RU2781195C2 - Combinations of anti-her2-antibody-drug conjugate and chemotherapeutic remedies, and application methods - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to a method for the treatment of malignant tumor expressing ErbB2, including the administration of a therapeutic combination, in the form of a combined composition or alternately, to a mammal, where the therapeutic combination contains trastuzumab-MCC-DM1 and docetaxel, where the administration of the therapeutic combination leads to an additive effect compared to the administration of trastuzumab-MCC-DM1 or docetaxel in the form of separate remedies, where the amount of trastuzumab-MCC-DM1 is from 0.3 to 15 mg/kg, and where the amount of docetaxel is about 30 mg/kg.

EFFECT: obtaining combination of an antibody-drug conjugate and chemotherapeutic means.

10 cl, 37 dwg, 21 tbl, 5 ex

Description

Relevant applications

This non-provisional application filed under 37 CFR § 1.53(b) claims priority under 35 USC § 119(e) of U.S. Provisional Application No. 61/037410, filed March 18, 2008, which is hereby incorporated by reference in its entirety.

The field of technology to which the invention belongs

The invention mainly relates to pharmaceutical combinations of compounds having activity against hyperproliferative disorders such as cancer. The invention also relates to methods of using combinations of compounds for the diagnosis or treatment of mammalian cells in vitro, in situ and in vivo, or associated pathological conditions.

State of the art

HER2 (ErbB2) tyrosine kinase receptor is a member of the epidermal growth factor receptor (EGFR) family of transmembrane receptors. HER2 overexpression is noted in approximately 20% of human breast cancers and is associated with aggressive growth and poor prognosis in patients with these tumors (Slamon et al., 1987, Science, 235:177-182).

Trastuzumab (CAS number 180288-69-1, HERCEPTIN ^® , huMAb4D5-8, rhuMAb HER2, Genentech) is a recombinant DNA-derived humanized, IgG1 kappa, mouse anti-HER2 monoclonal antibody that selectively binds with high affinity to cell assay (Kd=5 nM) with the extracellular domain of human epidermal growth factor receptor 2 protein, HER2 (ErbB2) (U.S. Patent No. 5,677,171; U.S. Patent No. 5,821,337; U.S. Patent No. 6,054,297; U.S. Patent No. 6,165,464; U.S. Patent No. 6,339,142; U.S. Patent No. 6,407,213; U.S. Patent No. 6,639,055; U.S. Patent No. 6,719,971; U.S. Patent No. 6,800,738; U.S. Patent No. 7,074,404 Coussens et al. 1985 Science 230:1132-1139 Slamon et al. 707-712; Slamon et al., 2001, New Engl. J. Med., 344:783-792). Trastuzumab contains human framework regions with complementarity-determining regions of a mouse antibody (4D5) that binds to HER2. Trastuzumab binds to the HER2 antigen and thus inhibits the growth of tumor cells. In in vitro and animal tests, trastuzumab has been shown to inhibit the proliferation of human tumor cells overexpressing HER2 (Hudziak et al., 1989, Mol. Cell Biol., 9:1165-1172; Lewis et al., 1993, Cancer Immunol Immunother: 37:255-63; Baselga et al., 1998, Cancer Res., 58:2825-2831). Trastuzumab is a mediator of antibody-dependent cellular cytotoxicity, ADCC (Lewis et al., 1993, Cancer Immunol. Immunother., 37(4):255-263; Hotaling et al., 1996, [abstract]. Proc. Annual Meeting Am. Assoc. Cancer Res., 37:471; Pegram MD et al., 1997, [abstract] Proc. Annual Meeting Am. Assoc. Cancer Res., 38:602; Sliwkowski M. et al., 1999, Seminars in Oncology, 26 (4), Suppl. 12:60-70; Yarden Y. and Sliwkowski M., 2001, Nature Reviews: Molecular Cell Biology, Macmillan Magazines, Ltd., Vol. 2:127-137).

^Herceptin® was approved in 1998 for the treatment of patients with ErbB2 overexpressing metastatic breast cancer (Baselga et al., 1996, J. Clin. Oncol., 14:737-744) who had previously received intensive anticancer therapy, and has since been used on more than 300,000 patients (Slamon DJ et al., N. Engl. J. Med., 2001, 344:783-92; Vogel CL et al., J. Clin. Oncol., 2002, 20:719-26; Marty M. et al., J. Clin. Oncol., 2005, 23:4265-74; Romond EH et al., T N. Engl. J. Med., 2005, 353:1673- 84; Piccart-Gebhart MJ et al., N. Engl. J. Med., 2005, 353:1659-72; Slamon D. et al., [abstract] Breast Cancer Res. Treat., 2006, 100 (Suppl .1):52). In 2006, the FDA approved ^Herceptin® (trastuzumab, Genentech Inc.) as an integral component of a regimen containing doxorubicin, cyclophosphamide, and paclitaxel for the adjuvant treatment of patients with HER2-positive, metastatic breast cancer. Although ^Herceptin® , designed for patients with HER2-positive tumors, provided a higher therapeutic effect compared to chemotherapy alone, in the end, all patients with HER2-positive metastatic breast cancer (MBC) had disease progression on available drugs. There remained room for improvement in efficacy in the treatment of patients with MVS. Despite the different mechanisms of action of trastuzumab, a number of patients treated with trastuzumab did not respond to treatment or stopped responding after a positive period of treatment. Some HER2+ (HER2-positive) tumors did not respond to ^Herceptin® treatment and the majority of patients who had a tumor response eventually experienced disease progression. There is a significant clinical need to develop additional HER2-targeted anticancer agents for the treatment of patients with HER2 overexpressing tumors or other HER2-expressing diseases that do not respond or respond poorly to ^Herceptin® treatment.

An alternative approach for antibody-targeted therapy is the use of antibodies to deliver cytotoxic drugs specifically to antigen-expressing tumor cells. Maytansinoids, derivatives of the antimitotic agent maytansine, bind to microtubules in a manner similar to vinca alkaloid drugs (Issell BF et al., 1978, Cancer Treat. Rev., 5:199-207; Cabanillas F. et al., 1979, Cancer Treat. Rep. 63:507-509). Antibody drug conjugates (ADCs) consisting of DM1 maytansinoid coupled to trastuzumab exhibit potent antitumor activity in HER2-expressing trastuzumab-responsive and trastuzumab-resistant tumor cell lines and in human breast cancer xenograft models. The potency of the maytansinoid conjugate coupled to the mouse anti-HER2 antibody, TA.1 via the MCC linker, is 200-fold higher than that of the corresponding disulfide linker conjugate (Chari et al., 1992, Cancer Res., 127-133). Antibody-drug conjugates (ADCs) consisting of maytansinoid, DM1, coupled to trastuzumab exhibit potent antitumor activity in trastuzumab-sensitive and trastuzumab-resistant tumor cell lines and in human breast cancer xenograft models. A trastuzumab-MCC-DM1 (T-DM1) conjugate is currently in phase II clinical trials in patients who are resistant to treatment with drugs that target HER2 (Beeram et al., 2007, “A phase I study of trastuzumab-MCC-DM1 (T-DM1), a first-in-class HER2 antibody-drug conjugate (ADC) in patients (pts) with HER2+ metastatic breast cancer (BC)”, American Society of Clinical ^Oncology 43rd :June 02 (Abs 1042; Krop et al., European Cancer Conference ECCO, Poster 2118, September 23-27, 2007, Barcelona; US Patent No. 7097840; US Patent Application No. 2005/0276812; US Patent Application No. 2005/0166993).

Combination therapy, in which two or more drugs are used in a specific regimen or form of administration, generally has one or more goals: (i) reducing the incidence of acquired resistance by combining drugs with minimal cross-resistance, (ii) reducing dosages drugs that are not toxic and have a similar therapeutic profile to achieve efficacy with fewer side effects, i.e. increasing the therapeutic index, (iii) sensitizing cells to the effects of one drug through the action of another drug, by changing the stage of the cell cycle or the ability to grow and (iv) achieving increased efficiency when using additive action or more than additive action, effects in biological activity two drugs (Pegram M. et al., 1999, Oncogene, 18:2241-2251; Konecny G. et al., 2001, Breast Cancer Res. and Treatment, 67:223-233; Pegram M. et al., 2004, J. Nat. Cancer Inst., 96(10):739-749; Fitzgarald et al., 2006, Nature Chem. Biol., 2(9):458-466; Borisy et al., 2003, Proc. Natl Acad Sci 100(13):7977-7982).

Levé additivity (Chou TC and Talalay P., 1977, J. Biol. Chem., 252:6438-6442; Chou TC and Talalay P., 1984, Adv. Enzyme Regul., 22:27-55; Berenbaum MC, 1989 , Pharmacol. Rev., 41:93-141) and Bliss independence/synergy (Bliss CI, 1956, Bacteriol. Rev., 20:243-258; Greco et al., 1995, Pharmacol. Rev., 47:331- 385) are methods used to calculate the expected dose-response relationship for combination therapy versus monotherapy based on parameters such as IC ₅₀ , the dose of drug required to inhibit 50% of the target and equal to Ki in the simplest case.

HER2 dimerization inhibitor antibodies and EGFR inhibitors have been reported for combination therapy in cancer (US Patent Application No. 2007/0020261). Trastuzumab-MCC-DM1 (T-DM1) and pertuzumab alone have shown efficacy in patients with MBC, and the combination of pertuzumab and trastuzumab has been shown to be active in patients with HER-positive MBC (Baselga J. et al., "A Phase II trial of trastuzumab and pertuzumab in patients with HER2-positive metastatic breast cancer that had progressed during trastuzumab therapy: full response data”, European Society of Medical Oncology, Stockholm, Sweden, September 12-16, 2008).

The essence of the invention

The invention primarily relates to an anti-HER2 antibody-drug conjugate, trastuzumab-MCC-DM1, which is administered in combination with one or more chemotherapeutic agents to inhibit the growth of tumor cells. Some combinations of trastuzumab-MCC-DM1 and a chemotherapeutic agent exhibit a synergistic effect in suppressing the growth of tumor cells in vitro and in vivo. The combinations and methods of the invention may be useful in the treatment of hyperproliferative disorders such as cancer. The combinations may inhibit the growth of tumors in mammals and may be useful in the treatment of human cancers.

In one aspect, the invention relates to a method of treating a hyperproliferative disorder comprising administering the therapeutic combination as a combination composition or alternately to a mammal, wherein the therapeutic combination comprises a therapeutically effective amount of trastuzumab-MCC-DM1 and a therapeutically effective amount of a chemotherapeutic agent selected from an antibody that inhibits HER2 dimerization , antibodies to VEGF, 5-FU, carboplatin, lapatinib, ABT-869, docetaxel, GDC-0941 and GNE-390.

A therapeutically effective amount of trastuzumab-MCC-DM1 and a therapeutically effective amount of a chemotherapeutic agent may be administered as a combined composition or alternately.

The invention also relates to methods of using compositions for the diagnosis or treatment of mammalian cells, organisms in vitro, in situ and in vivo, or associated pathological conditions.

The invention also relates to methods in which the administration of a therapeutic combination results in a synergistic effect.

Another aspect of the invention relates to pharmaceutical compositions containing trastuzumab-MCC-DM1, a chemotherapeutic agent selected from HER2 dimerization inhibitor antibody, anti-VEGF antibody, 5-FU, carboplatin, lapatinib, ABT-869, docetaxel, GDC-0941 and GNE -390; and one or more pharmaceutically acceptable carriers, flow regulators, diluents, or excipients.

Another aspect of the invention relates to methods of treating a hyperproliferative disease or disorder comprising administering to a mammal in need of such treatment a therapeutically effective amount of trastuzumab-MCC-DM1 and a chemotherapeutic agent. Trastuzumab-MCC-DM1 and the chemotherapeutic agent may be co-formulated for administration in combination as a pharmaceutical composition, or they may be administered alternately (alternate, consecutive administrations) as a therapeutic combination. In one embodiment, T-DM1 is administered by infusion and the chemotherapeutic agent is administered orally.

Another aspect of the invention relates to methods for predicting effective drug combinations in terms of effectiveness in vivo, where the combinations contain trastuzumab-MCC-DM1 and an antitumor, widely used chemotherapeutic agent. Efficacy data obtained in experiments to assess cell proliferation in vitro and on tumor xenografts in vivo are subjected to qualitative and quantitative analysis. Quantification methods may be based on the Chou & Talalay median effect principle and isobolograms, which determine the value of the combination index (CI) to establish synergy, antagonism or additive effect, or they may be based on the independence of the Bliss deviation.

Another aspect of the invention relates to a method of using a therapeutic combination of the invention for the treatment of a disease or condition, such as cancer, in a mammal, including a disease that is modulated by HER2 or KDR9 (VEGF receptor 1).

Another aspect of the invention relates to the use of a therapeutic combination of the invention in the manufacture of a medicament for the treatment of a disease or condition, such as cancer, in a mammal, including a disease that is modulated by HER2 or KDR9 (VEGF receptor 1).

Another aspect of the invention relates to articles or kits containing trastuzumab-MCC-DM1, a chemotherapeutic agent, a container, and optionally a package insert or label describing the treatment.

Another aspect of the invention relates to a method for determining compounds for use in combination for the treatment of cancer, comprising: (a) administering a therapeutic combination of trastuzumab-MCC-DM1 and a chemotherapeutic agent selected from an antibody that inhibits HER2 dimerization, an antibody to VEGF, 5-FU , carboplatin, lapatinib, ABT-869, docetaxel, GDC-0941 and GNE-390 into a tumor cell line in vitro and (b) determining synergistic or non-synergistic effect.

Additional advantages and novel features of the present invention will be set forth in part in the description which follows, and in part will become apparent to those skilled in the art upon reading the following application or learned by practice of the invention. The advantages of the invention can be realized and achieved through the instrumental performance, combinations, compositions and methods claimed in the attached claims.

Brief description of the figures

1 is a graph of day 3 in vitro viability of SK-BR-3 cells versus multiple IC ₅₀ concentrations of trastuzumab, trastuzumab-MCC-DM1 (T-DM1), and the combination of trastuzumab and T-DM1.

2 is a graph of day 3 in vitro viability of BT-474 EEI cells versus multiple IC ₅₀ concentrations of trastuzumab, trastuzumab-MCC-DM1 (T-DM1), and the combination of trastuzumab and T-DM1.

Figure 3 is a plot of in vitro MDA-MB-175 cell viability at Day 5 versus multiples of the IC ₅₀ concentrations of pertuzumab, trastuzumab-MCC-DM1 (T-DM1), and the combination of pertuzumab and T-DM1.

Figure 3a is a graph of in vitro MDA-MB-175 cell viability at day 5 versus multiples of the IC ₅₀ concentrations of pertuzumab, trastuzumab-MCC-DM1 (T-DM1), and the combination of pertuzumab and T-DM1.

4 is a graph of day 5 in vitro viability of BT-474 cells versus various fixed concentrations of pertuzumab in combination with trastuzumab-MCC-DM1 (T-DM1) response concentration and various concentrations of T-DM1 alone.

Figure 5 is a graph of day 5 in vitro viability of BT-474 cells versus various fixed concentrations of trastuzumab-MCC-DM1 (T-DM1) in combination with pertuzumab response concentration and various concentrations of pertuzumab alone.

Figure 6 is a graph of BT-474 cell viability in vitro at Day 5 versus multiples of the IC ₅₀ concentrations of pertuzumab, trastuzumab-MCC-DM1 (T-DM1), and the combination of pertuzumab and T-DM1.

Figure 7 shows a graph of the viability of SK-BR-3 cells in vitro on day 3 from various concentrations of T-DM1 in combination with fixed concentrations of lapatinib (4.5 nM; 14 nM; 41 nM; 123 nM) and various concentrations of one T-DM1 (0-1000 ng/ml).

Figure 7a is a plot of in vitro viability of SK-BR-3 cells at day 3 versus multiples of IC ₅₀ T-DM1, lapatinib, and fixed ratio combinations of T-DM1 and lapatinib.

Figure 8a is a graph of in vitro viability of BT-474 cells at day 3 versus multiples of IC ₅₀ T-DM1, lapatinib, and fixed ratio combinations of T-DM1 and lapatinib.

Figure 8 shows a graph of the viability of BT-474 cells in vitro on day 3 from various concentrations of T-DM1 in combination with fixed concentrations of lapatinib (1.5 nM; 4.5 nM; 14 nM; 41 nM; 123 nM ) and various concentrations of one T-DM1 (0-1000 ng/ml).

Figure 9 shows a graph of the viability of BT-474-EEI cells in vitro on day 3 from various concentrations of T-DM1 in combination with fixed concentrations of lapatinib (14 nM; 41 nM; 123 nM; 370 nM; 1111 nM) and different concentrations of one T-DM1 (0-1000 ng/ml).

Figure 10 is a plot of mean tumor volume over time in vivo for KPL-4 tumors implanted in the mammary fat pad of immunodeficient SCID mice (3 million cells in matrigel per mouse) following administration of: (1) ADC buffer; (2) pertuzumab 15 mg/kg; (3) T-DM1 at 0.3 mg/kg; (4) T-DM1 at 1 mg/kg; (5) T-DM1 at 3 mg/kg, (6) pertuzumab at 15 mg/kg + T-DM1 at 0.3 mg/kg; (7) pertuzumab 15 mg/kg + T-DM1 1 mg/kg; (8) pertuzumab 15 mg/kg + T-DM1 3 mg/kg. Buffer ADC and T-DM1 were administered once on day 0. Pretuzumab was administered at 0; 7 and 14 days.

Figure 11 is a plot of mean tumor volume over time in vivo for KPL-4 tumors implanted in the mammary fat pad of immunodeficient SCID mice (3 million cells in matrigel per mouse) following administration of: (1) ADC buffer; (2) 5-FU at 100 mg/kg; (3) pertuzumab at 40 mg/kg, the first dose of pertuzumab (groups 5, 7 and 9) was 2x the loading dose; (4) B20-4.1 at 5 mg/kg; (5) T-DM1 at 5 mg/kg; (6) 5-FU at 100mg/kg + T-DM1 at 5mg/kg; (7) pertuzumab 40 mg/kg + T-DM1 5 mg/kg; (8) B20-4.1 at 5mg/kg + T-DM1 at 5mg/kg; (9) B20-4.1 at 5 mg/kg + pertuzumab at 40 mg/kg. Buffer ADC and T-DM1 were injected intravenously once on day 0. Pretuzumab was administered at 0; Days 7 and 14 (once a week × 4). 5FU was injected at 0; 7 14 and 21 days (once a week ×3). B20-4.1 was injected at 0; 3; 7; ten; fourteen; 17; Days 21 and 24 (2 x/week x 8 in total).

12 is a plot of mean tumor volume over time in vivo for MMTV-Her2 Fo5 transgenic mammary tumors implanted in the mammary fat pad of CRL nu/nu mice after administration of: (1) diluent (ADC buffer) ; (2) B20-4.1 at 5 mg/kg; (3) T-DM1 at 3 mg/kg; (4) T-DM1 at 5 mg/kg; (5) T-DM1 at 10 mg/kg; (6) B20-4.1 at 5mg/kg + T-DM1 at 3mg/kg; (7) B20-4.1 at 5mg/kg + T-DM1 at 5mg/kg; (8) B20-4.1 at 5mg/kg + T-DM1 at 10mg/kg. Buffer ADC and T-DM1 were administered on days 0 and 21. B20-4.1 was injected at 0; 3; 7; ten; fourteen; 17; Days 21 and 24 (2 x/week x 4.8 in total).

13 is a plot of mean tumor volume over time in vivo for MMTV-Her2 Fo5 transgenic mammary tumors implanted in the mammary fat pad of CRL nu/nu mice after administration of: (1) diluent (ADC buffer) ; (2) T-DM1 at 10 mg/kg; (3) 5-FU at 100 mg/kg; (4) gemcitabine at 120 mg/kg; (5) carboplatin at 100 mg/kg; (6) 5-FU at 100mg/kg + T-DM1 at 10mg/kg; (7) gemcitabine 120 mg/kg + T-DM1 10 mg/kg; (8) carboplatin at 100mg/kg + T-DM1 at 10mg/kg. Buffer ADC, T-DM1 and carboplatin were administered once on day 0. 5-FU was administered once per 0; Days 7 and 14 (once a week ×3). Gemcitabine was administered at 0; 3; Days 6 and 9 (every 3 days ×4).

Figure 14 is a plot of mean tumor volume over time in vivo for MMTV-Her2 Fo5 transgenic mammary tumors implanted in the mammary fat pad of athymic Harlan mice after administration of: (1) diluent (PBS buffer) i.v. c, once a week ×4; (2) lapatinib 101 mg/kg po bid x21; (3) pertuzumab 40 mg/kg, IV, once a week ×4; (4) B20-4.1 at 5mg/kg, ip, 2×/wk×4; (5) T-DM1 at 15 mg/kg, IV, once every 3 weeks until the end; (6) lapatinib 101 mg/kg po bid ×21 + T-DM1 15 mg/kg iv once every 3 weeks until end; (7) pertuzumab 40 mg/kg IV once a week ×4 + T-DM1 15 mg/kg IV once every 3 weeks until the end; (8) B20-4.1 at 5mg/kg, ip, 2×/week ×4 + T-DM1 at 15mg/kg, iv, once every 3 weeks until the end.

15 is a plot of mean tumor volume over time in vivo for MMTV-Her2 Fo5 transgenic mammary tumors implanted in the mammary fat pad of athymic Harlan mice following administration of: (1) diluent (PBS buffer) orally, twice a day ×21; (2) T-DM1 at a dose of 7.5 mg/kg, IV, once a day ×1; (3) T-DM1 at a dose of 15 mg/kg, IV, once a day ×1; (4) ABT-869 at 5 mg/kg po twice a day ×21; (5) ABT-86 at 15 mg/kg PO twice a day ×21; (6) T-DM1 at 7.5 mg/kg IV, once a day ×1 + ABT-869 at a dose of 5 mg/kg, orally, twice a day ×21; (7) T-DM1 at 7.5 mg/kg IV, once a day ×1 + ABT-869 at a dose of 15 mg/kg, orally, twice a day ×21; (8) T-DM1 at 15mg/kg iv, once a day ×1 + ABT-869 at a dose of 5mg/kg, orally, twice a day ×21; (9) T-DM1 at 15mg/kg, iv, once a day ×1 + ABT-869 at a dose of 15mg/kg, orally, twice a day ×21.

Figure 16 is a plot of mean tumor volume over time in vivo for MMTV-Her2 Fo5 transgenic mammary tumors implanted in the mammary fat pad of athymic Harlan mice following administration of: (1) IV solvent, once per week ×3; (2) T-DM1 at 7.5 mg/kg IV once every 3 weeks ×2; (3) T-DM1 at 15mg/kg IV once every 3 weeks ×2; (4) docetaxel 30 mg/kg IV once a week ×3; (5) T-DM1 7.5mg/kg IV once every 3 weeks ×2 + docetaxel 30mg/kg IV once a week ×3; (6) T-DM1 15mg/kg IV once every 3 weeks ×2 + docetaxel 30mg/kg IV once a week ×3.

17 is a plot of mean in vivo tumor volume for MMTV-Her2 Fo5 transgenic tumors implanted in the mammary fat pad of athymic Harlan mice following administration of: (1) diluent po once a day x21; (2) T-DM1 at 7.5 mg/kg IV once every 3 weeks ×2; (3) T-DM1 at 15mg/kg IV once every 3 weeks ×2; (4) lapatinib 100 mg/kg orally twice daily ×21; (5) T-DM1 7.5 mg/kg IV once every 3 weeks ×2 + lapatinib 100 mg/kg po twice daily ×21; (6) T-DM1 15 mg/kg IV once every 3 weeks ×2 + lapatinib 100 mg/kg po twice daily ×21.

Figure 18 is a graph of in vitro viability of SK-BR-3 cells at Day 3 versus multiples of IC ₅₀ concentrations of 5-FU, trastuzumab-MCC-DM1 (T-DM1), and fixed ratio combinations of 5-FU and T-DM1.

Figure 19 shows a graph of the viability of BT-474 cells in vitro on day 3 from multiple concentrations of IC ₅₀ 5-FU, trastuzumab-MCC-DM1 (T-DM1) and combinations with a ratio of fixed concentrations of 5-FU and T- DM1.

FIG. 20 is a graph of day 3 in vitro viability of SK-BR-3 cells versus multiple IC ₅₀ concentrations of gemcitabine, trastuzumab-MCC-DM1 (T-DM1), and fixed ratio combinations of gemcitabine and T-DM1.

FIG. 21 is a plot of in vitro MDA-MD-361 cell viability at day 3 versus multiples of gemcitabine IC ₅₀ , trastuzumab-MCC-DM1 (T-DM1) and gemcitabine to T-DM1 fixed ratio combinations.

Figure 22 shows a graph of the viability (proliferation) of KPL4 cells in vitro on day 3 after treatment with T-DM1, GDC-0941 and combinations with a ratio of fixed concentrations of 1:10 T-DM1 and GDC-0941 (from 62.5 nm up to 1 μM) at multiple concentrations of IC ₅₀ from 0.25x to 4x. A graph of the prediction of the additive Bliss action was built in the form of a dotted line.

Figure 23 shows a graph of the viability (proliferation) of KPL4 cells in vitro on the 3rd day after treatment with T-DM1, GDC-0941 and combinations with a ratio of fixed concentrations of 1:25 (from 1.25 to 80 ng/ml) and GDC -0941 (from 31.25 nM to 2 μM) at multiples of IC ₅₀ concentrations from 0.0625x to 16x. A graph of the prediction of the additive Bliss action was built in the form of a dotted line.

Figure 24 shows a graph of the viability (proliferation) of KPL4 cells with amplification of Her2, resistant to Herceptin ^® , mutant for PIK3CA (H1047R) in vitro on day 3 after treatment with T-DM1, PI103, GDC-0941 and fixed ratio combinations concentrations of T-DM1+PI103 and T-DM1+GDC-0941 at multiple concentrations of IC ₅₀ from 0 to 16×.

Figure 25 shows a graph of the viability (proliferation) of cells KPL4 caspase 3/7 in vitro conditions 24 hours after treatment with T-DM1, GDC-0941 and combinations with a ratio of fixed concentrations of T-DM1 and GDC-0941 at concentrations of T-DM1 up to 160 ng/ml.

Figure 26 shows a graph of the viability (proliferation) of KPL4 cells in vitro conditions 3 days after treatment with T-DM1, GDC-0941 and combinations with a ratio of fixed concentrations of T-DM1 and GDC-0941 at concentrations of T-DM1 from 0 to 200 ng/ml.

Figure 27 shows a graph of the viability (proliferation) of MDA-0MB-361 cells in vitro on day 3 after treatment with T-DM1, GDC-0941 and combinations with a ratio of fixed concentrations of 1:20 T-DM1 (from 3.125 to 50 ng /ml) and GDC-0941 (from 62.5 nm to 1 μm) at multiple concentrations of IC ₅₀ from 0.125x to 8x. A graph of the prediction of the additive Bliss action was built in the form of a dotted line.

Figure 28 shows a graph of the viability (proliferation) of MDA-0MB-361 cells in vitro on day 3 after treatment with T-DM1, GDC-0941 and combinations with a ratio of fixed concentrations of 1:20 T-DM1 (from 3.125 to 100 ng /ml) and GDC-0941 (from 62.5 nm to 2 μm) at multiple concentrations of IC ₅₀ from 0.125x to 8x. A plot of the prediction of the additive Bliss action was built in the form of a dotted line.

Figure 29 shows a graph of the viability (proliferation) of BT-474 cells in vitro on day 3 after treatment with T-DM1, GDC-0941 and combinations with a ratio of fixed concentrations of 1:10 T-DM1 (from 3.125 to 100 ng / ml ) and GDC-0941 (from 31.25 nM to 1 μM) at multiple IC ₅₀ concentrations from 0.125x to 4x. A plot of the prediction of the additive Bliss action was built in the form of a dotted line.

Figure 30 shows a graph of the viability (proliferation) of BT-474 cells in vitro on day 3 after treatment with T-DM1, GDC-0941 and combinations with a ratio of fixed concentrations of 1:10 T-DM1 (from 6.25 to 100 ng /ml) and GDC-0941 (from 62.5 nm to 1 μm) at multiple concentrations of IC ₅₀ from 0.25x to 4x. A graph of the prediction of the additive Bliss action was built in the form of a dotted line.

Figure 31 shows a graph of the viability (proliferation) of AU565 cells with Her2 amplification, mutant without PIK3 under in vitro conditions on day 3 after treatment with T-DM1, PI103, GDC-0941 and combinations with a ratio of fixed concentrations of T-DM1 + PI103 and T -DM1+GDC-0941 at multiple IC ₅₀ concentrations from 0 to 16×.

Figure 32 shows a graph of the viability (proliferation) of EFM192A cells with Her2 amplification, mutant for PIK3CA (C420R) in vitro on day 3 after treatment with T-DM1, PI103, GDC-0941 and combinations with a ratio of fixed concentrations of T-DM1 + PI103 and T-DM1+GDC-0941 at multiple IC ₅₀ concentrations from 0 to 16×.

Figure 33 shows a graph of the viability (proliferation) of Her2-resistant HCC1954 cells with amplification of Herceptin ^® mutant for PIK3CA (H1047R) in vitro after treatment with T-DM1, PI103, GDC-0941 and combinations with a ratio of fixed concentrations of T- DM1+PI103 and T-DM1+GDC-0941 at multiple IC ₅₀ concentrations from 0 to 16×.

34 is a plot of mean tumor volume over time in vivo for MMTV-Her2 Fo5 transgenic tumors implanted in the mammary fat pad of CRL nu/nu mice following administration of: (1) diluent po, once daily ×21; (2) T-DM1 at 10 mg/kg IV once every 3 weeks; (3) 5-FU at 100mg/kg po once a week ×2; (4) T-DM1 5mg/kg iv once every 3 weeks + 5-FU 100mg/kg po once a week ×2.

35 is a plot of mean tumor volume over time in vivo for MMTV-Her2 Fo5 transgenic tumors implanted in the mammary fat pad of CRL nu/nu mice following administration of: (1) diluent po, once a day ×21; (2) T-DM1 at a dose of 5 mg/kg IV, once a day ×1; (3) GDC-0941 at 100 mg/kg po once a day ×21; (4) GDC-0152 dose of 50mg/kg orally once a week ×3; (5) T-DM1 at 5mg/kg iv once a day x1 + GDC-0941 at a dose of 100mg/kg po once a day x21; (6) T-DM1 5mg/kg iv once a day x1 + GDC-0152 50mg/kg po once a week x3.

36 is a plot of mean tumor volume over time in vivo for MDA-MB-361.1 tumors implanted in the mammary fat pad of CRL nu/nu mice following administration of: (1) diluent po, once daily ×21; (2) GDC-0941 at 25 mg/kg po once a day x21; (3) GDC-0941 at 50 mg/kg po once a day ×21; (4) GDC-0941 at 100 mg/kg po once a day ×21; (5) T-DM1 at a dose of 3 mg/kg IV, once a day ×1; (6) T-DM1 at a dose of 10 mg/kg IV, once a day ×1; (7) GDC-0941 at 25mg/kg po once a day x21 + T-DM1 at a dose of 3mg/kg iv once a day x1; (8) GDC-0941 at 50mg/kg po once a day x21 + T-DM1 at a dose of 3mg/kg iv once a day x1; (9) GDC-0941 at 100mg/kg po once a day x21 + T-DM1 at a dose of 3mg/kg iv once a day x1; (10) GDC-0941 at 25mg/kg po once a day x21 + T-DM1 at a dose of 10mg/kg iv once a day x1; (11) GDC-0941 at 50mg/kg po once a day x21 + T-DM1 at a dose of 10mg/kg iv once a day x1; (12) GDC-0941 at 100mg/kg po once a day x21 + T-DM1 at a dose of 10mg/kg iv once a day x1.

37 is a plot of mean tumor volume over time in vivo for MDA-MB-361.1 tumors implanted in the mammary fat pad of CRL nu/nu mice following administration of: (1) solvents [MCT (0.5 % methylcellulose/0.2% tween 80) + succinate buffer (100 mM sodium succinate, 100 mg/mL trehalose, 0.1% tween 80, pH 5.0)] po + iv once a day ×21 and once a day; (2) GNE-390 at 1.0 mg/kg po once a day x21; (3) GNE-390 at 2.5 mg/kg, po, once a day ×21; (4) T-DM1 at 3 mg/kg IV, once a day; (5) GNE-390 at 1.0 mg/kg po once a day ×21 + T-DM1 at 3 mg/kg iv once a day; (6) GNE-390 at 2.5mg/kg po once a day ×21 + T-DM1 at 3mg/kg iv once a day.

Detailed Description of the Preferred Embodiments

In the following, some embodiments of the invention will be described in detail, examples of which are given with the accompanying structures and formulas. While the invention will be described in conjunction with the listed embodiments, it is to be understood that they are not intended to limit the invention to these embodiments. On the contrary, the invention is intended to include all alternatives, modifications, and equivalents that may be included within the scope of the present invention as defined by the claims. Those skilled in the art will appreciate that there are many methods and materials similar or equivalent to those described herein that can be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described. In the event that one or more of the included literature, patents, and similar materials differ or conflict with this application, including, but not limited to, the definition of terms, the use of terms, the description of techniques, or the like, then this application controls this.

Definitions

The expressions "comprises", "comprising", "comprises", "comprising" and "includes" as used in this application and the claims are intended to identify the presence of the indicated features, integers, components or steps, but they do not preclude the presence or addition one or more features, integers, components or steps, or groups thereof.

The terms "treat" and "treatment" refer to therapeutic and prophylactic or preventive measures, the purpose of which is to prevent or slow (attenuate) an undesirable physiological change or disorder, such as the growth, development, or spread of a hyperproliferative condition, such as cancer. For the purposes of the present invention, beneficial or desirable clinical results include, but are not limited to, relief of symptoms, reduction in disease severity, maintenance of a stable (i.e., no worsening) disease state, reduction or slowing of disease progression, amelioration or alleviation of the disease state, and remission. (partial or complete), regardless of whether they are detectable or not detectable. Also, the term "treatment" can mean prolonged survival compared to the expected survival compared to the situation in the absence of treatment. Subjects in need of treatment include patients with an existing condition or disorder, as well as subjects prone to developing the condition or disorder, and subjects in whom the condition or disorder should be prevented.

The expression "therapeutically effective amount" means the amount of a compound of the present invention that can (i) treat a particular disease, condition or disorder, (ii) reduce, alleviate or eliminate one or more symptoms of a particular disease, condition or disorder, and (iii) prevent or delay the onset of one or more of the symptoms of a particular disease, condition, or disorder described herein. In the case of a malignant tumor, a therapeutically effective amount of a drug can result in a decrease in the number of tumor cells, a decrease in the size of the tumor, suppression (i.e., slowing to a certain extent and preferably stopping) infiltration of tumor cells into peripheral organs, suppression (i.e., slowing down to to a certain extent and preferably stop) tumor metastasis, inhibition to a certain extent of tumor growth and/or alleviation to a certain extent of one or more symptoms associated with a malignant tumor. The drug may, to a certain extent, prevent tumor growth and/or lead to the death of existing tumor cells, it may be cytostatic and/or cytotoxic. With respect to cancer therapy, efficacy can be assessed, for example, by time to disease progression (TTP) and/or rate of response (RR).

The term "hyperproliferative disorder" refers to tumors, cancers and neoplasms, including premalignant and non-malignant stages, and also includes psoriasis, endometriosis, polyps and fibroadenoma.

The terms "cancer" and "malignant tumor" refer to or describe a physiological condition in mammals that is characterized by uncontrolled cell growth. A "tumor" contains one or more tumor cells. Examples of a malignant tumor include, but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or malignant tumors of the lymphoid tissue. More specific examples of such malignancies include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma), lung cancer, including small cell lung carcinoma, non-small cell lung carcinoma ("NSCLC"), adenocarcinoma of the lung and squamous cell lung carcinoma, peritoneal cancer, hepatocellular carcinoma, stomach cancer, including malignant tumors of the digestive tract, pancreatic cancer, glioblastoma, cervical cancer, ovarian, liver, bladder cancer, hepatoma, breast, colon, rectal cancer, colorectal cancer, endometrial or uterine cancer , cancer of the salivary glands, kidney or excretory organs, vulva, thyroid, liver carcinoma, anal cancer, penile cancer, and malignant tumors of the head and neck.

The term "chemotherapeutic agent" is a chemical compound useful in the treatment of cancer, regardless of the mechanism of action. Groups of chemotherapeutic agents include, but are not limited to: alkylating agents, antimetabolites, plant alkaloids, which are mitotic spindle poisons, cytotoxic/antineoplastic antibiotics, topoisomerase inhibitors, antibodies, photosensitizers, and kinase inhibitors. Chemotherapeutic agents include compounds used in "targeted therapy" and conventional chemotherapy. Examples of chemotherapeutic agents include: erlotinib ( ^Tartceva® , Genentech/OSI Pharm.), docetaxel ( ^Taxotere® , Sanofi-Aventis), 5-FU (fluorouracil, 5-fluorouracil, CAS number 51-21-8), gemcitabine (Gemzar ^® , Lilly), PD-0325901 (CAS number 391210-10-9, Pfizer), cisplatin (dichloroplatinum(II) cis-diamine, CAS number 15663-27-1), carboplatin (CAS number 41575-94- 4), paclitaxel (Taxol ^® , Bristol-Myers Squibb Oncology, Princeton, NJ), trastuzumab (Herceptin ^® , Genentech), temozolomide (4-methyl-5-oxo-2,3,4,6,8-pentabicyclo[4.3 .0]nona-2,7,9-triene-9-carboxamide, CAS number 85622-93-1, temodar ^® , temodal ^® , Shering Plough), tamoxifen ((Z)-2-[4-(1, 2-diphenylbut-1-enyl)phenoxy]-N,N-dimethylethanamine, Nolvadex ^® , Itubal ^® , Valodex ^® ) and doxorubicin (adriamycin ^® ), acti-1/2, HPPD and rapamycin.

Other examples of chemotherapy agents include: oxaliplatin ( ^Eloxatin® , Sanofi), bortezomib ( ^Velcade® , Millennium Pharm.), sutent (Sunitinib®, ^SU11248 , Pfizer), letrozole ( ^Femara® , Novartis), imatinib mesylate ( ^Gleevec® , Novartis) , XL-518 (MEK inhibitor, Exelixis, WO 2007/044515), ARRY-886 (MEK inhibitor, AZD6244, Array BioPharma, Astra Zeneca), SF-1126 (PI3K inhibitor, Semafore Pharmaceuticals), BEZ-235 (PI3K inhibitor, Semafore Pharmaceuticals), PI3K, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787/ZK 222584 (Novartis), fulvestrant (Faslodex ^® , AstraZeneca), leucovorin (folinic acid), rapamycin (sirolimus, rapamune ^® , Wyeth), lapatinib (tikerb ^® , GSK572016, Glaxo Smith Kline), lonafarnib (Sarasar ^TM , SCH 66336, Shering Plough), sorafenib (Nexavar ^® , BAY43-9006, Bayer Labs), gefitinib (Iressa ^® , AstraZeneca), irinotecan (Camtosar ^® , CPT-11 , Pfizer), tipifarnib (Zarnestra ^TM , Johnson&Johnson), abraxane ^TM (cremophor-free), albumin-based nanoparticles with paclitaxel (American Pharmaceutical Partners, Schaumberg, Il), vandetanib (rINN, ZD6474, ^Zaktima® , AstraZeneca), chlorambucil, AG1478, AG1571 (SU 5271; Sugen), temsirolimus ( ^Torisel® , Wyeth), pazopanib (GlaxoSmithKline), canfosfamide ( ^Telsita® , Telik), thioteru, and cyclophosphamide ( ^Cytoxan® , ^Neosar® ); alkylsulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carbokwon, meturedopa and uredopa; ethyleneimines and methylamelamines, including altretamine; triethyleneamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamin; acetogenins (particularly bullatacin and bullatacinone); camptothecin (including the synthetic analog of topotecan); bryostatin; callistatin; CC-1065 (including its synthetic analogues adozelesin, carzelesin and bizelesin); cryptophycins (in particular, cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including synthetic analogues of KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphasine, chlorphosphamide, estramustine, ifosfamide, mechlorethamine, mechlortamine oxide hydrochloride, melphalan, novembichin, fenestrin, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorzotocin, fotemustine, lomustine, nimustine and ranimnustine; antibiotics such as enediine antibiotics (eg calicheamicin, calicheamycin gamma 1I, calicheamycin omegaI1 (Angew Chem. Intl. Ed. Engl., 1994, 33:183-186); dynemycin, dynemycin A; bisphosphonates such as clodronate; esperamycin; as well as neocarzinostatin chromophore and related chromoprotein enediin antibiotics chromophores), aclacinomysins, actinomycin, autramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolin-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rhodorubicin , streptozocin, tubercidin, ubenimes, zinostatin, zorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprin, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxyfluridine, enocytabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; adrenal inhibitors such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraksat; defofamine; demecolcin; diaziquon; elfornitrin; elliptinium acetate; epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainin; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitaerin; pentostatin; phenamet; pararubicin; losoxantrone; podophyllic acid; 2-ethylhydrazide; procarbazine; polysaccharide complex PSK ^® (JHS Natural Products, Eugene, OR); razoxane; rhizoxin; sisofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichloroethylamine; trichothecenes (T-2 toxin, verracurin A, roridin A and anguidin); urethane; vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; hacytosine; arabinoside (Ara-C); cyclophosphamide; thiotepa; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine (navelbin ^® ); novantron; teniposide; etatrexate; daunomycin; aminopterin; capecitabine ( ^Xeloda® , Roche); ibandronate; SRT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid; and pharmaceutically acceptable salts, acids, and derivatives of any of the products listed above.

The term "chemotherapeutic agent" also includes: (i) antihormonal agents that regulate or inhibit the action of hormones on tumors, such as antiestrogen and selective estrogen receptor modulators (SERMs), including, for example, tamoxifen (nolvadex ^® ; tamoxifen citrate), raloxifene, droloxifene, 4-hydroxy tamoxifen, trioxifene, keoxifene, LY117018, onapristone and ^fareston® (toremifin citrate); (ii) aromatase inhibitors that inhibit the enzyme aromatase, which regulates estrogen production in the adrenal glands, such as 4(5)-imidazoles, aminoglutethimide, ^Megaza® (megestrol acetate), ^Aromasin® (exemestane, Pfizer), formestane, fadrozole, rivizor ^® (Vorozol), Femara ^® (Letrozole, Novartis) and Arimidex ^® (Anastrozole, AstraZeneca); (iii) antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide, and goserelin, as well as troxcitabine (an analog of the cytosine nucleoside 1,3-dioxolane); (iv) protein kinase inhibitors such as MEK inhibitors (WO 2007/044515); (v) lipid kinase inhibitors; (vi) antisense oligonucleotides, in particular, which inhibit the expression of genes in signaling pathways involved in abnormal cell proliferation, for example, PKC-alpha, Raf and H-Ras, such as oblimersen (genasens ^® , Genta Inc.); (vii) ribozymes such as inhibitors of VEGF expression (eg ^angiozyme® ) and inhibitors of HER2 expression; (viii) vaccines such as gene therapy based vaccines, eg ^allovectin® , ^leuvectin® and ^vaxid® ; ^proleukin® rIL-2; topoisomerase 1 inhibitors such as lurtothecan ^® ; abarelix ^® rmRH; (ix) anti-angiogenic agents such as bevacizumab ( ^Avastin® , Genentech), and pharmaceutically acceptable salts, acids and derivatives of any of the above products.

Also included in the term "chemotherapeutic agent" are therapeutic antibodies such as alemtuzumab (Campath), bevacizumab ( ^Avastin® , Genentech); cetuximab (Erbitux ^® , Imclone); panitumumab (Vectibix ^® , Amgen), rituximab (Rituxan ^® , Genentech/Biogen Idec), pertuzumab (Omnitarg ^TM , 2C4, Genentech), trastuzumab (Herceptin ^® , Genentech), tositumomab (Bexxar, Corixia), and an antibody-drug conjugate, gemtuzumab ozogamicin ( ^Mylotarg® , Wyeth).

Humanized monoclonal antibodies with therapeutic activity as chemotherapeutic agents in combination with trastuzumab-MCC-DM1 include: alemtuzumab, aplizumab, azelizumab, atlizumab, bapinyuzumab, bevacizumab, bivatuzumab mertansine, cantuzumab mertansine, cedelizumab, sertolizumab pegol, citfusituzumab, cidakulizumab , эфализумаб, эптатузумаб, эрлизумаб, фелвизумаб, фонтолизумаб, гемтузумаб озогамицин, инотузумаб озогамицин, ипилимумаб, лабетузумаб, линтузумаб, матузумаб, меполизумаб, мотавизумаб, мотовизумаб, натализумаб, нимоиузумаб, ноловизумаб, нумавизумаб, окрелизумаб, омализумаб, паливизумаб, пасколизумаб, пекфуситузумаб, пектузумаб , пертузумаб, пекселизумаб, раливизумаб, ранибизумаб, ресливизумаб, реслизумаб, резивизумаб, ровелизумаб, руплизумаб, сибротузумаб, сиплизумаб, сонтузумаб, такатузумаб тетраксетан, тадоцизумаб, тализумаб, тефибазумаб, тоцилизумаб, торазумаб, трастузумаб, тукотузумаб целмолейкин, тукузитузумаб, умавизумаб, уртоксазумаб и ви zilizumab.

The term "metabolite" means a product synthesized as a result of the metabolism of a particular compound or its salt in the body. Compound metabolites can be identified using conventional methods known in the art and their activity assessed using the tests described herein. Such products may be formed, for example, from the oxidation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, enzymatic cleavage, and the like, of the administered compound. Therefore, the invention includes metabolites of the compounds of the invention, including compounds produced by a process comprising contacting a compound of the present invention with a mammal for a period of time sufficient to form its metabolite.

In the sense in which the term "package insert" is used in this document, it refers to the instructions that are usually included in commercial packages of medicinal products containing information about the indications, use, dosage, administration, contraindications and / or warnings regarding the use such medicinal products.

As used herein, the term "pharmaceutically acceptable salt" refers to pharmaceutically acceptable organic or inorganic salts of a compound of the invention. Exemplary salts include, but are not limited to, sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, hydrogen phosphate, isonicotinate, lactate, salicylate, hydrocitrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucuronate, saccharate, formate, benzoate, glutamate, methanesulfonate, "mesylate", ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate (i.e. 1,1' -methylene-bis-(2-hydroxy-3-naphthoate)). Pharmaceutically acceptable salts may contain the inclusion of another molecule such as an acetate ion, a succinate ion, or other counterion. The counterion can be any organic or inorganic group that stabilizes the charge in the parent compound. In addition, pharmaceutically acceptable salts may contain more than one charged atom in their structure. Where multiple charged atoms are part of a pharmaceutically acceptable salt, they may contain multiple counterions. Therefore, a pharmaceutically acceptable salt may contain one or more charged atoms and/or one or more counterions.

Where the compound of the invention is a base, the desired pharmaceutically acceptable salt may be prepared by any suitable method known in the art, for example, by treating the free base with an inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid. , methanesulfonic acid, phosphoric acid and the like, or an organic acid such as acetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, pyruvic acid, oxalic acid, glycolic acid, salicylic acid, pyranosidyl acid, such such as glucuronic acid or galacturonic acid, alpha hydroxy acid such as citric acid or tartaric acid, amino acid such as aspartic acid or glutamic acid, aromatic acid such as benzoic acid or cinnamic acid, sulfonic acid such as p-toluenesulfonic acid or ethanesulfonic acid, or the like. General aspects on acids suitable for preparing pharmaceutically acceptable or acceptable salts from basic pharmaceutical compounds are discussed in, for example, P. Stahl et al., Camille G. (eds.) Handbook of Pharmaceutical Salts. Properties, Selection and Use, 2002, Zurich: Wiley-VCH; S. Berge et al., Journal of Pharmaceutical Sciences, 1977, 66(1), 119; P. Gould, International J. of Pharmaceutics, 1986, 33:201-217; Anderson et al., the Practice of Medicinal Chemistry, 1996, Academic Press, New York; Remington's Pharmaceutical Sciences, 18th ^ed ., 1995, Mack Publishing Co., Easton PA; and in The Orange Book (Food&Drug Administration, Washington, DC on their website). These sources are included in this document for information.

Where the compound of the invention is an acid, the desired pharmaceutically acceptable salt may be obtained by any suitable method, for example, by treating the free acid with an inorganic or organic base such as an amine (primary, secondary, or tertiary), an alkali metal hydroxide, or hydroxide alkaline earth metal or the like. Illustrative examples of suitable salts include, but are not limited to, organic salts derived from amino acids such as glycine and arginine, ammonia, primary, secondary and tertiary amines and cyclic amines such as piperidine, morpholine and piperazine, and inorganic salts derived from sodium. , calcium, potassium, magnesium, manganese, iron, copper, zinc, aluminum and lithium.

The phrase "pharmaceutically acceptable" indicates that the compound or composition may be chemically and/or toxicologically compatible with the other ingredients in the composition or with the mammal being treated with them.

The term "solvate" refers to a physical association or complex of one or more solvent molecules and a compound of the invention. The compounds of the invention may be in both unsolvated form and solvated form. Examples of solvents that form solvates include, but are not limited to, water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid, and ethanolamine. The term "hydrate" refers to a complex in which the solvent molecule is water. This physical association has various degrees of ionic and covalent bonding, including hydrogen bonds. In some cases, the solvate can be isolated, for example, when one or more solvent molecules enter the crystal lattice of a crystalline solid. In general, the preparation of solvates is known, for example, it is described by M. Caira et al., J. Pharmaceutical Sci., 93(3), 601-611, 2004. Similar methods for the preparation of solvates, hemisolvates, hydrates and the like are described by E.C. van Tonder et al., AAPS Pharm. sci. Tech., 5(1), article 12, 2004 and A.L. Bingham et al., Chem. Commun., 603-604, 2001. A typical, non-limiting method involves dissolving a compound of the invention in the desired amount of the desired solvent (organic solvent or water, or mixtures thereof) at a temperature higher than room temperature and cooling the solution at a rate sufficient to the formation of crystals, which are then isolated by conventional methods. Analytical methods, such as IR spectroscopy, determine the presence of a solvent (or water) in the crystals in the form of a solvate (or hydrate).

In the sense in which the term "synergistic" is used in this document, it refers to a therapeutic combination that is more effective than the additive action of two or more agents alone. The determination of a synergistic interaction between trastuzumab-MCC-DM1 and one or more chemotherapeutic agents may be based on the results of the tests described herein. The results of these tests are analyzed using the Chou and Talalay combination method and dose-response analysis using the CalcuSyn program to determine the combination index "CI" (Chou and Talalay, 1984, Adv. Enzyme Regul., 22:27-55). The combinations of the present invention have been evaluated in several assay systems and the data can be processed using a standard program to quantify synergy, additive effect and antagonism in antineoplastic agents. Preferably, the program described by Chou and Talalay in "New Avenues in Developmental Cancer Chemotherapy", Academic Press, 1987, chapter 2, is used. Combination Index (CI) values below 0.8 indicate synergism, values above 1.2 indicate antagonism and values ranging from 0.8 to 1.2 indicate an additive effect. Combination therapy can provide "synergy" and be "synergistic", ie. when the effect achieved when the active ingredients are used together is higher than the sum of the effects provided using the compounds separately. A synergistic effect can be achieved when the active ingredients are: (1) formulated together and administered or delivered simultaneously in a combined single dosage form; (2) administered sequentially as separate dosage forms, or (3) administered in a different schedule. When administered sequentially, a synergistic effect can be achieved when the compounds are administered or delivered sequentially, for example by different injections in separate syringes. In general, during "sequential" therapy, an effective dose of each active ingredient is administered sequentially, for example, alternately in time.

Trastuzumab-MCC-DM1

The present invention relates to therapeutic combinations containing trastuzumab-MCC-DM1(T-DM1), an antibody drug conjugate (CAS number 139504-50-0) having the formula:

where Tr is trastuzumab linked via an MCC linker to the maytansinoid drug moiety, DM1 (US Pat. No. 5,208,020; US Pat. No. 6,441,163). The drug to antibody ratio or drug load is represented by the "p" value in the above structure of trastuzumab-MCC-DM1, and the range of integer values is from 1 to about 8. The "p" drug load value is 1-8. Trastuzumab-MCC-DM1 contains all mixtures of variably loaded and attached antibody-drug conjugates, where 1, 2, 3, 4, 5, 6, 7, and 8 drug groups are covalently attached to the trastuzumab antibody (U.S. Patent No. 7,097,840; application for US Patent No. 2005/0276812; US Patent Application No. 2005/0166993). The trastuzumab-MCC-DM1 conjugate can be prepared following the method described in Example 1.

Trastuzumab is produced in suspension culture of mammalian cells (Chinese hamster ovary cells, CHO). The HER2 (or c-erbB2) proto-oncogene encodes a 185 kDa transmembrane receptor protein that is structurally similar to the epidermal growth factor receptor. Overexpression of the HER2 protein is noted in 25-30% of primary breast cancers and can be detected using an immunohistochemical method based on the analysis of fixed tumor blocks (Press MF et al., 1993, Cancer Res., 53:4960-70). Trastuzumab is an antibody containing antigen-binding residues or derived from the mouse antibody 4D5 (ATCC CRL 10463, which is deposited with the American Type Culture Collection, 12301 Parklawn Drive, Rockville, Md. 20852 under the Budapest Agreement of May 24, 1990). Exemplary humanized 4D5 antibodies include huMAb4D5-1, huMAb4D5-2, huMAb4D5-3, huMAb4D5-4, huMAb4D5-5, huMAb4D5-6, huMAb4D5-7, and huMAb4D5-8 (Herceptin®) described in US Pat. No. ^5,821,337 .

In phase I clinical trials, the maximum tolerated dose (MTD) of T-DM1, given by intravenous infusion every 3 weeks, was found to be 3.6 mg/kg. The DLT (dose-limiting toxicity) value for grade 4 thrombocytopenia in 2 out of 3 patients is 4.8 mg/kg. Grade ≥2 side effects observed at the 3.6 mg/kg dose were rare and minor. This treatment regimen was well tolerated by patients and provided a good clinical effect, as described previously. In phase II clinical trials, the dose of 3.6 mg/kg was shown to be well tolerated when administered every 3 weeks with a small percentage of patients (3 out of 112 patients) requiring dose reduction of the agent. Thus, a dose of T-DM1 of 3.6 mg/kg every 3 weeks was chosen for testing in the present study based on the following: (1) T-DM1 at a dose of 3.6 mg/kg every 3 weeks and (2) for this group of patients, a regimen once every 3 weeks is convenient.

Chemotherapeutic agents

Several chemotherapeutic agents have been shown to exhibit surprising and unexpected properties in combination with trastuzumab-MCC-DM1 in inhibiting cell proliferation in vitro and in vivo. Such chemotherapeutic agents include HER2 dimerization inhibitor antibody, anti-VEGF antibody, 5-FU, carboplatin, lapatinib, ABT-869, docetaxel, GDC-0941 and GNE-390.

Pertuzumab (CAS number 380610-27-5, ^Omnitarg® , 2C4, Genentech) is a recombinant, humanized monoclonal antibody that inhibits HER2 dimerization (US Pat. No. 6,054,297; US Pat. No. 6,407,213; US Pat. No. 6,800,738; US Pat. US patent No. 7041292). Pertuzumab and trastuzumab target different extracellular regions of the HER-2 tyrosine kinase receptor (Nahta et al., 2004, Cancer Res., 64:2343-2346). A hybridoma cell line expressing 2C4 (pertuzumab) is deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, Va. 20110-2209, USA as ATCC HB-12697 April 8, 1999. Pertuzumab blocks the ability of the HER2 receptor to bind to other members of the HER2 receptor family, ie. HER1/EGFR, HER3 and HER4 (Agus et al. 2002 Cancer Cell 2:127-137; Jackson et al. 2004 Cancer Res. 64:2601-2609; Takai et al. 2005 Cancer, 104:2701-2708; US Pat. No. 6,949,245). In tumor cells, disruption of the ability of HER2 to interact with other members of the HER receptor family leads to blockage of cell signaling and can ultimately lead to suppression of tumor cell growth and tumor cell death. HDIs, due to their unique mechanism of action, are able to function in a wide range of tumors, including tumors without HER2 overexpression (Mullen et al., 2007, Molecular Cancer Therapeutics, 6:93-100).

Pertuzumab is based on human IgG1 (κ) framework region sequences. It consists of two heavy chains and two light chains. Like trastuzumab, pertuzumab targets the extracellular domain of HER2. However, it differs from trastuzumab in its light chain and heavy chain epitope-binding regions. As a result, pertuzumab binds to an epitope within that is known as subdomain 2 of HER2, while the epitope from trastuzumab resides in subdomain 4 (Cho et al., 2003; Franklin et al., 2004). Pertuzumab functions by blocking the association of HER2 with other members of the HER family, including HER1 (epidermal growth factor receptor; EGFR), HER3, and HER4. This association is required for signaling in the presence of a ligand via MAP kinase and PI3 kinase. As a result, pertuzumab inhibits ligand-initiated intracellular signaling. Inhibition of these signaling pathways can lead to growth arrest and apoptosis, respectively (Hanahan and Weinberg, 2000). As a result of pertuzumab and trastuzumab binding to different epitopes at the HER2 receptor, ligand-activated signaling downstream is blocked by pertuzumab, but not by trastuzumab. Therefore, pertuzumab does not require HER2 overexpression to exhibit its anticancer activity. In addition, as a result of their complementary mechanisms of action, the combination of pertuzumab and T-DM1 has the potential to be used in the treatment of diseases in which there is overexpression of HER2.

Pertuzumab was evaluated as a single agent in five Phase II clinical trials in a variety of cancers, including low HER2-expressing MBC, non-small cell lung carcinoma, hormone-resistant prostate cancer, and ovarian cancer. Phase II clinical trials evaluated the efficacy of pertuzumab as a standalone drug in second or third line chemotherapy in patients with metastatic breast cancer (MBC) with normal expression of HER2 (Cortes et al., 2005, J. Clin. Oncol., 23:3068 ). The effectiveness of pertuzumab in phase II clinical trials in combination with trastuzumab was evaluated (Baselga J. et al., “A Phase II trial of trastuzumab and pertuzumab in patients with HER-2-positive metastatic breast cancer that had progressed during trastuzumab therapy: full response data ", European Society of Medical Oncology, Stockholm, Sweden, September 12-16, 2008; Gelmon et al., 2008, J. Clin. Oncol., 26:1026). The first study included 11 HER-positive MBC patients who had previously received up to three courses of treatment including trastuzumab (Portera et al., 2007).

Bevacizumab (CAS number 216974-75-3, Avastin ^® , Genentech) is a monoclonal antibody to VEGF, to vascular endothelial growth factor (US patent No. 7227004; US patent No. 6884879; US patent No. 7060269; US patent No. 7169901; patent US No. 7297334), the drug is used to treat malignant tumors, where it inhibits their growth by blocking the formation of new blood vessels. Bevacizumab was the first US clinical angiogenesis inhibitor approved by the FDA in 2004 for use in combination with conventional chemotherapy for the treatment of metastatic colon cancer and most forms of metastatic non-small cell lung carcinoma. Several end-stage clinical trials have been conducted to evaluate its safety and efficacy in patients with adjuvant/non-metastatic colon cancer, metastatic breast cancer, metastatic kidney cancer, metastatic glioblastoma multiforme, metastatic ovarian cancer, metastatic hormone-resistant prostate cancer, and metastatic or inoperable locally advanced pancreatic cancer.

Typically, an anti-VEGF antibody will not bind to other VEGF homologues, such as VEGF-B or VEGF-C, and to other growth factors, such as PIGF, PDGF, or bFGF. Preferred anti-VEGF antibodies include a monoclonal antibody that binds to the same epitope as the anti-VEGF monoclonal antibody A4.6.1 produced by ATCC HB 10709 hybridoma; recombinant humanized anti-VEGF monoclonal antibody prepared as described by Presta et al. 1997, Cancer Res., 57:4593-4599, including but not limited to bevacizumab. Bevacizumab contains mutant human IgG1 framework regions and antigen-binding complementarity-determining regions from the mouse monoclonal anti-hVEGF antibody A4.6.1, which blocks the binding of human VEGF to its receptors. Approximately 93% of the amino acid sequence of bevacizumab, including most of the framework regions, is derived from human IgG1 and approximately 7% of the sequence is derived from the mouse antibody A4.6.1. Bevacizumab has a molecular weight of approximately 149,000 daltons and is glycosylated. Bevacizumab and other humanized anti-VEGF antibodies are detailed in US Pat. international application WO2005/012359. In one embodiment, the B20 series antibody binds to a functional epitope in human VEGF containing residues F17, M18, D19, Y21, Y25, Q89, I91, K101, E103, and C104.

Hybridoma lines expressing anti-VEGF antibodies A4.6.1 (ATCC HB 10709) and B2.6.2 (ATCC HB 10710) have been deposited with the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110-2209, USA. A clone expressing a VEGF-E polypeptide (U.S. Patent No. 6,391,311) encoded by the ATCC deposit nucleotide sequence insert DNA29101-1276 deposited March 5, 1998 and stored at ATCC 209653 at the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas , Va. 20110-2209, USA.

5-FU (fluorouracil, 5-fluorouracil, CAS number 51-21-8) is a thymidylate synthase inhibitor and has been used for decades in the treatment of cancer, including colorectal and pancreatic cancer (U.S. Patent No. 2,802,005; U.S. Patent No. 2885396; Barton et al., 1972, Jour. Org. Chem., 37:329; Hansen, R. M., 1991, Cancer Invest., 9:637-642). 5-FU is 5-fluoro-1H-pyrimidine-2,4-dione and it has the formula:

Carboplatin (CAS number 41575-94-4) is a chemotherapeutic agent that is used to treat malignant tumors of the ovaries, lungs, head and neck (US patent No. 4140707). Carboplatin is Azanide; cyclobutane-1,1-dicarboxylic acid platinum and has the formula:

Lapatinib (CAS number 388082-78-8, tickerb ^® , GW572016, Glaxo SmithKline) is approved for use in combination with capecitabine (Xeloda ^® , Roche) for the treatment of patients with locally advanced or metastatic breast cancer with HER2 overexpression ( ErbB2) and patients previously treated with anthracycline, taxane, and trastuzumab. Lapatinib is an ATP-competitive epidermal growth factor (EGFR) and dual Her2/neu (ErbB-2) tyrosine kinase inhibitor US No. 7157466; US patent No. 7141576), which inhibits autophosphorylation and activation of receptors through binding to the ATP-binding site of the EGFR/HER2 protein kinase domain. Lapatinib is N-(3-chloro-4-(3-fluorobenzyloxy)phenyl)-6-(5-((2-(methylsulfonyl)ethylamino)methyl)furan-2-yl)quinazolin-4-amine and it has the formula :

ABT-869 (Abbott and Genentech) is a multi-target inhibitor of the VEGF and PDGF tyrosine kinase receptor family for potential oral cancer treatment (US Patent No. 7297709; US Patent Application No. 2004/235892; US Patent Application No. 2007/104780). Started clinical trials of the drug in the treatment of non-small cell lung cancer (NSCLC), hepatocellular carcinoma (HCC) and renal cell carcinoma (RCC). Chemically, ABT-869 is 1-(4-(3-amino-1H-indazol-4-yl)phenyl)-3-(2-fluoro-5-methylphenyl)urea (CAS number 796967-16-3 ) and has the formula:

Docetaxel ( ^Taxotere® , Sanofi-Aventis) is used to treat breast, ovarian, and NSCLC cancer (US Patent No. 4,814,470; US Patent No. 5,438,072; US Patent No. 5,968,582; US Patent No. 5,714,512; US Patent No. 5,750,561). Docetaxel is (2R,3S)-N-carboxy-3-phenylisoserine N-tert-butyl ester, 5,20-epoxy-1,2,4,7,10,13-hexahydroxytax-11-ene-9 13-ester -one 4-acetate-2-benzoate trihydrate (US patent No. 4814470; European patent 253738; CAS number 114977-28-5) and has the formula:

GDC-0941 (Genentech Inc.) is a selective, good oral bioavailability thienopyrimidine PI3K inhibitor with promising pharmacokinetic and pharmaceutical properties (Folkes et al., 2008, J. Med. Chem., 51(18):5522-5532 ; US Patent Application No. 2008/0076768; US Patent Application No. 2008/0207611; Belvin et al., American Association for Cancer Research Annual Meeting 2008, 99 ^th :April 15, Abstract 4004; Folkes et al., American Association for Friedman et al., American Association for Cancer Research Annual Meeting 2008, ^99th : ^April 15, Abstract LB-110). GDC-0941 demonstrates synergistic activity in vitro and in vivo in combination with certain chemotherapeutic agents in solid tumor cell lines (U.S. Patent Application No. 12/208227, Belvin et al. "Combinations of phosphoinositide 3-kinase inhibitor compounds and chemotherapeutic agents and methods of use", filed September 10, 2008). Chemically, GDC-0941 is 4-(2-(1H-indazol-4-yl)-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno[3,2-d]pyrimidin-4 -yl)morpholine (CAS number 957054-30-7) and has the formula:

GNE-390 (Genentech Inc.) is a selective, good oral bioavailability thienopyrimidine PI3K inhibitor with promising pharmacokinetic and pharmaceutical properties (U.S. Patent Application No. 2008/0242665; International Application WO 2008/070740). GNE-390 demonstrates synergistic activity in vitro and in vivo in combination with certain chemotherapeutic agents in solid tumor cell lines (U.S. Patent Application No. 12/208227, Belvin et al. "Combinations of phosphoinositide 3-kinase inhibitor compounds and chemotherapeutic agents and methods of use", filed September 10, 2008). GDC-390 is (S)-1-(4-((2-(2-aminopyrimidin-5-yl)-7-methyl-4-morpholinothieno[3,2-d]pyrimidin-6-yl)methyl)piperazine -1-yl)-2-hydroxypropan-1-one and has the formula:

Assessment of biological activity

In vitro cell culture studies using trastuzumab-MCC-DM1(T-DM1) in combination with various chemotherapeutic agents or biologically targeted agents were performed on a range of HER2 amplifying cell lines. Data were analyzed using the Chou & Talalay method to determine the combination index (CI) for each combination at multiples of the IC ₅₀ values for each drug. CI values below 0.7 indicate synergy; CI in the range of 0.7-1.3 is an additive effect and CI values above 1.3 are antagonism. With respect to combinations with chemotherapeutic agents, T-DM1 in combination with docetaxel or 5-FU resulted in additive or synergistic antiproliferative activity, while combinations with gemcitabine or carboplatin were inactive or antagonistic against T-DM1. In experiments with mouse xenografts, similar results were obtained, where T-DM1 in combination with docetaxel or 5-FU provided significantly higher antitumor activity compared to these agents alone. T-DM1 in combination with carboplatin provided higher efficacy compared to drugs alone, while the combination of T-DM1 with gemcitabine was not more effective than T-DM1 alone. T-DM1 in combination with pertuzumab, lapatinib, or GDC-0941 resulted in an additive or synergistic antiproliferative effect on cell cultures, and antitumor activity was significantly increased in vivo compared to treatment with the drugs alone. In contrast, unconjugated trastuzumab was T-DM1 antagonistic by binding to the same epitope on HER2. In vivo experiments with T-DM1 in combination with anti-angiogenic agents such as the B20-4.1 antibody or the small molecule inhibitor ABT-869 have been shown to enhance antitumor efficacy with all combinations tested except for the highest dose of T-DM1 (10 or 15 mg/kg) in combination with B20-4.1.

The effectiveness of combinations of trastuzumab-MCC-DM1(T-DM1) and numerous antitumor agents was evaluated by determining their antiproliferative activity in in vitro models on HER2 overexpressing breast tumor cells and by determining in vivo antitumor efficacy in breast cancer xenografts. glands. In these experiments, trastuzumab-MCC-DM1 was added to cytotoxic chemotherapeutic agents, antibodies or small molecule kinase inhibitors.

Combination of mouse anti-VEGF antibody B20-4.1 (Liang et al., 2006, J. Biol. Chem., 281:951-961), a bevacizumab analog, and trastuzumab-MCC-DM1 in a breast cancer xenograft model tested in mice showed higher antitumor activity compared to B20-4.1 alone. The results of these studies provide a predictive basis for synergistic effects and rationale for future clinical trials of treatment regimens that include trastuzumab-MCC-DM1 with various anticancer agents in HER-2-positive breast cancer.

Synergistic effects have been observed with combinations of agents targeting HER2 such as trastuzumab-DM1 plus lapatinib or trastuzumab-DM1 plus the anti-HER2 antibody pertuzumab (an inhibitor of HER2 dimerization).

Trastuzumab-MCC-DM1 in combination with carboplatin or 5-FU showed increased activity compared with treatment with these agents alone, while combination treatment with gemcitabine did not lead to an increase in antitumor activity.

Blockade of the PI3 kinase pathway using GDC-0941, a small molecule p110 isoform kinase inhibitor (WO 2007/129161), enhanced the activity of trastuzumab-DM1.

The use of T-DM1 in combination with the PI3K inhibitor GDC-0941 led to an increase in antitumor activity on HER2-expressing PI3K mutant breast cancer cell lines: BT-474 (K111N), MDA-361.1 (E545K), and KPL4 (H1047R). Combined in vitro treatment resulted in additive or synergistic inhibition of cell proliferation as well as increased apoptosis. Similarly, in vivo efficacy was enhanced by combined drug treatment compared to the activity of drugs alone in MDA-MB-361.1 models and Fo5 xenografts with HER2 amplification. The results of biochemical determination of biomarkers for each drug showed that there was an inhibition of phospho-Akt and phospho-ERK by both T-DM1 and GDC-0941, a decrease in Rb and PRAS40 phosphorylation by GDC-0941, and an increase in the level of mitotic markers of phospho-histone H3 and cyclin B1 after treatment with T-DM1. In addition, administration of T-DM1 resulted in cell apoptosis in these breast cancer models as measured by the 23 kDa PARP cleavage fragment, decreased Bcl-XL levels, and activated caspases 3 and 7. Addition of GDC-0941 to T-DM1 additionally induced an increase in apoptosis. These studies provided an evidence base for the use of rational drug combinations in the treatment of HER2-amplifying breast cancer and proposals for additional therapeutic approaches for patients who experience disease progression on trastuzumab or lapatinib therapy.

In vitro cell proliferation tests

The activity of trastuzumab-MCC-DM1 combinations with chemotherapeutic agents was evaluated in vitro using the cell proliferation assay described in Example 2; commercially available CellTiter- ^Glo® Luminescent Cell Viability Test from Promega Corp., Madison, WI. This homogeneous assay is based on recombinant expression of Coleoptera luciferase (US Pat. No. 5,583,024; US Pat. No. 5,674,713; US Pat. No. 5,700,670) and determination of the number of viable cells in culture by the amount of ATP present, an indicator of metabolically active cells (Crouch et al., 1993, J Immunol Meth 160:81-88, US Pat. No. 6,602,677). The CellTiter- ^Glo® assay was run in 96- or 384-well plate format, making them suitable for automated high throughput screening (HTS) (Cree et al., 1995, AntiCancer Drugs, 6:398-404). Homogeneous assay involves adding one reagent (CellTiter- ^Glo® reagent) directly to cells cultured in serum-supplemented medium. Cell washing, media removal and multiple pipetting steps are not required. This system can detect as little as 15 cells/well in a 384-well plate within 10 minutes of reagent addition and mixing.

The homogeneous add-mix-detect format results in cell lysis and generation of a luminescent signal that is directly proportional to the amount of ATP present. The amount of ATP is directly proportional to the number of cells in culture. The CellTiter-Glo ^® assay reproduces a luminescent luminescence-type signal produced by a reaction involving luciferase, which has a half-life greater than 5 hours, depending on the cell type and medium used. Viable cells are reflected in relative luminescence units (RLU). The beetle luciferin substrate undergoes oxidative decarboxylation, which is catalyzed by recombinant firefly luciferase, with simultaneous conversion of ATP to AMP and generation of photons. As a result of the long half-life, the need for injectors is eliminated and flexibility is provided for continuous or batch processing of multiple plates. This cell proliferation assay can be used in a variety of multi-well formats, such as 96- or 384-well format. Data can be recorded using a luminometer or an imaging device with a CCD camera. The luminescence output is expressed in relative light units (RLU) over time.

The antiproliferative effects of trastuzumab-MCC-DM1 and combinations with chemotherapeutic agents were evaluated using the CellTiter- ^Glo® test (Example 2) on tumor cell lines, the results are shown in FIG. 1-9 and 18-33.

Exemplary embodiments include a method for identifying compounds for use in combination for the treatment of cancer, comprising: (a) administering a therapeutic combination of trastuzumab-MCC-DM1 (T-DM1) and a chemotherapeutic agent to a tumor cell line in vitro, and (b ) determination of synergistic or non-synergistic effect. A combination index (CI) value above 1.3 indicates antagonism; a CI value ranging from 0.7 to 1.3 indicates an additive effect, and a CI value below 0.7 indicates a synergistic drug interaction.

Figure 1 shows the antagonistic effect of trastuzumab in combination with trastuzumab-MCC-DM1 (T-DM1) at various concentrations at multiples of individual IC ₅₀ values (Table 1) on trastuzumab-responsive SK-BR-3 cells. Built a graph of the number of viable cells against multiple values of IC ₅₀ . The combination index (CI) in the range from IC ₁₀ to IC ₉₀ for each combination is greater than 2, indicating the presence of an antagonistic effect in vitro. However, the T-DM1+trastuzumab combination did not show antagonistic activity in vivo.

Table 1 Proliferation of SK-BR-3 cells - 3 days Multiples of IC ₅₀ Trastuzumab ng/ml T-DM1 ng/ml Effect (%) CI 0.5 X 20.57 2.28 5.1 >2 1 x 61.72 6.86 26.2 >2 2 X 185.19 20.58 36.3 >2 4 X 555.56 61.73 43.6 >2 8 X 1666.67 185.19 45.0 >2 16 X 5000 555.56 41.7 >2

Figure 2 shows the antagonistic effect of trastuzumab in combination with trastuzumab-MCC-DM1 (T-DM1) at various concentrations at multiples of individual IC ₅₀ values (Table 2) on BT-474 EEI cells that are resistant to trastuzumab. Built a graph of the number of viable cells from multiple values of IC ₅₀ . The combination index (CI) in the range from IC ₁₀ to IC ₉₀ for each combination is above 2, indicating an antagonistic effect.

table 2 Proliferation of BT-474-EEI cells - 3 days Multiples of IC ₅₀ Trastuzumab ng/ml T-DM1 ng/ml Effect (%) CI 0.125 X 1.52 1.52 9.5 >2 0.25 X 4.57 4.57 4.5 >2 0.5 X 13.71 13.71 3.1 >2 1 X 41.15 41.15 12.1 >2 2 X 123.46 123.46 10.8 >2 4X 370.4 370.4 11.6 >2 8 X 1111.1 1111.1 18.4 >2

Figure 3 shows the synergistic effect of pertuzumab in combination with trastuzumab-MCC-DM1 (T-DM1) at various concentrations at multiples of individual IC ₅₀ values (Table 3) on MDA-MB-175 cells. Built a graph of the number of viable cells from multiple values of IC ₅₀ . The combination index (CI) in the range from IC ₁₀ to IC ₉₀ for each combination is below 1, indicating the presence of a synergistic effect.

Table 3 Proliferation of MDA-MB-175 cells - 5 days Multiples of IC ₅₀ Pertuzumab ng/ml T-DM1 ng/ml Effect (%) CI 0.0625 X 39.06 31.25 21.1 0.2 0.125 X 78.13 62.5 33.3 0.107 0.25 X 156.3 125 21.9 .766 0.5 X 312.5 250 33.6 0.597 1 x 625 500 50.7 0.391 2 X 1250 1000 67.7 0.259

Figure 3a is a graph of day 5 in vitro cell viability versus multiple IC ₅₀ concentrations of pertuzumab, trastuzumab-MCC-DM1 (T-DM1), and the combination of pertuzumab and T-DM1. Built a graph of the number of viable cells from multiple values of IC ₅₀ . The combination index (CI) ranging from IC ₁₀ to IC ₉₀ for each combination is below 1 with a mean CI=0.096 indicating synergistic effect (Table 3a).

Table 3a Proliferation of MDA-MB-175 cells - 5 days Multiples of IC ₅₀ Effect (%) CI 0.0625x 21.3 0.093 0.125x 37.5 0.037 0.25x 40.1 0.060 0.5x 50.3 0.052 1x 53.9 0.078 2x 57.0 0.120 4x 65.5 0.117 8x 66.8 0.208

4 is a graph of day 5 in vitro viability of BT-474 cells versus various fixed concentrations of pertuzumab in combination with trastuzumab-MCC-DM1 (T-DM1) response concentration and various concentrations of T-DM1 alone. Figure 4 shows the effect of fixed concentrations of T-DM1 in combination with various concentrations of pertuzumab. The addition of pertuzumab to T-DM1 results in slightly higher antiproliferative activity compared to T-DM1 alone.

Figure 5 is a graph of day 5 in vitro viability of BT-474 cells versus various fixed concentrations of trastuzumab-MCC-DM1 (T-DM1) in combination with pertuzumab response concentration and various concentrations of pertuzumab alone. Figure 5 shows the effect of fixed concentrations of pertuzumab in combination with various concentrations of T-DM1 on the proliferation of BT-474 cells. The addition of T-DM1 to pertuzumab results in an enhanced effect of pertuzumab alone.

Figure 6 shows the synergistic effect of pertuzumab in combination with trastuzumab-MCC-DM1 at various concentrations at multiples of individual IC ₅₀ values (Table 4) on BT-474 cells. Built a graph of the number of viable cells from multiple values of IC ₅₀ . The combination index (CI) in the range from IC ₁₀ to IC ₉₀ ranges from 0.198 to 1.328. Mean CI for this limit = 0.658, indicating a synergistic effect.

Table 4 Proliferation of BT-474 cells - 5 days Multiples of IC ₅₀ Pertuzumab ng/ml T-DM1 ng/l Effect (%) CI 0.25 X 34.29 11.43 3.9 >2 0.5 X 102.88 34.29 2.0 >2 1 x 308.64 102.88 58.9 0.198 2 X 925.93 308.64 64.6 0.449 4 X 2777 926 64.9 1.328

Figure 7 shows a graph of the viability of SK-BR-3 cells in vitro on day 3 from various concentrations of T-DM1 in combination with fixed concentrations of lapatinib (4.5 nM; 14 nM; 41 nM; 123 nM) and various concentrations of one T-DM1 (0-1000 ng/ml). The addition of T-DM1 to lapatinib resulted in some increased antiproliferative activity compared to T-DM1 alone.

Figure 7a is a graph of day 3 in vitro viability of SK-BR-3 cells versus multiples of IC ₅₀ T-DM1, lapatinib, and T-DM1 to lapatinib fixed-ratio combinations as shown in Table 7a. Mean CI between IC ₁₀ and IC ₉₀ = 0.793 indicating an additive effect.

Table 7a Proliferation of SK-BR-3 cells - 3 days Multiples of IC ₅₀ Lapatinib nM T-DM1 ng/ml Effect (%) CI 0.25x 4.57 1.52 3.5 >2 0.5x 13.72 4.57 22.0 1.384 1x 41.15 13.72 52.5 0.596 2x 123.44 41.15 75.9 0.406 4x 370.33 123.44 81.1 0.787 8x 1111 370.33 80.1 >2

Figure 8a is a graph of day 3 in vitro viability of BT-474 cells versus multiple _IC50 concentrations of T-DM1, lapatinib, and fixed-ratio combinations of T-DM1 and lapatinib, as shown in Table 8a. Mean CI between IC ₁₀ and IC ₉₀ =0.403 indicating synergy.

Table 8a Proliferation of BT-474 cells - 3 days Multiples of IC ₅₀ Lapatinib nM T-DM1 ng/ml Effect (%) CI 0.125x 0.51 1.52 1.4 >2 0.25x 1.52 4.57 1.2 >2 0.5x 4.57 13.72 26.8 0.493 lx 13.72 41.15 62.2 0.201 2x 41.15 123.44 73.9 0.293 4x 123.44 370.33 84.1 0.390 8x 370.33 1111 89.3 0.638

Figure 8 shows a graph of the viability of BT-474 cells in vitro on day 3 from various concentrations of T-DM1 in combination with fixed concentrations of lapatinib (1.5 nM; 4.5 nM; 14 nM; 41 nM; 123 nM ) and various concentrations of one T-DM1 (0-1000 ng/ml). The addition of lapatinib to T-DM1 results in an increased antiproliferative effect compared to drug alone.

Figure 9 shows a graph of the viability of BT-474-EEI cells in vitro on day 3 from various concentrations of T-DM1 in combination with fixed concentrations of lapatinib (14 nM; 41 nM; 123 nM; 370 nM; 1111 nM) and different concentrations of one T-DM1 (0-1000 ng/ml). The addition of lapatinib to T-DM1 results in an increased antiproliferative effect compared to drug alone.

Figure 18 is a graph of in vitro viability of SK-BR-3 cells at Day 3 versus multiples of IC ₅₀ concentrations of 5-FU, trastuzumab-MCC-DM1 (T-DM1), and fixed ratio combinations of 5-FU and T-DM1 (table 18). The combination of 5-FU and T-DM1 has an additive effect on SK-BR-3 cells with a mean CI ranging from IC ₁₀ to IC ₉₀ =0.952.

Table 18 5-FU+T-DM1: proliferation of SK-BR-3 cells - 3 days Multiples of IC ₅₀ 5-FU (µM) T-DM1 ng/ml Effect (%) CI 0.5x 62.5 1.95 38.9 1.035 1x 125 3.91 60.3 0.647 2x 250 7.81 69.2 0.835 4x 500 15.625 74.3 1.292

Figure 19 shows a graph of the viability of BT-474 cells in vitro on day 3 from multiple concentrations of IC ₅₀ 5-FU, trastuzumab-MCC-DM1 (T-DM1) and combinations with a ratio of fixed concentrations of 5-FU and T- DM1 (table 19). The combination of 5-FU and T-DM1 has a synergistic effect on BT-474 cells with a mean CI of 0.623.

Table 19 5-FU + T-DM1: proliferation of BT-474 cells - 3 days Multiples of IC ₅₀ 5-FU (µM) T-DM1 ng/ml Effect (%) CI 0.25x 0.488 3.90 17.1 0.508 0.5x 0.976 7.81 26.8 0.494 1x 1.95 15.62 38.2 0.513 2x 3.91 31.25 46.8 0.661 4x 7.81 62.5 53.6 0.941

Figure 20 shows a graph of the viability of SK-BR-3 cells in vitro on day 3 from multiple concentrations of the IC _{50 of} gemcitabine, trastuzumab-MCC-DM1 (T-DM1) and combinations with the ratio of fixed concentrations of gemcitabine and T-DM1 ( table 20). Gemcitabine in combination with T-DM1 resulted in an antagonistic drug interaction with a mean CI>1.3 for all combinations tested.

Table 20 Gemcitabine (GEM) + T-DM1: proliferation of SK-BR-3 cells - 3 days Multiples of IC ₅₀ GEM (nM) T-DM1 ng/ml Effect (%) CI 0.5x 3.12 6.25 28.7 1.308 1x 6.25 12.5 61.4 1,500 2x 12.5 25 69.9 2.588 4x 25 fifty 72.2 4.957

Figure 21 shows a graph of the viability of MDA-MD-361 cells in vitro on day 3 from multiple concentrations of the IC _{50 of} gemcitabine, trastuzumab-MCC-DM1 (T-DM1) and combinations with the ratio of fixed concentrations of gemcitabine and T-DM1 ( table 21). The combination of drugs gives an antagonistic effect with an average CI value of 1.706.

Table 21 Gemcitabine (GEM) + T-DM1: proliferation of MDA-MD-361 cells - 3 days Multiples of IC ₅₀ GEM (nM) T-DM1 ng/ml Effect (%) CI 0.125x 0.39 3.12 4.5 1.420 0.25x 0.78 6.25 10.3 1.584 0.5x 1.56 12.5 30.7 1.336 1x 3.12 25 59.2 1.280 2x 6.25 fifty 76.3 1.581 4x 12.5 100 80.3 2.747

Figure 22 shows a graph of the viability (proliferation) of KPL4 cells in vitro on day 3 after treatment with T-DM1, GDC-0941 and combinations with a ratio of fixed concentrations of T-DM1 (from 6.25 to 100 ng/ml) and GDC -0941 (from 62.5 nM to 1 μM) at multiples of IC ₅₀ concentrations from 0.25x to 4x. Table 22 shows the effect in the 10-90% inhibition range with calculated CI values and a mean CI value of 1.111.

The additive Bliss action prediction was plotted as a dotted line in FIG. The Bliss independence plot shows the calculated additive action of the combination of two separate compounds.

Table 22 GDC-0941 + T-DM1: KPL4 cell proliferation - 3 days Multiples of IC ₅₀ GDC-0941 (nM) T-DM1 ng/ml Effect (%) CI 0.25x 62.5 6.25 1.0 6.319 0.5x 125 12.5 33.9 1.229 1x 250 25 71.8 1.053 2x 500 fifty 91.1 1.051 4x 1000 100 93.7 1.753

Figure 23 shows a graph of the viability (proliferation) of KPL4 cells in vitro on the 3rd day after treatment with T-DM1, GDC-0941 and combinations with a ratio of fixed concentrations of T-DM1 (from 1.25 to 80 ng/ml) and GDC -0941 (from 31.25 nM to 2 μM) at multiples of IC ₅₀ concentrations from 0.0625x to 16x. A plot of the prediction of the additive Bliss action was built in the form of a dotted line. Table 23 shows the effect in the 10-90% inhibition range with calculated CI values and a mean CI value of 0.802. The combination of T-DM1 and GDC-0941 shows an additive effect on the KPL4 cell line.

Table 23 GDC-0941 + T-DM1: KPL4 cell proliferation - 3 days Multiples of IC ₅₀ GDC-0941 (nM) T-DM1 ng/ml Effect (%) CI 0.125x 31.25 1.25 12.6 1,100 0.25x 62.5 2.5 20.6 1.344 0.5x 125 5 39.2 1.263 1x 250 ten 84.5 0.452 2x 500 twenty 94.9 0.350 4x 1000 40 97.1 0.440 8x 2000 80 97.9 0.668

Figure 24 shows a graph of the viability (proliferation) of KPL4 cells with amplification of Her2, resistant to Herceptin ^® , mutant for PIK3CA (H1047R) in vitro after 3 days after treatment with T-DM1, PI103, GDC-0941 and fixed ratio combinations concentrations of T-DM1+PI103 and T-DM1+GDC-0941 at multiple concentrations of IC ₅₀ from 0 to 16×. Table 24 shows the values of the combination index. Based on the results obtained, it can be assumed that there is synergy between T-DM1 and GDC-0941 in vitro, since CI values are in the range from 0.5 to 1, and there is an additive effect between T-DM1 and PI103, t .to. CI values are close to 1.

Table 24 Combinations: KPL4 cell proliferation CI value at: T-DM1+GDC-0941 T-DM1+PI103 ED50 0.74303 1.04069 ED75 0.63448 0.9721 ED90 0.54179 0.91094

PI3K is a selective inhibitor of PI103 (Hayakawa et al., 2007, Bioorg. Med. Chem. Lett., 17:2438-2442; Raynaud et al., 2007, Cancer Res., 67:5850; Fan et al., 2006, Cancer Cell, 9:341-349; US patent No. 6608053) and has the formula:

Figure 25 is a graph of apoptosis (programmed cell death) of KPL4 caspase 3/7 cells in vitro 24 hours after treatment with T-DM1, GDC-0941 and fixed ratio combinations of T-DM1 and GDC-0941. The combination of T-DM1 and GDC-0941 leads to a significant increase in apoptosis compared to a single drug.

Figure 26 shows a graph of apoptosis (programmed cell death) of KPL4 cells in vitro on day 3 after treatment with T-DM1, GDC-0941 and fixed ratio combinations of T-DM1 and GDC-0941 in fixed concentration ratios. The combination of T-DM1 and GDC-0941 resulted in a significant increase in apoptosis compared to a single drug.

Figure 27 shows a graph of the viability (proliferation) of MDA-MB-361 cells in vitro on day 3 after treatment with T-DM1, GDC-0941 and combinations with a ratio of fixed concentrations of T-DM1 (from 3.125 to 50 ng / ml) and GDC-0941 (from 62.5 nM to 1 μM) at multiple IC ₅₀ concentrations from 0.125x to 8x. A graph of the prediction of the additive Bliss action was built in the form of a dotted line. Table 27 shows the effect in the 10-90% inhibition range with calculated CI values and a mean CI value of 0.888. The combination of T-DM1 and GDC-0941 results in an additive antiproliferative effect on MDA-MB-361 cells with a mean CI of 0.889.

Table 27 GDC-0941 + T-DM1: proliferation of MDA-MB-361 cells - 3 days Multiples of IC ₅₀ GDC-0941 (nM) T-DM1 ng/ml Effect (%) CI 0.25x 62.5 3.125 21.9 1.003 0.5x 125 6.25 37.3 0.862 1x 250 12.5 51.8 0.920 2x 500 25 73.1 0.742 4x 1000 fifty 82.3 0.917

Figure 28 shows a graph of the viability (proliferation) of MDA-MB-361 cells in vitro on day 3 after treatment with T-DM1, GDC-0941 and combinations with a ratio of fixed concentrations of T-DM1 (from 3.125 to 100 ng / ml) and GDC-0941 (from 62.5 nM to 2 μM) at multiple IC ₅₀ concentrations from 0.125x to 8x. A plot of the prediction of the additive Bliss action was built in the form of a dotted line. Table 28 shows the effect in the 10-90% inhibition range with calculated CI values and a mean CI value of 0.813. T-DM1 in combination with GDC-0941 results in an additive antiproliferative effect on MDA-MB-361 cells with a mean CI of 0.813.

Table 28 GDC-0941 + T-DM1: proliferation of MDA-MB-361 cells - 3 days Multiples of IC ₅₀ GDC-0941 (nM) T-DM1 ng/ml Effect (%) CI 0.25x 62.5 3.125 28.6 0.785 0.5x 125 6.25 36.7 0.960 1x 250 12.5 48.5 1.026 2x 500 25 66.6 0.807 4x 1000 fifty 83.2 0.590 8x 2000 100 87.7 0.709

Figure 29 shows a graph of the viability (proliferation) of BT-474 cells in vitro on day 3 after treatment with T-DM1, GDC-0941 and combinations with a ratio of fixed concentrations of T-DM1 (from 3.125 to 100 ng/ml) and GDC -0941 (from 31.25 nM to 1 μM) at multiples of IC ₅₀ concentrations from 0.125x to 4x. A graph of the prediction of the additive Bliss action was built in the form of a dotted line. Table 29 shows the effect in the 10-90% inhibition range with calculated CI values and a mean CI value of 1.2122. At the concentration ratios used, GDC-0941 in combination with T-DM1 did not show a combined effect on BT-474 cells.

Table 29 GDC-0941 + T-DM1: proliferation of BT-474 cells - 3 days Multiples of IC ₅₀ GDC-0941 (nM) T-DM1 ng/ml Effect (%) CI 0.125x 31.25 3.125 8.0 >2 0.25x 62.5 6.25 22.7 1.032 0.5x 125 12.5 31.4 1.178 1x 250 25 43.9 1.207 2x 500 fifty 53.9 1.473 4x 1000 100 71.5 1.171

Figure 30 shows a graph of the viability (proliferation) of BT-474 cells in vitro on day 3 after treatment with T-DM1, GDC-0941 and combinations with a ratio of fixed concentrations of T-DM1 (from 6.25 to 100 ng / ml) and GDC-0941 (from 62.5 nM to 1 μM) at multiple IC ₅₀ concentrations from 0.25x to 4x. A graph of the prediction of the additive Bliss action was built in the form of a dotted line. Table 30 shows the effect in the 10-90% inhibition range with calculated CI values and a mean CI value of 0.997 indicating an additive effect.

Table 30 GDC-0941 + T-DM1: proliferation of BT-474 cells - 3 days Multiples of IC ₅₀ GDC-0941 (nM) T-DM1 ng/ml Effect (%) CI 0.25x 62.5 6.25 19.7 1.338 0.5x 125 12.5 31.5 1.167 1x 250 25 49.0 0.886 2x 500 fifty 66.0 0.708 4x 1000 100 73.9 0.886

Figure 31 shows a graph of the viability (proliferation) of AU565 cells with Her2 amplification, mutant without PI3K under in vitro conditions on day 3 after treatment with T-DM1, PI103, GDC-0941 and combinations with a ratio of fixed concentrations of T-DM1 + PI103 and T -DM1+GDC-0941 at multiple IC ₅₀ concentrations from 0 to 16×. Table 31 shows the values of the combination index. Based on the results obtained, it can be assumed that there is an in vitro antagonism between T-DM1 and GDC-0941, since the CI values are >1, and there is an additive effect or little antagonism between T-DM1 and PI103, because CI values are close or slightly higher than 1.

Table 31 Combinations: AU565 cell proliferation CI values at: T-DM1+GDC-0941 T-DM1 + PI 103 ED50 1.19123 1.12269 ED75 1.36342 0.97338 ED90 1.56063 0.84956

Figure 32 shows a graph of the viability (proliferation) of EFM192A cells with Her2 amplification, mutant for PIK3CA (C420R) in vitro on the 3rd day after treatment with T-DM1, PI103, GDC-0941 and combinations with a ratio of fixed concentrations of T-DM1 + PI103 and T-DM1+GDC-0941 at multiple IC ₅₀ concentrations from 0 to 16×. Table 32 shows the values of the combination index. Based on the results obtained, it can be assumed that there is a moderate synergy between T-DM1 and GDC-0941 in vitro, since the CI values are in the range from 0.5 to 1, and synergy between T-DM1 and PI103, because CI values are close to 0.5.

Table 32 Combinations: EFM192A cell proliferation CI value at: T-DM1 + GDC-0941 T-DM1 + PI 103 ED50 0.80379 0.53861 ED75 0.66352 0.52087 ED90 0.5485 0.52001

Figure 33 shows a graph of the viability (proliferation) of HCC1954 cells with amplification of Her2, resistant to Herceptin ^® , mutant for PIK3CA (H1047R) in vitro on day 3 after treatment with T-DM1, PI103, GDC-0941 and fixed ratio combinations concentrations of T-DM1+PI103 and T-DM1+GDC-0941 at multiple concentrations of IC ₅₀ from 0 to 16×. Table 33 shows the values of the combination index. Based on the results obtained, it can be assumed that there is an additive effect or little synergy between T-DM1 and GDC-0941 in vitro, since the CI values are close to 1, and there is little synergy between T-DM1 and PI103, because CI values <1.

Table 33 Combinations: HCC1954 cell proliferation CI value at: T-DM1 + GDC-0941 T-DM1 + PI 103 ED50 1.15864 0.78902 ED75 0.92365 0.78684 ED90 0.74198 0.80771

Efficacy on tumor xenografts

in vivo

The effectiveness of the combinations of the invention can be evaluated in vivo when allografts or xenografts of tumor cells are implanted in rodents and tumors are treated with the combinations. Different results are expected to be obtained depending on the cell line, the presence or absence of certain mutations in tumor cells, the sequence of administration of trastuzumab-MCC-DM1 and the chemotherapeutic agent, the regimen of administration, and other factors. Mice were treated with drug(s) or control solution (solvent) and observed for several weeks or more to assess the time period required for a twofold increase in tumor, log cell death and suppression of tumor growth (example 3). 10-17 and 34-37 show the efficacy of trastuzumab-MCC-DM1 in combination with chemotherapeutic agents in suppressing the growth of tumor xenografts in mice.

Figure 10 is a plot of mean tumor volume over time in vivo for KPL-4 tumors implanted in the mammary fat pad of immunodeficient SCID mice following administration of: (1) ADC buffer; (2) pertuzumab 15 mg/kg; (3) T-DM1 at 0.3 mg/kg; (4) T-DM1 at 1 mg/kg; (5) T-DM1 at 3 mg/kg, (6) pertuzumab at 15 mg/kg + T-DM1 at 0.3 mg/kg; (7) pertuzumab 15 mg/kg + T-DM1 1 mg/kg; (8) pertuzumab 15 mg/kg + T-DM1 3 mg/kg. Animals injected with ADC buffer (1) had 0 PR and 0 CR. Animals treated with pertuzumab at 15 mg/kg (2) had 0 PR and 0 CR. Animals treated with T-DM1 alone at 0.3 mg/kg (3) had 0 PR and 0 CR. Animals treated with T-DM1 alone at 1 mg/kg (4) had 1 PR and 0 CR. Animals treated with T-DM1 alone at 3 mg/kg (5) had 7 PRs and 0 CRs. Animals treated with the combination of pertuzumab 15 mg/kg and T-DM1 0.3 mg/kg (6) had 5 PR and 0 CR. Animals treated with the combination of pertuzumab 15 mg/kg and T-DM1 1 mg/kg (7) had an 8 PR and 0 CR. Animals treated with the combination of pertuzumab 15 mg/kg and T-DM1 3 mg/kg (8) had 8 PRs and 0 CRs. The combination of pertuzumab and T-DM1 provides superior antitumor activity on KPL4 xenografts compared to drug alone.

FIG. 11 is a plot of mean tumor volume over time in vivo for KPL-4 tumors implanted in the mammary fat pad of immunodeficient SCID mice following administration of: (1) ADC buffer; (2) 5-FU at 100 mg/kg; (3) pertuzumab 40 mg/kg; (4) B20-4.1 at 5 mg/kg; (5) T-DM1 at 5 mg/kg; (6) 5-FU at 100mg/kg + T-DM1 at 5mg/kg; (7) pertuzumab 40 mg/kg + T-DM1 5 mg/kg; (8) B20-4.1 at 5mg/kg + T-DM1 at 5mg/kg; (9) B20-4.1 at 5 mg/kg + pertuzumab at 40 mg/kg. At the end of the experiment, a histological examination of all remaining tumors that had a volume of less than 50 mm ³ was performed, and it was found that in 8 tumor samples from mice treated with T-DM1 alone at a dose of 5 mg/kg (5), 5 samples from mice in group treated with a combination of 5-FU at a dose of 100 mg/kg + T-DM1 at a dose of 5 mg/kg (6) and 8 samples from mice treated with a combination of pertuzumab at a dose of 40 mg/kg + T-DM1 at a dose of 5 mg/kg kg (7), there were no viable tumor cells.

12 is a plot of mean tumor volume over time in vivo for MMTV-Her2 Fo5 transgenic mammary tumors implanted in the mammary fat pad of CRL nu/nu mice after administration of: (1) diluent (ADC buffer) ; (2) B20-4.1 at 5 mg/kg; (3) T-DM1 at 3 mg/kg; (4) T-DM1 at 5 mg/kg; (5) T-DM1 at 10 mg/kg; (6) B20-4.1 at 5mg/kg + T-DM1 at 3mg/kg; (7) B20-4.1 at 5mg/kg + T-DM1 at 5mg/kg; (8) B20-4.1 at 5mg/kg + T-DM1 at 10mg/kg. The combination of T-DM1 and B20-4.1 provides enhanced tumor growth inhibition at T-DM1 doses of 3 and 5 mg/kg, but not 10 mg/kg.

13 is a plot of mean tumor volume over time in vivo for MMTV-Her2 Fo5 transgenic mammary tumors implanted in the mammary fat pad of CRL nu/nu mice after administration of: (1) diluent (ADC buffer) ; (2) T-DM1 at 10 mg/kg; (3) 5-FU at 100 mg/kg; (4) gemcitabine at 120 mg/kg; (5) carboplatin at 100 mg/kg; (6) 5-FU at 100mg/kg + T-DM1 at 10mg/kg; (7) gemcitabine 120 mg/kg + T-DM1 10 mg/kg; (8) carboplatin at 100mg/kg + T-DM1 at 10mg/kg. The combination of T-DM1 with 5-FU, carboplatin, or gemcitabine provides superior antitumor activity compared to single drug treatment.

14 is a plot of mean tumor volume over time in vivo for MMTV-Her2 Fo5 transgenic mammary tumor xenografts implanted in the mammary fat pad of athymic Harlan mice after administration of: (1) diluent (PBS buffer) in /v, once a week ×4; (2) lapatinib 101 mg/kg po bid x21; (3) pertuzumab 40 mg/kg, IV, once a week ×4; (4) B20-4.1 at 5mg/kg, ip, 2×/wk×4; (5) T-DM1 at 15 mg/kg, IV, once every 3 weeks until the end; (6) lapatinib 101 mg/kg po bid x21 + T-DM1 15 mg/kg iv once every 3 weeks until end; (7) pertuzumab 40 mg/kg IV once a week ×4 + T-DM1 15 mg/kg IV once every 3 weeks until the end; (8) B20-4.1 at 5mg/kg po 2x/week x4 + T-DM1 at 15mg/kg ip once every 3 weeks until the end.

T-DM1 alone at a dose of 15 mg/kg (5) does not differ significantly in efficacy from the combination of T-DM1 at a dose of 15 mg/kg and B20-4.1 at a dose of 5 mg/kg (8). In the present experiment, lapatinib and pertuzumab did not differ from the vehicle. For B20-4.1, there was a tendency to increase efficiency compared to control. T-DM1 was effective as a single preparation (p<0.01). The combination of T-DM1 with lapatinib was significantly more effective than lapatinib alone (p<0.01), but was not different from T-DM1 alone. The efficacy of the combination of T-DM1 with pertuzumab was significantly higher compared with pertuzumab alone (p<0.01), but did not differ compared with T-DM1 alone. The combination of T-DM1 with B20-4.1 was statistically significantly more effective than B20-4.1 alone (p<0.01), but did not differ from that for T-DM1 alone.

Figure 15 is a plot of mean tumor volume over time in vivo for MMTV-Her2 Fo5 transgenic mammary tumor xenografts implanted in the mammary fat pad of athymic Harlan mice following administration of: (1) diluent (PBS buffer) orally , twice a day ×21; (2) T-DM1 at a dose of 7.5 mg/kg, IV, once a day ×1; (3) T-DM1 at a dose of 15 mg/kg, IV, once a day ×1; (4) ABT-869 at 5 mg/kg po twice a day ×21; (5) ABT-869 at 15 mg/kg PO twice a day ×21; (6) T-DM1 at 7.5 mg/kg IV, once a day ×1 + ABT-869 at a dose of 5 mg/kg, orally, twice a day ×21; (7) T-DM1 at 7.5 mg/kg IV, once a day ×21 + ABT-869 at a dose of 5 mg/kg, orally, twice a day ×21; (8) T-DM1 at 15mg/kg iv, once a day ×1 + ABT-869 at a dose of 5mg/kg, orally, twice a day ×21; (9) T-DM1 at 15mg/kg, iv, once a day ×1 + ABT-869 at a dose of 15mg/kg, orally, twice a day ×21.

The combination of T-DM1 and ABT-869 at 5 mg/kg showed two partial responses (8) and was not statistically significantly more effective than ABT-869 alone at 5 mg/kg (4). The combination of T-DM1 and ABT-869 at 15 mg/kg (9) is slightly more effective than ABT-869 alone at 15 mg/kg (5). ABT-869 at a dose of 5 mg/kg was significantly more effective than vehicle until the endpoint time (p<0.01), but its effectiveness was not different by the time of tumor doubling. The efficacy of ABT-869 at a dose of 15 mg/kg and T-DM1 at a dose of 7.5 or 15 mg/kg was statistically significantly higher compared to the solvent at the time of doubling the volume of the tumor and at the time of the end point of the tumor (p<0.01) . The combination of T-DM1 at 7.5 mg/kg and ABT-869 at 5 mg/kg was not significantly different in efficacy compared to T-DM1 alone at 7.5 mg/kg. The efficacy of the combination of T-DM1 at a dose of 7.5 mg/kg and ABT-869 at a dose of 5 mg/kg was significantly higher compared to ABT-869 at a dose of 5 mg/kg alone at the time of tumor doubling (p<0.01 ), but did not differ by endpoint time. The combination of T-DM1 at a dose of 7.5 mg/kg and ABT-869 at a dose of 15 mg/kg was significantly better in efficacy compared to either drug alone (p<0.01). The combination of T-DM1 at 15 mg/kg + ABT-869 at 5 mg/kg did not differ in efficacy with T-DM1 at 15 mg/kg alone. Compared with ABT-869 alone at 5 mg/kg, the efficacy of the combination of T-DM1 at 15 mg/kg + ABT-869 at 5 mg/kg did not differ by endpoint but was significantly higher at time of tumor doubling (p<0.01). The combination of T-DM1 at 15 mg/kg + ABT-869 at 15 mg/kg was significantly superior to ABT-869 alone at 15 mg/kg and was superior to T-DM1 at 15 alone. mg/kg by time of tumor doubling (p<0.01). At the time of the endpoint, the antitumor activity of T-DM1 at a dose of 15 mg/kg and T-DM1 at a dose of 15 mg/kg + ABT-869 at a dose of 15 mg/kg did not differ from each other.

Figure 16 is a plot of mean tumor volume over time in vivo for MMTV-Her2 Fo5 transgenic mammary tumor xenografts implanted in the mammary fat pad of athymic Harlan mice after administration of: (1) an IV solvent, one once a week ×3; (2) T-DM1 at 7.5 mg/kg IV once every 3 weeks ×2; (3) T-DM1 at 15mg/kg IV once every 3 weeks ×2; (4) docetaxel 30 mg/kg IV once a week ×3; (5) T-DM1 7.5mg/kg IV once every 3 weeks ×2 + docetaxel 30mg/kg IV once a week ×3; (6) T-DM1 15mg/kg IV once every 3 weeks ×2 + docetaxel 30mg/kg IV once a week ×3.

Animals treated with T-DM1 alone at 15 mg/kg (3) had 6 partial responses (PR) and 1 complete response (CR). Animals treated with docetaxel alone at 30 mg/kg (4) had 2 PRs. Animals treated with the combination of T-DM1 at 7.5 mg/kg and docetaxel at 30 mg/kg (5) had 10 PR. Animals treated with the combination of T-DM1 at 15 mg/kg and docetaxel at 30 mg/kg (6) had 7 PRs and 3 CRs. All groups with the introduction of some drugs significantly differed from the results obtained on animals that were injected with the solvent (p<0.01). The efficacy of the combination of T-DM1 at a dose of 7.5 mg/kg and docetaxel was significantly higher compared to each drug alone at the time of doubling the tumor volume and at the time of the end point (p<0.01). There were no objective responses in the T-DM1 7.5 mg/kg group of mice and there were 2 partial responses (PR) in the docetaxel alone group. The introduction of a combination of T-DM1 at a dose of 7.5 mg/kg and docetaxel resulted in 9 PR and 1 complete response (CR). The efficacy of the combination of T-DM1 at a dose of 15 mg/kg + docetaxel was significantly higher compared to each drug alone at the time of doubling the tumor volume and at the time of the end point (p<0.01). The introduction of one T-DM1 at a dose of 15 mg/kg gave 5 PR and 2 CR. The combined administration of T-DM1 at a dose of 15 mg/kg + docetaxel increased the number of objective responses to treatment to 7 PR and 3 CR. All mice in this combination regimen group had an objective response to treatment.

17 is a plot of mean in vivo tumor volume for MMTV-Her2 Fo5 transgenic tumor xenografts implanted in the mammary fat pad of athymic Harlan mice following administration of: (1) diluent po once a day x21; (2) T-DM1 at 7.5 mg/kg IV once every 3 weeks ×2; (3) T-DM1 at 15mg/kg IV once every 3 weeks ×2; (4) lapatinib 100 mg/kg orally twice daily ×21; (5) T-DM1 7.5 mg/kg IV once every 3 weeks ×2 + lapatinib 100 mg/kg po twice daily ×21; (6) T-DM1 15 mg/kg IV once every 3 weeks ×2 + lapatinib 100 mg/kg po twice daily ×21.

Animals treated with T-DM1 alone at 15 mg/kg (3) had 6 partial responses (PR) and 3 complete responses (CR). Animals treated with the combination of T-DM1 at 7.5 mg/kg and lapatinib at 100 mg/kg (5) experienced 4 PRs and 5 CRs. In animals treated with a combination of T-DM1 at a dose of 15 mg/kg and lapatinib at a dose of 100 mg/kg (6), the response to treatment was 8 CR. All single drug groups were significantly different from vehicle treated animals (p<0.01) at time of tumor doubling and at end point. The efficacy of the combination of T-DM1 at a dose of 7.5 mg/kg with lapatinib was significantly higher compared with lapatinib and T-DM1 at a dose of 7.5 mg/kg when administered as a single drug (p<0.01). The efficacy of T-DM1 at a dose of 15 mg/kg in combination with lapatinib was significantly higher compared with lapatinib alone (p<0.01). This combination did not differ in efficacy from T-DM1 alone at a dose of 15 mg/kg.

Tumor volume doubling time was determined using Kaplan-Meier statistical analysis as 2×Vo. Tumor volume doubling time and survival analysis were quantified by log-rank-p values. Progression time was defined as the time elapsed until a tumor volume of 1000 mm ³ was reached or survival time if a tumor volume of 1000 mm ³ was not reached. T-DM1 in combination with lapatinib provided a significantly higher antitumor efficacy compared to treatment with a single drug.

34 is a plot of mean tumor volume over time in vivo for MMTV-Her2 Fo5 transgenic tumor xenografts implanted in the mammary fat pad of CRL nu/nu mice following administration of: day ×21; (2) T-DM1 at 10 mg/kg IV once every 3 weeks; (3) 5-FU at 100mg/kg po once a week ×2; (4) (5) T-DM1 5mg/kg iv once every 3 weeks + 5-FU 100mg/kg po once a week ×2. Vehicle-treated animals had 0 partial responses (PR) and 0 complete responses (CR). In animals treated with T-DM1 alone, 1 PR and 0 CR were detected. Animals treated with 5-FU alone had 0 PR and 0 CR. Animals treated with the combination of T-DM1 and 5-FU had 3 PRs and 0 CRs at the 42 day time point. Treatment with T-DM1 and 5-FU resulted in increased antitumor activity compared to either formulation alone.

35 is a plot of mean tumor volume over time in vivo for MMTV-Her2 Fo5 transgenic tumor xenografts implanted in the mammary fat pad of CRL nu/nu mice following administration of: day ×21; (2) T-DM1 at 5 mg/kg IV once every 3 weeks; (3) GDC-0941 at 100 mg/kg orally twice a day ×21; (4) GDC-0152 dose of 50 mg/kg orally once a week ×2; (5) T-DM1 5mg/kg iv once every 3 weeks + GDC-0941 100mg/kg po bid x21; (6) T-DM1 5mg/kg iv once every 3 weeks + GDC-0152 50mg/kg po once a week ×2. Treatment with T-DM1 and GDC-0941 resulted in increased antitumor activity compared to treatment alone, while the combination of T-DM1 and GDC-0152 was not more effective than T-DM1 alone.

GDC-0152 is an inhibitor of caspases that suppress apoptotic proteins (Call et al., 2008, The Lancet Oncology, 9(10):1002-1011; Deveraux et al., 1999, J. Clin. Immunol., 19:388- 398).

36 is a plot of mean tumor volume over time in vivo for MDA-MB-361.1 tumors implanted in the mammary fat pad of CRL nu/nu mice following administration of: (1) diluent po, once daily ×21; (2) GDC-0941 at 25 mg/kg po once a day x21; (3) GDC-0941 at 50 mg/kg po once a day ×21; (4) GDC-0941 at 100 mg/kg po once a day ×21; (5) T-DM1 at 3 mg/kg IV once every 3 weeks; (6) T-DM1 at 10 mg/kg IV once every 3 weeks; (7) GDC-0941 at 25mg/kg po once a day ×21 + T-DM1 at 3mg/kg iv once every 3 weeks; (8) GDC-0941 at 50 mg/kg po once a day ×21 + T-DM1 at 3 mg/kg iv once every 3 weeks; (9) GDC-0941 at 100 mg/kg po once a day ×21 + T-DM1 at 3 mg/kg iv once every 3 weeks; (10) GDC-0941 at 25 mg/kg po once a day ×21 + T-DM1 at 10 mg/kg iv once every 3 weeks; (11) GDC-0941 at 50mg/kg po once a day ×21 + T-DM1 at 10mg/kg iv once every 3 weeks; (12) GDC-0941 at 100mg/kg po once a day ×21 + T-DM1 at 10mg/kg iv once every 3 weeks.

Animals treated with solvent (1) had 0 partial responses (PR) and 0 complete responses (CR). Animals treated with GDC-0941 alone at 25 mg/kg (2) had 0 PR and 0 CR. Animals treated with GDC-0941 alone at 50 mg/kg (3) had 1 PR and 0 CR. Efficacy in animals treated with GDC-0941 alone at 100 mg/kg (4) was reported as 0 PR and 0 CR. Animals treated with T-DM1 alone at 3 mg/kg (5) had 1 PR and 1 CR. Animals treated with T-DM1 alone at 10 mg/kg (6) had 8 PRs and 1 CR. Animals treated with the combination of T-DM1 at 3 mg/kg and GDC-0941 at 25 mg/kg (7) experienced 5 PRs and 0 CRs. Animals treated with the combination of T-DM1 at 3 mg/kg and GDC-0941 at 50 mg/kg (8) experienced 3 PRs and 0 CRs. Animals treated with the combination of T-DM1 at 3 mg/kg and GDC-0941 at 100 mg/kg (9) experienced 3 PRs and 1 CR. Animals treated with the combination of T-DM1 at 10 mg/kg and GDC-0941 at 50 mg/kg (10) had 9 PR and 0 CR. Animals treated with the combination of T-DM1 at 10 mg/kg and GDC-0941 at 50 mg/kg (11) had 7 PRs and 2 CRs. In animals treated with a combination of T-DM1 at 10 mg/kg and GDC-0941 at 100 mg/kg (12), its efficacy was 9 PR and 1 CR.

37 is a plot of mean tumor volume over time in vivo for MDA-MB-361.1 tumors implanted in the mammary fat pad of CRL nu/nu mice following administration of: (1) solvents [MCT (0.5 % methylcellulose/0.2% tween 80) + succinate buffer (100 mM sodium succinate, 100 mg/mL trehalose, 0.1% tween 80, pH 5.0)] po + iv once a day ×21 and once a day; (2) GNE-390 at 1.0 mg/kg po once a day x21; (3) GNE-390 at 2.5 mg/kg, po, once a day ×21; (4) T-DM1 at 3 mg/kg IV, once a day; (5) GNE-390 at 1.0 mg/kg po once a day ×21 + T-DM1 at 3 mg/kg iv once a day; (6) GNE-390 at 2.5mg/kg po once a day ×21 + T-DM1 at 3mg/kg iv once a day.

Animals treated with vehicle (1) had 0 partial responses (PR) and 0 complete responses (CR). Animals treated with GNE-390 alone at 1.0 mg/kg (2) had 0 PR and 0 CR. Efficacy in animals treated with GNE-390 alone at 2.5 mg/kg (3) was 1 PR and 0 CR. Animals treated with T-DM1 alone at 3 mg/kg (5) had 1 PR and 1 CR. Animals treated with T-DM1 alone at 3 mg/kg (4) had 0 PR and 0 CR. Animals treated with the combination of T-DM1 at 3 mg/kg and GNE-390 at 25 mg/kg (5) experienced 3 PRs and 0 CRs. The effectiveness of the combination of T-DM1 at a dose of 3 mg/kg and GNE-390 at a dose of 2.5 mg/kg (6) was 5 PR and 1 CR. The combination of GNE-390 with T-DM1 resulted in a significant increase in antitumor activity compared to GNE-390 or T-DM1 alone in the MB-361.1 breast cancer xenograft model.

Pharmaceutical compositions

Pharmaceutical compositions or dosage forms of the present invention contain combinations of trastuzumab-MCC-DM1, a chemotherapeutic agent, and one or more pharmaceutically acceptable carriers, flow regulators, diluents, or excipients.

Trastuzumab-MCC-DM1 and the chemotherapeutic agents of the present invention can be used in unsolvated form as well as solvated forms with pharmaceutically acceptable solvents such as water, ethanol and the like, and the invention is intended to include solvated and unsolvated forms.

Trastuzumab-MCC-DM1 and the chemotherapeutic agents of the present invention may be in various tautomeric forms, and all such forms are within the scope of the invention. The terms "tautomer" or "tautomeric form" refer to structural isomers with different energies that are converted into each other through a low energy barrier. For example, proton tautomers (also known as prototropic tautomers) include proton migration interconversions such as keto-enol and imine-enamine isomerizations. Valence tautomers include interconversions that occur through the reorganization of some of the electrons involved in the formation of bonds.

Pharmaceutical compositions include bulk compositions and single unit dosage forms containing more than one (e.g., two) pharmaceutically active substances, including trastuzumab-MCC-DM1 and a chemotherapeutic agent selected from the list of additional drugs described herein, along with with any pharmaceutically inactive excipients, diluents, carriers or flow regulators. The bulk composition and each individual unit dosage form may contain fixed amounts of the above pharmaceutically active substances. A bulk composition is a substance that has not been formulated into separate single dosage forms. An exemplary dosage form is an oral dosage form such as tablets, pills, capsules and the like. Similarly, the method of treating a patient with a pharmaceutical composition described herein is also intended to include the administration of a bulk composition and separate unit dosage forms.

Pharmaceutical compositions also include isotopically labeled compounds of the present invention, which are similar to those already described herein, but differ in that one or more atoms are replaced by an atom having an atomic mass or mass number different from the atomic mass or mass number that is usually found in nature. All isotopes of any particular atom or element, as already mentioned, are included within the scope of the compounds of the invention and their uses. Exemplary isotopes that can be included in the compounds of the invention include hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine, chlorine and iodine, such as ² H, ³ H, ¹¹ C, ¹³ C, ¹⁴ C, ¹³ N, ¹⁵ N, ¹⁵ O, ¹⁷ O, ¹⁸ O, ³² P, ³³ P, ³⁵ S, ¹⁸ F, ^{36 Cl} , ¹²³ I and ^{125 I} C) are suitable for setting up an analysis of the distribution of the compound and/or substrate in tissues. The isotopes tritium ( ³ H) and carbon-14 ( ¹⁴ C) are suitable for easy production and detection of compounds. In addition, substitution with heavier isotopes such as deuterium ( ² H) may confer some therapeutic benefits as a result of higher metabolic stability (eg, increased half-life or somewhat infrequent administration) and may therefore be preferred in some situations. Positron emitting isotopes such as ¹⁵ O, ¹³ N, ¹¹ C and ¹⁸ F are suitable for positron emission tomography (PET) studies to study substrate receptor occupancy. Generally, the isotopically labeled compounds of the present invention can be prepared using the following methods, analogous to those described in the Schemes and/or Examples provided herein below, by substituting a non-isotopically labeled reagent with an isotopically labeled reagent.

Trastuzumab-MCC-DM1 and chemotherapeutic agents can be formulated according to conventional pharmaceutical practice for use in a therapeutic combination for the medical treatment (including prophylactic treatment) of hyperproliferative disorders in mammals, including humans. The invention relates to a pharmaceutical composition containing trastuzumab-MCC-DM1 in association with one or more pharmaceutically acceptable carriers, flow regulators, diluents or excipients.

Suitable carriers, diluents and excipients are well known to those skilled in the art and include substances such as carbohydrates, waxes, water soluble and/or swellable polymers, hydrophilic or hydrophobic substances, gelatin, oils, solvents, water and the like. The specific carrier, diluent and excipient to be used will depend on the methods and purpose for which the compound of the present invention is being used. Generally, the solvents chosen are solvents known to those skilled in the art to be safe (GRAS) for administration to a mammal. In general, safe solvents are non-toxic aqueous solvents such as water and other non-toxic solvents that are soluble or miscible with water. Suitable solvents include water, ethanol, propylene glycol, polyethylene glycols (eg PEG 400, PEG 300), etc. and their mixtures. The compositions may also contain one or more of buffers, stabilizers, surfactants, wetting agents, lubricants, emulsifiers, suspending agents, preservatives, antioxidants, opacifying agents, flow regulators, processing agents, colorants, sweeteners, flavoring agents, flavoring agents. and other known additives to provide an elegant presentation of a drug (ie, a compound of the present invention or a pharmaceutical composition thereof) or ancillaries used in the manufacture of a pharmaceutical product (ie, a drug).

Compositions can be prepared using conventional dissolution and mixing. For example, a bulk drug (ie, a compound of the present invention or a stabilized form of a compound) (eg, a complex with a cyclodextrin or other complexing agent) is dissolved in a suitable solvent in the presence of one or more of the excipients mentioned above. Generally, the compound of the present invention is formulated into pharmaceutical dosage forms to allow easy controlled administration of the drug and for patient convenience in a prescribed manner.

The pharmaceutical composition (or dosage form) for use can be packaged in a variety of ways depending on the route of administration used. Typically, the subject for introduction includes a container containing the pharmaceutical composition in the appropriate form. Suitable containers are well known to those skilled in the art and include materials such as vials (plastic or glass), sachets, ampoules, plastic bags, metal cylinders, and the like. The container may also include a secure connection to prevent inadvertent access to the contents of the package. In addition, the container also has a label on it that describes the contents of the container. There may also be appropriate warnings on the label.

Pharmaceutical compositions of the compounds of the present invention can be formulated for various routes and types of administration with pharmaceutically acceptable diluents, carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences, 1995, ^18th edition, Mack Publ. Co., Easton, PA) as a lyophilized composition, crushed powder or aqueous solution. The composition can be prepared by mixing at room temperature, at an appropriate pH and at the desired degree of purity, with physiologically acceptable carriers, i. carriers that are not toxic to recipients at the doses and concentrations used. The pH value of the composition will generally depend on the particular application and concentration of the compound, but may range from about 3 to about 8.

Preferably the pharmaceutical composition is sterile. In particular, compositions for in vivo administration must be sterile. Such sterilization is easily accomplished by filtration through sterile membrane filters.

Typically, the pharmaceutical composition may be stored as a solid composition, a lyophilized composition, or as an aqueous solution.

Pharmaceutical compositions of the invention should be dosed and administered in this way, i. in amounts, concentrations, schemes, courses, solvents and routes of administration consistent with good medical practice. Factors to be considered in this regard include the particular disorder being treated, the particular mammal being treated, the clinical condition of the individual patient, the cause of the disorder, site of administration, route of administration, schedule of administration, and other factors known to practitioners. When determining a "therapeutically effective amount" of a compound of the invention to administer, the above factors should be considered and is the minimum amount necessary to prevent, ameliorate, or treat a coagulation factor-mediated disorder. Preferably, the amount is below the amount that is toxic to the host or makes the host substantially more susceptible to bleeding.

As a general guideline, the initial pharmaceutically effective amount of trastuzumab-MCC-DM1 administered per dose is in the range of about 0.01-1000 mg/kg, more specifically about 0.1 to 20 mg/kg of patient body weight per day, with typical the initial limits of the compound used are from 0.3 to 15 mg/kg.

Acceptable diluents, carriers, excipients, and stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butylethanol or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight polypeptides (less than about 10 residues in length); proteins such as serum albumin, gelatin or immunoglobulin; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrins; complexing agents such as EDTA; sugars such as sucrose, mannitol, trehalose or sorbitol; salt-forming counterions such as sodium; metal complexes (eg Zn-protein complexes) and/or non-ionic surfactants such as Tween ^TM including Tween 80, Pluronics ^TM or polyethylene glycol (PEG) including PEG400. Active pharmaceutical ingredients can also be included in microcapsules prepared, for example, by coacervation or interfacial polymerization, for example, microcapsules, respectively, of hydroxymethylcellulose or gelatin and poly(methyl methacrylate), in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsion. Such methods are disclosed in Remington's Pharmaceutical Sciences 18 ^th edition, 1995, Mack Publ. Co., Easton, PA.

Pharmaceutical compositions include compositions suitable for administration by the routes detailed herein. The compositions may suitably be presented in unit dosage form and may be prepared by any of the methods well known in the art of pharmacy. A general description of methods and compositions can be found in Remington's Pharmaceutical Sciences 18 ^th edition, 1995, Mack Publ. Co., Easton, PA. Such methods include the step of bringing into contact the active ingredient with the carrier which represents one or more excipients. In general, the compositions are prepared by uniformly and intimately bringing into association the active ingredient with liquid carriers or finely divided solid carriers, or both, and then shaping the product if necessary.

Compositions of a chemotherapeutic agent suitable for oral administration may be formulated as discrete unit dosage forms such as pills, hard or soft, e.g. gelatin capsules, cachets, lozenges, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, syrups or elixirs, each containing a predetermined amount of a trastuzumab-MCC-DM1 compound and/or a chemotherapeutic agent. Such compositions may be prepared using any method known in the art for the manufacture of pharmaceutical compositions, and such compositions may contain one or more additives, including sweeteners, flavoring agents, coloring agents, and preservatives, to provide a palatable formulation. Compressed tablets can be prepared by compressing, using a suitable apparatus, the active ingredient in free-flowing form such as powder or granules, optionally in admixture with a binder, flow regulator, inert diluent, preservative, surfactant or dispersing agent. Molded tablets can be prepared by shaping, on a suitable machine, a mixture of powdered active ingredient moistened with an inert liquid diluent. Tablets may optionally be coated or scored, and optionally formulated to provide sustained or controlled release of the active ingredient therefrom.

Excipients in tablets of a pharmaceutical composition of the invention may include: an excipient (or diluent) to increase the volume of a powder formulation for tablet preparation; disintegrators to ensure that the tablet breaks up into small fragments when taken orally, ideally individual particles of the drug, and rapid dissolution and absorption of the drug; a binder for obtaining granules and tablets with the necessary mechanical force and maintaining the integrity of the tablets after pressing, preventing its destruction into constituent powders during packaging, transportation and normal handling of the drug; a flow regulator to ensure the flow of the powder during the production of tablets; a lubricant to ensure that the tablet powder does not stick to the equipment that is used to press the tablets in production. These substances increase the flowability of powder mixtures when passing through the presses and minimize attrition and brittleness during the extraction of the final tablets from the equipment; an anti-adhesive agent with a function similar to a flow regulator that reduces adhesion between the tablet powder and the apparatus used to punch tablets in production; a flavoring agent which is included in tablets to enhance their palatability or mask an unpleasant taste; and a coloring agent to provide identification and convenience to the patient.

Tablets containing the active ingredient in admixture with a non-toxic pharmaceutically acceptable excipient which is suitable for the manufacture of tablets are acceptable. These excipients may, for example, be inert diluents such as calcium or sodium carbonate, lactose, calcium or sodium phosphate; granulating and loosening aids such as corn starch or alginic acid; binders such as starch, gelatin or gum; and lubricants such as magnesium stearate, stearic acid or talc. Tablets may be uncoated or may be coated using known techniques, including microencapsulation, to slow down disintegration and absorption in the gastrointestinal tract and thereby provide a delayed action over a longer period of time. For example, an absorption delay agent such as glyceryl monostearate or glyceryl distearate alone or with wax can be used.

For the treatment of the eyes or other external tissues, such as the mouth and skin, the compositions are preferably applied as an ointment or topical cream containing the active ingredient(s) in an amount of, for example, 0.075 to 20% w/w. When formulated as an ointment, the active ingredients can be used with a paraffinic or water-miscible ointment base. Alternatively, the active ingredients may be formulated in a cream with an oil-in-water cream base.

If desired, the aqueous phase of the cream base may contain a polyhydric alcohol, i. an alcohol having two or more hydroxyl groups such as propylene glycol, butane-1,3-diol, mannitol, sorbitol, glycerin and polyethylene glycol (including PEG 400) and mixtures thereof. For topical compositions, it may be desirable to include a compound that enhances the absorption or penetration of the active ingredient through the skin or other affected areas. Examples of such skin penetration enhancers include dimethyl sulfoxide and related analogues.

The oil phase of the emulsions of this invention may consist of known components in known proportions, including a mixture of at least one emulsifier with fat or oil, or with fat or oil together. Preferably, a hydrophilic emulsifier is included along with a lipophilic emulsifier which functions as a stabilizer. The emulsifier(s) with or without stabilizer(s) provide an emulsifying wax, and the wax, together with oil and fat, creates an emulsifying ointment base that forms the oily dispersed phase of the cream compositions. Emulsifiers and emulsion stabilizers suitable for use in the composition of the invention include Tween® 60, Span® 80, cetostearyl alcohol, benzyl alcohol, myristyl alcohol, glyceryl monostearate, and sodium lauryl sulfate.

Aqueous suspensions of the pharmaceutical compositions of the invention contain the active substances in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients include a suspending agent such as sodium carboxymethylcellulose, croscarmellose, povidone, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth, and gum arabic, and dispersing or wetting agents such as natural phosphatide (e.g., lecithin), an alkylene oxide condensate. with a fatty acid (for example, polyethylene stearate), a condensation product of ethylene oxide with a long chain aliphatic alcohol (for example, heptaneethyleneoxycetanol), a condensation product of ethylene oxide with a partial ester derived from a fatty acid and hexitol anhydride (for example, polyoxyethylene sorbitan monooleate). The aqueous suspension may also contain one or more preservatives such as ethyl or n-propyl p-hydroxybenzoate, one or more colorants, one or more flavors, and one or more sweeteners such as sucrose or saccharin.

The pharmaceutical compositions may be in the form of a sterile injectable preparation, such as a sterile injectable aqueous or oily suspension. This suspension may be formulated according to methods known in the art using the appropriate dispersing or wetting agents and suspending agents as indicated above. The sterile injectable preparation may be a solution or suspension in a non-toxic, parenterally acceptable diluent or solvent, such as a solution in 1,3-butanediol, or prepared from a lyophilized powder. Among the acceptable vehicles or solvents that may be used are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils, usually used as a solvent or suspending medium, can be used. Any bland fixed oil can be used for this purpose, including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid can also be used to prepare injection solutions.

The amount of active ingredient that can be combined with the carrier to prepare a single dosage form may vary depending on the host being treated and the particular route of administration. For example, a sustained release dosage form intended for oral administration to humans may contain from about 1 to 1000 mg of the active substance formulated with an appropriate and appropriate amount of carrier, the content of which can range from about 5 to about 95% by weight of the total composition. (mass:mass). The pharmaceutical composition can be prepared in such a way that the amount to be administered can be easily determined. For example, an aqueous solution for intravenous infusion may contain from about 3 to 500 micrograms of the active ingredient per milliliter of solution to provide a suitable volume for infusion at a rate of administration of about 30 ml/hour.

Compositions suitable for parenteral administration include aqueous or non-aqueous sterile injectable solutions, which may contain antioxidants, buffers, bacteriostatics, and solutes that render the composition isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions, which may include suspending agents and thickeners.

Compositions suitable for topical application to the eye include eye drops in which the active ingredient is dissolved or suspended in a suitable carrier, in particular an aqueous solvent for the active ingredient. Preferably, the active ingredient is present in such compositions at a concentration of from about 0.5 to 20% w/w, for example, from about 0.5 to 10% w/w, for example, it is 1.5% w/w. wt.

Compositions suitable for topical use in the oral cavity include medicated lozenges containing the active ingredient in a flavored base, typically sucrose and gum arabic or tragacanth; lozenges containing the active ingredient in an inert base such as gelatin and glycerin, or sucrose and acacia; and mouthwashes comprising the active ingredient in a suitable liquid carrier.

Compositions for rectal administration may be presented as a suppository with a suitable base containing, for example, cocoa butter or salicylate.

Compositions suitable for intrapulmonary or nasal administration have a particle size, for example, ranging from 0.1 to 500 microns (including particle sizes ranging from 0.1 to 500 microns in micron increments such as 0.5; 1; 30; 35 microns, etc.), which are administered by rapid inhalation through the nasal passages or by inhalation through the mouth in such a way as to reach the alveolar sacs. Suitable compositions include aqueous or oily solutions of the active ingredient. Compositions suitable for administration as an aerosol or dry powder may be prepared using conventional methods and may be administered with other drugs, such as compounds used in the treatment or prevention of the diseases referred to herein.

Compositions suitable for intravaginal administration may be presented as vaginal suppositories, tampons, creams, gels, pastes, foams or sprays containing, in addition to the active ingredient, suitable carriers as are known in the art.

The compositions can be packaged in single dosage forms or multi-dose containers, such as sealed ampoules and vials, and they can be stored in a freeze-dried (lyophilized) state, when it is only necessary to add a sterile liquid carrier, such as water for injection, immediately before use. Prepared extemporo injection solutions and suspensions are prepared from sterile powders, granules and tablets of the type described earlier. Preferred single dosage forms are those containing the daily dose or partial daily dose of the active ingredient mentioned above, or their appropriate fraction.

The invention also relates to veterinary compositions containing at least one of the active ingredients mentioned above, together with a carrier for it, used in veterinary medicine. Veterinary carriers are substances suitable for administering the composition and may be solid, liquid or gaseous substances which are inert or acceptable for veterinary use and are compatible with the active ingredient. These veterinary compositions may be administered parenterally, orally, or by any other route.

Combination Therapy

Trastuzumab-MCC-DM1 can be used in combination with other chemotherapeutic agents to treat a hyperproliferative disease or disorder, including tumors, malignancies, and neoplastic tissue, as well as premalignant and non-neoplastic or benign hyperproliferative diseases. In some embodiments, trastuzumab-MCC-DM1 is combined in a pharmaceutical combination composition or combination therapy regimen with a second compound that has anti-hyperproliferative properties or is useful in the treatment of a hyperproliferative disorder. The second compound in the pharmaceutical combination composition or administration schedule preferably has complementary activities for trastuzumab-MCC-DM1 and thus they do not adversely affect each other. Such compounds are suitably in combination in amounts that are effective for the intended purpose. In one embodiment, the composition of this invention contains trastuzumab-MCC-DM1 in combination with a chemotherapeutic agent described herein. Examples 4 and 5 provide clinical protocols for T-DM1 + pertuzumab and T-DM1 + GDC-0941, respectively.

Therapeutic combinations of the invention include a composition, regimen, or other treatment comprising administering trastuzumab-MCC-DM1 and a chemotherapeutic agent selected from a HER2 dimerization inhibitor antibody, an anti-VEGF antibody, 5-FU, carboplatin, lapatinib, ABT-869, and docetaxel , as a combined preparation for separate, simultaneous or sequential use in the treatment of a hyperproliferative disorder.

Combination therapy can be carried out in the form of a simultaneous or sequential scheme. When administered sequentially, the combination may be administered in two or more doses. Combined administration includes co-administration, the use of separate dosage forms or a single pharmaceutical form, and sequential administration in any order, where preferably there is a period of time when both (or all) active compounds simultaneously exhibit their biological activity.

Suitable doses of any of the above drugs for co-administration are currently used and can be reduced due to the combined action (synergy) of the newly identified drug and other chemotherapeutic agents or treatments.

In a specific embodiment, antitumor therapy, trastuzumab-MCC-DM1 can be combined with a chemotherapeutic agent, including hormonal agents or antibodies, such as described above, as well as combined with surgery and radiation therapy. The amounts of trastuzumab-MCC-DM1 and other pharmaceutically active chemotherapeutic agent(s) and the relative time of administration are chosen to achieve the desired combined therapeutic effect.

Introduction of Pharmaceutical Compositions

The compounds of the invention may be administered by any route that is appropriate for the condition being treated. Suitable routes include oral, parenteral (including subcutaneous, intramuscular, intravenous, intraarterial, inhalation, intradermal, intrathecal, epidural, and infusion), transdermal, rectal, nasal, topical (including buccal or sublingual), vaginal, intraperitoneal, intrapulmonary, and intranasal. Topical administration may also include the use of transdermal administration using transdermal patches or iontophoresis devices. Drug formulation is discussed in Remington's Pharmaceutical Sciences, 1995, ^18th edition, Mack Publ. Co., Easton, PA. Other examples of dosage forms can be found in Liberman HA and Lachman L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, Vol. 3, 2nd ^Ed ., New York, NY. For topical immunosuppressive therapy, the compounds can be administered to the site of the lesion, including perfusion or otherwise contacting the graft with the suppressor prior to transplantation. Those skilled in the art will obviously understand that the preferred route may vary depending on, for example, the condition of the recipient. When the compound is administered orally, it may be formulated as a pill, capsule, tablet, etc. with a pharmaceutically acceptable carrier, flow regulator or excipient. Where a compound is administered parenterally, it may be formulated with a pharmaceutically acceptable parenteral vehicle or vehicle and in a single injectable dosage form, as detailed below.

The dose of trastuzumab-MCC-DM1 for the treatment of humans may range from about 100 mg to about 500 mg. The dose of trastuzumab-MCC-DM1 can be administered once every 6 weeks, once every 3 weeks, once a week, or more frequently depending on the pharmacokinetic (PK) and pharmacodynamic (PD) properties of the drug, including absorption, distribution, metabolism and excretion. The dose of the chemotherapeutic agent used in combination with trastuzumab-MCC-DM1 may range from about 10 mg to about 1000 mg. The chemotherapeutic agent can be administered once every 6 weeks, once every 3 weeks, once a week, or more frequently, such as once or twice a day. In addition, toxic factors may influence the dose and administration schedule. For oral administration, the pill, capsule or tablet may be taken daily or less frequently over a period of time. The scheme can be repeated over a number of therapeutic courses.

Methods of treatment

Therapeutic combinations: (1) trastuzumab-MCC-DM1 and (2) a chemotherapeutic agent are useful in the treatment of diseases, conditions and/or disorders, including, but not limited to, diseases characterized by activation of the HER2 pathway. Therefore, another aspect of this invention relates to methods of treating diseases or conditions that can be treated by targeting HER2 or VEGR receptor 1. Therapeutic combinations of (1) trastuzumab-MCC-DM1 and (2) a chemotherapeutic agent can be used to treat a hyperproliferative disease or disorder, including tumors, malignancies, and neoplastic tissue, along with premalignant and non-neoplastic or benign hyperproliferative diseases.

Cancers that can be treated with the methods of this invention include, but are not limited to, breast, ovarian, cervical, prostate, testicular, genitourinary, esophagus, larynx, glioblastoma, neuroblastoma, stomach, skin, keratoacanthoma, lung cancer, squamous cell carcinoma, large cell carcinoma, non-small cell lung carcinoma (NSCLC), small cell carcinoma, lung adenocarcinoma, bone cancer, colon cancer, adenoma, pancreatic cancer, adenocarcinoma, thyroid cancer, follicular carcinoma, undifferentiated carcinoma, papillary carcinoma, seminoma , melanoma, sarcoma, bladder carcinoma, liver and bile duct carcinoma, renal carcinoma, myeloid disorders, lymphoid disorders, hairy cell cancer, buccal pocket and pharynx (oral cavity), cancer of the lips, tongue, mouth, pharynx, small intestine , cancer of the colon-rectum, large intestine, rectum, brain and center nervous system, Hodgkin's lymphoma and leukemia.

Another aspect of this invention relates to a pharmaceutical composition or therapeutic combination for use in the treatment of diseases or conditions mentioned in this document, in a mammal, for example, a human suffering from such a disease or condition. Also provided is the use of a pharmaceutical composition in the manufacture of a medicament for the treatment of the diseases and conditions set forth herein in a warm-blooded animal, such as a mammal, such as a human, suffering from such a disorder.

Products

In another embodiment, the invention provides an article or "kit" containing trastuzumab-MCC-DM1, useful in the treatment of diseases or disorders described above. In one embodiment, the kit includes a container containing trastuzumab-MCC-DM1. The kit may further comprise a label or package insert on or associated with the container. The term "package insert" is used to refer to instructions commonly included in commercial packaging of therapeutic products that contain information about the indications, use, dosage, administration, contraindications and/or warnings regarding the use of such therapeutic products. Suitable containers include, for example, bottles, vials, syringes, blister packs, and the like. The container can be made from various materials such as glass or plastic. The container may contain trastuzumab-MCC-DM1 or a composition thereof that is effective for treating the condition and may have a sterile entry port (for example, the container may be a bag of intravenous solution or a vial with a stopper pierced by an intradermal injection needle). At least one active compound in the composition is trastuzumab-MCC-DM1. The label or package insert indicates that the composition is used to treat a condition of choice such as cancer. In one embodiment, the label or package insert indicates that the composition containing trastuzumab-MCC-DM1 can be used to treat a disorder resulting from abnormal cell growth. Also, the label or package insert may indicate that the composition can be used to treat other disorders. Alternatively or additionally, the product may also contain a second container containing a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and dextrose solution. Additionally, it may contain other substances desirable from a commercial and consumer point of view, including buffers, diluents, filters, needles and syringes.

The kit may further contain instructions for administering trastuzumab-MCC-DM1 and, if present, a second pharmaceutical composition. For example, if the kit includes a first composition containing trastuzumab-MCC-DM1 and a second pharmaceutical composition, then the kit may further contain instructions for the simultaneous, sequential or separate administration of the first and second pharmaceutical compositions to a patient in need thereof.

In another embodiment, the kits are suitable for administering oral solid dosage forms of trastuzumab-MCC-DM1, such as tablets or capsules. Preferably, such a kit contains a number of single dosage forms. Such kits may include a card with doses arranged in order for the intended use. An example of such a kit is a "blister pack". Blister packs are well known in the packaging industry and are widely used for the packaging of pharmaceutical unit dosage forms. If desired, the kit can be provided with a reminder, for example in the form of numbers, letters or other markings, or a calendar insert showing the treatment schedule in days on which doses can be administered.

In one embodiment, the kit contains (a) a first container with trastuzumab-MCC-DM1 in it, and optionally (b) a second container with a second pharmaceutical composition in it, wherein the second pharmaceutical composition contains a second compound having antihyperproliferative activity. Alternatively or additionally, the kit may also include a third container containing a pharmaceutically acceptable buffer such as bacteriostatic water for injection (BWFI), phosphate buffered saline, Ringer's solution and dextrose solution. Additionally, it may contain other substances desirable from a commercial and consumer point of view, including buffers, diluents, filters, needles and syringes.

In the event that the kit contains a composition of trastuzumab-MCC-DM1 and a second drug, i.e. chemotherapeutic agent, then the kit may contain a container for individual compositions, such as a divided bottle or a divided foil pack, however, individual compositions can also be contained in a single, undivided container. As a rule, the kit contains instructions for the introduction of individual components. The kit form is particularly advantageous when the individual components are preferably administered in different dosage forms (eg, orally and parenterally), administered at different dosing intervals, or when it is desired for the physician to titrate the individual components of the combination.

Examples

The following examples are given to illustrate the invention. However, it is obviously clear that these examples do not limit the invention and only mean suggestions for a way to practice the invention.

Example 1 Preparation of trastuzumab-MCC-DM1

Trastuzumab was isolated from ^Herceptin® by buffer exchange at a concentration of 20 mg/ml in a buffer consisting of 50 mM potassium phosphate/50 mM sodium chloride/2 mM EDTA, pH 6.5 and treated with 7.5-10 mole equivalents of succinimidyl-4 -(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC, Pierce Biotechnology, Inc), 20 mM in DMSO or DMA (dimethylacetamide), 6.7 mg/mL (US Patent Application 2005/0169933; US Patent Application 2005 /0276812). After stirring for 2-4 hours under argon at room temperature, the reaction mixture was passed through a Sephadex G25 column equilibrated with 50 mM potassium phosphate/50 mM sodium chloride/2 mM EDTA, pH 6.5 buffer. Alternatively, the reaction mixture was filtered through a gel with 30 mM citrate and 150 mM sodium chloride at pH 6. Fractions containing antibody were pooled and analyzed. The recovery of trastuzumab-SMCC was 88%.

The drug-linker intermediate, trastuzumab-MCC above, was diluted with 50 mM potassium phosphate/50 mM sodium chloride/2 mM EDTA pH 6.5 buffer to a final concentration of 10 mg/mL and reacted with 10 mM DM1 (1.7 eq, assuming a ratio of 5 SMCC/trastuzumab, 7.37 mg/ml) in dimethylacetamide. DM1 can be prepared from ansamitocin fermentation product (US Pat. No. 6,790,954; US Pat. No. 7,432,088) and derivatized for conjugation (US Pat. No. 6,333,410; RE 39151). The reaction mixture was stirred at room temperature under argon for about 4-16 hours. The conjugation reaction mixture was passed through a Sephadex G25 (1.5×4.9) filter column 1× PBS pH 6.5. Alternatively, the reaction mixture was passed through a gel with 10 mM citrate and 150 mM sodium chloride at pH 5. The DMI/trastuzumab ratio (p) was 3.1 as measured by absorbance at 252 nm and 280 nm. The ratio of drug to antibody (p) can be determined using mass spectrometry. Also, the progress of conjugation can be followed using SDS polyacrylamide gel electrophoresis. Aggregation can be assessed by analyzing the scattering of a laser beam of light.

Alternatively, trastuzumab-MCC-DM1 can be prepared by preparing the MCC-DM1 linker-drug reagent and then reacting with trastuzumab.

Typically, the DM1 conjugation reaction of trastuzumab-MCC results in a heterogeneous mixture containing antibodies with varying amounts of DM1 conjugated drug attached, i.e. with different drug loading, where p represents a distribution from 1 to about 8. There is an additional dimension of heterogeneity with different attachment sites of SMCC to trastuzumab, where many different nucleophiles in trastuzumab, for example, lysine amino terminal groups, can interact with SMCC. Thus, trastuzumab-MCC-DM1 includes isolated, purified molecules, as well as mixtures with an average drug load of 1 to 8, and where the MCC-DM1 is attached through any site of the trastuzumab antibody.

The average number of DM1 drug moieties per trastuzumab antibody in trastuzumab-MCC-DM1 conjugation preparations can be determined by conventional methods such as mass spectroscopy, ELISA, electrophoresis, and HPLC. It is also possible to determine the quantitative distribution of trastuzumab-MCC-DM1 by p value. ELISA can determine the average p value in a particular ADC preparation (Hamblett et al., 2004, Clinical Cancer Res., 10:7063-7070; Sanderson et al., 2005; Clinical Cancer Res., 11:843-852). However, the distribution of p (drug) values is not distinguishable by antibody-antigen binding and ELISA detection limit. Also, using ELISA for antibody-drug conjugates, it is not possible to determine in which region of the drug groups are attached to the antibody, for example, to fragments of the heavy chain or light chain, or to specific amino acid residues. In some cases it is possible to isolate, purify and characterize homogeneous trastuzumab-MCC-DM1, where p has defined values, from trastuzumab-MCC-DM1 loaded with other drugs by reverse phase HPLC or electrophoresis.

Example 2 In Vitro Cell Proliferation Test

The efficacy of the combinations of the invention can be determined by a cell proliferation assay using the following protocol (Promega Corp. Technical Bulletin TB288; Mendoza et al., 2002, Cancer Res., 62:5485-5488). The reagents and protocol for performing the Cell-Titer Glo assay are commercially available (Promega). With this test, the ability of compounds to penetrate cells and affect cell proliferation can be assessed. The principle of the method is to determine the number of available viable cells by quantitative analysis of ATP. Cell-Titer Glo is a reagent used for quantitative analysis. This is a homogeneous assay where the addition of the Cell-Titer Glo reagent results in cell lysis and generation of a luminescent signal in reaction with luciferase. The luminescence signal is directly proportional to the amount of ATP present.

DMSO and media plates: 96-well polypropylene plates manufactured by Nunc (cat.#249946).

Cell plates: 384-well black, transparent bottom (microclear) TC plates with lid manufactured by Falcon (353962).

Cell culture medium: RPMI or high glucose DMEM; Ham's F-12 medium (50:50), 10% fetal bovine serum, 2 mM L-glutamine.

Cell-Titer Glo: Promega (Cat.#G7572).

Method protocol:

1 day - cells are seeded in cell plates, cells are harvested, cells are seeded with a titer of 1000-2000 cells in 54 μl per well in 384-well cell plates for 3 days. Incubate overnight (approximately 16 h) at 37°C, in an atmosphere with 5% CO ₂ .

Day 2 - a drug, a diluted compound, and DMSO plates are added to the cells (serial dilutions 1:2 for 9 points). Transfer 20 μl of compounds (10 mM small molecule drug stock) to the second row of a 96-well plate. Serial 1:2 dilutions are made on the plate (10 µl + 10 µl 100% DMSO) for a total of 9 points using Precision Media plates (1:50 dilution). Dispense 147 µl of media into all wells of a separate 96-well media plate. From each well in the DMSO plate, transfer 3 μl of DMSO+compound to the corresponding well in the media plate using a Rapidplate. In two drug combination experiments, one drug in 1.5 µl DMSO+compound from each well in the DMSO plate was transferred to each corresponding well in the media plate using a Rapidplate. Then transfer 1.5 µl of the other preparation to the media plate.

Drugs are added to the cells, a cell plate (1:10 dilution), 6 µl of medium + compound are added directly to the cells (54 µl of medium is already on the cells). Incubate for 3 days in a thermostat at 37°C, in an atmosphere with 5% CO ₂ , which should not be opened frequently.

5 days - develop tablets, thaw buffer for Cell-Titer Glo at room temperature. Remove the cell plates from the 37° C. incubator and equilibrate to room temperature over about 30 minutes. Cell-Titer Glo buffer (bottle per bottle) is added to the Cell-Titer Glo substrate. Add 30 µl of Cell Titer-Glo reagent to each cell well. Place on a plate shaker for approximately 30 minutes. Determine the luminescence on a PerkinElmer Envision plate reader (0.1 sec per well) or Analyst HT (half a second per well).

Cell Viability Assay and Combination Tests: Cells are seeded at a titer of 1000-2000/well in 384-well plates at 16 hours. On day 2, nine 1:2 serial dilutions of the compound in DMSO are made in a 96-well plate. Compounds are then diluted in culture media using an automated Rapidplate device (Zymark Corp., Hopkinton, MA). The diluted compounds are then added to four wells in 384-well cell plates and incubated at 37° C. and 5% CO ₂ atmosphere. After 4 days, relative viable cell counts are determined by luminescence using a Cell-Titer Glo (Promega) following the manufacturer's instructions and the signal is recorded on an Envision or Wallac Multilabel reader (PerkinElmer, Foster City). Calculate EC ₅₀ using Kaleidagraph 4.0 (Synergy Software) or Prism 4.0 (GraphPad, San Diego) software. Drugs in combination tests are dosed starting at a concentration of 8×EC ₅₀ . Where the drug EC ₅₀ value was >2.5 μM, the highest concentration of 10 μM was used. In all tests, trastuzumab-MCC-DM1 and chemotherapeutic agents were added simultaneously or separately at an interval of 4 hours (one before the other).

In a further exemplary description of the in vitro cell proliferation test, the assay includes the following steps:

1. A 100 μl aliquot of cell culture containing approximately 10 ⁴ cells (see FIG. 1 for cell lines and tumor type) in medium is placed in each well of a 384-well plate with opaque walls.

2. Prepare control wells containing medium and without cells.

3. Compound is added to test wells and incubated for 3-5 days.

4. The plates are equilibrated to room temperature over about 30 minutes.

5. Add Cell-Titer Glo reagent to each well in a volume equal to the volume of cell culture medium contained in each well.

6. The contents are stirred for 2 minutes on an orbital shaker to induce cell lysis.

7. The plate is incubated at room temperature for 10 minutes to stabilize the luminescence signal.

8. Record luminescence and build graphs in RLU=relative luminescence units.

Alternatively, cells were seeded at optimal density in a 96-well plate and incubated for 4 days in the presence of the test compound. Then alamar blue ^TM was added to the medium for setting up the test, and the cells were incubated for 6 h before recording the signal at an excitation wavelength of 544 nm and an emission length of 590 nm. EC ₅₀ values were calculated using a sigmoidal dose-response curve.

Example 3

Tumor xenografts in vivo

Animals suitable for transgenic experiments can be obtained from conventional commercial nurseries. Immunodeficient female CB-17 SCID mice (Charles River Laboratory) were implanted with 3 million KPL-4 (overexpressing Her2) breast cancer cells in matrigel in the mammary fat pad. Nude female athymic mice (Charles River Laboratory or Harlan) were implanted with fragments of transgenic 2x2 mm ³ mammary tumors MMTV-Her2 Fo5 in the mammary fat pad. On day 0, xenografts in mice were injected with a drug, combination of drugs or vehicle according to the scheme corresponding to each tumor model. 5-FU, gemcitabine, carboplatin, and B20-4.1 were administered ip, pertuzumab was administered iv or ip as appropriate, trastuzumab-MCC-DM1 and docetaxel were administered iv, lapatinib, GDC-0941, and ABT-869 were administered orally via gastric tube. Tumor sizes were measured twice a week throughout the experiment. Also, twice a week, the weight of mice was determined and the condition of the animals was regularly monitored. Tumor volume was determined in two dimensions (length and width) using an Ultra Cal IV caliper (Model 54-10-111; Fred V. Fowler Co., Inc.; Newton, MA) and analyzed using Excel v. 11.2 (Microsoft Corporation; Redmond, WA). Tumor inhibition graphs were generated using KaleidaGraph 3.6 (Synergy Software; Reading, PA). The volume of tumors was calculated by the formula: tumor size (mm ³ ) = (longer dimension × shorter dimension ² ) ×0.5.

Animal weights were analyzed using the Adventurera Pro AV812 software (Ohaus Corporation; Pine Brook, NJ). Graphs were built using the KaleidaGraph Version 3.6 program. Percentage body weight change was calculated using the formula: Percentage weight change by group = (1-(initial weight/new weight)) × 100.

Mice with a tumor volume greater than 2000 mm ³ or with a loss of body weight of more than 20% of the original, were euthanized according to accepted practice.

Percentage tumor growth retardation (% TGD) at the end of the trial (EOS) was calculated using the formula: % TGD=100 × (mean time to endpoint in treatment group – mean time to endpoint in control group)/mean time to endpoint in the control group.

Tumor incidence (TI) was determined based on the number of detectable tumors remaining in each group at the end of the trial. Partial response (PR) was defined as greater than 50% but less than 100% reduction in tumor volume from baseline tumor volume for three consecutive measurements. Complete response (CR) was defined as a 100% reduction in tumor volume compared to baseline tumor volume for three consecutive measurements. Data were analyzed and p-values were determined using Dunnett's t-test with JMP statistical program, version 5.1.2 (SAS Institute; Cary, NC). The volume of individual tumors at the end of the experiment and the average volume of tumors±SEM were processed using the statistical program JMP, version 5.1.2. Build a graph according to the determination of body weight based on the average percentage change compared to the values of the initial body weight±SEM.

Example 4 Clinical Study of Trastuzumab-MCC-DM1

(T-DM1) in combination with pertuzumab

A phase 1b/II open clinical study of the safety, tolerability and efficacy of trastuzumab-MCC-DM1 (T-DM1) in combination with pertuzumab when administered intravenously to patients with HER2-positive locally advanced or metastatic breast cancer is being conducted to assess the safety and tolerability of the combination. The combination is administered once every 3 weeks to patients with HER2-positive locally advanced or metastatic breast cancer who have previously received trastuzumab in any line of therapy, received chemotherapy in combination with HER2-targeted therapy for disease in advanced stages or who have progressed disease when receiving the last therapy. Another goal is to evaluate the pharmacokinetics of T-DM1 when the combination of T-DM1 and pertuzumab is administered according to this regimen. Another study objective is to pre-evaluate the efficacy of the combination of T-DM1 and pertuzumab in this regimen based on investigator-based objective responses using the Response Evaluation Criteria for Solid Tumors (RECIST), version 1.0. The secondary objectives of this study are: (1) to establish progression-free survival (PFS) in patients treated with the combination of T-DM1 and pertuzumab when administered according to this regimen; (2) assessment of the duration of response to the combination of T-DM1 and pertuzumab when administered according to this scheme and (3) assessment of the production of antibodies to T-DM1.

T-DM1 is given by intravenous (IV) infusion in combination with pertuzumab, which is also given by intravenous (IV) infusion, to patients with HER2-positive locally advanced or metastatic breast cancer who have previously received trastuzumab and who have progressed disease after or during the last therapy. Patients receive a combination of T-DM1 and pertuzumab in repeated courses with a minimum interval of 3 weeks.

Patients are monitored at this dose for DLT (dosage-limiting toxicity) during the DLT observation period (21 days from the first T-DM1 administration) after the first doses of the test agents before administering higher doses to any patient. In the event that these patients do not observe DLT during the observation period for DLT, then the next dose can be increased.

DLT is defined as the occurrence of any of the following treatment-related toxic effects during the DLT follow-up period: (1) a non-haematological Grade ≥3 adverse effect that occurs not as a result of disease progression or other clearly identifiable cause, except alopecia of any degree; (2) grade ≥3 diarrhea that meets standard of care; (3) Grade 3 nausea or vomiting in the absence of premedication that meets the standard of care; (4) Elevation of bilirubin, liver transaminase activity (ALT or AST) or alkaline phosphatase (AP) in serum grade ≥3, lasting for 72 hours, except for patients with liver transaminase activity and grade 2 ALP at baseline (≤ 5 upper limit of normal [ULN] due to presence of liver and bone metastases Transaminase activity or ALP grade ≥10 ULN will be considered as a manifestation of DLT (5) thrombocytopenia grade ≥4 lasting 24 hours (6) neutropenia grade ≥4 (absolute neutrophil count <500 cells/mm ³ ) lasting for 4 days or accompanied by fever (temperature measured in the mouth and tympanic temperature of 100.4°F or 38°C) (7) any subjectively assessed side effect, reported by the investigator associated with administration of each test compound; (8) any treatment-related toxicity that precludes initiation of a second course of treatment.

After a decision has been made to continue treatment at the next higher dose, an increase in the dose for patients is allowed; patients in this study initially receive a reduced dose of T-DM1 (3.0 mg/kg) along with a full dose of pertuzumab. These patients are allowed to increase their doses of both drugs to full doses on further courses if they have completed their DLT follow-up period. However, the safety of the 3.6 mg/kg dosage will be based on the DLT assessment. Patients (including patients enrolled in a study during the dose escalation phase of a study) are considered to be evaluable for efficacy if they remain in the study until the first subsequent tumor evaluation. At the end of the first course and then every three courses during treatment, echocardiography (ECHO) or MUGA-scan (multi-entry arteriography) should be performed.

Formulating T-DM1

T-DM1 can be formulated as a lyophilized single use formulation in a 20 ml USP/EP Type I glass vial with a 20 mm fluoro rubber laminated stopper and a rolled aluminum cap with a dark gray removable plastic cap. After reconstitution with 8.0 ml of sterile water for injection (SWFI), the resulting product contains T-DM1 at a concentration of 20 mg/ml in 10 mM sodium succinate, pH 5.0, 6% (w/v) sucrose and 0.02 % (w/v) polysorbate 20. Each 20 ml vial contains approximately 172 mg of T-DM1, allowing 160 mg of T-DM1 to be administered. The indicated volume of T-DM1 solution is withdrawn from the vial(s) and transferred to an intravenous bag. Reconstituted T-DM1 is reconstituted in polyvinyl chloride (PVC) or latex-free, PVC-free polyolefin (PO) bags with 0.45% or 0.9% sodium chloride injection (minimum volume 250 ml). PVC or PO bags containing 0.45% sodium chloride are preferred. In the case of PVC or PO bags containing 0.9% sodium chloride, it is recommended to use built-in 0.22 µm filters. The bag is carefully inverted to mix the solution. T-DM1 solution for infusion, reconstituted in polyvinyl chloride (PVC) or latex-free, PVC-free polyolefin (PO) bags with 0.9% or 0.45% sodium chloride injection USP, can be stored at 2°-8° C (36°-46°F) for a short period of time.

Formulation of pertuzumab

Pertuzumab can be formulated as a single use formulation containing pertuzumab at a concentration of 30 mg/ml in a buffer of 20 mM L-histidine (pH 6.0), 120 mM sucrose and 0.02% polysorbate 20. Each 20 cm ³ vial contains approximately 420 mg of pertuzumab (14.0 ml/vial). The indicated volume of pertuzumab solution is withdrawn from the vials and transferred to a 250 cm ³ intravenous bag containing 0.9% sodium chloride injection. The bag is gently inverted to mix the solution and visually inspected for particles and discoloration prior to administration. Pertuzumab infusion solution reconstituted in polyethylene or non-PVC polyolefin bags with 0.9% sodium chloride injection can be stored at 2°-8°C (36°-46°F) for a short period of time.

Definition of safety indicators

The safety and tolerability of T-DM1 and pertuzumab is assessed by the following key safety indicators: (1) frequency, origin, and severity of side effects; (2) side effects or changes in physical condition and clinical laboratory results during and after administration of the test agents that result in dose modification, dose reduction or discontinuation of T-DM1 and pertuzumab treatment and (3) changes in cardiac function (i.e. e. left ventricular ejection fraction [LVEF], segmental wall abnormalities), including ECHO or MUGA data.

Determination of pharmacokinetic and

pharmacodynamic parameters

In all patients receiving test treatment, the pharmacokinetic parameters of T-DM1 and pertuzumab are determined using non-compartmental and/or population methods, when appropriate, as the following parameters: (1) T-DM1 (conjugate) serum concentration, trastuzumab as a whole (free and conjugated to DM1); (2) free DM1 plasma concentration; (3) total exposure (area under the concentration-time curve [AUC]; (4) maximum serum concentration (Cmax); (5) minimum concentration (Cmin); (6) clearance; (7) volume of distribution; (8) ) terminal half-life (9) antibodies to T-DM1.

Definition of effectiveness

Objective responses are determined as performance evaluation data using the RECIST v. 1.0. In this study, efficacy is assessed by the following secondary measures: (1) PFS, defined as the time from the start of the study treatment to the first manifestation of disease progression or death during the study period (within 30 days after the last dose of the study treatment) from any cause for Tumor assessment report data using RECIST v. 1.0 and (2) duration of response, defined as the first occurrence of a documented objective response to the time of onset of disease progression as measured by a tumor assessment report using modified RECIST v. 1.0 or death during the study period (within 30 days of the last dose of the study treatment) from any cause.

Trial treatment

T-DM1 is administered no more than once every 3 weeks at doses of 2.4; 3.0 or 3.6 mg/kg IV. Any patient can be switched to a lower dose of T-DM1 equal to 2.4 mg/kg. Depending on the toxicity observed in the group of patients who started therapy at a dose of 3.0 mg/kg, and if it is confirmed that T-DM1 is well tolerated at a dose of 3.0 mg/kg, then in subsequent courses, patients can be transferred to a higher dose of 3.6 mg/kg IV once every 3 weeks. Pertuzumab is administered at a loading dose of 840 mg IV for 1 day, 1 course, then 420 mg IV every 3 weeks in subsequent cycles.

Statistical Methods

The primary efficacy endpoint in this study is the investigator-assessed objective response, defined as a complete or incomplete response in two consecutive surveys ≥4 weeks apart. The value of the frequency of manifestation of the main response is obtained using a computer program, as well as the corresponding 95% confidence interval. In terms of objective response, patients without a reliable tumor assessment at baseline are considered to be non-responders. With respect to duration of response and PFS, data from patients lost to follow-up are considered to the most recent time when there was evidence of no disease progression. Data for patients without tumor evaluation after treatment or at the time of death are considered at the time of treatment initiation plus 1 day.

Example 5 Clinical Study of Trastuzumab-MCC-DM1

(T-DM1) in combination with GDC-0941

An open-label Phase Ib clinical study is underway on the combination of IV T-DM1 and oral GDC-0941 in patients with HER2-positive locally advanced or metastatic breast cancer who have experienced disease progression on prior trastuzumab therapy. , to evaluate the safety, tolerability, pharmacokinetics, and efficacy of the combination. The main objectives of this study are: to assess the safety and tolerability of GDC-0941 when administered in combination with T-DM1; determination of MTD GDC-0941 when administered in combination with T-DM1; identification of the recommended phase II dose for GDC-0941 in combination with T-DM1 and evaluation of any observed antitumor activity of GDC-0941 when administered in combination with T-DM1. The pharmacokinetic objectives are to characterize the pharmacokinetics of GDC-0941 in the absence and presence of T-DM1 and to characterize the pharmacokinetics of T-DM1 in the relative absence and presence of GDC-0941.

Formulation of GDC-0941

GDC-0941 is a dry powder intended for oral administration. The prepared drug product is placed in hard gelatin capsules with two strengths of action (15 and 50 mg), which are encapsulated in size 0 shells and differentiated by color. The excipients included in the capsules are Microcrystalline Cellulose NF/EP, Sodium Lauryl Sulfate NF/DP (only in 50 mg potency capsules), Citric Acid Anhydrous USP/EP, Croscarmellose Sodium NF/EP, Colloidal Silicon Dioxide NF/EP and magnesium stearate (non-bovine) NF/EP. GDC-0941 capsules should be stored refrigerated between 36°F and 46°F (2°C and 8°C). Patients should be instructed on the required refrigeration storage regimen between 36°F and 46°F (2°C and 8°C).

Definition of effectiveness

The parameters of safety, pharmacokinetics, pharmacodynamics and efficacy are determined and evaluated, including statistical methods, as described in example 4.

Trial treatment

The test treatment is carried out in 3-week courses. Patients who experience clinical benefit as a result of a trial treatment may be able to receive more courses of therapy, which may take place in a single study, depending on the stage of drug development, drug availability and other factors.

During the escalation phase of the study, study patients receive a single dose of GDC-0941 on day 1 of fasted course 1 for pre- and post-dose sampling for pharmacokinetic studies and observation of inter-patient variability. The starting dose of GDC-0941 is 60 mg once daily, which has been determined in a phase I clinical study to be safe when given alone with the drug without dose-limiting toxicity. On the 2nd day of the 1st course, a full dose of T-DM1 is administered, which is 3.6 mg/kg IV for 90 minutes without a loading dose. This is followed by a dose of GDC-0941. The condition of the patients is monitored for 90 minutes after the first infusion of T-DM1. GDC-0941 is then administered once a day for a total of 14 days followed by a 1 week interval after the first cycle.

Increasing the dose of GDC-0941 in subsequent patients is continued until disease progression or drug intolerance occurs. The duration of subsequent courses of study treatment will be 3 weeks at a dose of T-DM1 equal to 3.6 mg/kg IV for 30 minutes on day 1 of each course, and after T-DM1 infusion, GDC-0941 is administered and treatment is continued for 2 weeks and make an interval of 1 week. The introduction is continued until the progression of the disease or the manifestation of intolerance to the drug. T-DM1 should be administered over a 30-90 (±10) minute IV infusion, depending on how T-DM1 is tolerated in the initial study. If the 90-minute infusion is well tolerated, subsequent infusions may be given over 30 (±10) minutes.

The above description should be considered only as illustrative in relation to the principles of the invention. In addition, since numerous modifications and changes are obvious to those skilled in the art, it is not desirable to limit the invention to the exact design and method described herein. Therefore, all suitable modifications and equivalent variations are within the scope of the invention as defined by the claims that follow.

Claims (11)

1. A method of treating an ErbB2-expressing cancer comprising administering a therapeutic combination as a combination composition or alternately to a mammal, wherein the therapeutic combination comprises trastuzumab-MCC-DM1 and docetaxel, wherein administration of the therapeutic combination results in an additive effect compared to administering trastuzumab-MCC- DM1 or docetaxel as separate agents, where the amount of trastuzumab-MCC-DM1 is from 0.3 to 15 mg/kg, and where the amount of docetaxel is approximately 30 mg/kg. 2. The method of claim 1 wherein trastuzumab-MCC-DM1 and docetaxel are administered as a combination formulation. 3. The method of claim 1 wherein trastuzumab-MCC-DM1 and docetaxel are administered alternately. 4. The method of claim 3 wherein docetaxel is administered to the mammal and then trastuzumab-MCC-DM1 is subsequently administered. 5. The method according to any one of paragraphs. 1-4, wherein the therapeutic combination is administered to a human suffering from ErbB2 expressing cancer at intervals of approximately three weeks. 6. The method of claim 3 wherein trastuzumab-MCC-DM1 is administered to a human afflicted with ErbB2 expressing cancer at intervals of about one week to three weeks. 7. The method of claim 3 wherein trastuzumab-MCC-DM1 is administered no more frequently than every 3 weeks at a dosage of 2.4, 3.0, or 3.6 mg/kg IV. 8. The method according to any one of paragraphs. 1-7, where the amount of trastuzumab-MCC-DM1 and the amount of docetaxel are each from about 1 mg to about 1000 mg, and the amount of trastuzumab-MCC-DM1 and the amount of docetaxel are in a ratio of from about 1:10 to about 10:1 mass. 9. The method of claim 1 wherein the mammal is a HER2 positive patient. 10. The method of claim 9, wherein the HER2 positive patient was treated with trastuzumab or lapatinib.

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