for simple administration of precise dosages. A
particular route of administration is oral, particularly one in which a convenient daily dosage
regimen can be adjusted according to the degree of severity of the disease to be treated.
[0071] n another aspect, the instant application is directed to a composition that includes
the MEK inhibitor shown in Formula (1) and a PI3K inhibitor selected from the compounds
shown in Formulas (2a) and (2b). In some embodiments, the composition includes only the
MEK and PI3K inhibitors described above. In other embodiments, the composition is in the
form of a solid (e.g., a powder or tablet) including the MEK and PI3K inhibitors in solid form,
and optionally, one or more auxiliary (e.g., adjuvant) or pharmaceutically active compounds
in solid form. In other embodiments, the composition further includes any one or combination
of pharmaceutically acceptable carriers (i.e., vehicles or excipients) known in the art, thereby
providing a liquid dosage form.
[0072] Auxiliary and adjuvant agents may include, for example, preserving, wetting,
suspending, sweetening, flavoring, perfuming, emulsifying, and dispensing agents.
Prevention of the action of microorganisms is generally provided by various antibacterial and
antifungal agents, such as, parabens, chlorobutanol, phenol, sorbic acid, and the like. Isotonic
agents, such as sugars, sodium chloride, and the like, may also be included. Prolonged
absorption of an injectable pharmaceutical form can be brought about by the use of agents
delaying absorption, for example, aluminum monostearate and gelatin. The auxiliary agents
also can include wetting agents, emulsifying agents, pH buffering agents, and antioxidants,
such as, for example, citric acid, sorbitan monolaurate, triethanolamine oleate, butylated
hydroxytoluene, and the like.
[0073] Dosage forms suitable for parenteral injection may comprise physiologically
acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions,
and sterile powders for reconstitution into sterile injectable solutions or dispersions.
Examples of suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include
water, ethanol, polyols (propyleneglycol, polyethyleneglycol, glycerol, and the like), suitable
mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl
oleate. Proper fluidity can be maintained, for example, by the use of a coating such as
lecithin, by the maintenance of the required particle size in the case of dispersions and by the
use of surfactants.
|0074] Solid dosage forms for oral administration include capsules, tablets, pills, powders,
and granules. In such solid dosage forms, the active compound is admixed with at least one
inert customary excipient (or carrier) such as sodium citrate or dicalcium phosphate or (a)
fillers or extenders, as for example, starches, lactose, sucrose, glucose, mannitol, and silicic
acid, (b) binders, as for example, cellulose derivatives, starch, alignates, gelatin,
polyvinylpyrrolidone, sucrose, and gum acacia, (c) humectants, as for example, glycerol, (d)
disintegrating agents, as for example, agar-agar, calcium carbonate, potato or tapioca starch,
alginic acid, croscarmellose sodium, complex silicates, and sodium carbonate, (e) solution
retarders, as for example paraffin, (f) absorption accelerators, as for example, quaternary
ammonium compounds, (g) wetting agents, as for example, cetyl alcohol, and glycerol
monostearate, magnesium stearate and the like (h) adsorbents, as for example, kaolin and
bentonite, and (i) lubricants, as for example, talc, calcium stearate, magnesium stearate, solid
polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules,
tablets, and pills, the dosage forms also may comprise buffering agents.
[0075] Solid dosage forms as described above can be prepared with coatings and shells,
such as enteric coatings and others well-known in the art. They can contain pacifying agents
and can be of such composition that they release the active compound or compounds in a
certain part of the intestinal tract in a delayed manner. Examples of embedded compositions
that can be used are polymeric substances and waxes. The active compounds also can be in
microencapsulated form, if appropriate, with one or more of the above-mentioned excipients.
[0076] Liquid dosage forms for oral administration include pharmaceutically acceptable
emulsions, solutions, suspensions, syrups, and elixirs. Such dosage forms are prepared, for
example, by dissolving, dispersing, etc., a MEK or PI3K inhibitor compound described herein,
or a pharmaceutically acceptable salt thereof, and optional pharmaceutical adjuvants in a
carrier, such as, for example, water, saline, aqueous dextrose, glycerol, ethanol and the like;
solubilizing agents and emulsifiers, as for example, ethyl alcohol, isopropyl alcohol, ethyl
carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propyleneglycol, 1,3-
butyleneglycol, dimethyl formamide; oils, in particular, cottonseed oil, groundnut oil, corn
germ oil, olive oil, castor oil and sesame oil, glycerol, tetrahydrofurfuryl alcohol,
polyethyleneglycols and fatty acid esters of sorbitan; or mixtures of these substances, and the
like, to thereby form a solution or suspension.
[0077] Suspensions, in addition to the active compounds, may contain suspending agents, as
for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters,
microcrystalline cellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth, or
mixtures of these substances, and the like.
[0078] Compositions for rectal administrations are, for example, suppositories that can be
prepared by mixing the compounds described herein with, for example, suitable non- irritating
excipients or carriers such as cocoa butter, polyethyleneglycol or a suppository wax, which are
solid at ordinary temperatures but liquid at body temperature and therefore, melt while in a
suitable body cavity and release the active component therein.
[0079] Dosage forms for topical administration may include, for example, ointments,
powders, sprays, and inhalants. The active component is admixed under sterile conditions
with a physiologically acceptable carrier and any preservatives, buffers, or propellants as can
be required. Ophthalmic formulations, eye ointments, powders, and solutions also can be
employed.
[0080] Generally, depending on the intended mode of administration, the pharmaceutically
acceptable compositions will contain about 1% to about 99% by weight of the compounds
described herein, or a pharmaceutically acceptable salt thereof, and 99% to 1% by weight of a
pharmaceutically acceptable excipient. In one example, the composition will be between
about 5% and about 75% by weight of a compounds described herein, or a pharmaceutically
acceptable salt thereof, with the rest being suitable pharmaceutical excipients.
[0081] Actual methods of preparing such dosage forms are known, or will be apparent, to
those skilled in this art. Reference is made, for example, to Remington's Pharmaceutical
Sciences, 18th Ed., (Mack Publishing Company, Easton, Pa., 1990).
[0082] In some embodiments, the composition does not include one or more other ant i
cancer compounds. In other embodiments, the composition includes one or more other ant i
cancer compounds. For example, administered compositions can comprise standard of care
agents for the type of tumors selected for treatment.
[0083] n another aspect, kits are provided. Kits according to the invention include
package(s) comprising compounds or compositions of the invention. In one embodiment, kits
comprise Compound ( 1), or a pharmaceutically acceptable salt thereof, and a compound
selected from the group consisting of Compound (2a) and Compound (2b), or a
pharmaceutically acceptable salt thereof.
[0084] The phrase "package" means any vessel containing compounds or compositions
presented herein. In some embodiments, the package can be a box or wrapping. Packaging
materials for use in packaging pharmaceutical products are well-known to those of skill in the
art. Examples of pharmaceutical packaging materials include, but are not limited to, bottles,
tubes, inhalers, pumps, bags, vials, containers, syringes, bottles, and any packaging material
suitable for a selected formulation and intended mode of administration and treatment.
[0085] The kit also can contain items that are not contained within the package but are
attached to the outside of the package, for example, pipettes.
[0086] Kits can contain instructions for administering compounds or compositions of the
invention to a patient. Kits also can comprise instructions for approved uses of compounds
herein by regulatory agencies, such as the United States Food and Drug Administration. Kits
also can contain labeling or product inserts for the inventive compounds. The package(s)
and/or any product insert(s) may themselves be approved by regulatory agencies. The kits can
include compounds in the solid phase or in a liquid phase (such as buffers provided) in a
package. The kits also can include buffers for preparing solutions for conducting the methods,
and pipettes for transferring liquids from one container to another.
[0087] Examples have been set forth below for the purpose of illustration and to describe
certain specific embodiments of the invention. However, the scope of the claims is not to be
in any way limited by the examples set forth herein.
Example 1. In vitro activity of Compound (1) in combination with Compound (2b)
[0088] This study describes the activity of individual anticancer agents Compound ( 1) and
Compound (2b), as well as their combination, in a panel of 81 cancer cell lines. Cell lines
were selected to represent 17 different indications with many different genetic variations and
biochemical characteristics. In addition, the study included resting Peripheral Blood
Mononuclear Cells, PBMC, as a model for non-proliferating cells. The results of individual
activity profiles were further used to perform a combination study of Compound ( 1) and
Compound (2b) using a panel of 8 1 cell lines. The study also compared the activity profiles
of Compound ( 1) and Compound (2b) with profiles of more than 300 known anticancer
agents.
(0089 Prior to in vitro combination studies, the activity of individual agents was
investigated using a panel of 82 cell lines. T e purpose of testing individual agents was to
determine the independence of their action. In addition, comparison to an activity profile of
known anticancer agents may help form a hypothesis regarding potential mechanisms of the
compounds' action.
Materials and methods
[0090] Cell lines were purchased directly from the ATCC, NCI, CLS, and DSMZ cell line
collections. A master bank and working aliquots were prepared. Cells used for the study had
undergone less than 20 passages. To ensure the absence of potential contamination and wrong
assignment, all cell lines were tested on the Whole Genome Array (Agilent, USA) and by STR
analysis. Absence of mycoplasma and SMRV contamination was confirmed for all cell lines
used in the studies.
[0091 ] The cell lines were grown in the media recommended by the suppliers in the
presence of 100 U/ml penicillin G and 100 g/ml streptomycin supplied with 10% FCS (PAN,
Germany). The RPM 1640, DMEM, and MEM Earle's medium were from Lonza (Cologne,
Germany), supplements 2mM L-glutamine, 1 mM Na-pyruvate and 1% NEAA were from
PAN (Aidenbach, Germany), 2.5% horse serum and 1 unit/ml insulin from Sigma-Aldrich
(Munich, Germany). RPMI medium was used for culturing the following cell lines: 5637,
22RV 1, 7860, A2780, A43 1, A549, ACHN, ASPC 1, BT20, BXPC3, CAKI , CLS439,
COLO205, COL0678, DLD1 , DU145, EF02 1, EJ28, HCT1 , HS578T, IGROV 1, JAR,
LOVO, MCF7, MDAMB23 1, MDAMB435, MDAMB436, MDAMB468, MHHES 1, MT3,
NCIH292, NCIH358M, NCIH460, NCIH82, OVCAR3, OVCAR4, PANC 1005 (addition of
insulin), PBMC, PC3, RDES, SF268, SF295, S BR3, SKMEL28, SKMEL5, S OV3,
SW620, U20S, UMUC3, and U03 1.
[00921 DMEM was used for A204, A375, A673, C33A, CASKI, HCTl 16, HEPG2, HS729,
HT29, J82, MG63, MIAPACA2 (addition of horse serum), PANCl, PLCPRF5, RD, SAOS2,
SKLMS1, SKNAS, SNB75, T24, and TE671.
10093] MEM Earle's medium was used for CAC02, CALU6, HEK293, HELA, HT1 080,
IMR90, JEG3, J1MT1 , SKHEP1 , SKNSH, and U87MG.
[0094] Cells were grown in 5% C02 atmosphere in a HeraCell 150 incubator (Thermo
Scientific, Germany).
[0095] The following is a list of compounds used in the studies:
[0096) The stock solutions of Compound (1) and Compound (2b) were prepared in DMSO
(Sigma-Aldrich, Germany) as indicated in table above. Stock solutions were further aliquoted
and stored under argon at -20°C.
[0097] 10% w/v of trichloracetic acid, TCA (Sigma-Aldrich, Germany), was prepared in
distilled water. 0.08% wt v sulforhodamine B, SRB (Sigma-Aldrich, Germany) solution was
prepared in 1% acetic acid (Sigma-Aldrich). Tris base was purchased from Karl Roth
(Germany).
]0098] Cell growth and treatment were performed in 96-well microtitre plates
CELLSTAR®(Greiner Bio-One, Germany). Cells harvested from exponential phase cultures
by trypsinization were plated in 150 of media at optimal seeding densities. The optimal
seeding densities for each cell line were determined to ensure exponential growth for the
duration of the experiment. All cells growing without anticancer agents were sub-confluent
by the end of the treatment as determined by visual inspection.
[0099] Compound dilutions in DMSO were performed in 96-well rigid PCR plates.
Compounds were then diluted 1:250 in RPMI medium.
[00100] 150 ΐ of cells, after a 24-hour pre-growth period, were treated by mixing with 50 ΐ
of the compound containing media (resulting in a final DMSO concentration of 0.1%). The
cells were allowed to grow at 37°C for 72 hours. In addition, all experiments contained a few
plates with cells that were processed for measurement immediately after the 24 hours recovery
period. These plates contained information about the cell number that existed before
treatment, at time zero, and served to calculate the cytotoxicity.
[00101] After treatment, cells were precipitated by addition of 10% TCA. Prior to fixation,
the media was aspirated as described. After an hour of incubation at 4°C, the plates were
washed two times with 400 ΐ of deionized water. Cells were then stained with 100 ΐ of a
0.08% wt v SRB. The plates were allowed to sit for at least 30 min. and washed six times
with 1% acetic acid to remove unbound stain. The plates were left to dry at room temperature
and bound SRB was solubilized with 100 ΐ of 10 m Tris base. Measurement of optical
density was performed at 560 nm on a Victor 2 plate reader (Perkin Elmer, Germany). The
SRB values for A375 and H460 cell lines were near to saturation (2.5 OD units) due to the
high protein content of these cells, but not cell confluence. The measurements for these cells
were performed at 520 nm instead of 560 nm.
[00102] Prior to in vitro combination studies, the activity of individual agents was
investigated using a panel of 80 cell lines. The purpose of testing individual agents was to
determine the independence of their action. In addition, comparison to an activity profile of
known anticancer agents may help form a hypothesis regarding potential mechanisms of the
compounds' action.
(00103J The calculations used nomenclature introduced by DTP NCI. Unprocessed optical
density data from each microtitre plate were stored in MS Excel or as a text file in a databank.
The first step of data processing was calculating an average background value for each plate,
derived from wells containing medium without cells. The average background optical density
was then subtracted from the appropriate control values (containing cells without addition of a
drug), from values representing the cells treated with an anticancer agent, and from values of
wells containing cells at time zero. Thus the following values were obtained for each
experiment: control cell growth, C; cells in the presence of an anticancer agent Tj and cells
prior to compound treatment at time zero, Tz (or To, in some publications).
|00104] The Z -factor is a parameter commonly used to assess quality of the assay
performance and was calculated according to the following equation:
Z ' = 1 - ÷ + - )
where +and - are denoted for the means of positive and negative control signals and c+
and _ are their standard deviation. In a way, the Z '-factor reflects the significance of the
dynamic range of the measurements recorded and should be >0.5. In this study, Z '-factor
was applied to determine the significance of signals over background for Tz and C values.
The results of the screening were accepted only if the Z-factor was above 0.5 for each case.
[00105] The non-linear curve fitting calculations were performed using in-house developed
algorithms and visualization tools. The algorithms are similar to those previously described
and were complemented with the mean square error or MSE model. This can be compared to
commercial applications, e.g. XLfit (ID Business Solutions Ltd., Guild-ford, UK) algorithm
"205". The calculations included the dose response curves with the best approximation line, a
95% confidence interval for the 50% effect (see below).
[00106] A common way to express the effect of an anticancer agent is to measure cell
viability and survival in the presence of the test agent as % T/C 100. The relationship
between viability and dose is called a dose response curve. Two major values are used to
describe this relationship without needing to show the curve: the concentration of test agents
giving a % T/C value of 50%, or 50% growth inhibition (IC50), and a % T/C value of 10%, or
90% growth inhibition (IC90) .
[00107] Using these measurements, cellular responses can be calculated for incomplete
inhibition of cell growth (GI), complete inhibition of cell growth (T GI) and net loss of cells
(LC) due to compound activity. Growth inhibition of 50% (GI50) is calculated as 100 * [(Tj -
TZ)/(C - T )] = 50. This is the drug concentration causing a 50% reduction compared to the
net protein increase in control cells during the drug incubation period. In other words, GI50 is
IC50 corrected for time zero. Similar to IC90, calculated GI90 values are also reported for all
compounds tested. TGI was calculated from T, = Tz. LC50 is the concentration of drug
causing a 50% reduction in the measured protein at the end of the drug incubation period
compared to that at the beginning. It was calculated as 100 [(Tj - Tz)/Tz] = -50. However,
due to 72 hours treatment, low cell seeding density was required and LC50 could rarely be
achieved.
[00108j The IC50, IC90, GI50, GI90 and T GI values were computed automatically. Visual
analysis of all dose response curves was performed to check the quality of the fitting
algorithm. In cases where the effect was not reached or exceeded, the values were either
approximated or expressed as In this study all values were greater than the maximum drug
concentration tested. In these cases, the values were either excluded from the analysis, or
approximation of C o and GI10 were used for analysis.
[00109] All values were loglO-transformed for analysis. This transformation ensures better
data fitting to the normal distribution, a prerequisite to apply any statistical tool. Statistical
analyzes were performed using proprietary software developed at Oncolead integrated as a
database analysis tool. However, except for database comparison, the analysis can be
reproduced using either MS Excel or STATISTICA®(StatSoft, Hamburg). Using MS Excel:
identification of mean, e.g. mean GI50 (function: "Average"); calculation of ,, delta (GI50 -
mean GI50 ); and z-score (function "Standardize"). Comparison of the activity profile of
Compound (1) and Compound (2b) cross-correlation could be performed using Pearson and
Spearman correlations (for example by using STATISITCA(R)). In addition, Pearson
pairwise and Spearman pairwise comparisons were used to increase the confidence of the
results. Pairwise comparison was calculated based on pairwise similarity of the agents to all
tested agents in the database.
[00110] Z-score is a way to report standard deviations rather than absolute deltas and mean
values. t indicates how far the value deviated from its mean in units of standard deviation:
z - 6
where X is a single measured value, e.g. G o, and is a mean of all measured values (mean
GI50) and is a standard deviation of X.
[00111] The concept of the mean graph introduced by NCI permits visualization of a cell
activity parameter for a given anticancer drug in all cells. This graph yields a characteristic
pattern that provides rich information for visual comparison. The values are plotted as
horizontal bars from the mean values. Each bar, therefore, represents the relative activity of
the compound in the given cell lines deviating from the mean in all cell lines. In contrast to
NCI, z-score values were plotted rather than absolute delta. In statistical terms, z-values
represent a standard deviation that provides a kind of normalization and simplifies comparison
between compounds with different activity distributions. In addition, an averaged combined
z-score was calculated for cell lines of the same origin.
[00112] Z-score values as well as the range of tested concentrations were included in all
visualizations. The applicability of z-score graphs should be considered with precaution if the
agent's activity does not follow the normal distribution.
(00113] The most sensitive and non-sensitive cell lines were visualized by using either a boxplot
graph or by selecting the eight most and least sensitive cell lines using the z-score for
each agent. This also applied to the cell lines where activity of an agent could not be
determined. Box plots were constructed from five values: the smallest value (the lowest
whisker), the first quartile (the lowest border of the box), the median (square in the middle),
the third quartile (the upper border of the box), and the largest value (the highest whisker).
[00114] The screening was designed to determine potential synergistic combinations. All
and/or part of the 5x5 or 7x7 matrix were used to design the study. Bliss independence was
used as a basis for calculations, unless otherwise stated. The following parameters were
calculated:
5, - ea u e 'value,- —Theoretical v i
where i = [1..n] is one of the values of the matrix used and theoretical valuej calculated as
described for the Bliss Independence method. Vector sum was determined as:
Vector sum = Sign(Ef f ct )Ef f ct
= 1
in this term the Vector Sum rather represents scalar:
1 "
Vector sum average = — Effecti = Mean{ Effecti)
n . ,
[001 15] The average values below -0.5 indicate a strong synergy effect: (-0.5, -.02) -
Synergy effect, (-0.2, .02) -Zero effect (additivism), (0.02, 0.5) - potential antagonism, and
above 0.5 -strong antagonism. However, it is possible that the effect of the combination is not
synergistic (or even antagonistic) but still better than each of the agents alone. Moreover, in
vivo, any effect better than a single agent is considered clinically positive (or synergistic). In
this case, one considers a potential interaction of two agents that can be determined by the
highest single agent, HSA, model. This model determines the difference between the larger
effects produced by one of the single agents at the same concentrations as in the mixture.
Single Best; —Best of [Agent 1,•; agent 2,]
and delta HSAj for two agents can be determined as:
del HS.Ai = SAi - s red al ei—Single Best;
d
_, oHSAi
Average H Effect =
Summary of in vitro results
[001 16] Efficacy of Compound ( 1) varies broadly from 4-5 n in sensitive cell lines to
minimal activity at 50 in the most non-sensitive cell lines. Under the conditions tested,
minimal activity could be determined for cancer cell lines: A673, HE 293, J82, JAR, JEG3,
MDAMB436, MDAMB468, MHHES l , NCIH82, PANC l , PLCPRF5, and SF268. For cell
lines CLS439, EF02, 1 PC3, SAOS2, SF295, and S OV3, activity was estimated above the
highest tested concentration of 50 . At the same time, 50% of the cell lines tested
exhibited a sensitivity below 500 nM (the median is 490 nM), and 27 of 82 cell lines were
found to be sensitive below 100 nM of Compound (1). Action of Compound (1) and
Compound (2b) was synergistic in a larger number of human cancer cell lines, which suggests
that the mechanisms of compound action are complementary. A673 cells are non-sensitive to
the action of Compound (1) or Compound (2b) alone, but can show strong synergy in
combination. A549 and MCF7 cells show some sensitivity to both agents, which can be
further potentiated with their combination. SK.BR3 cell line is very sensitive to Compound
(2b). However, the effect can be further increased by the combination of both agents. These
findings may be related to the all breast cancer cell lines with overexpression of the HER2
gene.
[00117] The most sensitive cell lines were HT29, COLO205, TE671, A375; S MEL5,
COL0678, SKNAS, and NC1H292, where Compound (1) showed activity between 4.8 and 8
nM. The difference between the most and least sensitive cell lines was as large as 10,000-
fold. Due to such a large window of activity, the activity distribution is broad and does not
follow a normal distribution. In such a case, z-score has little statistical meaning; however, it
can still be applicable, for example, to group activities according to therapeutic indications.
[00118] The rank of Compound (1) activity (or rank of z-score values) is another tool that
can be applied. These properties of Compound (1) stress the necessity of using diverse
analysis tools and covering a broad concentration range to test anticancer agents. One
possibility is that Compound (1) has a specific mechanism of action and acts only on a subpopulation
of tumor cells.
[00119] The 8 human cancer cell lines represented 17 different tumor origins. Figures 15A
and 15B show individual z-scores within one tumor origin group, as well as combined zscores
for each therapeutic indication as an average value (green triangle). As in the case of
individual z-scores, direction to the left points towards sensitivity to the compound action. A
zeroline corresponds to average activity. The data suggest that lung, pancreas, colon, and
melanoma cell lines are generally more sensitive to Compound (1), since the average value of
z-scores are on the left. All but one pancreas (PANC1) cell line are very sensitive to
Compound (1) action. HT1 080 is also a very sensitive cell line. .
00120 Activity, GI50 values, of Compound (2b) in cell lines ranged between < 500 nM in
A204, 1MR90, MDAMB468, S BR3, CA I , and IGROV1 (most sensitive, as determined
by z-score < -1.5) and > 4 in SW620, COL0678, and HCT1 16 (non-sensitive cell lines, z
score > 1.5). These results may indicate that cell lines showing the strongest negative
deviation of z-scores from the mean will also show activity in other biological systems, e.g.,
mouse xenograft models. The average Glso value in all 8 1 cell lines was 1.3-1 .4 ,
calculated based on log 10-transformed data. No activity was shown in arrested PBMC
suggesting that Compound (2b) may act preferably on proliferating cells. Figures 16A and
16B show that the activity distribution is narrow, but sensitive cell lines can be welldiscriminated.
[00121] Comparison of the Compound (2b) activity profile with an internal databank
containing more than 300 different anticancer agents identified a number of agents. The most
similar agent (average similarity above 0.8) is MSC2208382A. Weaker similarity (above 0.7)
is detected with GDC-0941 bismesylate and ZSTK474, and some degree of similarity to
MSC2313080A. GDC-0941 bismesylate is an analog of PI-103, a dual PI3K/mTOR inhibitor
and considered to be a relatively specific inhibitor of class I PI3 enzymes as well as
ZSTK474. It could be suggested that Compound (2b) belongs to the class of PI3 inhibitors.
[00122] As in the case of individual z-scores, the direction to the left points towards
sensitivity to the compound action. A zero-line corresponds to average activity. Ovarian and
prostate tumors could be specific therapeutic areas. At least for all cell lines tested, the z-score
is below zero. Applications for breast, lung, and renal tumors also could be considered.
However, each of the indications contains cell lines either very sensitive or non-sensitive to
Compound (2b) action.
[00123] Although most of the cell lines showed potential synergy for in vitro combination of
Compound (1) and Compound (2b), the results with a vector sum of below - 1 can be
considered significant. Table 1 and Figure 17 summarize the results. Cell line A673 is nonsensitive
to the action of Compound (1) or Compound (2b) alone, but shows strong synergy in
combination. However, from in vivo or clinical perspectives, cell line groups four and five are
probably more relevant. Activity (GI50) of Compound (1) is 300 nM and 150 nM in A549 and
MCF7 cells, respectively, which is comparable with 100 nM activity in the most sensitive cell
lines. Activity (GI50) of Compound (2b) is 1.15 and 1.6 in A549 and MCF7 cells,
respectively, below or close to the average activity of 1.3-1.4 for this agent. The
combination index for these cell lines is close to - , which is indicative of synergy. Another
example is S BR3. This cell line is very sensitive to Compound (2b) and non-sensitive to
Compound (1). However, the effect can be further increased by the combination of both
agents.
[00124] Compound (1) and Compound (2b) act on proliferating cells and showed no activity
in resting PBMC. However, these agents differ in their activity. The difference between the
most and least sensitive cell lines for Compound (1) was as large as 10,000-fold. For the most
insensitive cell lines, resistance extends beyond the tested concentration range > 50.
[00125] Thus, it appears that Compound (1) may have a specific mechanism of action and
acts only on a sub-population of tumor cells. Selection of therapeutic indications in the clinic
can be complemented by the mutational analysis. In contrast, Compound (2b) shows narrow
activity in cell lines. The separation between sensitive and insensitive cell lines is statistically
significant but the differences in activity are in the range of 10-20-fold. The activity profile of
Compound (2b) has similarities to the PI3 inhibitors, e.g. PI-103 or its pharmalog GDC-
0941 . No prediction could be made about the agent's activity and the mutational status of
genes involved in activation of the PI3K pathway, e.g. EGFR, PTEN, and PI3 . Some
markers may be predictive for induction of apoptosis upon action of this PI3 inhibitor:
EGFR (mutation), HER2 (amplification), MET (mutation/amplification). Indirectly, this fact
can be supported by the observation that S BR3 cells (HER2 amplification) were among the
most sensitive cell lines.
[00126] Compound (1) and Compound (2b) were further tested in combination in all cell
lines using a 7x7 matrix, with variation around GI50 averaged in al cell lines for each of the
agents. The rationale for selecting this concentration was as follows. First, this concentration
is a reference concentration that describes efficacy of the anticancer agents in cellular models,
i.e. only cell lines that show significant effects below mean G o. Second, it is known that
efficacy of anticancer agents is limited, based on citations reporting 10-30%. Therefore,
selection of mean GI50 would correspond to the expected efficacy of approximately 50%.
Third, the variation spanned by the 7 x 7 matrix (almost ten-fold in both directions from the
mean GI50) allows enough coverage to address the question of whether there are any potential
interactions between the two agents.
[00127] In almost all cases, Compound (1) and Compound (2b) in combination showed
potential to be synergistic (Figure 17), as determined by the Bliss Independence model (see,
for example, Yan et al., BMC Systems Biology, 4:50 (2010)). See also Figures 18A, 18B,
18C, 18D, 18E, 18F, 19A, 19B, 20A, 20B.
[00128] However, the strongest synergistic effect was detected when the activity of either
agent was weak. This may be attributed, at least in part, to experimental set-up, i.e., any effect
of combination is considered significant if the agents alone mediate little, if any effect on the
cells. Alternatively, the effect of a single agent can be too strong to detect increasing effects.
In the later case, the HSA model provides a better view of the potential interaction between
two agents.
Example 2 . In vivo activity of Compound (1) in combination with Compound (2b) or
Compound (2a) against subcutaneous human colon carcinoma HCT 16 bearing SCID mice
[00129] To evaluate the antitumor activity of the ME inhibitor Compound (1) in
combination with the pan-PI3K inhibitor Compound (2a) or the dual pan-PI3K / mTOR
inhibitor Compound (2b), experiments were conducted using female SCID mice bearing
human colon carcinoma HCT 116 ( RAS and PIK.3CA mutant) xenografts. Four studies
were performed:
[00130] In a first study, a low dose of Compound (1) at 5 mg kg was tested in combination
with Compound (2b) at 30 mg/kg and Compound (2a) at 50 and 75 mg/kg.
[00131] In a second study, the dose of Compound (1) was increased to 10 and 20 mg/kg in
combination with Compound (2b) at 20 mg/kg, and Compound (1) at 10 mg/kg was combined
with Compound (2a) at 50 and 75 mg/kg.
[00132] In a third study, used as a confirmation study, the dose of Compound (1) was used at
10 and 20 mg/kg in combination with Compound (2a) at 50 and 75 mg/kg.
[00133] In a fourth study, used as a confirmation stiidy, the dose of Compound (1) was used
at 10 and 20 mg/kg in combination with Compound (2b) at 20 mg/kg.
Materials and methods
[00134] CB 7/lCR-Prkdc severe combined immunodeficiency (SCID) /Crl mice, at 8- 10
weeks old, were bred at Charles River France (Domaine des Oncins, 69210 L'Arbresle,
France) from strains obtained from Charles River, USA. Mice were over 18 g at start of
treatment after an acclimatization time of at least 5 days. The mice had free access to food
(UAR reference 113, Villemoisson, 9 1160 Epinay sur Orge, France) and sterile water. The
mice were housed on a 12 hours light/dark cycle. Environmental conditions including animal
maintenance, room temperature (22°C ± 2°C), relative humidity (55% ± 15%) and lighting
times were recorded by the supervisor of laboratory animal sciences and welfare (LASW) and
archived. j
[00135] Human colon carcinoma HCT 116 cells were purchased at American^Type Culture
Collection [(ATCC), Rockville, MD, USA). The HCT 1 6 cells were cultured in Dulbecco's
modified Eagle's medium (DMEM) (Invitrogen). The tumor model was established by
implanting (SC) 3x1 06 cells mixed with 50% matrigel (Reference 356234, Becton Dickinson
Biosciences) per SCID female mice.
[00136] Compound (1) formulation was prepared by incorporating the ME inhibitor into
0.5% CMC 0.25% Tween 20. The preparation was stored at 4°C and resuspended by
vortexing before use. The oral form of the compound was prepared every 3 days. The volume
of administration per mouse was 0 mL/kg.
[00137] Compound (2a) formulation was prepared in water for injection. The stock solution
was chemically stable 7 days in the dark at 4°C. The volume of administration per mouse was
10 mL/kg.
[00138] Compound (2b) formulation was prepared in IN HC1 and water for injection
followed by five cycles of vortexing and sonicating. The pH of the final solution was 3. The
stock solution was chemically stable 7 days in the dark at 4°C. The volume of PO
administration per mouse was 10 mL/kg.
[00139] For subcutaneous implantation of tumor cells, skin in the flank of the mice was
disinfected using alcohol or Betadine® solution (Alcyon) and a suspension of tumor cells was
inoculated SC unilaterally under a volume of 0.2 mL using a 23 G needle.
[00140] The activity on tumor growth of Compound (1), Compound (2a) and Compound (2b)
used as single agent or in combination was evaluated in four different studies. The dosages
and schedule of administration for each study are described in the results section and detailed
in the tables that follow.
[00141 ] The animals required to begin a given experiment were pooled and implanted
monolaterally on day 0. Treatments were administered on measurable tumors. The solid
tumors were allowed to grow to the desired volume range (animals with tumors not in the
desired range were excluded). The mice were then pooled and unselectively distributed to the
various treatment and control groups. Treatment started days post HCT 16 tumor cell
implantation as indicated in the results section and in each table. The dosages are expressed in
mg/kg, based on the body weight at start of therapy. Mice were checked daily, and adverse
clinical reactions noted. Each group of mice was weighed as a whole daily until the weight
nadir was reached. Then, groups were weighed once to thrice weekly until the end of the
experiment. Tumors were measured with a caliper 2 to 3 times weekly until final sacrifice for
sampling time, tumor reached 2000 mm3 or until the animal died (whichever comes first).
Solid tumor volumes were estimated from two-dimensional tumor measurements and
calculated according to the following equation:
(00142) Tumor weight (mg) = Length (mm) x Width2 (mm )/2
[00143] The day of death was recorded. Surviving animals were sacrificed and macroscopic
examination of the thoracic and abdominal cavities was performed.
[00144] A dosage producing a 15% body weight loss (BWL) during three consecutive days
(mean of group), 20% BWL during 1 day or 0% or more drug deaths was considered an
excessively toxic dosage. Animal body weights included the tumor weight.
[00145] The primary efficacy end points are AT/AC, percent median regression, partial and
complete regressions (PR and CR).
100146] Changes in tumor volume for each treated (T) and control (C) group were calculated
for each tumor by subtracting the tumor volume on the day of first treatment (staging day)
from the tumor volume on the specified observation day. The median AT is calculated for the
treated group, and the median AC is calculated for the control group. Then the ratio AT/AC is
calculated and expressed as a percentage. The dose is considered as therapeutically active
when AT/AC is lower than 40% and very active when AT/AC is lower than 10%. If AT/AC is
equal to or lower than 0, the dose is considered as highly active and the percentage of
regression is dated.
[00147] The percent of tumor regression is defined as the % of tumor volume decrease in the
treated group at a specified observation day compared to its volume on the first day of
treatment. At a specific time point and for each animal, % regression is calculated. The
median % regression is then calculated for the group using the following equation:
[00148] % regression (at t) = (volume at to - volume at t)/volume at to) x 100
[00149] Partial regression: Regressions are defined as partial if the tumor volume decreases
to 50 % of the tumor volume at the start of treatment. ,
[00150] Complete regression: The CR is achieved when tumor volume = 0 mm3 (CR is
considered when tumor volume cannot be recorded).
[00151] The term "therapeutic synergy" is used when the combination of two products at
given doses is more efficacious than the best of the two products alone considering the same
doses. n order to study therapeutic synergy, each combination was compared to the best
single agent using estimates obtained from a two-way analysis of variance with repeated
measurements (Time factor) on parameter tumor volume.
[00152] Statistical analyses were performed on SAS system release 8.2 for SUN4 via
Everstat V5 software and SAS 9.2 software. A probability less than 5% (p<0.05) was
considered as significant.
Results of in vivo studies
First study: antitumor activity of Compound ( 1) (5 mg/kg) in combination with Compound
(2b) (30 mg/kg) or Compound (2a) (50 and 75 mg/kg) against HCT 16 bearing SCID mice
[00153] The median tumor burden at start of therapy was 198 to 22 1mm3. As single agents,
Compound ( 1) (5 mg/kg/administration (Adm)), Compound (2b) (30 mg kg adm) and
Compound (2a) (50 and 75 mg/kg/adm) were administered PO daily from days 1to 18 post
tumor implantation. n the combination groups, the dose of Compound ( 1) was combined
with each dose of Compound (2a) and Compound (2b), as shown in Table 2.
[00154] As single agents or used in combination, Compound (1) and Compound (2a) were
well-tolerated, inducing minimal BWL (Figure 1 and Table 2). As single agents, Compound
(1), Compound (2a) and Compound (2b) achieved a AT/AC>40%) under these test
conditions.
[00155] In combination, treatment with Compound (1) at 5 mg/kg/adm and Compound (2b)
at 30 mg/kg/adm achieved a AT/AC of 27 % (Figure 2 and Table 1), but as shown by Table 3,
therapeutic synergy was not reached (p = 0.0606 for global analysis). Treatment with
Compound (1) at 5 mg/kg/adm and Compound (2a) at 50 and 75 mg/kg/adm achieved a
AT/AC of 22 % and 2 %, respectively (Figure 3 and Table 2). As shown by Table 2,
therapeutic synergy was achieved for both combinations (p=0.0091 and p<0.0001 globally,
respectively). See also Tables A and 1IB.
[00156] Second study: antitumor activity of Compound (1) (10 and 20 mg/kg) in
combination with Compound (2b) (20 mg/kg) and Compound ( (10 mg/kg) in combination
with Compound (2a) (50 and 75 mg/kg) against HCT 6 bearing SOD mice
[00157] The median tumor burden at start of therapy was 80 to 198 mm3. As single agents,
Compound (1) (10 and 20 mg/kg/adm), Compound (2b) (20 mg/kg/adm) and Compound (2a)
(50 and 75 mg/kg/adm) were administered PO daily from days 1 to 18 post tumor
implantation. In the combination groups, the dose of Compound ( 1) was combined with each
dose of Compound (2a) and Compound (2b), as shown in Table 3.
(00158] As single agents, Compound ( 1), Compound (2a) and Compound (2b) were welltolerated,
inducing minimal BWL (Figure 4 and Table 4).
[00159] As single agents, Compound (1) (10 and 20 mg/kg/adm) achieved a AT/ACof 20 %
and 22 %, respectively, while Compound (2b) at 20 mg/kg/adm achieved a AT/AC>40 %. As
shown in Table 4, Compound (2a) at both doses tested achieved a AT/AC>40%.
[00160] In combination, treatment with Compound (1) at 10 or 20 mg/kg/adm and
Compound (2b) at 20 mg/kg/adm achieved a AT/AC of 0, and therapeutic synergy was
reached with Compound (1) at 10 mg/kg/adm (p=0.0004 globally). As shown by Table 5,
therapeutic synergy was not reached with Compound (1) at 20 mg/kg/adm (p=0.2169
globally). Partial regression (PR) was observed in 2/7 mice for the combination treatment of
Compound (1) at 0 mg/kg/adm and Compound (2b) at 20 mg/kg/adm (Figure 5 and Table 4).
When Compound (1) was used at 0 mg kg adm, the combinations with Compound (2a) at 75
and 50 mg/kg/adm achieved, respectively a AT/AC of 5 % and AT/AC<0, with 1/7 PR
occurring for both combination treatments (Figure 6 and Table 4). As shown by Table 5, both
combinations (p=0.0063 and p=0.0019 globally, respectively) achieved therapeutic synergy.
In all combination groups, tumor stasis was achieved (Figure 5 and Figure 6). See also Tables
12A and 12B below.
[00161] Third study: antitumor activity of Compound (1) (10 and 20 mg/kg) in combination
with Compound (2a) (50 and 75 mg/kg) against HCT 6 bearing SCID mice
[00162] The median tumor burden at start of therapy was 87 to 189 mm3. As single agents,
Compound (1) (10 and 20 mg/kg/adm) and Compound (2a) (50 and 75 mg/kg/adm) were
administered PO daily from days 11to 20 post tumor implantation. In the combination
groups, the dose of Compound (1) was combined with each dose of Compound (2a), as shown
in Table 6.
[00163] As single agents, Compound (1) and Compound (2a) were well-tolerated, inducing
minimal BWL (Figure 7 and Table 6).
[00164] As a single agent, Compound (1) achieved a AT/AC of 34 % at a dose of 20
mg/kg/adm and AT/AC>40% at a dose of 10 mg/kg/adm (Figure 7). As shown in Table 6,
Compound (2a) at both doses tested achieved a AT/AC>40%.
[00165] In the combination, treatment with Compound (1) at 10 or 20 mg/kg/adm and
Compound (2a) at 75 mg/kg/adm achieved AT/ACof 8 % and 9 %, respectively) (Figure 10
and Table 6), and therapeutic synergy was reached (p=0.0109 and p=0.0003 globally,
respectively) (Table 6). The treatment with Compound (1) at 10 or 20 mg/kg/adm and
Compound (2a) at 50 mg/kg/adm achieved AT/ACof 19 % and 22 %, respectively) (Figure 10
and Table 6). Therapeutic synergy was reached only for the combination with Compound (1)
at 10 mg/kg (p=0.0088 globally) (Table 7). As shown by Table 7, therapeutic synergy was not
reached with Compound (1) at 20 mg/kg/adm (p=0.0764 globally). In all combination groups,
tumor stasis was achieved (Figure 8). See also Table 13 below.
[00166] Fourth study: antitumor activity of Compound (1) (10 and 20 mg/kg) in combination
with Compound (2b) (20 mg/kg) against HCT 1 6 bearing SCID mice
[00167] The median tumor burden at start of therapy was 189 to 6 mm3. As single agents,
Compound (1) (10 and 20 mg/kg/adm) and Compound (2b) (20 mg/kg/adm) were
administered PO daily from days 11 to 20 post tumor implantation. In the combination
groups, the dose of Compound (2b) was combined with each dose of Compound (1), as shown
in Table 8.
[00168] As single agents, Compound (l) ' and Compound (2b) were well-tolerated, inducing
minimal BWL (Figure 9 and Table 8).
[00169] As single agents, Compound (1) (10 and 20 mg/kg/adm) and Compound (2b) at 20
mg/kg achieved a /40 % (Figure 10 and Table 8).
[00170] n the combination, the treatment with Compound (1) at 10 or 20 mg/kg/adm and
Compound (2b) at 20 mg/kg/adm achived a AT/AC of 30 % and 15 %, respectively (Figure 10
and Table 8), and therapeutic synergy was reached (p=0.0002 and p=0.0008 globally,
respectively) (Table 9). See also Table 4 below.
Example 3. In vivo activity of Compound (1) in combination with Compound (2a) or
Compound (2b) against subcutaneous human pancreatic MiaPaCa-2 bearing nude mice
[00171] To evaluate the antitumor activity of the ME inhibitor Compound (1) (5 mg/kg) in
combination with the pan-PI3K inhibitor Compound (2a) (50 mg/kg) or the dual pan-P13K /
mTOR inhibitor Compound (2b) (30 mg/kg), experiments were conducted using female nude
mice bearing human pancreatic MiaPaCa-2 (KRAS mutant) xenografts.
[00172] A low dose of Compound (1) at 5 mg/kg was tested in combination with Compound
(2b) at 30 mg/kg and Compound (2a) at 50 mg/kg.
Materials and methods
[00173] The human pancreatic cancer cell line MiaPaCa-2 (American Type Culture
Collection, Manassas VA), was cultured in MEM medium containing 10% fetal bovine serum,
1% essential amino acid, 1% sodium pyruvate (Life Technologies, Carlsbad, CA). Cells were
trypsonized during the log phase of growth at 60-85% confluence, collected and washed once
with PBS. Cells were re-suspended in PBS (Life Technologies, Carlsbad, CA) and then mixed
1: 1 with Matrigel (BD Biosciences, San Jose, CA). Cells were stored at 4°C until
implantation.
[00174] MiaPaCa-2 cells (lOxlO6 in a 2001PBS:Matrigel ( :1) suspension) were
subcutaneously injected into the right flank area of female nude (Crl:NU-Foxnlnu) mice (6-8
weeks old, Charles River Laboratories, Wilmington, MA). All mice in this study were used
according to the guidelines approved by the EMD-Serono Institutional Care and Animal Use
Committee (1ACUC), #07-003.
[00175] A solution of 0.5% CMC (carboxymethylcellulose; Sigma-Aldrich, St. Louis, MO)
and 0.25% Tween 20 (Acros Organics, Morris Plains, NJ) in water was used as the vehicle for
this study. Compound (1) (Lot #27) was prepared by suspending 10 mg of compound in 20
mL of 0.5% CMC 0.25% Tween 20 in water to make a 0.5 mg/mL (5.0 mg/kg) dosing
solution.
[00176] Compound (2a) was weighed (5 mg for 1mL of solution) and water added for
injection (60% of final volume i.e. 0.60 ml). Solution was mixed via five cycles of vortexing
and sonicating in a sonicating water bath for 1 min each. Completed with water for dosing.
Compound (2b) was weighed (3 mg for 1mL of solution), 10 L• HCl IN was added and then
water was added for injection (60% of final volume i.e. 0.60 ml). Solution was mixed via five
cycles of vortexing and sonicating in a sonicating water bath for 1 min each. I NaOH was
added to adjust the pH up to 3 and finally completed with water for injection.
[00177J Developing tumors located in the right flank area of female nude mice were
measured over time with digital calipers. Seven days after cell implantation, the tumors had
reached an average volume of 165 mm3 in an ample number of mice to begin the study. Mice
bearing a tumor that was significantly different from the average tumor volume were excluded
from the study. The remaining tumor-bearing mice were randomized into seven experimental
groups (n=9), so that each group had the same mean tumor volume.
[00178] In all combination groups, both agents were administered to the animals at the same
time, within approximately 5-10 minutes of each other. The treatments began on the seventh
day following implantation of the Miapaca-2 cells, which was designated as Day 0 for data
evaluation purposes. Animals underwent 2 1 days of treatment. Body weights and tumor
volumes were assessed twice per week post treatment initiation. On Day 22, all animals were
euthanized via progressive hypoxia with C0 2.
[00179] Efficacy was determined by analyzing tumor volumes and the percent /
(%/). Tumor volume was determined by using the tumor length (1) and width (w)-
measurements and calculating the volume with the equation l*w /2. The length was measured
along the longest axis of the tumor and width was measured perpendicular to that length. The
mean percent of actual tumor growth inhibited by the treatments was calculated as follows:
[%AVAC= ( (TV - TVj/TVfctn- TViQri)) x 100%], where TV=tumor volume, /=final, /^initial
and Ctrl=control group. Tolerability was assessed by regarding percent body weight
difference during the treatment period. Percent body weight difference was calculated as
follows: [%Body weight difference = (BW -BW,) / BW, x 100%], where BW = body weight,
c = current, = initial.
[00180] Tumor volume data and percent body weight differences were analyzed by Repeated
Measures Analysis of Variance (RM-ANOVA) followed by Tukey's post-hoc multiple pairwise
comparisons (a = 0.05).
Results of in vivo studies
[00181] No groups experienced more than 5% body weight loss during the study. No clinical
signs were noted (Figure 1) for the combination with Compound (2a) or (Figure 12) for the
combination with Compound (2b).
[00182] As single agents, Compound (1) (5 mg/kg/adm), Compound (2a) (50 mg/kg) and
Compound (2b) (30 mg/kg) achieved />40 % in these assays (Figure 1 and 14 and
Table 10).
[00183] In combination, treatment with Compound (1) at 5 mg/kg/adm and Compound (2b)
at 30 mg/kg/adm achieved AT/AC = 27.3 % (Figure 1 and Table 0), and therapeutic synergy
was reached (p<0.05) (Table 10). In contrast, the treatment with Compound (1) at 5
mg/kg/adm and Compound (2a) at 50 mg/kg/adm achieved /^40 % (Figure 13 and
Table 10), and therapeutic synergy was not reached (p>0.05) (Table 10).
Summary of in vivo results
[00184] The in vivo work presented here reports the in vivo antitumor activity of combining
Compound (1), an oral potent and selective allosteric inhibitor of MEK1/2, with oral, potent,
and specific inhibitors of class I PI3 lipid kinases Compound (2a), a pan-PI3K inhibitor, and
Compound (2b), a dual pan-PI3K and mTOR inhibitor. This work has been performed against
human colon carcinoma HCT 116 xenografts harboring a G13D activating mutation of KRAS
and an activating mutation of PIKC3A known to reduce the sensitivity to MEK inhibition and
against human pancreatic MiaPaCa-2 xenografts harboring a KRAS mutation.
[00185] In the studies described above, combination treatment was highly effective in
inducing a sustained tumor stasis during the treatment phase and realizing therapeutic synergy.
[00186] In conclusion, a potent antitumor activity with therapeutic synergy has been achieved
in PIKC3A and KRAS mutant HCT 16 driven xenograft model when combining the inhibitor
of MEK1/2 Compound (1) with Compound (2a), a pan-PI3K inhibitor, and in both PIKC3A
and KRAS mutant HCT 16 driven xenograft model and KRAS mutant MiaPaCa-2 driven
xenograft model, when combining Compound (1) with Compound (2b), a dual pan-PI3K and
mTOR inhibitor.
Example 4. Fluorescence molecular tomography study of combination of Compound (1) with
Compound (2b) or Compound (2b) against subcutaneous human colon carcinoma HCT 1 6
bearing SCID mice
[00187] To evaluate the apoptotic activity of the MEK inhibitor Compound (1) in
combination with the pan-PI3K inhibitor Compound (2a) or the dual pan-PI3K / mTOR
inhibitor Compound (2b), experiments were conducted using female SCID mice bearing
human colon carcinoma HCT 116 (KRAS and PIK3CA mutant) xenografts in which apoptosis
induction was monitored non-invasively using fluoresence molecular tomography (FMT).
Methods
[00188] HCT1 16 tumor cells were implanted subcutaneously in the intra-scapular region in
SCID mice. Implanted animals received 50 mg/kg Compound (2a) or 20 mg/kg Compound
(2b) from day 1 to day 17, as single agents or combined with lOmg/kg Compound (1). Each
agent was given by oral route on a daily schedule. Tumor growth was monitored throughout
the experiment by callipering the tumors. To quantify apoptosis, fluorescent Annexin-Vivo-
750 was injected intravenously one hour post daily treatment on days three and seven after
start of treatment. Animals were imaged by FMT three hours post probe injection to
document fluorescent Annexin uptake in the tumor. Ex vivo apoptosis was assessed on tumor
lysates using Meso Scale Discovery assays for cleaved caspase-3 and cleaved-PARP
detection.
Results
[00189] Under these regimens, Compound (1), Compound (2a) and Compound (2b) used as
single agents showed marginal activity on HCT1 16 tumor growth with AT/AC = 40% (NS),
36% (p= 0.023) and 80% (NS) respectively at the end of study (Figure 28). Conversely, both
Compound (2a) and Compound (2b) in combination with Compound ( 1) induced strong tumor
growth inhibition (AT/AC <0, associated with 23% median tumor regression (pO.000 1) for
Compound (2a)/ Compound ( 1) and (AT/AC <0 with 5% median tumor regression (p= 0.0009)
for Compound (2b)/ Compound ( 1)). Both combination therapies were associated with a clear
enhancement of ex vivo cleaved caspase-3 (3.7 & 5.2 fold) (Figure 27B) and cleaved-PARP
(8.4 & 12.8 fold) (Figure 27A) after four days treatment. Compound (2a)/ Compound ( 1)
combination therapy was associated with a significant enhancement of Annexin-V-750 uptake
in the tumor, reflecting apoptosis induction after three and seven days of combined therapy
(p=0.005 and <0.000 1) (Figure 26B). The ratios of Annexin fluorescence in treated animal
groups relative to control were respectively 2.1 after 3 days and 3.8 after 7 days of
combination therapy (Figure 26A).
Summary
[00190] The combination of the E 1/2 inhibitor Compound ( 1) with the Pan-P13
inhibitor Compound (2a) or the Pan-PI3K/mTOR Compound (2b) resulted in significantly
enhanced anti-tumor activity in a dual RAS P1 3CA mutated tumor xenograft model, with
synergistic induction of tumor apoptosis as demonstrated ex vivo for both combinations and in
vivo using longitudinal FMT imaging for the Compound (2a)/ Compound ( 1) combination.
Example 5. In vivo activity of Compound ( 1) in combination with Compound (2b) or
Compound (2a) against subcutaneous human colon tumors CR-LRB-009C bearing SCID
female mice
[00191 ] To evaluate the antitumor activity of the MEK inhibitor Compound ( 1) in
combination with the pan-P13 inhibitor Compound (2a) or the dual pan-P13K / mTOR
inhibitor Compound (2b), experiments were conducted using female SCID mice bearing
human primary colon tumors CR-LRB-009C ( RAS and PI 3CA mutant) xenografts. In this
study, Compound (1) at 20 mg/kg was tested in combination with Compound (2b) at 20 mg kg
and Compound (2a) at 75 mg/kg.
Materials and methods
[00192] CB17/1CR-Prkdc severe combined immunodeficiency (SCID) /Crl mice, at 8-10
weeks old, were bred at Charles River France (Domaine des Oncins, 69210 L'Arbresle,
France) from strains obtained from Charles River, USA. Mice were over 18 g at start of
treatment after an acclimatization time of at least 5 days. The mice had free access to food
(UAR reference 113, Villemoisson, 9 1160 Epinay sur Orge, France) and sterile water. The
mice were housed on a 12 hours light/dark cycle. Environmental conditions including animal
maintenance, room temperature (22°C ± 2°C), relative humidity (55% ± 15%) and lighting
times were recorded by the supervisor of laboratory animal sciences and welfare (LASW) and
archived.
[00193] The human primary colon carcinoma CR-LRB-009C tumor model was established
by implanting (SC) small tumor fragments and was maintained in SCID female mice using
serial passages.
[00194] Compound (1) formulation was prepared by incorporating the ME inhibitor into
0.5% CMC 0.25% Tween 20. The preparation was stored at 4°C and resuspended by
vortexing before use. The oral form of the compound was prepared every 3 days. The volume
of administration per mouse was 10 mL/kg.
[00195] Compound (2a) formulation was prepared in water for injection. The stock solution
was chemically stable 7 days in the dark at 4°C. The volume of administration per mouse was
10 mL/kg.
[00196] Compound (2a) and Compound (2b) formulations were prepared in IN HC1 and
water for injection, Final pH was 3, followed by five cycles of vortexing and sonicating. The
stock solution was chemically stable 7 days in the dark at 4°C. The volume of PO
administration per mouse was 10 mL/kg.
[00197] For subcutaneous implantation of tumor cells, skin in the flank of the mice was
disinfected using alcohol or Betadine® solution (Alcyon) and a suspension of tumor cells was
inoculated SC unilaterally under a volume of 0.2 mL using a 23 G needle.
100198) The dosages and schedule of administration of Compound (1), Compound (2a) and
Compound (2b) used as single agent or in combination are described in the results section and
detailed in Tables 15-17.
[00199] The animals required to begin a given experiment were pooled and implanted
monolaterally on day 0. Treatments were administered on measurable tumors. The solid
tumors were allowed to grow to the desired volume range (animals with tumors not in the
desired range were excluded). The mice were then pooled and unselectively, distributed to the
various treatment and control groups. Treatment started 11 days post CR-LRB-009C tumor
fragment implantation as indicated in the results section and in each table. The dosages are
expressed in mg/kg, based on the body weight at start of therapy. Mice were checked daily,
and adverse clinical reactions noted. Each group of mice was weighed as a whole daily until
the weight nadir was reached. Then, groups were weighed once to thrice weekly until the end
of the experiment. Tumors were measured with a caliper 2 to 3 times weekly until final
sacrifice for sampling time, tumor reached 2000 mm'or until the animal died (whichever
comes first). Solid tumor volumes were estimated from two-dimensional tumor measurements
and calculated according to the following equation:
[00200] Tumor weight (mg) = Length (mm) x Width 2 (mm )/2
[00201) The day of death was recorded. Surviving animals were sacrificed and macroscopic
examination of the thoracic and abdominal cavities was performed.
[00202] A dosage producing a 5% body weight loss (BWL) during three consecutive days
(mean of group), 20% BWL during 1 day or 10% or more drug deaths was considered an
excessively toxic dosage. Animal body weights included the tumor weight.
[00203] The primary efficacy end points are /AC, percent median regression, partial and
complete regressions (PR and CR). Statistical analyses were performed on SAS system
release 8.2 for SLTN4 via Everstat V5 software and SAS 9.2 software. A probability less than
5% (p<0.05) was considered as significant.
Results of in vivo studies
[00204] The median tumor burden at start of therapy was 126 to 4 mm3. As single agents,
Compound ( 1) (20 mg/kg/administration (Adm)), Compound (2b) (20 mg/kg/adm) and
Compound (2a) (75 mg kg adm) were administered PO daily from days 11 to 21 post tumor
implantation. In the combination groups, the dose of Compound (1) was combined with each
dose of Compound (2a) and Compound (2b), as shown in Table 15.
[00205J As single agents or used in combination, Compound ( 1), Compound (2b) and
Compound (2a) were tolerated, inducing some BWL but not reaching toxicity (Figure 21 and
Table 15). As single agents, Compound ( 1) and Compound (2b) achieved a AT/AC>40 %,
while Compound (2a) achieved a /AC of 39 % under these test conditions.
[00206] In the combination, the treatment with Compound ( 1) at 20 mg/kg/adm and
Compound (2b) at 20 mg/kg/adm achieved a /AC of 4 % (Figure 22 and Table 15), and as
shown by Table 16, therapeutic synergy was reached (p < 0.0001 for global analysis). The
treatment with Compound ( 1) at 20 mg/kg/adm and Compound (2a) at 75 mg/kg/adm
achieved a /AC of 21% (Figure 22 and Table 15), and as shown by Table 16, therapeutic
synergy was achieved (p=0.0386 globally). See also Table 17.
Summary of in vivo results
[00207] The in vivo work presented here reports the in vivo antitumor activity of combining
Compound ( 1), an oral potent and selective allosteric inhibitor of ME 1/2, with oral, potent,
and specific inhibitors of class I PI3 lipid kinases Compound (2a), a pan-PI3 inhibitor, and
Compound (2b), a dual pan-PI3K and mTOR inhibitor. This work has been performed against
human primary colon carcinoma CR-LRB-009C xenografts harboring a dual KRAS and
PIK.C3A mutation known to reduce the sensitivity to MEK inhibition.
[00208] In the study, combination treatment induced a sustained tumor stasis during the
treatment phase and reached therapeutic synergy.
[00209] Accordingly, a potent antitumor activity with therapeutic synergy has been achieved
in a PI C3A- and KRAS-mutant CR-LRB-009C driven xenograft model when combining the
inhibitor of MEK1/2 Compound ( 1) with Compound (2a), a pan-PI3K inhibitor or Compound
(2b), a dual pan-PI3K and mTOR inhibitor.
Example 6. In vivo activity of Compound ( 1) in combination with Compound (2a) or
Compound (2b) against subcutaneous human colon tumors CR-LRB-0 13P bearing SCID
female mice
[00210] To evaluate the antitumor activity of the ME inhibitor Compound ( 1) in
combination with the pan-PI3 inhibitor Compound (2a) or the dual pan-PI3K / mTOR
inhibitor Compound (2b), experiments were conducted using female SCID mice bearing
human primary colon tumors CR-LRB-0 13P (KRAS mutant) xenografts. In this study,
Compound (1) at 20 mg kg was tested in combination with Compound (2b) at 20 mg/kg or
Compound (2a) at 75 mg/kg.
Materials and methods
[0021 1] CB17/1CR-Prkdc severe combined immunodeficiency (SCID) /Crl mice, at 8- 10
weeks old, were bred at Charles River France (Domaine des Oncins, 692 10 L'Arbresle,
France) from strains obtained from Charles River, USA. Mice were over 18 g at start of
treatment after an acclimatization time of at least 5 days. The mice had free access to food
(UAR reference 11 , Villemoisson, 9 160 Epinay sur Orge, France) and sterile water. The
mice were housed on a 12 hours light/dark cycle. Environmental conditions including animal
maintenance, room temperature (22°C ± 2°C), relative humidity (55% ± 15%) and lighting
times were recorded by the supervisor of laboratory animal sciences and welfare (LASW) and
archived.
[00212] The human primary colon carcinoma CR-LRB-0 3P tumor model was established
by implanting (SC) small tumor fragments and was maintained in SCID female mice using
serial passages.
[00213] Compound ( 1) formulation was prepared by incorporating the MEK inhibitor into
0.5% CMC 0.25% Tween 20. The preparation was stored at 4°C and resuspended by
vortexing before use. The oral form of the compound was prepared every 3 days. The volume
of administration per mouse was 10 mL/kg.
[00214] Compound (2a) formulation was prepared in water for injection. The stock solution
was chemically stable 7 days in the dark at 4°C. The volume of administration per mouse was
10 mL/kg.
[00215] Compound (2a) and Compound (2b) formulations were prepared in IN HC1 and
water for injection, final pH was 3, followed by five cycles of vortexing and sonicating. The
stock solution was chemically stable 7 days in the dark at 4°C. The volume of PO
administration per mouse was 0 mL/kg.
[00216] For subcutaneous implantation of tumor cells, skin in the flank of the mice was
disinfected using alcohol or Betadine® solution (Alcyon) and a suspension of tumor cells was
inoculated SC unilaterally under a volume of 0.2 mL using a 23 G needle.
[00217] The dosages and schedule of administration of Compound (1), Compound (2a) and
Compound (2b) used as single agent or in combination are described in the results section and
detailed in the tables that follow.
[00218] The animals required to begin a given experiment were pooled and implanted
monolaterally on day 0. Treatments were administered on measurable tumors. The solid
tumors were allowed to grow to the desired volume range (animals with tumors not in the
desired range were excluded). The mice were then pooled and unselectively distributed to the
various treatment and control groups. Treatment started 33 days post CR-LRB-013P tumor
fragment implantation as indicated in the results section and in each table. The dosages are
expressed in mg/kg, based on the body weight at start of therapy. Mice were checked daily,
and adverse clinical reactions noted. Each group of mice was weighed as a whole daily until
the weight nadir was reached. Then, groups were weighed once to thrice weekly until the end
of the experiment. Tumors were measured with a calliper 2 to 3 times weekly until final
sacrifice for sampling time, tumor reached 2000 mm' or until the animal died (whichever
comes first). Solid tumor volumes were estimated from two-dimensional tumor measurements
and calculated according to the following equation:
[00219] Tumor weight (mg) = Length (mm) x Width2 (mm )/2
[00220] The day of death was recorded. Surviving animals were sacrificed and macroscopic
examination of the thoracic and abdominal cavities was performed.
[00221] A dosage producing a 15% body weight loss (BWL) during three consecutive days
(mean of group), 20% BWL during 1 day or 10% or more drug deaths was considered an
excessively toxic dosage. Animal body weights included the tumor weight.
[00222] The primary efficacy end points are /, percent median regression, partial and
complete regressions (PR and CR). Statistical analyses were performed on SAS system release
8.2 for SU 4 via Everstat V5 software and SAS 9.2 software. A probability less than 5%
(p<0.05) was considered as significant.
Results of in vivo studies
[00223) The median tumor burden at start of therapy was 144 to 162 mm3. As single agents,
Compound (1) (20 mg/kg/administration (Adm)), Compound (2b) (20 mg/kg/adm) and
Compound (2a) (75 mg/kg/adm) were administered PO daily from days 33 to 50 post tumor
implantation. In the combination groups, the dose of Compound (1) was combined yvith each
dose of Compound (2a) and Compound (2b), as shown in Table 8. <
[00224J As single agents or used in combination, Compound (1), Compound (2b) and
Compound (2a) were tolerated, inducing some BWL but not reaching toxicity (Figure 23 and
Table 18). As single agents under these test conditions, Compound (2a) and Compound (2b)
achieved a AT/AC>40 %, while Compound (1) achieved a /AC of 30 %.
[00225] In combination, treatment with Compound (1) at 20 mg/kg/adm and Compound (2b)
at 20 mg/kg/adm achieved a /AC of 26 % (Figure 24 and Table 18) with 1/7 partial
regression, and as shown by Table 19, therapeutic synergy was reached (p = 0.0302 for global
analysis). The treatment with Compound (1) at 20 mg/kg/adm and Compound (2a) at 75
mg/kg/adm achieved a /AC of -5 % (Figure 24 and Table 18) with 5/7 partial regression,
and as shown by Table 19, therapeutic synergy was achieved (pO.0001 globally). See also
Table 20.
Summary of in vivo results
[00226] The in vivo work presented here reports the in vivo antitumor activity of combining
Compound (1), an oral potent and selective allosteric inhibitor of E 1/2, with oral, potent,
and specific inhibitors of class I PI3K lipid kinases Compound (2a), a pan-PI3K inhibitor, and
Compound (2b), a dual pan-PI3K and mTOR inhibitor. This work has been performed against
human primary colon carcinoma CR-LRB-013P xenografts harboring a K.RAS mutation.
[00227] In the study, combination treatment induced a sustained tumor stasis or partial
regressions during the treatment phase and reached therapeutic synergy.
[00228] Accordingly, a potent antitumor activity with therapeutic synergy has been achieved
in RAS mutant CR-LRB-013P driven xenograft model when combining the inhibitor of
MEKl/2 Compound (I) with Compound (2a), a pan-PI3K inhibitor or Compound (2b), a dual
pan-PI3K and mTOR inhibitor.
Example 7. Evaluation of tumor permeability
[00229] The following experiment was conducted to evaluate the impact of Compound (2a)
and Compound (2b), alone or in combination with Compound (1), on tumor vascular
permeability.
Methods
[00230] HCT1 16 tumor cells were implanted subcutaneously in the intra-scapular region in
SCID mice. Implanted animals received Compound (2a) 50mg/kg or Compound (2b)
20mg/kg from day 11 to day 13, as single agents or combined with Compound (1) mg kg
(five animals per group). Each agent was given by oral route on a daily schedule. Tumor
growth was monitored throughout the experiment by callipering the tumors. To quantify
tumor vascular permeability, tumors were excised under ketamine/Xylazine (120/6 mg/kg ip)
anesthesia at day 13, 4 hours post last treatment, 30 min after 0.5% Evans Blue iv injection,
and 2 min post Dextran-Fitc lOOmg/kg iv injection. Tumors were then snap frozen, and 25
sections obtained for fluorescence quantification. Tumors sections were imaged with Icyte at
488 nm for vascular Dextran-Fitc determination and at 633 nm for Evans-Blue extravasation
determination. Respective fluorescence were quantified as the sum of integral phantoms of
fluorescence intensity and expressed as the mean ratio of Evans-Blue signal / Dextran-Fitc
Signal.
Results
[00231] Under these test conditions in advanced subcutaneously grafted HCT1 16 human
RAS PI3 CA mutated colon carcinoma, Compound (1) and Compound (2a) used as single
agents and the combination of Compound (2a)/Compound (1) did not significantly modify
tumor permeability, showing -9%, -8% and 4% decrease, respectively, of the Evans-
Blue/Dextran-Fitc ratio compared to control. On the other hand, 3 days of treatment with
Compound (2b) or the combination of Compound (2b)/Compound (1) induced clear
modulation of Evans-Blue/Dextran Fitc ratio, producing a 50% decrease for the single agent
and 45% decrease for the combination. See Figure 25.
Summary
[00232] Compound (2b) used as a single agent or in combination with Compound (1) alters
tumor vascular permeability after 3 days of treatment in advanced subcutaneously grafted
HCTl 16 human RAS/PI3 CA mutated colon carcinoma. This alteration in HCTl 16 tumor
vascular permeability disrupts in vivo fluorescent-Annexin tumor distribution for FMT
imaging and precludes apoptosis detection by this method.
Table 1. Results of Compound ( 1) and Compound (2b) in vitro combination separated into 5 different groups
Table 2. Antitumor activity of Compound (1) (5 mg/kg) in combination with Compound (2b) (30 mg/kg) or Compound (2a) (50 and 75
against human HCT 116 bearing SCID female mice
Tumor size at start of therapy was 162-352 mm , with a median tumor burden per group of 198-221 mm . Drug formulation: Compound (1) =
carboxymethylcellulose 0.5%, tween 20 0.25% in water; Compound (2b) = water, pH3, Compound (2a) = water. Treatment duration: Compound
(1), Compound (2b), Compound (2a) and combination = 8 days. Abbreviations used: BWL = body weight loss, AT/AC=Ratio of change in tumor
volume from baseline median between treated and control groups (TVday - TVO) / (CVday - CVO) * 100, HNTD = highest non toxic dose, HDT
= highest dose tested.
Table 3. Antitumor activity of Compound ( 1) (5 mg/kg) in combination with Compound (2b) (30 mg/kg) or Compound (2a) (50 and 75 mg/kg)
against human HCT 16 bearing SCID female mice: Therapeutic synergy determination
a Each combination was compared to the best single agent using estimates obtained from a 2-way analysis of variance with repeated
measurements (Time factor) on parameter tumor volume (proc mixed of SAS 9.2 software). A probability less than 5% (p<0.05) was considered
as significant.
Table 4. Antitumor activity of Compound ( 1) ( 10 and 20 mg/kg) in combination with Compound (2b) (20 mg/kg) or Compound (2a) (50 and 75
mg/kg) against human HCT 116 bearing SCID female mice
Tumor size at start of therapy was 126 - 294 mm , with a median tumor burden per group of 180- 198 mm . Drug formulation: Compound ( 1) =
carboxymethy!cellulose 0.5%, Tween 20 0.25% in water; Compound (2b) = water, pH3; Compound (2a) = water. Treatment duration:
Compound ( 1), Compound (2b), Compound (2a) and combination = 8 days. Abbreviations used: BWL = body weight loss, AT/AC= Ratio of
change in tumor volume from baseline median between treated and control groups (TVday - TVO) / (CVday - CVO) * 100, H TD = highest non
toxic dose, HDT = highest dose tested.
a On day 17, mice received 20 mg/kg instead of 10 mg kg.
Table 5. Antitumor activity of Compound (1) (10 and 20 mg/kg) in combination with Compound (2b) (20 mg/kg) or Compound (2a) (50 and 75
mg/kg) against human HCT 1 bearing SCID female mice: therapeutic synergy determination
a Each combination was compared to the best single agent using estimates obtained from a two-way analysis of variance with repeated
measurements (Time factor) on parameter tumor volume (proc mixed of SAS 9.2 software). A probability less than 5% (p<0.05) was considered
as significant.
Table 6. Antitumor activity of Compound ( ) ( 10 and 20 mg/kg) in combination with Compound (2a) (50 and 75 mg/kg) against human HCT
bearing SCID female mice
Tumor size at start of therapy was 112-319 mm3, with a median tumor burden per group of 187-1 89 mm3. Drug formulation: Compound (1) =
carboxymethylcellulose 0.5%, Tween 20 0.25% in water; Compound (2a) = water. Treatment duration: Compound (1), Compound (2a) and
combination = 10 days. Abbreviations used: bwl = body weight loss, AT/AC= (TVday - TVO) / (CVday - CVO) * 100, NTD = highest non toxic
dose, HDT = highest dose tested.
Table 7. Antitumor activity of Compound (1) (10 and 20 mg/kg) in combination with Compound (2a) (50 and 75 mg kg) against human HCT 116
bearing SCID female mice: therapeutic synergy determination
a Each combination was compared to the best single agent using estimates obtained from a 2-way analysis of variance with repeated
measurements (Time factor) on parameter tumor volume (proc mixed of SAS 9.2 software). A probability less than 5% (p<0.05) was considered
as significant.
Table 8. Antitumor activity of Compound (1) (10 and 20 mg/kg) in combination with Compound (2b) (20 mg/kg) against human HCT 116
bearing SCID female mice
Tumor size at start of therapy was 144-294 mm , with a median tumor burden per group of 189-196 mm3. Drug formulation: Compound (1) =
carboxymethylcellulose 0.5%, Tween 20 0.25% in water ; Compound (2b) = water, pH3. Treatment duration: Compound (1), Compound (2b) and
combination = 10 days . Abbreviations used: bwl = body weight loss, AT/AC=(TVday - TVO) / (CVday - CVO) * 100, H TD = highest non toxic
dose, HDT = highest dose tested
Table 9 . Antitumor activity of Compound (1) (10 and 20 mg/kg) in combination with Compound (2b) (20 mg/kg) against human HCT 16
bearing SC1D female mice: therapeutic synergy determination
a Each combination was compared to the best single agent using estimates obtained from a 2-way analysis of variance with repeated
measurements (Time factor) on parameter tumor volume (proc mixed of SAS 9.2 software). A probability less than 5% (p<0.05) was
as significant.
Table 10. Percent /AC and statistical analysis in MiaPaCa-2 tumor-bearing mice treated with Compound (1), Compound (2a), and Compound
(2b) alone or in combination.
The mean percent of actual Miapaca-2 tumor growth inhibited by the treatments was calculated as follows: [%AT/AC= (TV f - TV;/TV fc r -
c ri) x 100%], where TV=tumor volume,/= final, = initial and Ctrl = control group.
Table 1A.
Table 1 B.
Table 12A.
Table 12B.
AT/AC (%)
on dl8
Compound (1)
lOmpk 20
Compound (2a)
75mpk 56
Compound (2a)
50mpk 52 ,
Compound (2a)
75mpk
Compound 5 (1)
lOmpk
Compound (2a)
50mpk
Compound (1) -4
lOmpk
Table 3.
Table 14.
Table 1 . Antitumor activity of Compound ( 1) (20 mg g) in combination with Compound (2b) (20 mg/kg) or Compound (2a) (75 mg/kg) against
human primary colon CR-LRB-009C tumors bearing SCID female mice
Average Regressions
body
Drug weight
Route/Dosage Dosage in Median
death change in
Agent in mL/kg mg/kg per Schedule /(Day % per
(batch) per administration in days in % day
of mouse at administration (total dose) 21 Partial Complete death) nadir
(day of
nadir)
Compound ( 1) PO
20 (220) 11-21 0/7 -7.7 (20) 53 0/7 0/7
(VAC.HAL1 .166) 10 mL kg
Compound (2b) PO
20 (220) 11-21 0/7 -7.4 ( 19 ) 5 1 0/7 0/7
(T1 007388) 10 mL/kg
Compound (2a)
O
(T1 007032 75 (825) 11-21 0/7 - 15.8(21 ) 39 0/7 0/7
10 mL/kg
M022906)
Compound (1) PO 20 (220)
11-21 0/7 - 13.7 (21 ) 4 1/7 0/7
Compound (2b) 10 mL/kg 20 (220)
Compound (1) PO 20 (220)
11-21 0/7 - 14.0 (21 ) 2 1 0/7 0/7
Compound (2a) 10 mL/kg 75 (825)
Control 0/7 -7.8 (20) 100
Tumor size at start of therapy was 100-22 1mm3, with a median tumor burden per group of 126- 144 mm3. Drug formulation: Compound ( 1) =
carboxymethylcellulose 0.5%, tween 20 0.25% in water; Compound (2b) and Compound (2a) = water, pH3. Treatment duration: Compound
( 1), Compound (2a) and Compound (2b) and combination = 1 days. Abbreviations used: BWL = body weight loss, AT/AC=Ratio of change in
tumor volume from baseline median between treated and control groups (TVday - TVO) / (CVday - CVO) * 00, HDT = highest dose tested.
Table 16. Antitumor activity of Compound ( 1) (20 mg/kg) in combination with Compound (2b) (20 mg/kg) or Compound (2a) (75 mg/kg) against
human primary colon CR-LRB-009C tumors bearing SCID female mice: Therapeutic synergy determination
Each combination was compared to the best single agent using estimates obtained from a 2-way analysis of variance with repeated
measurements (Time factor) on parameter tumor volume (proc mixed of SAS 9.2 software). A probability less than 5% (p<0.05) was
considered as significant.

Table . Antitumor activity of Compound ( 1) (20 mg/kg) in combination with Compound (2b) (20 mg/kg) or Compound (2a) (75 mg kg) against
human primary colon CR-LR.B-0 13P tumors bearing SCID female mice
Average Regressions
body
Drug
Route/Dosage in Dosage in mg/kg weight Median
death
Agent mL/kg per Schedule change in /
(Day
(batch) per administration in days % per C in %
of Partial Complete
administration (total dose) mouse at day 5 0
death)
nadir (day
of nadir)
Compound ( 1) PO
20 (360) 33-50 0/7 -4.5 (50) 30 0/7 0/7
(VAC.HAL1. 166) 10 mL/kg
Compound (2b) PO .
20 (360) 33-50 0/7 -5.2 (50) 83 0/7 0/7
(T1 007388) 10 mL/kg
Compound (2a) PO
75 ( 1350) ' 33-50 0/7 -9.2 (50) 53 0/7 0/7
(200901 50) 10 mL/kg
Compound (1) PO 20 (360)
33-50 0/7 -3.7 (43) 26 1/7 0/7
Compound (2b) 10 mL/kg 20 (360)
Compound ( 1) PO 20 (360)
33-50 0/7 - 10.2 (38) -5 5/7 0/7
Compound (2a) 10 mL/kg 75 ( 1350)
Control 0/7 -3.5 (50) 100
Tumor size at start of therapy was 108-245 mm3 , with a median tumor burden per group of 144- 162 mm3 . Drug formulation: Compound ( 1) =
carboxymethylcellulose 0.5%, tween 20 0.25% in water; Compound (2b) and Compound (2a) = water, pH3. Treatment duration: Compound ( 1),
Compound (2a) and Compound (2b) and combination = 18 days. Abbreviations used: BWL = body weight loss, AT/AC=Ratio of change in
tumor volume from baseline median between treated and control groups (TVday - TVO) / (CVday - CVO) * 100, HDT = highest dose tested.
Table 1 . Antitumor activity of Compound (1) (20 mg/kg) in combination with Compound (2b) (20 mg/kg) or Compound (2a) (75 mg/kg) against
human primary colon CR-LRB-013P tumors bearing SCID female mice: Therapeutic synergy determination
a Each combination was compared to the best single agent using estimates obtained from a 2-way analysis of variance with repeated
measurements (Time factor) on parameter tumor volume (proc mixed of SAS 9.2 software). A probability less than 5% (p<0.05) was
as significant.
Table 20.
[00233] While there have been shown and described what are at present considered the
preferred embodiments of the invention, those skilled in the art may make various changes
and modifications which remain within the scope of the appended claims.
WE CLAIM:
1. A composition comprising a compound having the following structural formula:
or a pharmaceutically acceptable salt thereof, and a compound having a structural formula
selected from the group consisting of
and
or a pharmaceutically acceptable salt thereof.
2. The composition of claim 1, further comprising a pharmaceutically acceptable carrier.
3. The composition of claim 1, wherein said compound according to formula (1) and said
compound according to formula (2a) or (2b) are in amounts that produce a synergistic effect in
reducing tumor volume in a patient when said composition is administered to a patient.
4. A method of treating a patient with cancer, comprising administering to said patient a
therapeutically effective amount of the compound of Formula (1), or a pharmaceutically
acceptable salt thereof, in combination with the compound of Formula (2a) or Formula (2b),
or a pharmaceutically acceptable salt thereof.
5. The method of claim 4, wherein the effective amount achieves a synergistic effect in
reducing a tumor volume in said patient.
6. The method of claim 4, wherein the effective amount achieves tumor stasis in said
patient.
7. The method of claim 4, wherein said cancer is selected from the group consisting of
non-small cell lung cancer, breast cancer, pancreatic cancer, liver cancer, prostate cancer,
bladder cancer, cervical cancer, thyroid cancer, colorectal cancer, liver cancer, muscle cancer,
hematological malignancies, melanoma, endometrial cancer and pancreatic cancer.
8. The method of claim 4, wherein the cancer is selected from the group consisting of
colorectal cancer, endometrial cancer, hematological malignancies, thryoid cancer, breast
cancer, melanoma, pancreatic cancer and prostate cancer.
9. The method of claim 4, wherein said method comprises administering the compound
of Formula (2a).
10. The method of claim 4, wherein said method comprises administering the compound
of Formula (2b).
11. A combination for use in treating cancer, the combination comprising a therapeutically
effective amount of (A) the compound of Formula (1), or a pharmaceutically acceptable salt
thereof, and (B) the compound of Formula (2a) or Formula (2b), or a pharmaceutically
acceptable salt thereof.
12. A kit comprising: (A) the compound of Formula (1), or a pharmaceutically acceptable
salt thereof; (B) the compound of Formula (2a) or Formula (2b), or a pharmaceutically
acceptable salt thereof; and (C) instructions for use.
13. Use of a combination comprising a therapeutically effective amount of (A) the
compound of Formula (1), or a pharmaceutically acceptable salt thereof, and (B) the
compound of Formula (2a) or Formula (2b), or a pharmaceutically acceptable salt thereof, for
the preparation of a medicament for use in treatment of cancer.