MASITINIB COMBINATION FOR USE IN TREATING BREAST CANCER

Abstract

Disclosed is a method for treating breast cancer in a subject in need thereof, including administering to the subject a therapeutically effective amount of a tyrosine kinase inhibitor or a pharmaceutically acceptable salt or solvate thereof, optionally in combination with a therapeutically effective amount of a chemotherapeutic agent.

Claims

1-27. (canceled)

28. A method for treating inflammatory breast cancer (IBC) in a subject in need thereof, comprising administering to said subject a therapeutically effective amount of masitinib or a pharmaceutically acceptable salt or solvate thereof in combination with a therapeutically effective amount of at least one chemotherapeutic agent.

29. The method according to claim 28, wherein the pharmaceutically acceptable salt or solvate of masitinib is masitinib mesilate.

30. The method according to claim 28, for improving survival and/or life expectancy of the subject.

31. The method according to claim 28, comprising sensitizing to a chemotherapeutic agent or restoring sensitivity to chemotherapy in the subject.

32. The method according to claim 28, wherein inflammatory breast cancer (IBC) is advanced IBC, locally advanced IBC or metastatic IBC.

33. The method according to claim 28, wherein inflammatory breast cancer is triple-negative inflammatory breast cancer.

34. The method according to of claim 28, wherein inflammatory breast cancer (IBC) is relapsed IBC or is refractory IBC.

35. The method according to claim 28, wherein the subject is naïve to anti-breast cancer treatments, or wherein inflammatory breast cancer relapsed after at least one anti-breast cancer treatment, or after two or more anti-breast cancer treatments.

36. The method according to claim 35, wherein anti-breast cancer treatments are selected from the group consisting of treatment with one or more chemotherapeutic agent, surgery, radiotherapy and any combination thereof.

37. The method according to claim 28, wherein said at least one chemotherapeutic agent is selected from the group consisting of: abitrexate (Methotrexate), abraxane (Paclitaxel Albumin-stabilized Nanoparticle Formulation), ado-Trastuzumab Emtansine, adrucil (Fluorouracil), afinitor (Everolimus), anastrozole, aredia (Pamidronate Disodium), arimidex (Anastrozole), aromasin (Exemestane), carboplatin, capecitabine, cisplatin, Clafen (Cyclophosphamide), Cyclophosphamide, Cytoxan (Cyclophosphamide), Docetaxel, Doxorubicin Hydrochloride, Efudex (Fluorouracil), Ellence (Epirubicin Hydrochloride), Epirubicin Hydrochloride, Eribulin Mesylate, Everolimus, Exemestane, Fareston (Toremifene), Faslodex (Fulvestrant), Femara (Letrozole), Fluoroplex (Fluorouracil), Fluorouracil, Folex (Methotrexate), Folex PFS (Methotrexate), Fulvestrant, Gemcitabine Hydrochloride, Gemzar (Gemcitabine Hydrochloride), Goserelin Acetate, Halaven (Eribulin Mesylate), Herceptin (Trastuzumab), Ixabepilone, Ixempra (Ixabepilone), Kadcyla (Ado-Trastuzumab Emtansine), Lapatinib Ditosylate, Letrozole, Megace (Megestrol Acetate), Megestrol Acetate, Methotrexate, Methotrexate LPF (Methotrexate), Mexate (Methotrexate), Mexate-AQ (Methotrexate), mitoxantrone, Neosar (Cyclophosphamide), Nolvadex (Tamoxifen Citrate), Paclitaxel, Paclitaxel Albumin-stabilized Nanoparticle Formulation, Pamidronate Disodium, Perjeta (Pertuzumab), Pertuzumab, Tamoxifen Citrate, Taxol (Paclitaxel), Taxotere (Docetaxel), Trastuzumab, Toremifene, Tykerb (Lapatinib Ditosylate), Velban (Vinblastine Sulfate), Velsar (Vinblastine Sulfate), Vinblastine Sulfate, Xeloda (Capecitabine), Zoladex (Goserelin Acetate), and Irinotecan.

38. The method according to claim 28, wherein said at least one chemotherapeutic agent is selected from anthracyclines, taxanes, platinum based chemotherapeutic agents, antimetabolites, and mixtures thereof.

39. The method according to claim 28, wherein said at least one chemotherapeutic agent is selected from anthracyclines.

40. The method according to claim 39, wherein said anthracycline is selected from the group consisting of doxorubicin, epirubicin, mitoxantrone, pixantrone, losoxantrone and daunorubicin, and any mixtures thereof.

41. The method according to claim 28, wherein said at least one chemotherapeutic agent is selected from the group consisting of gemcitabine, capecitabine, cisplatin, carboplatin, doxorubicin, mitoxantrone and any mixtures thereof.

42. The method according to claim 28, wherein the therapeutically effective amount of masitinib or a pharmaceutically acceptable salt or solvate thereof ranges from about 6 mg/kg/day to about 9 mg/kg/day.

43. The method according to claim 28, wherein masitinib or a pharmaceutically acceptable salt or solvate thereof is orally administered.

44. A method for inhibiting tyrosine kinases selected from the group consisting of c-Kit, LYN, FYN and PDGFR α and β and for inducing an anti-tumoral Th1 immune response, in an inflammatory breast cancer patient, thereby treating inflammatory breast cancer, wherein said method comprises administering a therapeutically effective amount of masitinib or a pharmaceutically acceptable salt or solvate thereof in combination with a therapeutically effective amount of a chemotherapeutic agent.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0194] FIG. 1 is a histogram showing the sensitivity of resistant Mia Paca-2 cells to masitinib, imatinib and dasatinib.

[0195] FIG. 2 is a histogram showing the sensitivity of Mia Paca-2 cells to the combination treatment of gemcitabine plus masitinib, imatinib or dasatinib.

EXAMPLES

[0196] The present invention is further illustrated by the following examples.

Example 1: Masitinib Sensitized Breast Cancer Cell Lines to Chemotherapies

Materials and Methods

Compounds

[0197] Masitinib mesilate (having the molecular formula C.sub.28H.sub.30N.sub.6OS.CH.sub.4O.sub.3S) presents as a white powder. Stock solution of 20 mM in DMSO was stored at −80° C. Gemcitabine (2′,2′,-dofluoro-2′,-deoxycytidine) was from Eli Lilly and is a nucleoside analogue of deoxycytidine that interferes with DNA synthesis. The other agents were purchased from Sigma Aldrich Corporation: anti-topoisomerase I (Irinotecan), alkylant agents such as platinum salt (Carboplatin), and doxorubicin (anthracycline antibiotic). These agents are commonly used as treatment for various tumor types either as single agent or in combination regimen.

Cell Culture

[0198] Breast cancer cell lines (generous gift from Dr. Patrice Dubreuil, UMR 599 INSERM, Marseille, France) were cultured as monolayers in RPMI 1640 medium, containing L-glutamine supplemented with 100 U/mL penicillin and 100 μg/mL streptomycin, and 10% v/v heat-inactivated fetal calf serum (AbCys Lot S02823S1800) under standard culture conditions (5% CO2, 95% air in humidified chamber at 37° C.). During proliferation assay, all cells were grown in medium containing 1% FCS.

Experimental Design

[0199] Colorimetric cell proliferation and viability assay (reagent WST-1 purchased from Roche cat N.sup.o 1644807)—Cells were washed once and resuspended in RPMI 1% FCS. Cells were plated at 1×10.sup.4/50 μl per well of a 96 well plate. Drug dilutions were prepared in a 96 well plate and obtained by sequential dilutions of masitinib or gemcitabine in RPMI 1. Treatment was started by the addition of 50 μl of a 2× concentrated drug solution to a final volume of 100 μl. For treatment with combination of masitinib mesilate and cytotoxic agents, the cells were first resuspended in medium RPMI 1% FCS containing masitinib at the concentrations of 0, 2, 5 and 10 μM. 1×10.sup.4 cells/50 μl were plated per well of a 96 wells plate and the plates placed in the incubator o/n before treatment with cytotoxic agents. Cytotoxic agent treatment was initiated by addition of 50 μl of a 2× drug dilution (and containing the respective masitinib drug concentration) to a final volume of 100 μl. Masitinib final concentrations remained 0, 2, 5 and 10 μM. After incubating for 72 hours at 37° C., 10 μl of a ½ dilution of WST-1 reagent was added to each well and the plates were returned to the incubator for an additional 4 hours. The absorbance of the samples was measured at 490 nm using an EL800 Universal microplate reader (Bio-Tek Instruments Inc.). A background control without cells was used as a blank. The positive control of the assay corresponds to the cell proliferation obtained in the absence of drug treatment (100% proliferation). Each sample was done in duplicate, the absorbance values were transferred to an excel file, the average and standard deviation of the duplicates were calculated and expressed as a percentage of the proliferation obtained in absence of treatment. The results presented are representative of a minimum of four experiments. The sensitization factor/Index is calculated by dividing the IC.sub.50 of the chemotherapeutic agent alone by the IC.sub.50 of the chemotherapeutic agent used in combination with masitinib mesilate.

Results

[0200] In order to assess the benefits of using masitinib mesilate in combination therapy for cancer treatment, preclinical studies involving tumor cell lines were performed. The project consisted to evaluate the ability of masitinib mesilate to sensitize breast cancer cell lines to cytotoxic agents using in vitro proliferation assays.

[0201] We used a large panel of cytotoxic agents that exert their cytotoxicity through different mechanisms. These agents included the conventional chemotherapies gemcitabine (GCB) and doxorubicin (DOX) as well as non-standard chemotherapeutic agents such as irinotecan (CPT-11) and platinum salts carboplatin (CPT).

Masitinib Mesilate is not Active as Single Agent

[0202] Breast cancer cell lines were first analyzed for their sensitivity to masitinib mesilate when used as single agent. This analysis showed that breast cancer cell lines were not sensitive to masitinib mesilate (IC.sub.50>6 μM) suggesting that proliferation/survival of the cell lines examined may not be dependent on the expression masitinib mesilate main targets PDGFRα/β and c-Kit. Based on these data, masitinib mesilate was used at concentrations of 5 and 10 μM in the following combinatory experiments.

Masitinib Mesilate Sensitizes Breast Cancer Cells to Gemcitabine

[0203] To determine the IC.sub.50 of gemcitabine as single agent or in association with masitinib mesilate, breast cancer cell lines grown in 1% FCS were pre-treated with solvent control (DMSO) or masitinib mesilate for about 12-16 hours before being exposed to different doses of the chemotherapeutic agent.

[0204] Results are shown in Table 1.

TABLE-US-00001 TABLE 1 Masitinib mesilate sensitizes breast tumor cell lines to gemcitabine (GCB) IC.sub.50 μM GCB GCB plus Cell lines μM Masitinib mesilate SI BT20 50  5-10 5-10 BT474 100 10-50 2-10 MDAMB134 100 10-50 2-10 MDAMB231 100 50 2 SI = Sensitization Index

[0205] A good masitinib mesilate sensitization to gemcitabine is observed in these cell lines resistant to gemcitabine. Moreover, the addition of masitinib mesilate lowers the IC.sub.50 of gemcitabine to clinically achievable concentrations.

Masitinib Mesilate Sensitizes Breast Cancer Cells to Doxorubicin

[0206] The ability of masitinib mesilate to sensitize breast cancer cell lines to the action of the anthracycline doxorubicin (Adriamycin) was next assessed. Summary of the results is presented in Table 2.

TABLE-US-00002 TABLE 2 Masitinib mesilate sensitizes breast tumor cell lines to doxorubicin (DOX) IC.sub.50 (μM) DOX DOX plus Cell lines μM Masitinib mesilate SI MDAMB231 1 0.1 10 MDAMB134 1 0.1-0.5 2-10 HCC1937 1 0.1-0.5 2-10 MCF-7 1 0.25-0.1  4-10 SI = Sensitization Index

[0207] Interestingly these four cell lines appear to be resistant to doxorubicin and a good sensitization is observed when masitinib mesilate was added. The presence of masitinib mesilate lowers the IC.sub.50 of doxorubicin to clinically achievable concentrations.

Masitinib Mesilate Sensitizes Breast Cancer Cell Lines to Carboplatin

[0208] We next tested the ability of masitinib mesilate to sensitize breast cancer cell lines to the action of carboplatin. Summary of the results is presented in Table 3.

TABLE-US-00003 TABLE 3 Masitinib mesilate sensitizes breast tumor cell lines to carboplatin (CPT) IC.sub.50 (μM) CPT CPT plus Cell lines μM Masitinib mesilate SI MDAMB134 >100 10 >10 MDAMB231 >100 30 >3 BT474 >100 50 >2 BrCa-MZ-01 100 5 20 BT20 100 20 5 SI = Sensitization Index

[0209] The cell lines MDAMB134 and 231, BT474, BrCa-MZ-01 and BT20 are resistant to carboplatin and the addition of masitinib mesilate significantly enhances the sensitivity of these cell lines to carboplatin.

Masitinib Mesilate Sensitizes Breast Cancer Cells to Irinotecan

[0210] We next assessed the ability of masitinib mesilate to sensitize breast cancer cell lines to the action of an inhibitor of topoisomerase I irinotecan. Summary of the results is presented in Table 4.

TABLE-US-00004 TABLE 4 Masitinib mesilate sensitizes breast tumor cell lines to irinotecan (CPT-11) IC.sub.50 (μM) CPT-11 CPT-11 plus Cell lines μM Masitinib mesilate SI MCF-7 100 5-20 5-20 MDAMB134 30 10 3 MDAMB231 30 10 3 SI = Sensitization Index

[0211] Masitinib mesilate efficiently sensitizes MCF-7 cell line to irinotecan while sensitizing to a lesser extent MDAMB134 and 231 cell lines.

[0212] These results thus demonstrate that, surprisingly, masitinib mesilate is able to sensitize breast cancer cell lines to cytotoxic agents in vitro, despite its absence of activity when used alone. Therefore, these results highlight the synergistic effect of the combination of masitinib mesilate and cytotoxic agents.

Example 2: Masitinib Sensitized Breast Cancer Cell Lines to Chemotherapies

[0213] Mitoxantrone is an anthracycline analog and type II topoisomerase inhibitor. Mitoxantrone is an intravenous treatment for advanced-stage breast cancer. Other drugs of this class include doxorubicin, epirubicin, pixantrone, losoxantrone, daunorubicin and other anthracyclines.

Materials and Methods

[0214] Two canine mammary tumor (CMT) cell lines were evaluated, CMT-U27 and CMT-U309. CMT-U27 is a highly metastatic canine mammary carcinoma cell line while CMT-U309 is a non-metastatic spindle-cell cell line. Cell lines were maintained in Dulbecco's modified Eagles Medium-F12 supplemented with 10% fetal bovine serum and antimicrobials (penicillin [100 U/mL] and streptomycin [0.1 mg/mL]). Cell lines were maintained in a humidified atmosphere of 5% carbon dioxide at 37° C.

[0215] Masitinib mesilate was dissolved in 100% DMSO at a concentration of 20 mM to create a stock solution, which was then protected from light and kept at −20° C. The stock solution was further diluted with culture media prior to use in tissue culture, DMSO concentration did not exceed 0.2% volume per condition. Mitoxantrone dihydrochloride was dissolved in Dulbecco's modified Eagles Medium-F12 at a concentration of 3.2 mM to create a stock solution. Solutions were prepared newly prepared from the stock solution for each experiment.

[0216] Cells were seeded at a density of 1.0×10.sup.4 cells/well (final volume, 100 μL/well) in three replicates. After 24 hours, 100 μL of medium containing (DMSO) (control wells) or various concentrations of masitinib and mitoxantrone were added to each well to create final masitinib and mitoxantrone concentrations of 0.25, 0.5, 1, 2, 4, 8, 16 and 32 μM, and the cells were incubated for an additional 24, 48 and 72 hours. At the end of treatment, 10 μl of MTT solution (5 mg/ml in PBS) was added to each well. The plates were incubated for 4 hours in a humidified atmosphere at 37° C. with 5% CO.sub.2. After that incubation, cell proliferation was assessed by use of a commercial cell proliferation MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltrazolium bromide] kit. All samples were assayed in triplicate, and the mean for each experiment was calculated. The effect of each compound on growth inhibition was assessed as percent cell viability where vehicle-treated cells were taken as 100% viable. Cell proliferation was expressed as a percentage of the control wells by use of the following equation: (absorbance of sample wells/absorbance of control wells)×100. The mean of triplicate experiments for each dose was used to calculate IC.sub.50 as determined by CalcuSyn software. Proliferation assays for the combined incubation of masitinib with mitoxantrone used the same procedure except that the cells were incubated for 72 hours.

[0217] The multiple drug effect analysis of Chou and Talalay, which is based on the median-effect principle, was used to examine the nature of the interaction observed between masitinib and mitoxantrone (Chou T C, Talalay P. Adv Enzyme Regul. 1984; 22:27-55) (Chou T C. Cancer Res. 2010 Jan. 15; 70(2):440-6). Determination of the synergistic versus additive versus antagonistic cytotoxic effects of the combined treatment of cells with masitinib and mitoxantrone were assessed by the combination index (CI) where CI<1, CI=1 and CI>1 indicate synergism, additive and antagonism, respectively.

Results

[0218] After 24, 48 and 72 hours of treatment, the determination of cell number showed that mitoxantrone inhibited cell growth in a time- and dose-dependent manner. After 72 hours incubation the mitoxantrone IC.sub.50 for CMT-U27 and CMT-U309 were 0.22 and 0.75 μM, respectively. Conversely, masitinib showed weak inhibition of these cell lines (IC.sub.50 of 7.5 and 8.5 μM, respectively). Summary of the results is presented in Table 5.

[0219] The concentration of agents for use in the combination assays were based on their 72 hour single-agent IC.sub.50 data. The combination of masitinib and mitoxantrone produced strong synergistic growth inhibition of both CMT-U27 and CMT-U309 cell lines with CIs of 0.549, and 0.809, respectively. Summary of the results is presented in Table 6.

[0220] These results show that masitinib mesilate strongly enhances the sensitivity of these cell lines to mitoxantrone. Taken together with those data showing masitinib mesilate sensitizes breast cancer cells to doxorubicin these results, surprisingly, show that masitinib mesilate is synergistic with anthracycline agents in vitro for the inhibition of breast cancer cell lines.

TABLE-US-00005 TABLE 5 Growth inhibitory activity of masitinib and mitoxantrone on CMT-U27 and CMT-U309 cells Cell line Treatment Time IC.sub.50 (μM) CMT-U27 Masitinib 24 h 9.129 48 h 7.607 72 h 7.498 CMT-U27 Mixoxantrone 24 h 3.091 48 h 1.108 72 h 0.219 CMT-U309 Masitinib 24 h 15.032 48 h 8.871 72 h 8.545 CMT-U309 Mitoxantrone 24 h 2.774 48 h 1.00 72 h 0.751

TABLE-US-00006 TABLE 6 Growth inhibitory activity of masitinib and mitoxantrone after 72 hours incubation and combination index (CI) values of the masitinib plus mitoxantrone combination in CMT-U27 and CMT-U309 cell lines. Masitinib and mitoxantrone doses at single- agent IC.sub.50 concentrations Viability Interpre- Cell line Treatment (%) CI tation CMT-U27 Masitinib alone 77.3 Mixoxantrone alone 42.0 Masitinib + mixoxantrone 28.2 0.549 Synergism CMT-U309 Masitinib alone 66.0 Mixoxantrone alone 56.8 Masitinib + mixoxantrone 42.7 0.809 Synergism

Example 3: Masitinib but not Imatinib or Dasatinib can Resensitize Gemcitabine Resistant Cancer Cell Lines

[0221] As shown in Examples 1 and 2, masitinib can reverse resistance to chemotherapy and generate synergistic growth inhibition in various human and canine breast cancer cell lines. The potential of masitinib over other tyrosine kinase inhibitors (TKI) such as imatinib and dasatinib to enhance gemcitabine cytotoxicity was also investigated. Findings showed a surprising and unexpected synergy by the combination of masitinib with gemcitabine over the combination of other known c-Kit inhibitors such as imatinib or dasatinib with gemcitabine for treating solid tumors. Specifically, it was found that only masitinib is able to restore sensitivity to gemcitabine in Mia Paca-2-cells.

[0222] Imatinib (Gleevec, STI-571; Novartis) is a TKI targeting ABL, PDGFR, and c-Kit. Dasatinib (Sprycel, Bristol-Myers Squibb) is a TKI targeting SRC, ABL, PDGFR, and c-Kit. The structures of imatinib and dasatinib appear below:

##STR00005##

[0223] The potential of masitinib, imatinib, or dasatinib to enhance gemcitabine cytotoxicity is assessed by pretreating gemcitabine-resistant cell lines (Mia Paca-2) with these tyrosine kinase inhibitors (TKI) overnight and then exposing them to different doses of gemcitabine to record the IC.sub.50 concentrations.

Methodology

[0224] The antiproliferative activity of each TKI and gemcitabine is assessed using a WST-1 proliferation/survival assay in growth medium containing 1% fetal calf serum (FCS). Treatment is started with the addition of the relevant drug. For combination treatment (TKI plus gemcitabine), cells are re-suspended in medium (1% FCS) containing 0, 5 or 10 μM of the relevant TKI and incubated overnight before gemcitabine addition. After 72 hours, WST-1 reagent is added and incubated with the cells for 4 hours before absorbance measurement at 450 nm in an EL800 Universal Microplate Reader (Bio-Tek Instruments Inc.). Media alone is used as a blank and proliferation in the absence of drug served as a positive control. A TKI sensitization index is calculated as the ratio of the IC.sub.50 of gemcitabine against the IC.sub.50 of the drug combination.

Results

[0225] The results for the sensitivity of resistant Mia Paca-2 cells to various single agent treatment confirmed that these cells exhibit resistance (IC.sub.50 superior to 10 μM) to gemcitabine. Masitinib alone does not significantly affect the growth of Mia Paca-2 cells (IC.sub.50 of 5 to 10 μM), with more than 50% of the cells remaining resistant at a masitinib concentration of 10 μM (FIG. 1). Mia Paca-2 cell proliferation is affected by a similar extent when exposed to dasatinib (10 μM) as a single agent (FIG. 1).

[0226] However, a partial inhibition in the presence of low concentrations (superior to 0.1 μM) is also observed for this TKI. Mia Paca-2 cell proliferation is only weakly inhibited by imatinib (10 μM) as a single agent (FIG. 1).

[0227] Considering the sensitivity of Mia Paca-2 cells to the combination treatment of gemcitabine plus a given TKI, it is revealed that Mia Paca-2 cells are significantly sensitized with masitinib at 5 and 10 μM, as evidenced by the substantial reductions in cell proliferation (FIG. 2) and in gemcitabine IC.sub.50. Specifically, the IC.sub.50 of gemcitabine as a single agent is superior to 10 μM compared with an IC.sub.50 of 0.025 μM for the masitinib (10 μM) plus gemcitabine combination, producing a sensitization factor superior to 400.

[0228] This antiproliferative action is also confirmed via microscopic observation, which clearly revealed cells to be dying rather than being arrested in the cell cycle. Conversely, pre-incubation of cells with 10 μM of imatinib or dasatinib does not result in an increased response of Mia Paca-2 cells to gemcitabine as compared to masitinib (FIG. 2).

Example 4: Case Reports Demonstrating that Masitinib is Active in the Treatment of Inflammatory Breast Cancer (IBC)

Case 1

[0229] This patient was initially diagnosed with invasive ductal carcinoma, T2N0M0, TNM-based stage I, and Scarff-Bloom-Richardson (SBR) grade II. No nodes were involved out of 9 sampled nodes. Immuno-histochemistry revealed positive status for estrogen receptors (ER+), negative status for progesterone receptors (PR−), and negative HER2 status.

[0230] At screening for treatment with masitinib, the patient was in failure to first-line of treatment including taxane treatment with clinically classified metastatic inflammatory breast cancer (lung).

TABLE-US-00007 TABLE 7 Treatment of a patient suffering from metastatic inflammatory breast cancer (Case 1) Diagnosis Metastatic inflammatory breast cancer Treatment Masitinib (6 mg/kg/day) administered orally in two daily doses plus gemcitabine (1250 mg/m.sup.2 intravenous on days 1 and 8, over a 3-week cycle) Masitinib exposure 12 weeks Best response (Modified- Stable disease RECIST criteria v1.1) Median overall survival 20.4 months (deceased) Age 53 Previous treatment failure Taxane Clinical classification at Metastatic (lung) screening (location) Histology at initial diagnosis Invasive ductal carcinoma Progesterone receptor (PR) Negative status Estrogen receptor (ER) status Positive HER2 status Negative

Case 2

[0231] This patient was initially diagnosed with invasive ductal carcinoma, T2N0M0, TNM-based stage III, and Scarff-Bloom-Richardson (SBR) grade III. Eleven nodes were involved out of 14 sampled nodes. Immuno-histochemistry revealed positive status for estrogen receptors (ER+), negative status for progesterone receptors (PR−), and positive HER2 status (2+).

[0232] At screening for treatment with masitinib, the patient was in failure to first-line of treatment including taxane treatment with clinically classified metastatic inflammatory breast cancer (bone, mediastinal, and lung).

TABLE-US-00008 TABLE 8 Treatment of a patient suffering from metastatic inflammatory breast cancer (Case 2) Diagnosis Metastatic inflammatory breast cancer Treatment Masitinib (6 mg/kg/day) administered orally in two daily doses plus carboplatin (5AUC intravenous on day 1 over a 3- week cycle) Dose reduction after week 3 Masitinib (4.5 mg/kg/day) administered orally in two daily doses plus carboplatin (5AUC intravenous on day 1 over a 3- week cycle) Masitinib exposure 18 weeks Best response (Modified- Stable disease RECIST criteria v1.1) Median overall survival 20.9 months (deceased) Age 43 Previous treatment failure Taxane Clinical classification at Metastatic (bone, mediastinal, lung) screening (location) Histology at initial diagnosis Invasive ductal carcinoma Progesterone receptor (PR) Negative status Estrogen receptor (ER) status Positive HER2 status Positive

[0233] Taken together, these case reports demonstrate that masitinib has a surprising and unexpected positive treatment-effect in refractory inflammatory breast cancer as compared with the benchmark of 11.2 months for lapatinib in this population (Kaufman B, et al. Lancet Oncol. 2009 June; 10(6):581-8).

Example 5: Treatment of TNBC with a Combination of Masitinib and Cisplatin (Phase 1/2 Study)

[0234] A prospective, multicenter, open-label, uncontrolled, phase 1/2 clinical study has been conducted to evaluate efficacy and safety of masitinib mesilate in association with cisplatin after a first-line of cytotoxic chemotherapy of patients suffering from TNBC (either metastatic or locally advanced).

Methodology

[0235] Sixteen TNBC patients resistant to at least one first line of chemotherapy including anthracycline or taxane were enrolled. In this open-label study, masitinib mesilate was administered orally at the daily dose of 9 mg/kg in two intakes, in combination with cisplatin given at the dose of 75 mg/m.sup.2 by intravenous infusion every 3 weeks.

Results

[0236] In the first phase 2 study, overall survival with masitinib mesilate in combination with cisplatin is 8.9 months and comparable to the existing standard survival in TNBC.

[0237] Overall survival (OS) is defined as the time from first treatment intake to the date of documented death. If death was not observed, data on OS were censored at the last date patient was known to be alive. OS was analyzed using Kaplan-Meier and was given with its confidence interval (CI) of 95%.

[0238] The median OS for patients with already one previous chemotherapy line was 10 months with a 95% CI [6.8-16.0]. In comparison, the treatment with cisplatin alone leads to a median OS of 9.4 (Baselga J, et al: J Clin Oncol 31:2586-2592, 2013)). Moreover, Kaplan Meier analysis of OS performed on patients with more than two previous chemotherapy lines showed that patients already treated with more than two previous chemotherapy lines had a higher OS than patients with less than 2 previous chemotherapy lines (median overall survival were: 13 versus 10.0 months). Summary of the results is presented in Table 9.

TABLE-US-00009 TABLE 9 Overall Survival Median OS (months) [95% CI] Cisplatin (N = 58) 9.4 Masitinib plus cisplatin: 1 previous chemotherapy line (N = 8) 10 [7.2; 19] >2 previous chemotherapy lines (N = 4) 13 [6.8; 16]

[0239] In conclusion, patients with metastatic and locally advanced TNBC and treated with the combination of masitinib mesilate plus cisplatin showed an improved efficacy compared to single-agent cisplatin when they had received at least one previous chemotherapy line.

Example 6: Treatment of TNBC with a Combination of Masitinib, Gemcitabine and Carboplatin (Phase 1/2 Study)

[0240] The objective of this prospective, multicenter, central allocation, open-label, 3-parallel groups, phase 1/2 study is to evaluate efficacy and safety of masitinib mesilate at 6 or 9 mg/kg/day in association with gemcitabine plus carboplatin in patients with a triple negative breast cancer (either metastatic or locally advanced).

Methodology

[0241] Twenty-one patients suffering from a metastatic or locally advanced triple-negative breast cancer have been enrolled. In this open-label study, masitinib mesilate was administered orally at a dose of 6 or 9 mg/kg/day in two intakes. The chemotherapeutic agents of gemcitabine and carboplatin were administered by intravenous injection, gemcitabine at a dose of 750 or 1000 mg/m.sup.2 at day 1 and 8 of a cycle of 3 weeks and carboplatin at a dose of AUC 5 at day 1 of a cycle of 3 weeks.

Preliminary Results

[0242] In the on-going phase 2 study, preliminary results show that current median overall survival on this study is 10.2 months for masitinib mesilate plus gemcitabine plus carboplatin. Results of OS are presented in Table 10.

TABLE-US-00010 TABLE 10 Overall Survival Median OS (months) [95% CI] Benchmark* 6-7 Masitinib plus gemcitabine plus 10.2 [9.2-13.2] carboplatin (N = 21) *O'Shaughnessy et al. N Engl J Med. 2011 Jan. 20; 364(3): 205-14. O'Shaughnessy J, et al. J Clin Oncol 27: 18s, 2009 (suppl; abstr 3)

[0243] The treatment with masitinib mesilate plus gemcitabine plus carboplatin increases the OS of patients suffering from triple negative metastatic or locally advanced breast cancer and compared favorably to the benchmark of gemcitabine plus carboplatin estimated from the BSI-201 study's 2009 interim analysis with minimal treatment cross-over (about 5.7 months) and final 2011 study results that would be confounded by treatment-arm crossover (about 7.7 months).

Example 7: Treatment of Metastatic or Locally Advanced Breast Cancer with a Combination of Masitinib and Capecitabine or Gemcitabine (Phase 1/2 Study)

[0244] The objective of this prospective, multicenter, open-label, randomized, uncontrolled, 3-parallel groups, phase 1/2 study was to evaluate efficacy and safety of masitinib mesilate at 6 or 9 mg/kg/day in association with capecitabine or gemcitabine in patients with a metastatic or locally advanced breast cancer (all hormonal status except triple negative tumor) except with a triple negative tumor.

Methodology

[0245] Twenty-seven patients suffering from metastatic or locally advanced breast cancer (with all hormonal status tumor except triple negative tumor) and resistant to at least one first line of chemotherapy including anthracycline or taxane have been enrolled for treatment with the combinations of masitinib plus gemcitabine, or masitinib plus capecitabine. In this open-label study, masitinib mesilate was administered orally at the daily dose of 6 or 9 mg/kg in two intakes, in combination with capecitabine orally administered at the dose of 1250 mg/m.sup.2 twice daily for 2 weeks followed by a 7-day rest period (in cycles of 3 weeks) or with gemcitabine infused at the dose of 1000 or 1250 mg/m.sup.2 at days 1 and 8 of a cycle of 3 weeks.

Results

[0246] Preliminary results are presented in the Table 11 below. The data of the literature indicate that the median OS for patients resistant to at least one first-line of chemotherapy is 14.5 months with single-agent capecitabine and about 12.6 months for single-agent gemcitabine.

TABLE-US-00011 TABLE 11 Overall Survival Median OS (months) [95% CI] Benchmark for single-agent 14.5 — capecitabine (L2)* Benchmark for single-agent 12.6  .sup. [3.9-30.8] gemcitabine (L2) ** Masitinib plus gemcitabine (N = 13) 20.9 [19.9-NR] Masitinib plus capecitabine (N = 14) 22.5 [13.7-NR] *Miller et al. J Clin Oncol 23 (4): 792-9, 2005. ** Brodowicz et al. Breast. 2000 December; 9(6): 338-42.

[0247] These preliminary results show that the treatment of patients suffering from metastatic or locally advanced breast cancer with a combination of masitinib mesilate and capecitabine or gemcitabine increases their survival.