U.S. patent application number 14/166519 was filed with the patent office on 2014-05-22 for combinations of an anti-her2 antibody-drug conjugate and chemotherapeutic agents, and methods of use.
This patent application is currently assigned to Genentech, Inc.. The applicant listed for this patent is Genentech, Inc.. Invention is credited to Gail Lewis Phillips, Leanne Berry Ross, Mark X. Sliwkowski.
Application Number | 20140140993 14/166519 |
Document ID | / |
Family ID | 40858064 |
Filed Date | 2014-05-22 |
United States Patent
Application |
20140140993 |
Kind Code |
A1 |
Ross; Leanne Berry ; et
al. |
May 22, 2014 |
COMBINATIONS OF AN ANTI-HER2 ANTIBODY-DRUG CONJUGATE AND
CHEMOTHERAPEUTIC AGENTS, AND METHODS OF USE
Abstract
Combinations of the antibody-drug conjugate trastuzumab-MCC-DM1
and chemotherapeutic agents, including stereoisomers, geometric
isomers, tautomers, solvates, metabolites and pharmaceutically
acceptable salts thereof, are useful for inhibiting tumor cell
growth, and for treating disorders such as cancer mediated by HER2
and KDR (VEGFR receptor 1). Methods of using such combinations for
in vitro, in situ, and in vivo diagnosis, prevention or treatment
of such disorders in mammalian cells, or associated pathological
conditions, are disclosed.
Inventors: |
Ross; Leanne Berry; (South
San Francisco, CA) ; Phillips; Gail Lewis; (South San
Francisco, CA) ; Sliwkowski; Mark X.; (South San
Francisco, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Genentech, Inc. |
South San Francisco |
CA |
US |
|
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
40858064 |
Appl. No.: |
14/166519 |
Filed: |
January 28, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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12400988 |
Mar 10, 2009 |
8663643 |
|
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14166519 |
|
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61037410 |
Mar 18, 2008 |
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Current U.S.
Class: |
424/133.1 ;
424/181.1; 435/32; 435/8 |
Current CPC
Class: |
A61P 35/02 20180101;
A61K 2039/507 20130101; G01N 33/5044 20130101; A61P 43/00 20180101;
A61K 31/517 20130101; G01N 2800/52 20130101; A61P 35/00 20180101;
G01N 33/5011 20130101; G01N 2500/10 20130101; G01N 33/57415
20130101; G01N 2333/71 20130101; A61K 31/555 20130101; A61K 9/0019
20130101; A61K 47/6803 20170801; A61K 31/5377 20130101; A61K 45/06
20130101; A61K 31/337 20130101; C07K 16/32 20130101; A61K 31/513
20130101; A61K 39/39558 20130101; A61K 47/6855 20170801; A61K
31/5365 20130101; A61K 31/416 20130101; A61K 31/5355 20130101; A61P
35/04 20180101; A61K 31/337 20130101; A61K 2300/00 20130101; A61K
31/416 20130101; A61K 2300/00 20130101; A61K 31/513 20130101; A61K
2300/00 20130101; A61K 31/517 20130101; A61K 2300/00 20130101; A61K
31/5355 20130101; A61K 2300/00 20130101; A61K 31/555 20130101; A61K
2300/00 20130101; A61K 39/39558 20130101; A61K 2300/00 20130101;
A61K 31/5377 20130101; A61K 2300/00 20130101 |
Class at
Publication: |
424/133.1 ;
424/181.1; 435/32; 435/8 |
International
Class: |
A61K 47/48 20060101
A61K047/48; G01N 33/50 20060101 G01N033/50; A61K 31/517 20060101
A61K031/517; A61K 31/337 20060101 A61K031/337; A61K 39/395 20060101
A61K039/395; A61K 31/5377 20060101 A61K031/5377 |
Claims
1. A method for the treatment of a hyperproliferative disorder
comprising administering a therapeutic combination as a combined
formulation or by alternation 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 a HER2 dimerization inhibitor
antibody, lapatinib, and docetaxel.
2. The method of claim 1 wherein the HER2 dimerization inhibitor
antibody is pertuzumab.
3. The method of claim 1 wherein the chemotherapeutic agent is
lapatinib.
4. The method of claim 1 wherein the chemotherapeutic agent is
docetaxel.
5. The method of claim 1 wherein the therapeutically effective
amount of trastuzumab-MCC-DM1 and the therapeutically effective
amount of the chemotherapeutic agent are administered as a combined
formulation.
6. The method of claim 1 wherein the therapeutically effective
amount of trastuzumab-MCC-DM1 and the therapeutically effective
amount of the chemotherapeutic agent are administered by
alternation.
7. The method of claim 6 wherein the mammal is administered the
chemotherapeutic agent and then subsequently administered
trastuzumab-MCC-DM1.
8. The method of claim 5 wherein the combined formulation is
administered at about three week intervals to a human subject with
the hyperproliferative disorder.
9. The method of claim 6 wherein trastuzumab-MCC-DM1 is
administered at intervals from about one week to three weeks to a
human subject with the hyperproliferative disorder.
10. The method of claim 6 wherein trastuzumab-MCC-DM1 is
administered at about three week intervals to a human subject with
the hyperproliferative disorder.
11. The method of claim 1 wherein administration of the therapeutic
combination results in a synergistic effect.
12. The method of claim 1 wherein the hyperproliferative disorder
is cancer.
13. The method of claim 12 wherein the hyperproliferative disorder
is a cancer expressing ErbB2.
14. The method of claim 13 wherein the cancer is breast, ovary,
cervix, prostate, testis, genitourinary tract, esophagus, larynx,
glioblastoma, neuroblastoma, stomach, skin, keratoacanthoma, lung,
epidermoid carcinoma, large cell carcinoma, non-small cell lung
carcinoma (NSCLC), small cell carcinoma, lung adenocarcinoma, bone,
colon, adenoma, pancreas, adenocarcinoma, thyroid, follicular
carcinoma, undifferentiated carcinoma, papillary carcinoma,
seminoma, melanoma, sarcoma, bladder carcinoma, liver carcinoma and
biliary passages, kidney carcinoma, pancreatic, myeloid disorders,
lymphoma, hairy cells, buccal cavity, nasopharyngeal, pharynx, lip,
tongue, mouth, small intestine, colon-rectum, large intestine,
rectum, brain and central nervous system, Hodgkin's or
leukemia.
15. The method of claim 14 wherein the cancer is breast, ovary or
stomach cancer.
16. The method of claim 15 wherein the cancer is breast cancer.
17. The method of claim 1 wherein the amount of trastuzumab-MCC-DM1
and the amount of chemotherapeutic agent are each from about 1 mg
to about 1000 mg.
18. The method of claim 1 wherein the amount of trastuzumab-MCC-DM1
and the amount of chemotherapeutic agent are in a ratio of about
1:10 to about 10:1 by weight.
19. The method of claim 1 wherein the mammal is a HER2 positive
human subject.
20. The method of claim 19 wherein the hyperproliferative disorder
is HER2 positive cancer.
21. The method of claim 20 wherein the cancer is HER2 positive
breast, ovary or stomach cancer.
22. The method of claim 21 wherein the cancer is HER2 positive
breast cancer.
23. The method of claim 22 wherein the breast cancer is metastatic
breast cancer.
24. The method of claim 19 wherein the HER2 positive human subject
has received trastuzumab or lapatinib therapy.
25. The method of any one of claims 8-10 wherein the
trastuzumab-MCC-DM1 is administered at a dose of 2.4, 3.0 or 3.6
mg/kg intravenously.
26. The method of claim 19 wherein the trastuzumab-MCC-DM1 is
administered at a dose of 2.4, 3.0 or 3.6 mg/kg intravenously.
27. The method of claim 21 or claim 22 wherein the
trastuzumab-MCC-DM1 is administered at a dose of 2.4, 3.0 or 3.6
mg/kg intravenously.
28. The method of claim 27 wherein the trastuzumab-MCC-DM1 is
administered at a dose of 3.6 mg/kg intravenously.
29. A pharmaceutical composition comprising trastuzumab-MCC-DM1, a
chemotherapeutic agent selected from a HER2 dimerization inhibitor
antibody, lapatinib, and docetaxel, and one or more
pharmaceutically acceptable carrier, glidant, diluent, or
excipient.
30. The pharmaceutical composition of claim 29 comprising a
pharmaceutically acceptable glidant selected from silicon dioxide,
powdered cellulose, microcrystalline cellulose, metallic stearates,
sodium aluminosilicate, sodium benzoate, calcium carbonate, calcium
silicate, corn starch, magnesium carbonate, asbestos free talc,
stearowet C, starch, starch 1500, magnesium lauryl sulfate,
magnesium oxide, and combinations thereof.
31. The pharmaceutical composition of claim 29 wherein the amount
of trastuzumab-MCC-DM1 and the amount of chemotherapeutic agent are
each present from about 1 mg to about 1000 mg.
32. The pharmaceutical composition of claim 29 wherein the amount
of trastuzumab-MCC-DM1 and the amount of chemotherapeutic agent are
present in a ratio of about 1:10 to about 10:1 by weight.
33. An article of manufacture for treating a hyperproliferative
disorder comprising: a) a therapeutic combination administered to a
mammal as a combined formulation or by alternation, and comprising
a therapeutically effective amount of trastuzumab-MCC-DM1, and a
therapeutically effective amount of a chemotherapeutic agent
selected from a HER2 dimerization inhibitor antibody, lapatinib,
and docetaxel; and b) instructions for use.
34. A method for determining compounds to be used in combination
for the treatment of cancer comprising: a) administering a
therapeutic combination of trastuzumab-MCC-DM1 and a
chemotherapeutic agent selected from a HER2 dimerization inhibitor
antibody, lapatinib, and docetaxel, to HER2-amplified breast cancer
cells, and b) measuring inhibition of cellular proliferation
wherein nonmalignant and malignant mammary cells are discriminated
by cell viability.
35. The method of claim 34 wherein the HER2-amplified breast cancer
cells are BT-474.
36. A method for determining a synergistic therapeutic combination
to be used for the treatment of cancer comprising: a) treating an
in vitro tumor cell line with a combination of trastuzumab-MCC-DM1,
and a chemotherapeutic agent selected from a HER2 dimerization
inhibitor antibody, lapatinib, and docetaxel, and b) measuring a
synergistic or non-synergistic effect; whereby a synergistic
therapeutic combination for the treatment of cancer is
determined.
37. A method for the treatment of a cancer expressing ErbB2
comprising administering trastuzumab-MCC-DM1 intravenously once
every three weeks to a patient having the cancer at a dose of 2.4
mg/kg, 3.0 mg/kg, or 3.6 mg/kg.
38. The method of claim 37, wherein the trastuzumab-MCC-DM1 is
administered at a dose of 3.6 mg/kg intravenously.
39. The method of claim 37, wherein the cancer expressing ErbB2 is
breast cancer.
40. The method of claim 39, wherein the breast cancer is metastatic
breast cancer.
41. The method of claim 37, wherein the patient is a HER2 positive
patient.
42. The method of claim 41, wherein the HER2 positive patient has
received trastuzumab or lapatinib therapy.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a divisional application of U.S. patent
application Ser. No. 12/400,988 filed Mar. 10, 2009, which claims
priority under 35 U.S.C. Section 119(e) and the benefit of U.S.
Provisional Application Ser. No. 61/037,410 filed Mar. 18, 2007,
the entire disclosures of which are incorporated herein by
reference in their entireties.
FIELD OF THE INVENTION
[0002] The invention relates generally to pharmaceutical
combinations of compounds with activity against hyperproliferative
disorders such as cancer. The invention also relates to methods of
using the combinations of compounds for in vitro, in situ, and in
vivo diagnosis or treatment of mammalian cells, or associated
pathological conditions.
BACKGROUND OF THE INVENTION
[0003] The HER2 (ErbB2) receptor tyrosine is a member of the
epidermal growth factor receptor (EGFR) family of transmembrane
receptors. Overexpression of HER2 is observed in approximately 20%
of human breast cancers and is implicated in the aggressive growth
and poor clinical outcomes associated with these tumors (Slamon et
al (1987) Science 235:177-182).
[0004] Trastuzumab (CAS 180288-69-1, HERCEPTIN.RTM., huMAb4D5-8,
rhuMAb HER2, Genentech) is a recombinant DNA-derived humanized,
IgG1 kappa, monoclonal antibody version of the murine HER2 antibody
which selectively binds with high affinity in a cell-based assay
(Kd=5 nM) to the extracellular domain of the human epidermal growth
factor receptor2 protein, HER2 (ErbB2) (U.S. Pat. No. 5,677,171;
U.S. Pat. No. 5,821,337; U.S. Pat. No. 6,054,297; U.S. Pat. No.
6,165,464; U.S. Pat. No. 6,339,142; U.S. Pat. No. 6,407,213; U.S.
Pat. No. 6,639,055; U.S. Pat. No. 6,719,971; U.S. Pat. No.
6,800,738; U.S. Pat. No. 7,074,404; Coussens et al (1985) Science
230:1132-9; Slamon et al (1989) Science 244:707-12; Slamon et al
(2001) New Engl. J. Med. 344:783-792). Trastuzumab contains human
framework regions with the complementarity-determining regions of a
murine antibody (4D5) that binds to HER2. Trastuzumab binds to the
HER2 antigen and thus inhibits the growth of cancerous cells.
Trastuzumab has been shown, in both in vitro assays and in animals,
to inhibit the proliferation of human tumor cells that overexpress
HER2 (Hudziak et al (1989) Mol Cell Biol 9:1165-72; 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 M D, et al (1997) [abstract]. Proc Am Assoc Cancer Res;
38:602; Sliwkowski et al (1999) Seminars in Oncology 26(4),
Supp112:60-70; Yarden Y. and Sliwkowski, M. (2001) Nature Reviews
Molecular Cell Biology, Macmillan Magazines, Ltd., Vol.
2:127-137).
[0005] HERCEPTIN.RTM. was approved in 1998 for the treatment of
patients with ErbB2-overexpressing metastatic breast cancers
(Baselga et al, (1996) J. Clin. Oncol. 14:737-744) that have
received extensive prior anti-cancer therapy, and has since been
used in over 300,000 patients (Slamon D J, et al. N Engl J Med
2001; 344:783-92; Vogel C L, et al. J Clin Oncol 2002; 20:719-26;
Marty M, et al. J Clin Oncol 2005; 23:4265-74; Romond E H, et al. T
N Engl J Med 2005; 353:1673-84; Piccart-Gebhart M J, 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.RTM. (trastuzumab, Genentech Inc.) as part of a treatment
regimen containing doxorubicin, cyclophosphamide and paclitaxel for
the adjuvant treatment of patients with HER2-positive,
node-positive breast cancer. While the development of
HERCEPTIN.RTM. provided patients with HER2-positive tumors a
markedly better outcome than with chemotherapy alone, virtually all
HER2-positive, metastatic breast cancer (MBC) patients will
eventually progress on available therapies. Opportunities remain to
improve outcomes for patients with MBC. Despite trastuzumab's
diverse mechanisms of action, a number of patients treated with
trastuzumab show either no response or stop responding after a
period of treatment benefit. Some HER2+ (HER2 positive) tumors fail
to respond to HERCEPTIN.RTM. and the majority of patients whose
tumors respond eventually progress. There is a significant clinical
need for developing further HER2-directed cancer therapies for
patients with HER2-overexpressing tumors or other diseases
associated with HER2 expression that do not respond, or respond
poorly, to HERCEPTIN.RTM. treatment.
[0006] An alternative approach to antibody-targeted therapy is to
utilize antibodies for delivery of cytotoxic drugs specifically to
antigen-expressing cancer cells. Maytansinoids, derivatives of the
anti-mitotic drug maytansine, bind to microtubules in a manner
similar to vinca alkaloid drugs (Issell B F et al (1978) Cancer
Treat. Rev. 5:199-207; Cabanillas F et al. (1979) Cancer Treat Rep,
63:507-9. Antibody-drug conjugates (ADCs) composed of the
maytansinoid DM1 linked to trastuzumab show potent anti-tumor
activity in HER2-overexpressing trastuzumab-sensitive and
trastuzumab-resistant tumor cell lines, and xenograft models of
human breast cancer. A conjugate of maytansinoids linked to the
anti-HER2 murine breast cancer antibody TA.1 via the MCC linker was
200-fold less potent than the corresponding conjugate with a
disulfide linker (Chari et al (1992) Cancer Res. 127-133).
Antibody-drug conjugates (ADCs) composed of the maytansinoid, DM1,
linked to trastuzumab show potent anti-tumor activity in
HER2-overexpressing trastuzumab-sensitive and -resistant tumor cell
lines and xenograft models of human cancer. Trastuzumab-MCC-DM1
(T-DM1) is currently undergoing evaluation in phase II clinical
trials in patients whose disease is refractory to HER2-directed
therapies (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, Sep. 23-27, 2007, Barcelona; U.S. Pat. No. 7,097,840; US
2005/0276812; US 2005/0166993).
[0007] Combination therapy in which two or more drugs are used
together in some dosing regimen or administration form, typically
has one or more goals of: (i) reducing the frequency at which
acquired resistance arises by combining drugs with minimal
cross-resistance, (ii) lowering the doses of drugs with
non-overlapping toxicity and similar therapeutic profile so as to
achieve efficacy with fewer side effects, i.e. increase therapeutic
index, (iii) sensitizing cells to the action of one drug through
use of another drug, such as altering cell-cycle stage or growth
properties, and (iv) achieving enhanced potency by exploiting
additivity, or greater than additivity, effects in the biological
activity of 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. of the Nat.
Cancer Inst. 96(10):739-749; Fitzgerald et al (2006) Nature Chem.
Biol. 2(9):458-466; Borisy et al (2003) Proc. Natl. Acad. Sci.
100(13):7977-7982).
[0008] Loewe additivity (Chou, T. C. and Talalay, P. (1977) J.
Biol. Chem. 252:6438-6442; Chou, T. C. and Talalay, P. (1984) Adv.
Enzyme Regul. 22:27-55; Berenbaum, M. C. (1989) Pharmacol. Rev.
41:93-141) and Bliss independence/synergy (Bliss, C.I. (1956)
Bacteriol. Rev. 20:243-258; Greco et al (1995) Pharmacol. Rev.
47:331-385) are methods used for calculating the expected
dose-response relationship for combination therapy compared to
monotherapy based on parameters such as IC50, the dose of drug
needed to achieve 50% target inhibition and equal to Ki in the
simplest case.
[0009] HER2 dimerization inhibitor antibodies and EGFR inhibitors
have been reported for combination therapy against cancer (US
2007/0020261). Trastuzumab-MCC-DM1 (T-DM1) and pertuzumab have
individually demonstrated activity in MBC patients, and a
combination of pertuzumab and trastuzumab has been shown to be
active in HER-positive MBC patients (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, Sep. 12-16, 2008).
SUMMARY OF THE INVENTION
[0010] The invention relates generally to the anti-HER2
antibody-drug conjugate, trastuzumab-MCC-DM1, administered in
combination with one or more chemotherapeutic agents to inhibit the
growth of cancer cells. Certain combinations of trastuzumab-MCC-DM1
and a chemotherapeutic agent show synergistic effects in inhibiting
the growth of cancer 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 tumor growth in mammals and may be useful for treating
human cancer patients.
[0011] In one aspect, the invention includes a method for the
treatment of a hyperproliferative disorder comprising administering
a therapeutic combination as a combined formulation or by
alternation 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 a HER2 dimerization inhibitor
antibody, an anti-VEGF antibody, 5-FU, carboplatin, lapatinib,
ABT-869, docetaxel, GDC-0941, and GNE-390.
[0012] The therapeutically effective amount of trastuzumab-MCC-DM1
and the therapeutically effective amount of the chemotherapeutic
agent may be administered as a combined formulation or by
alternation.
[0013] The invention also relates to methods of using the
compositions for in vitro, in situ, and in vivo diagnosis or
treatment of mammalian cells, organisms, or associated pathological
conditions.
[0014] The invention also relates to methods wherein administration
of the therapeutic combination results in a synergistic effect.
[0015] Another aspect of the invention are pharmaceutical
compositions comprising trastuzumab-MCC-DM1, a chemotherapeutic
agent selected from a HER2 dimerization inhibitor antibody, an
anti-VEGF antibody, 5-FU, carboplatin, lapatinib, ABT-869,
docetaxel, GDC-0941, and GNE-390; and one or more pharmaceutically
acceptable carrier, glidant, diluent, or excipient.
[0016] Another aspect of the invention provides methods of treating
a hyperproliferative disease or disorder, comprising administering
to a mammal in need of such treatment effective amounts of
trastuzumab-MCC-DM1 and a chemotherapeutic agent.
Trastuzumab-MCC-DM1 and the chemotherapeutic agent may be
co-formulated for administration in a combination as a
pharmaceutical formulation or they may be administered separately
in alternation (alternating, sequential dosages) as a therapeutic
combination. In one embodiment, T-DM1 is delivered by infusion and
the chemotherapeutic agent is delivered orally.
[0017] Another aspect of the invention provides methods to predict
effective drug combinations for in vivo efficacy where the
combinations include trastuzumab-MCC-DM1 and an anti cancer,
standard-of-care, chemotherapeutic agent. Efficacy data from in
vitro cell proliferation and in vivo tumor xenograft experiments
are analyzed qualitatively and quantitatively. Quantitative
analysis methods may be based on the Chou & Talalay median
effect and isobolograms generating a combination index (CI) value
to denote synergy, antagonism, or additivity, or on the Bliss
Independence ribbon graph deflection.
[0018] Another aspect of the invention is a method of using a
therapeutic combination of the invention to treat a disease or
condition such as cancer, including one modulated by HER2 or KDR9
(VEGF receptor 1) in a mammal.
[0019] Another aspect of the invention is the use of a therapeutic
combination of the invention in the preparation of a medicament for
the treatment of a disease or condition such as cancer, including
one modulated by HER2 or KDR9 (VEGF receptor 1) in a mammal.
[0020] Another aspect of the invention includes articles of
manufacture or kits comprising trastuzumab-MCC-DM1, a
chemotherapeutic agent, a container, and optionally a package
insert or label indicating a treatment.
[0021] Another aspect of the invention includes a method for
determining compounds to be used in combination for the treatment
of cancer comprising: a) administering a therapeutic combination of
trastuzumab-MCC-DM1, and a chemotherapeutic agent selected from a
HER2 dimerization inhibitor antibody, an anti-VEGF antibody, 5-FU,
carboplatin, lapatinib, ABT-869, docetaxel, GDC-0941, and GNE-390
to an in vitro tumor cell line, and b) measuring a synergistic or
non-synergistic effect.
[0022] Additional advantages and novel features of this invention
shall be set forth in part in the description that follows, and in
part will become apparent to those skilled in the art upon
examination of the following specification or may be learned by the
practice of the invention. The advantages of the invention may be
realized and attained by means of the instrumentalities,
combinations, compositions, and methods particularly pointed out in
the appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0023] FIG. 1 shows a plot of SK-BR-3 in vitro cell viability at 3
days versus IC50 multiple concentrations of trastuzumab,
trastuzumab-MCC-DM1 (T-DM1), and the combination of trastuzumab and
T-DM1.
[0024] FIG. 2 shows a plot of BT-474 EEI in vitro cell viability at
3 days versus IC50 multiple concentrations of trastuzumab,
trastuzumab-MCC-DM1 (T-DM1), and the combination of trastuzumab and
T-DM1.
[0025] FIG. 3 shows a plot of MDA-MB-175 in vitro cell viability at
5 days versus IC50 multiple concentrations of pertuzumab,
trastuzumab-MCC-DM1 (T-DM1), and the combination of pertuzumab and
T-DM 1.
[0026] FIG. 3a shows a plot of MDA-MB-175 in vitro cell viability
at 5 days versus IC50 multiple concentrations of pertuzumab,
trastuzumab-MCC-DM1 (T-DM1), and the combination of pertuzumab and
T-DM 1.
[0027] FIG. 4 shows a plot of BT-474 in vitro cell viability at 5
days versus various fixed doses of pertuzumab in combination with
dose response of trastuzumab-MCC-DM1 (T-DM1), and various doses of
T-DM1 alone.
[0028] FIG. 5 shows a plot of BT-474 in vitro cell viability at 5
days versus various fixed doses of trastuzumab-MCC-DM1 (T-DM1) in
combination with dose response of pertuzumab, and various doses of
pertuzumab alone.
[0029] FIG. 6 shows a plot of BT-474 in vitro cell viability at 5
days versus IC50 multiple concentrations of pertuzumab,
trastuzumab-MCC-DM1 (T-DM1), and the combination of pertuzumab and
T-DM 1.
[0030] FIG. 7 shows a plot of SK-BR-3 in vitro cell viability at 3
days versus varying doses of T-DM1 in combination with fixed doses
of lapatinib (4.5 nM, 14 nM, 41 nM, 123 nM), and varying doses of
T-DM1 alone (0-1000 ng/ml).
[0031] FIG. 7a shows a plot of SK-BR-3 in vitro cell viability at 3
days versus T-DM1, lapatinib, and fixed dose ratio combinations of
T-DM1 and lapatinib.
[0032] FIG. 8a shows a plot of BT-474 in vitro cell viability at 3
days versus T-DM1, lapatinib, and fixed dose ratio combinations of
T-DM1 and lapatinib.
[0033] FIG. 8 shows a plot of BT-474 in vitro cell viability at 3
days versus varying doses of T-DM1 in combination with fixed doses
of lapatinib (1.5 nM, 4.5 nM, 14 nM, 41 nM, 123 nM), and varying
doses of T-DM1 alone (0-1000 ng/ml).
[0034] FIG. 9 shows a plot of BT-474-EEI in vitro cell viability at
3 days versus varying doses of T-DM1 in combination with fixed
doses of lapatinib (14 nM, 41 nM, 123 nM, 370 nM, 1111 nM), and
varying doses of T-DM1 alone (0-1000 ng/ml).
[0035] FIG. 10 shows a plot of the in vivo mean tumor volume change
over time in KPL-4 tumors inoculated into the mammary fat pad of
SCID beige mice (3 million cells in matrigel per mouse) after
dosing with: (1) ADC buffer, (2) pertuzumab 15 mg/kg, (3) T-DM1 0.3
mg/kg, (4) T-DM1 1 mg/kg, (5) T-DM1 3 mg/kg, (6) pertuzumab 15
mg/kg+T-DM1 0.3 mg, (7) pertuzumab 15 mg/kg+T-DM1 1 mg/kg, (8)
pertuzumab 15 mg/kg+T-DM1 3 mg/kg. ADC buffer and T-DM1 were dosed
once on day 0. Pertuzumab was dosed on days 0, 7, and 14.
[0036] FIG. 11 shows a plot of the in vivo mean tumor volume change
over time in KPL-4 tumors inoculated into the mammary fat pad of
SCID beige mice (3 million cells in matrigel per mouse) after
dosing with: (1) ADC buffer, (2) 5-FU 100 mg/kg, (3) pertuzumab 40
mg/kg, the first pertuzumab dose (groups 5, 7, and 9) was a
2.times. loading dose, (4) B20-4.1, 5 mg/kg, (5) T-DM1, 5 mg/kg,
(6) 5-FU, 100 mg/kg+T-DM1, 5 mg, (7) pertuzumab 40 mg/kg+T-DM1, 5
mg/kg, (8) B20-4.1, 5 mg/kg+T-DM1, 5 mg/kg, (9) B20-4.1, 5
mg/kg+pertuzumab, 40 mg/kg. ADC buffer and T-DM1 were dosed once on
day 0 by single iv injection. Pertuzumab was dosed on days 0, 7,
14, 21 (qwk.times.4. 5-FU was dosed on days 0, 7 and 14
(qwk.times.3). B20-4.1 was dosed on days 0, 3, 7, 10, 14, 17, 21
and 24 (2.times./wk.times.8 total).
[0037] FIG. 12 shows a plot of the in vivo mean tumor volume change
over time in MMTV-HER2Fo5 transgenic mammary tumors inoculated into
the mammary fat pad of CRL nu/nu mice after dosing with: (1)
Vehicle (ADC buffer), (2) B20-4.1, 5 mg/kg, (3) T-DM1, 3 mg/kg, (4)
T-DM1, 5 mg/kg, (5) T-DM1, 10 mg/kg, (6) B20-4.1, 5 mg/kg+T-DM1 3
mg/kg, (7) B20-4.1, 5 mg/kg+T-DM1 5 mg/kg, (8) B20-4.1, 5
mg/kg+T-DM1, 10 mg/kg. ADC buffer and T-DM1 were dosed on days 0
and 21. B20-4.1 was dosed on days 0, 3, 7, 10, 14, 17, 21 and 24
(2.times./wk.times.4 for 8 total).
[0038] FIG. 13 shows a plot of the in vivo mean tumor volume change
over time in MMTV-HER2Fo5 transgenic mammary tumors inoculated into
the mammary fat pad of CRL nu/nu mice after dosing with: (1)
Vehicle (ADC buffer), (2) T-DM1 10 mg/kg, (3) 5-FU 100 mg/kg, (4)
gemcitabine 120 mg/kg, (5) carboplatin 100 mg/kg, (6) 5-FU 100
mg/kg+T-DM1 10 mg/kg, (7) gemcitabine 120 mg/kg+T-DM1 10 mg/kg, (8)
carboplatin 100 mg/kg+T-DM1 10 mg/kg. ADC buffer, T-DM1 and
carboplatin were dosed on day 0; single injection. 5-FU was dosed
on day 0, 7 and 14 (qwk.times.3). Gemcitabine was dosed on days 0,
3, 6 and 9 (q3d.times.4).
[0039] FIG. 14 shows a plot of the in vivo mean tumor volume change
over time in MMTV-Her2 Fo5 transgenic mammary tumors inoculated
into the mammary fat pad of Harlan athymic nude mice after dosing
with: (1) Vehicle (PBS buffer) iv, qwk.times.4, (2) lapatinib 101
mg/kg, po, bid.times.21, (3) pertuzumab 40 mg/kg, iv, qwk.times.4,
(4) B20-4.1 5 mg/kg, ip, 2.times./wk.times.4, (5) T-DM1 15 mg/kg,
iv, q3wk to end, (6) lapatinib 101 mg/kg, po, bid.times.21+T-DM1 15
mg/kg, iv, q3wk to end (7) pertuzumab 40 mg/kg, iv,
qwk.times.4+T-DM1 15 mg/kg, iv, q3wk to end, (8) B20-4.1 5 mg/kg,
ip, 2.times./wk.times.4+T-DM1 15 mg/kg, iv, q3wk to end.
[0040] FIG. 15 shows a plot of the in vivo mean tumor volume change
over time on MMTV-Her2 Fo5 transgenic mammary tumor inoculated into
the mammary fat pad of Harlan athymic nude mice after dosing with:
(1) Vehicle (PBS buffer) po, bid.times.21 (2) T-DM1, 7.5 mg/kg, iv,
qd.times.1 (3) T-DM1, 15 mg/kg, iv, qd.times.1 (4) ABT-869, 5
mg/kg, po, bid.times.21 (5) ABT-869, 15 mg/kg, po, bid.times.21 (6)
T-DM1, 7.5 mg/kg, iv, qd.times.1+ABT-869, 5 mg/kg, po, bid.times.21
(7) T-DM1 7.5 mg/kg, iv, qd.times.1+ABT-869, 15 mg/kg, po,
bid.times.21 (8) T-DM1, 15 mg/kg, iv, qd.times.1+ABT-869, 5 mg/kg,
po, bid.times.21 (9) T-DM1, 15 mg/kg, iv, qd.times.1+ABT-869, 15
mg/kg, po, bid.times.21.
[0041] FIG. 16 shows a plot of the in vivo mean tumor volume change
over time on MMTV-Her2 Fo5 transgenic mammary tumor inoculated into
the mammary fat pad of Harlan athymic nude mice after dosing with:
(1) Vehicle, iv, qwk.times.3 (2) T-DM1, 7.5 mg/kg, iv, q3wk.times.2
(3) T-DM1, 15 mg/kg, iv, q3wk.times.2 (4) docetaxel, 30 mg/kg, iv,
qwk.times.3 (5) T-DM1, 7.5 mg/kg, iv, q3wk.times.2+docetaxel, 30
mg/kg, iv, qwk.times.3 (6) T-DM1, 15 mg/kg, iv,
q3wk.times.2+docetaxel, 30 mg/kg, iv, qwk.times.3
[0042] FIG. 17 shows a plot of the in vivo mean tumor volume change
over time on MMTV-Her2 Fo5 transgenic mammary tumor inoculated into
the mammary fat pad of Harlan athymic nude mice after dosing with:
(1) Vehicle, po, qd.times.21 (2) T-DM1, 7.5 mg/kg, iv,
q3wk.times.2, (3) T-DM1, 15 mg/kg, iv, q3wk.times.2 (4) lapatinib,
100 mg/kg, po, bid.times.21, (5) T-DM1, 7.5 mg/kg, iv,
q3wk.times.2+lapatinib, 100 mg/kg, po, bid.times.21, (6) T-DM1, 15
mg/kg, iv, q3wk.times.2+lapatinib, 100 mg/kg, po, bid.times.21
[0043] FIG. 18 shows a plot of SK-BR-3 in vitro cell viability at 3
days versus IC50 multiple concentrations of 5-FU,
trastuzumab-MCC-DM1 (T-DM1), and fixed dose ratio combinations of
5-FU and T-DM1.
[0044] FIG. 19 shows a plot of BT-474 in vitro cell viability at 3
days versus IC50 multiple concentrations of 5-FU,
trastuzumab-MCC-DM1 (T-DM1), and fixed dose ratio combinations of
5-FU and T-DM1.
[0045] FIG. 20 shows a plot of SK-BR-3 in vitro cell viability at 3
days versus IC50 multiple concentrations of gemcitabine,
trastuzumab-MCC-DM1 (T-DM1), and fixed dose ratio combinations of
gemcitabine and T-DM 1.
[0046] FIG. 21 shows a plot of MDA-MD-361 in vitro cell viability
at 3 days versus IC50 multiple concentrations of gemcitabine,
trastuzumab-MCC-DM1 (T-DM1), and fixed dose ratio combinations of
gemcitabine and T-DM1.
[0047] FIG. 22 shows a plot of KPL4 in vitro cell viability
(proliferation) at 3 days after treatment with T-DM1, GDC-0941, and
1:10 fixed dose ratio combinations of T-DM1 and GDC-0941 (62.5 nM
to 1 .mu.M) at IC50 multiple concentrations from 0.25.times. to
4.times.. The Bliss prediction of additivity is plotted as the
dotted line.
[0048] FIG. 23 shows a plot of KPL4 in vitro cell viability
(proliferation) at 3 days after treatment with T-DM1, GDC-0941, and
1:25 fixed dose ratio combinations of T-DM1 (1.25 to 80 ng/ml) and
GDC-0941 (31.25 nM to 2 .mu.M) at IC50 multiple concentrations from
0.0625.times. to 16.times.. The Bliss prediction of additivity is
plotted as the dotted line.
[0049] FIG. 24 shows a plot of Her2 amplified, HERCEPTIN.RTM.
resistant, PIK3CA (H1047R) mutant, KPL-4 cells in vitro cell
viability (proliferation) at 3 days after treatment with T-DM1,
PI103, GDC-0941, and fixed dose ratio combinations of T-DM1+PI103,
and T-DM1+GDC-0941, at IC50 multiple concentrations from 0 to
16.times..
[0050] FIG. 25 shows a plot of KPL4 Caspase 3/7 in vitro cell
viability (proliferation) at 24 hours after treatment with T-DM1,
GDC-0941, and fixed dose ratio T-DM1 and GDC-0941 combinations at
T-DM1 concentrations up to 160 ng/ml.
[0051] FIG. 26 shows a plot of KPL4 in vitro cell viability
(proliferation) at 3 days after treatment with T-DM1, GDC-0941, and
fixed dose ratio combinations of T-DM1 and GDC-0941 at T-DM1
concentrations from 0 to 200 ng/ml.
[0052] FIG. 27 shows a plot of MDA-OMB-361 in vitro cell viability
(proliferation) at 3 days after treatment with T-DM1, GDC-0941, and
1:20 fixed dose ratio combinations of T-DM1 (3.125 to 50 ng/ml) and
GDC-0941 (62.5 nM to 1 .mu.M) at IC50 multiple concentrations from
0.125.times. to 8.times.. The Bliss prediction of additivity is
plotted as the dotted line.
[0053] FIG. 28 shows a plot of MDA-OMB-361 in vitro cell viability
(proliferation) at 3 days after treatment with T-DM1, GDC-0941, and
1:20 fixed dose ratio combinations of T-DM1 (3.125 to 100 ng/ml)
and GDC-0941 (62.5 nM to 2 .mu.M) at IC50 multiple concentrations
from 0.125.times. to 8.times.. The Bliss prediction of additivity
is plotted as the dotted line.
[0054] FIG. 29 shows a plot of BT-474 in vitro cell viability
(proliferation) at 3 days after treatment with T-DM1, GDC-0941, and
1:10 fixed dose ratio combinations of T-DM1 (3.125 to 100 ng/ml)
and GDC-0941 (31.25 nM to 1 .mu.M) at IC50 multiple concentrations
from 0.125.times. to 4.times.. The Bliss prediction of additivity
is plotted as the dotted line.
[0055] FIG. 30 shows a plot of BT-474 in vitro cell viability
(proliferation) at 3 days after treatment with T-DM1, GDC-0941, and
1:10 fixed dose ratio combinations of T-DM1 (6.25 to 100 ng/ml) and
GDC-0941 (62.5 nM to 1 .mu.M) at IC50 multiple concentrations from
0.25.times. to 4.times.. The Bliss prediction of additivity is
plotted as the dotted line.
[0056] FIG. 31 shows a plot of Her2 amplified, non-PI3K mutant,
AU565 cells in vitro cell viability (proliferation) at 3 days after
treatment with T-DM1, PI103, GDC-0941, and fixed dose ratio
combinations of T-DM1+PI103, and T-DM1+GDC-0941 at IC50 multiple
concentrations from 0 to 16.times..
[0057] FIG. 32 shows a plot of Her2 amplified, PIK3CA (C420R)
mutant, EFM192A cells in vitro cell viability (proliferation) at 3
days after treatment with T-DM1, PI103, GDC-0941, and fixed dose
ratio combinations of T-DM1+PI103, and T-DM1+GDC-0941, at IC50
multiple concentrations from 0 to 16.times..
[0058] FIG. 33 shows a plot of Her2 amplified, HERCEPTIN.RTM.
resistant, PIK3CA (H1047R) mutant, HCC 1954 cells in vitro cell
viability (proliferation) after treatment with T-DM1, PI103,
GDC-0941, and fixed dose ratio combinations of T-DM1+PI103, and
T-DM1+GDC-0941, at IC50 multiple concentrations from 0 to
16.times..
[0059] FIG. 34 shows a plot of the in vivo mean tumor volume change
over time on MMTV-Her2 Fo5 transgenic mammary tumor inoculated into
CRL nu/nu mice after dosing with: (1) Vehicle, po, qd.times.21 (2)
T-DM1, 10 mg/kg, iv, q3wk, (3) 5-FU, 100 mg/kg, po, qwk.times.2,
(4) T-DM1, 5 mg/kg, iv, q3wk+5-FU, 100 mg/kg, po, qwk.times.2
[0060] FIG. 35 shows a plot of the in vivo mean tumor volume change
over time on MMTV-Her2 Fo5 transgenic mammary tumor inoculated into
CRL nu/nu mice after dosing with: (1) Vehicle, po, qd.times.21 (2)
T-DM1, 5 mg/kg, iv, qd.times.1, (3) GDC-0941, 100 mg/kg, po,
qd.times.21, (4) GDC-0152, 50 mg/kg, po, qwk.times.3, (5) T-DM1, 5
mg/kg, iv, qd.times.1+GDC-0941, 100 mg/kg, po, qd.times.21, (6)
T-DM1, 5 mg/kg, iv, qd.times.1+GDC-0152, 50 mg/kg, po,
qwk.times.3
[0061] FIG. 36 shows a plot of the in vivo mean tumor volume change
over time on MDA-MB-361.1 mammary tumor inoculated into CRL nu/nu
mice after dosing with: (1) Vehicle, po, qd.times.21 (2) GDC-0941,
25 mg/kg, po, qd.times.21, (3) GDC-0941, 50 mg/kg, po, qd.times.21,
(4) GDC-0941, 100 mg/kg, po, qd.times.21, (5) T-DM1, 3 mg/kg, iv,
qd.times.1, (6) T-DM1, 10 mg/kg, iv, qd.times.1, (7) GDC-0941, 25
mg/kg, po, qd.times.21+T-DM1, 3 mg/kg, iv, qd.times.1, (8)
GDC-0941, 50 mg/kg, po, qd.times.21+T-DM1, 3 mg/kg, iv, qd.times.1,
(9) GDC-0941, 100 mg/kg, po, qd.times.21+T-DM1, 3 mg/kg, iv,
qd.times.1, (10) GDC-0941, 25 mg/kg, po, qd.times.21+T-DM1, mg/kg,
iv, qd.times.1, (11) GDC-0941, 50 mg/kg, po, qd.times.21+T-DM1, 10
mg/kg, iv, qd.times.1, (12) GDC-0941, 100 mg/kg, po,
qd.times.21+T-DM1, 10 mg/kg, iv, qd.times.1
[0062] FIG. 37 shows a plot of the in vivo mean tumor volume change
over time on MDA-MB-361.1 mammary tumor inoculated into CRL nu/nu
mice after dosing with: (1) Vehicles [MCT (0.5%
methylcellulose/0.2% TWEEN80.TM.)+succinate buffer (100 mM sodium
succinate, 100 mg/ml trehalose, 0.1% TWEEN 80, pH 5.0)], po+IV,
qd.times.21 and qd (2) GNE-390, 1.0 mg/kg, po, qd.times.21, (3)
GNE-390, 2.5 mg/kg, po, qd.times.21, (4) T-DM1, 3 mg/kg, iv, qd,
(5) GNE-390, 1.0 mg/kg, po, qd.times.21+T-DM1, 3 mg/kg, iv, qd, (6)
GNE-390, 2.5 mg/kg, po, qd.times.21+T-DM1, 3 mg/kg, iv, qd
DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS
[0063] Reference will now be made in detail to certain embodiments
of the invention, examples of which are illustrated in the
accompanying structures and formulas. While the invention will be
described in conjunction with the enumerated embodiments, it will
be understood that they are not intended to limit the invention to
those embodiments. On the contrary, the invention is intended to
cover all alternatives, modifications, and equivalents which may be
included within the scope of the present invention as defined by
the claims. One skilled in the art will recognize many methods and
materials similar or equivalent to those described herein, which
could 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 incorporated
literature, patents, and similar materials differs from or
contradicts this application, including but not limited to defined
terms, term usage, described techniques, or the like, this
application controls.
DEFINITIONS
[0064] The words "comprise," "comprising," "include," "including,"
and "includes" when used in this specification and claims are
intended to specify the presence of stated features, integers,
components, or steps, but they do not preclude the presence or
addition of one or more other features, integers, components,
steps, or groups thereof.
[0065] The terms "treat" and "treatment" refer to both therapeutic
treatment and prophylactic or preventative measures, wherein the
object is to prevent or slow down (lessen) an undesired
physiological change or disorder, such as the growth, development
or spread of a hyperproliferative condition, such as cancer. For
purposes of this invention, beneficial or desired clinical results
include, but are not limited to, alleviation of symptoms,
diminishment of extent of disease, stabilized (i.e., not worsening)
state of disease, delay or slowing of disease progression,
amelioration or palliation of the disease state, and remission
(whether partial or total), whether detectable or undetectable.
"Treatment" can also mean prolonging survival as compared to
expected survival if not receiving treatment. Those in need of
treatment include those already with the condition or disorder as
well as those prone to have the condition or disorder or those in
which the condition or disorder is to be prevented.
[0066] The phrase "therapeutically effective amount" means an
amount of a compound of the present invention that (i) treats the
particular disease, condition, or disorder, (ii) attenuates,
ameliorates, or eliminates one or more symptoms of the particular
disease, condition, or disorder, or (iii) prevents or delays the
onset of one or more symptoms of the particular disease, condition,
or disorder described herein. In the case of cancer, the
therapeutically effective amount of the drug may reduce the number
of cancer cells; reduce the tumor size; inhibit (i.e., slow to some
extent and preferably stop) cancer cell infiltration into
peripheral organs; inhibit (i.e., slow to some extent and
preferably stop) tumor metastasis; inhibit, to some extent, tumor
growth; and/or relieve to some extent one or more of the symptoms
associated with the cancer. To the extent the drug may prevent
growth and/or kill existing cancer cells, it may be cytostatic
and/or cytotoxic. For cancer therapy, efficacy can be measured, for
example, by assessing the time to disease progression (TTP) and/or
determining the response rate (RR).
[0067] "Hyperproliferative disorder" is indicated by tumors,
cancers, and neoplastic tissue, including pre-malignant and
non-neoplastic stages, and also include psoriasis, endometriosis,
polyps and fibroadenoma.
[0068] The terms "cancer" and "cancerous" refer to or describe the
physiological condition in mammals that is typically characterized
by unregulated cell growth. A "tumor" comprises one or more
cancerous cells. Examples of cancer include, but are not limited
to, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or
lymphoid malignancies. More particular examples of such cancers
include squamous cell cancer (e.g., epithelial squamous cell
cancer), lung cancer including small-cell lung cancer, non-small
cell lung cancer ("NSCLC"), adenocarcinoma of the lung and squamous
carcinoma of the lung, cancer of the peritoneum, hepatocellular
cancer, gastric or stomach cancer including gastrointestinal
cancer, pancreatic cancer, glioblastoma, cervical cancer, ovarian
cancer, liver cancer, bladder cancer, hepatoma, breast cancer,
colon cancer, rectal cancer, colorectal cancer, endometrial or
uterine carcinoma, salivary gland carcinoma, kidney or renal
cancer, prostate cancer, vulval cancer, thyroid cancer, hepatic
carcinoma, anal carcinoma, penile carcinoma, as well as head and
neck cancer.
[0069] A "chemotherapeutic agent" is a chemical compound useful in
the treatment of cancer, regardless of mechanism of action. Classes
of chemotherapeutic agents include, but are not limited to:
alkylating agents, antimetabolites, spindle poison plant alkaloids,
cytotoxic/antitumor 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 (TARCEVA.RTM.,
Genentech/OSI Pharm.), docetaxel (TAXOTERE.RTM., Sanofi-Aventis),
5-FU (fluorouracil, 5-fluorouracil, CAS No. 51-21-8), gemcitabine
(GEMZAR.RTM., Lilly), PD-0325901 (CAS No. 391210-10-9, Pfizer),
cisplatin (cis-diamine,dichloroplatinum(II), CAS No. 15663-27-1),
carboplatin (CAS No. 41575-94-4), paclitaxel (TAXOL.RTM.,
Bristol-Myers Squibb Oncology, Princeton, N.J.), trastuzumab
(HERCEPTIN.RTM., Genentech), temozolomide
(4-methyl-5-oxo-2,3,4,6,8-pentazabicyclo[4.3.0]nona-2,7,9-triene-9-carbox-
amide, CAS No. 85622-93-1, TEMODAR.RTM., TEMODAL.RTM., Schering
Plough), tamoxifen
((Z)-2-[4-(1,2-diphenylbut-1-enyl)phenoxy]-N,N-dimethyl-ethanam-
ine, NOLVADEX.RTM., ISTUBAL.RTM., VALODEX.RTM.), and doxorubicin
(ADRIAMYCIN.RTM.), Akti-1/2, HPPD, and rapamycin.
[0070] More examples of chemotherapeutic agents include:
oxaliplatin (ELOXATIN.RTM., Sanofi), bortezomib (VELCADE.RTM.,
Millennium Pharm.), sutent (SUNITINIB.RTM., SU11248, Pfizer),
letrozole (FEMARA.RTM., Novartis), imatinib mesylate (GLEEVEC.RTM.,
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, Novartis), XL-147 (PI3K inhibitor, Exelixis), PTK787/ZK
222584 (Novartis), fulvestrant (FASLODEX.RTM., AstraZeneca),
leucovorin (folinic acid), rapamycin (sirolimus, RAPAMUNE.RTM.,
Wyeth), lapatinib (TYKERB.RTM., GSK572016, Glaxo Smith Kline),
lonafarnib (SARASAR.TM., SCH 66336, Schering Plough), sorafenib
(NEXAVAR.RTM., BAY43-9006, Bayer Labs), gefitinib (IRESSA.RTM.,
AstraZeneca), irinotecan (CAMPTOSAR.RTM., CPT-11, Pfizer),
tipifarnib (ZARNESTRA.TM., Johnson & Johnson), ABRAXANE.TM.
(Cremophor-free), albumin-engineered nanoparticle formulations of
paclitaxel (American Pharmaceutical Partners, Schaumberg, Il),
vandetanib (rINN, ZD6474, ZACTIMA.RTM., AstraZeneca),
chloranmbucil, AG1478, AG1571 (SU 5271; Sugen), temsirolimus
(TORISEL.RTM., Wyeth), pazopanib (GlaxoSmithKline), canfosfamide
(TELCYTA.RTM., Telik), thiotepa and cyclosphosphamide
(CYTOXAN.RTM., NEOSAR.RTM.); alkyl sulfonates such as busulfan,
improsulfan and piposulfan; aziridines such as benzodopa,
carboquone, meturedopa, and uredopa; ethylenimines and
methylamelamines including altretamine, triethylenemelamine,
triethylenephosphoramide, triethylenethiophosphoramide and
trimethylomelamine; acetogenins (especially bullatacin and
bullatacinone); a camptothecin (including the synthetic analog
topotecan); bryostatin; callystatin; CC-1065 (including its
adozelesin, carzelesin and bizelesin synthetic analogs);
cryptophycins (particularly cryptophycin 1 and cryptophycin 8);
dolastatin; duocarmycin (including the synthetic analogs, KW-2189
and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin;
spongistatin; nitrogen mustards such as chlorambucil,
chlornaphazine, chlorophosphamide, estramustine, ifosfamide,
mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,
novembichin, phenesterine, prednimustine, trofosfamide, uracil
mustard; nitrosoureas such as carmustine, chlorozotocin,
fotemustine, lomustine, nimustine, and ranimnustine; antibiotics
such as the enediyne antibiotics (e.g., calicheamicin,
calicheamicin gamma1I, calicheamicin omegaI1 (Angew Chem. Intl. Ed.
Engl. (1994) 33:183-186); dynemicin, dynemicin A; bisphosphonates,
such as clodronate; an esperamicin; as well as neocarzinostatin
chromophore and related chromoprotein enediyne antibiotic
chromophores), aclacinomysins, actinomycin, authramycin, azaserine,
bleomycins, cactinomycin, carabicin, caminomycin, carzinophilin,
chromomycinis, dactinomycin, daunorubicin, detorubicin,
6-diazo-5-oxo-L-norleucine, morpholino-doxorubicin,
cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin and
deoxydoxorubicin), epirubicin, esorubicin, idarubicin,
marcellomycin, mitomycins such as mitomycin C, mycophenolic acid,
nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,
quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,
ubenimex, zinostatin, zorubicin; anti-metabolites such as
methotrexate and 5-fluorouracil (5-FU); folic acid analogs such as
denopterin, methotrexate, pteropterin, trimetrexate; purine analogs
such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine;
pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine,
carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine,
floxuridine; androgens such as calusterone, dromostanolone
propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals
such as aminoglutethimide, mitotane, trilostane; folic acid
replenisher such as frolinic acid; aceglatone; aldophosphamide
glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil;
bisantrene; edatraxate; defofamine; demecolcine; diaziquone;
elfornithine; elliptinium acetate; an epothilone; etoglucid;
gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids
such as maytansine and ansamitocins; mitoguazone; mitoxantrone;
mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin;
losoxantrone; podophyllinic acid; 2-ethylhydrazide; procarbazine;
PSK.RTM. polysaccharide complex (JHS Natural Products, Eugene,
Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic
acid; triaziquone; 2,2',2''-trichlorotriethylamine; trichothecenes
(T-2 toxin, verracurin A, roridin A and anguidine); urethan;
vindesine; dacarbazine; mannomustine; mitobronitol; mitolactol;
pipobroman; gacytosine; arabinoside (Ara-C); cyclophosphamide;
thiotepa; 6-thioguanine; mercaptopurine; methotrexate; platinum
analogs such as cisplatin and carboplatin; vinblastine; etoposide
(VP-16); ifosfamide; mitoxantrone; vincristine; vinorelbine
(NAVELBINE.RTM.); novantrone; teniposide; edatrexate; daunomycin;
aminopterin; capecitabine (XELODA.RTM., Roche); ibandronate;
CPT-11; topoisomerase inhibitor RFS 2000; difluoromethylornithine
(DMFO); retinoids such as retinoic acid; and pharmaceutically
acceptable salts, acids and derivatives of any of the above.
[0071] Also included in the definition of "chemotherapeutic agent"
are: (i) anti-hormonal agents that act to regulate or inhibit
hormone action on tumors such as anti-estrogens and selective
estrogen receptor modulators (SERMs), including, for example,
tamoxifen (NOLVADEX.RTM.; tamoxifen citrate), raloxifene,
droloxifene, 4-hydroxytamoxifen, trioxifene, keoxifene, LY117018,
onapristone, and FARESTON.RTM. (toremifine citrate); (ii) aromatase
inhibitors that inhibit the enzyme aromatase, which regulates
estrogen production in the adrenal glands, such as, for example,
4(5)-imidazoles, aminoglutethimide, MEGASE.RTM. (megestrol
acetate), AROMASIN.RTM. (exemestane; Pfizer), formestanie,
fadrozole, RIVISOR.RTM. (vorozole), FEMARA.RTM. (letrozole;
Novartis), and ARIMIDEX.RTM. (anastrozole; AstraZeneca); (iii)
anti-androgens such as flutamide, nilutamide, bicalutamide,
leuprolide, and goserelin; as well as troxacitabine (a
1,3-dioxolane nucleoside cytosine analog); (iv) protein kinase
inhibitors such as MEK inhibitors (WO 2007/044515); (v) lipid
kinase inhibitors; (vi) antisense oligonucleotides, particularly
those which inhibit expression of genes in signaling pathways
implicated in aberrant cell proliferation, for example, PKC-alpha,
Raf and H-Ras, such as oblimersen (GENASENSE.RTM., Genta Inc.);
(vii) ribozymes such as VEGF expression inhibitors (e.g.,
ANGIOZYME.RTM.) and HER2 expression inhibitors; (viii) vaccines
such as gene therapy vaccines, for example, ALLOVECTIN.RTM.,
LEUVECTIN.RTM., and VAXID.RTM.; PROLEUKIN.RTM. rIL-2; topoisomerase
1 inhibitors such as LURTOTECAN.RTM.; ABARELIX.RTM. rmRH; (ix)
anti-angiogenic agents such as bevacizumab (AVASTIN.RTM.,
Genentech); and pharmaceutically acceptable salts, acids and
derivatives of any of the above.
[0072] Also included in the definition of "chemotherapeutic agent"
are therapeutic antibodies such as alemtuzumab (Campath),
bevacizumab (AVASTIN.RTM., Genentech); cetuximab (ERBITUX.RTM.,
Imclone); panitumumab (VECTIBIX.RTM., Amgen), rituximab
(RITUXAN.RTM., Genentech/Biogen Idec), pertuzumab (OMNITARG.TM.,
2C4, Genentech), trastuzumab (HERCEPTIN.RTM., Genentech),
tositumomab (Bexxar, Corixia), and the antibody drug-conjugate,
gemtuzumab ozogamicin (MYLOTARG.RTM., Wyeth).
[0073] Humanized monoclonal antibodies with therapeutic potential
as chemotherapeutic agents in combination with trastuzumab-MCC-DM1
include: alemtuzumab, apolizumab, aselizumab, atlizumab,
bapineuzumab, bevacizumab, bivatuzumab mertansine, cantuzumab
mertansine, cedelizumab, certolizumab pegol, cidfusituzumab,
cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab,
erlizumab, felvizumab, fontolizumab, gemtuzumab ozogamicin,
inotuzumab ozogamicin, ipilimumab, labetuzumab, lintuzumab,
matuzumab, mepolizumab, motavizumab, motovizumab, natalizumab,
nimotuzumab, nolovizumab, numavizumab, ocrelizumab, omalizumab,
palivizumab, pascolizumab, pecfusituzumab, pectuzumab, pertuzumab,
pexelizumab, ralivizumab, ranibizumab, reslivizumab, reslizumab,
resyvizumab, rovelizumab, ruplizumab, sibrotuzumab, siplizumab,
sontuzumab, tacatuzumab tetraxetan, tadocizumab, talizumab,
tefibazumab, tocilizumab, toralizumab, trastuzumab, tucotuzumab
celmoleukin, tucusituzumab, umavizumab, urtoxazumab, and
visilizumab.
[0074] A "metabolite" is a product produced through metabolism in
the body of a specified compound or salt thereof. Metabolites of a
compound may be identified using routine techniques known in the
art and their activities determined using tests such as those
described herein. Such products may result for example from the
oxidation, reduction, hydrolysis, amidation, deamidation,
esterification, deesterification, enzymatic cleavage, and the like,
of the administered compound. Accordingly, the invention includes
metabolites of compounds of the invention, including compounds
produced by a process comprising contacting a compound of this
invention with a mammal for a period of time sufficient to yield a
metabolic product thereof.
[0075] The term "package insert" is used to refer to instructions
customarily included in commercial packages of therapeutic
products, that contain information about the indications, usage,
dosage, administration, contraindications and/or warnings
concerning the use of such therapeutic products.
[0076] The phrase "pharmaceutically acceptable salt" as used
herein, 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, acid phosphate,
isonicotinate, lactate, salicylate, acid citrate, 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)) salts. A
pharmaceutically acceptable salt may involve the inclusion of
another molecule such as an acetate ion, a succinate ion or other
counter ion. The counter ion may be any organic or inorganic moiety
that stabilizes the charge on the parent compound. Furthermore, a
pharmaceutically acceptable salt may have more than one charged
atom in its structure. Instances where multiple charged atoms are
part of the pharmaceutically acceptable salt can have multiple
counter ions. Hence, a pharmaceutically acceptable salt can have
one or more charged atoms and/or one or more counter ion.
[0077] If the compound of the invention is a base, the desired
pharmaceutically acceptable salt may be prepared by any suitable
method available in the art, for example, treatment of 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 with 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, a
pyranosidyl acid, such as glucuronic acid or galacturonic acid, an
alpha hydroxy acid, such as citric acid or tartaric acid, an amino
acid, such as aspartic acid or glutamic acid, an aromatic acid,
such as benzoic acid or cinnamic acid, a sulfonic acid, such as
p-toluenesulfonic acid or ethanesulfonic acid, or the like. Acids
which are generally considered suitable for the formation of
pharmaceutically useful or acceptable salts from basic
pharmaceutical compounds are discussed, for example, by 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) 1 19; 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, 18.sup.th ed., (1995)
Mack Publishing Co., Easton Pa.; and in The Orange Book (Food &
Drug Administration, Washington, D.C. on their website). These
disclosures are incorporated herein by reference thereto.
[0078] If the compound of the invention is an acid, the desired
pharmaceutically acceptable salt may be prepared by any suitable
method, for example, treatment of the free acid with an inorganic
or organic base, such as an amine (primary, secondary or tertiary),
an alkali metal hydroxide or alkaline earth metal hydroxide, 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.
[0079] The phrase "pharmaceutically acceptable" indicates that the
substance or composition must be compatible chemically and/or
toxicologically, with the other ingredients comprising a
formulation, and/or the mammal being treated therewith.
[0080] A "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 exist in unsolvated as well as
solvated forms. 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 the complex where the solvent molecule is
water. This physical association involves varying degrees of ionic
and covalent bonding, including hydrogen bonding. In certain
instances the solvate will be capable of isolation, for example
when one or more solvent molecules are incorporated in the crystal
lattice of the crystalline solid. Preparation of solvates is
generally known, for example, M. Caira et al, J. Pharmaceutical
Sci., 93(3), 601 611 (2004). Similar preparations of solvates,
hemisolvate, hydrates and the like are described by E. C. van
Tonder et al, AAPS PharmSciTech., 5(1), article 12 (2004); and A.
L. Bingham et al, Chem. Commun., 603 604 (2001). A typical,
non-limiting, process involves dissolving the inventive compound in
desired amounts of the desired solvent (organic or water or
mixtures thereof) at a higher than ambient temperature, and cooling
the solution at a rate sufficient to form crystals which are then
isolated by standard methods. Analytical techniques such as, for
example I.R. spectroscopy, show the presence of the solvent (or
water) in the crystals as a solvate (or hydrate).
[0081] The term "synergistic" as used herein refers to a
therapeutic combination which is more effective than the additive
effects of the two or more single agents. A determination of a
synergistic interaction between trastuzumab-MCC-DM1, and one or
more chemotherapeutic agent may be based on the results obtained
from the assays described herein. The results of these assays are
analyzed using the Chou and Talalay combination method and
Dose-Effect Analysis with CalcuSyn software in order to obtain a
Combination Index "CI" (Chou and Talalay (1984) Adv. Enzyme Regul.
22:27-55). The combinations provided by this invention have been
evaluated in several assay systems, and the data can be analyzed
utilizing a standard program for quantifying synergism, additivism,
and antagonism among anticancer agents. The program preferably
utilized is that described by Chou and Talalay, in "New Avenues in
Developmental Cancer Chemotherapy," Academic Press, 1987, Chapter
2. Combination Index (CI) values less than 0.8 indicate synergy,
values greater than 1.2 indicate antagonism and values between 0.8
to 1.2 indicate additive effects. The combination therapy may
provide "synergy" and prove "synergistic", i.e., the effect
achieved when the active ingredients used together is greater than
the sum of the effects that results from using the compounds
separately. A synergistic effect may be attained when the active
ingredients are: (1) co-formulated and administered or delivered
simultaneously in a combined, unit dosage formulation; (2)
delivered by alternation as separate formulations; or (3) by some
other regimen. When delivered in alternation therapy, a synergistic
effect may be attained when the compounds are administered or
delivered sequentially, e.g., by different injections in separate
syringes. In general, during alternation therapy, an effective
dosage of each active ingredient is administered sequentially,
i.e., serially in time.
[0082] Trastuzumab-McC-DM1
[0083] The present invention includes therapeutic combinations
comprising trastuzumab-MCC-DM1 (T-DM1), an antibody-drug conjugate
(CAS Reg. No. 139504-50-0), which has the structure:
##STR00001##
[0084] where Tr is trastuzumab, linked through linker moiety MCC,
to the maytansinoid drug moiety, DM1 (U.S. Pat. No. 5,208,020; U.S.
Pat. No. 6,441,163). The drug to antibody ratio or drug loading is
represented by p in the above structure of trastuzumab-MCC-DM1, and
ranges in integer values from 1 to about 8. The drug loading value
p is 1 to 8. Trastuzumab-MCC-DM1 includes all mixtures of variously
loaded and attached antibody-drug conjugates where 1, 2, 3, 4, 5,
6, 7, and 8 drug moieties are covalently attached to the antibody
trastuzumab (U.S. Pat. No. 7,097,840; US 2005/0276812; US
2005/0166993). Trastuzumab-MCC-DM1 may be prepared according to
Example 1.
[0085] Trastuzumab is produced by a mammalian cell (Chinese Hamster
Ovary, CHO) suspension culture. The HER2 (or c-erbB2)
proto-oncogene encodes a transmembrane receptor protein of 185 kDa,
which is structurally related to the epidermal growth factor
receptor. HER2 protein overexpression is observed in 25%-30% of
primary breast cancers and can be determined using an
immunohistochemistry based assessment of fixed tumor blocks (Press
M F, et al (1993) Cancer Res 53:4960-70. Trastuzumab is an antibody
that has antigen binding residues of, or derived from, the murine
4D5 antibody (ATCC CRL 10463, deposited with American Type Culture
Collection, 12301 Parklawn Drive, Rockville, Md. 20852 under the
Budapest Treaty on 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.RTM.)
as in U.S. Pat. No. 5,821,337.
[0086] In a Phase I Study, the maximum tolerated dose (MTD) of
T-DM1 administered by IV infusion every 3 weeks was 3.6 mg/kg. A
DLT (Dose-Limiting Toxicity) consisted of Grade 4 thrombocytopenia
in 2 of 3 patients treated at 4.8 mg/kg. Related
Grade.gtoreq..quadrature.2 adverse events at 3.6 mg/kg were
infrequent and manageable. This treatment schedule was well
tolerated and associated with significant clinical activity as
described previously. A Phase II study has shown similar
tolerability at the 3.6 mg/kg dose level administered every 3
weeks, with only a small percentage of patients (3 out of 112
patients) requiring dose reduction. Thus, the T-DM1 dose of 3.6
mg/kg administered every 3 weeks was selected for testing in this
study based on 1) the demonstrated efficacy and safety of T-DM1 at
3.6 mg/kg every 3 weeks, and 2) the convenience of a 3-week regimen
for this patient population.
[0087] Chemotherapeutic Agents
[0088] Certain chemotherapeutic agents have demonstrated surprising
and unexpected properties in combination with trastuzumab-MCC-DM1
in inhibiting cellular proliferation in vitro and in vivo. Such
chemotherapeutic agents include a HER2 dimerization inhibitor
antibody, an anti-VEGF antibody, 5-FU, carboplatin, lapatinib,
ABT-869, docetaxel, GDC-0941, and GNE-390.
[0089] Pertuzumab (CAS Reg. No. 380610-27-5, OMNITARG.RTM., 2C4,
Genentech) is a recombinant, humanized monoclonal antibody that
inhibits dimerization of HER2 (U.S. Pat. No. 6,054,297; U.S. Pat.
No. 6,407,213; U.S. Pat. No. 6,800,738; U.S. Pat. No. 6,627,196,
U.S. Pat. No. 6,949,245; U.S. Pat. No. 7,041,292). Pertuzumab and
trastuzumab target different extracellular regions of the HER-2
tyrosine kinase receptor (Nahta et al (2004) Cancer Res.
64:2343-2346). The hybridoma cell line expressing 2C4 (pertuzumab)
was deposited with the American Type Culture Collection (ATCC),
10801 University Boulevard, Manassas, Va. 20110-2209, USA as ATCC
HB-12697 on Apr. 8, 1999. Pertuzumab blocks the ability of the HER2
receptor to collaborate with other HER receptor family members,
i.e. HER1/EGFR, HER3, and HER4 (Agus et al (2002) Cancer Cell
2:127-37; Jackson et al (2004) Cancer Res 64:2601-9; Takai et al
(2005) Cancer 104:2701-8; U.S. Pat. No. 6,949,245). In cancer
cells, interfering with the ability of HER2 to collaborate with
other HER family receptors blocks cell signaling and may ultimately
lead to cancer cell growth inhibition and death of the cancer cell.
HDIs, because of their unique mode of action, have the potential to
work in a wide variety of tumors, including those that do not
overexpress HER2 (Mullen et al (2007) Molecular Cancer Therapeutics
6:93-100).
[0090] Pertuzumab is based on the human IgG1 (.kappa.) framework
sequences. It consists of two heavy chains and two light chains.
Like trastuzumab, pertuzumab is directed against the extracellular
domain of HER2. However, it differs from trastuzumab in the
epitope-binding regions of the light chain and heavy chain. As a
result, pertuzumab binds to an epitope within what is known as a
sub-domain 2 of HER2, while the epitope from trastuzumab is
localized to sub-domain 4 (Cho et al. 2003; Franklin et al. 2004).
Pertuzumab acts by blocking the association of HER2 with other HER
family members, including HER1 (epidermal growth factor receptor;
EGFR), HER3, and HER4. This association is required for signaling
in the presence of ligand via MAP-kinase and PI3-kinase. As a
result, pertuzumab inhibits ligand-initiated intracellular
signaling Inhibition of these signaling pathways can result in
growth arrest and apoptosis, respectively (Hanahan and Weinberg
2000). Because pertuzumab and trastuzumab bind at distinct epitopes
on the HER2 receptor, ligand-activated downstream signaling is
blocked by pertuzumab but not by trastuzumab. Pertuzumab,
therefore, may not require HER2 overexpression to exert its
activity as an anti-tumor agent. In addition, because of their
complementary modes of action, the combination of pertuzumab and
T-DM1 may have a potential role in HER2-overexpressing
diseases.
[0091] Pertuzumab has been evaluated as a single agent in five
Phase II studies conducted in various cancer types, including MBC
expressing low levels of HER2, non-small cell lung cancer,
hormone-refractory prostate cancer, and ovarian cancer. A Phase II
trial evaluated pertuzumab as a single agent in the second- or
third-line treatment of metastatic breast cancer (MBC) patients
with normal HER2 expression (Cortes et al. (2005) J. Clin. Oncol.
23:3068). Pertuzumab has been evaluated in two Phase II studies in
combination with trastuzumab (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, Sep. 12-16, 2008; Gelmon et al (2008) J. Clin.
Oncol. 26:1026). The first study enrolled 11 patients with
HER2-positive MBC who previously received up to three prior
trastuzumab-containing regimens (Portera et al. 2007).
[0092] Bevacizumab (CAS Reg. No. 216974-75-3, AVASTIN.RTM.,
Genentech) is an anti-VEGF monoclonal antibody against vascular
endothelial growth factor (U.S. Pat. No. 7,227,004; U.S. Pat. No.
6,884,879; U.S. Pat. No. 7,060,269; U.S. Pat. No. 7,169,901; U.S.
Pat. No. 7,297,334) used in the treatment of cancer, where it
inhibits tumor growth by blocking the formation of new blood
vessels. Bevacizumab was the first clinically available
angiogenesis inhibitor in the United States, approved by the FDA in
2004 for use in combination with standard chemotherapy in the
treatment of metastatic colon cancer and most forms of metastatic
non-small cell lung cancer. Several late-stage clinical studies are
underway to determine its safety and effectiveness for patients
with: adjuvant/non-metastatic colon cancer, metastatic breast
cancer, metastatic renal cell carcinoma, metastatic glioblastoma
multiforme, metastatic ovarian cancer, metastatic
hormone-refractory prostate cancer, and metastatic metastatic or
unresectable locally advanced pancreatic cancer.
[0093] An anti-VEGF antibody will usually not bind to other VEGF
homologues such as VEGF-B or VEGF-C, nor other growth factors such
as P1GF, PDGF or bFGF. Preferred anti-VEGF antibodies include a
monoclonal antibody that binds to the same epitope as the
monoclonal anti-VEGF antibody A4.6.1 produced by hybridoma ATCC HB
10709; a recombinant humanized anti-VEGF monoclonal antibody
generated according to Presta et al. (1997) Cancer Res.
57:4593-4599, including but not limited to bevacizumab. Bevacizumab
includes mutated human IgG1 framework regions and antigen-binding
complementarity-determining regions from the murine anti-hVEGF
monoclonal antibody A.4.6.1 that blocks 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 about 7% of the sequence is derived from the
murine antibody A4.6.1. Bevacizumab has a molecular mass of about
149,000 daltons and is glycosylated. Bevacizumab and other
humanized anti-VEGF antibodies are further described in U.S. Pat.
No. 6,884,879. Additional anti-VEGF antibodies include the G6 or
B20 series antibodies (e.g., G6-31, B20-4.1), as described in any
one of FIGS. 27-29 of W02005/012359. In one embodiment, the B20
series antibody binds to a functional epitope on human VEGF
comprising residues F17, M18, D19, Y21, Y25, Q89, I91, K101, E103,
and C104
[0094] The A 4.6.1 (ATCC HB 10709) and B 2.6.2 (ATCC HB 10710)
anti-VEGF expressing hybridoma cell lines have been deposited and
maintained with the American Type Culture Collection (ATCC), 10801
University Boulevard, Manassas, Va. 20110-2209 USA. The clone
expressing VEGF-E polypeptide (U.S. Pat. No. 6,391,311) encoded by
the nucleotide sequence insert of the ATCC deposit identified as
DNA29101-1276 was deposited on Mar. 5, 1998 and maintained as ATCC
209653 with the American Type Culture Collection, 10801 University
Boulevard, Manassas, Va. 20110-2209, USA.
[0095] 5-FU (fluorouracil, 5-fluorouracil, CAS Reg. No. 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. Pat. No. 2,802,005, U.S. Pat. No. 2,885,396; Barton et al
(1972) Jour. Org. Chem. 37:329; Hansen, R. M. (1991) Cancer Invest.
9:637-642). 5-FU is named as 5-fluoro-1H-pyrimidine-2,4-dione, and
has the structure:
##STR00002##
[0096] Carboplatin (CAS Reg. No. 41575-94-4) is a chemotherapeutic
drug used against ovarian carcinoma, lung, head and neck cancers
(U.S. Pat. No. 4,140,707). Carboplatin is named as azanide;
cyclobutane-1,1-dicarboxylic acid platinum, and has the
structure:
##STR00003##
[0097] Lapatinib (CAS Reg. No. 388082-78-8, TYKERB.RTM., GW572016,
Glaxo SmithKline) has been approved for use in combination with
capecitabine (XELODA.RTM., Roche) for the treatment of patients
with advanced or metastatic breast cancer whose tumors over-express
HER2 (ErbB2) and who have received prior therapy including an
anthracycline, a taxane and trastuzumab. Lapatinib is an
ATP-competitive epidermal growth factor (EGFR) and HER2/neu
(ErbB-2) dual tyrosine kinase inhibitor (U.S. Pat. No. 6,727,256;
U.S. Pat. No. 6,713,485; U.S. Pat. No. 7,109,333; U.S. Pat. No.
6,933,299; U.S. Pat. No. 7,084,147; U.S. Pat. No. 7,157,466; U.S.
Pat. No. 7,141,576) which inhibits receptor autophosphorylation and
activation by binding to the ATP-binding pocket of the EGFR/HER2
protein kinase domain. Lapatinib is named as
N-(3-chloro-4-(3-fluorobenzyloxy)phenyl)-6-(5-(2-(methylsulfonyl)ethylami-
no)methyl)furan-2-yl)quinazolin-4-amine, and has the structure:
##STR00004##
[0098] ABT-869 (Abbott and Genentech) is a multi-targeted inhibitor
of VEGF and PDGF family receptor tyrosine kinases, for the
potential oral treatment of cancer (U.S. Pat. No. 7,297,709; US
2004/235892; US 2007/104780). Clinical trials have been initiated,
treating non-small cell lung cancer (NSCLC), hepatocellular
carcinoma (HCC), and renal cell carcinoma (RCC). ABT-869 is named
as
1-(4-(3-amino-1H-indazol-4-yl)phenyl)-3-(2-fluoro-5-methylphenyl)urea
(CAS No. 796967-16-3), and has the structure:
##STR00005##
[0099] Docetaxel (TAXOTERE.RTM., Sanofi-Aventis) is used to treat
breast, ovarian, and NSCLC cancers (U.S. Pat. No. 4,814,470; U.S.
Pat. No. 5,438,072; U.S. Pat. No. 5,698,582; U.S. Pat. No.
5,714,512; U.S. Pat. No. 5,750,561). Docetaxel is named as
(2R,3S)--N-carboxy-3-phenylisoserine, N-tert-butyl ester, 13-ester
with 5,20-epoxy-1,2,4,7,10,13-hexahydroxytax-11-en-9-one 4-acetate
2-benzoate, trihydrate (U.S. Pat. No. 4,814,470; EP 253738; CAS
Reg. No. 114977-28-5) and has the structure:
##STR00006##
[0100] GDC-0941 (Genentech Inc.), is a selective, orally
bioavailable thienopyrimidine inhibitor of PI3K with promising
pharmacokinetic and pharmaceutical properties (Folkes et al (2008)
Jour. of Med. Chem. 51(18):5522-5532; US 2008/0076768; US
2008/0207611; Belvin et al, American Association for Cancer
Research Annual Meeting 2008, 99th:April 15, Abstract 4004; Folkes
et al, American Association for Cancer Research Annual Meeting
2008, 99th:April 14, Abstract LB-146; Friedman et al, American
Association for Cancer Research Annual Meeting 2008, 99th:April 14,
Abstract LB-110). GDC-0941, shows synergistic activity in vitro and
in vivo in combination with certain chemotherapeutic agents against
solid tumor cell lines (U.S. Ser. No. 12/208,227, Belvin et al
"Combinations Of Phosphoinositide 3-Kinase Inhibitor Compounds And
Chemotherapeutic Agents, And Methods Of Use", filed 10 Sep. 2008).
GDC-0941 is named as
4-(2-(1H-indazol-4-yl)-6-((4-(methylsulfonyl)piperazin-1-yl)methyl)thieno-
[3,2-d]pyrimidin-4-yl)morpholine (CAS Reg. No. 957054-30-7), and
has the structure:
##STR00007##
[0101] GNE-390 (Genentech Inc.), is a selective, orally
bioavailable thienopyrimidine inhibitor of PI3K with promising
pharmacokinetic and pharmaceutical properties (US 2008/0242665; WO
2008/070740). GNE-390 shows synergistic activity in vitro and in
vivo in combination with certain chemotherapeutic agents against
solid tumor cell lines (U.S. Ser. No. 12/208,227, Belvin et al
"Combinations Of Phosphoinositide 3-Kinase Inhibitor Compounds And
Chemotherapeutic Agents, And Methods Of Use", filed 10 Sep. 2008).
GNE-390 is named as
(S)-1-(4-((2-(2-aminopyrimidin-5-yl)-7-methyl-4-morpholinothieno[3,2-d]py-
rimidin-6-yl)methyl)piperazin-1-yl)-2-hydroxypropan-1-one, and has
the structure:
##STR00008##
[0102] Biological Evaluation
[0103] In vitro cell culture studies using trastuzumab-MCC DM1
T-DM1) combined with different chemotherapeutic or biologically
targeted agents were performed on a number of HER2-amplified cell
lines. Data were analyzed using the Chou & Talalay method to
determine the Combination Index (CI) value for each combination,
set up in multiples of the IC50 for each drug. CI values less than
0.7 denote synergy; CI values between 0.7-1.3 denote additivity;
and CI values greater than 1.3 denote antagonism. For combinations
with chemotherapeutic agents, T-DM1 combined with docetaxel or 5-FU
resulted in additive or synergistic anti-proliferative activity,
while combinations with either gemcitabine or carboplatin had no
effect or were antagonistic with T-DM 1. Mouse xenograft studies
showed similar results where T-DM1 combined with docetaxel or 5-FU
resulted in greatly enhanced anti-tumor efficacy compared to
treatment with individual agents. T-DM1 combined with carboplatin
resulted in enhanced efficacy compared to either drug alone whereas
the combination of T-DM1 with gemcitabine was not more efficacious
than T-DM1 alone. T-DM1 combined with either pertuzumab, lapatinib
or GDC-0941 resulted in additive or synergistic anti-proliferative
activity in cell culture experiments, and in greatly enhanced
anti-tumor efficacy in vivo compared to treatment with individual
agents. In contrast, unconjugated trastuzumab antagonized the
activity of T-DM1 due to binding of the same epitope on HER2. In
vivo studies using combinations of T-DM1 with anti-angiogenic
agents such as the antibody B20-4.1 or the small molecule inhibitor
ABT-869 resulted in enhanced anti-tumor efficacy with all
combinations tested, with the exception of the highest dose of
T-DM1 (10 or 15 mg/kg) given with B20-4.1.
[0104] Combinations of trastuzumab-MCC-DM1 (T-DM1) with numerous
anti-cancer drugs were studied by measuring both the in vitro
anti-proliferative activity in HER2-overexpressing breast tumor
cells and in vivo anti-tumor efficacy in breast cancer xenograft
models. In these studies, trastuzumab-MCC-DM1 was added to either
cytotoxic chemotherapeutic agents, antibodies, or small molecule
kinase inhibitors.
[0105] The combination of anti-VEGF murine antibody B20-4.1 (Liang
et al (2006) Jour. Biol. Chem. 281:951-961), a bevacizumab
surrogate, and trastuzumab-MCC-DM1 in breast cancer mouse xenograft
models resulted in greater anti-tumor activity than B20-4.1 alone.
The results of these studies provide predictive basis of
synergistic effects and rationale for future clinical evaluation of
treatment regimens which include trastuzumab-MCC-DM1 in combination
with different anti-tumor therapies in HER2-positive breast
cancer.
[0106] Synergistic drug effects were observed with combinations of
HER2-targeted agents, such as trastuzumab-DM 1 plus lapatinib, or
trastuzumab-DM 1 combined with the HER2 antibody pertuzumab (a HER2
dimerization inhibitor).
[0107] Trastuzumab-MCC-DM1 combined with carboplatin or 5-FU showed
enhanced activity compared to treatment with individual agents
alone, whereas combination treatment with gemcitabine did not
result in increased anti-tumor activity.
[0108] Blockade of the PI3 kinase pathway with GDC-0941, a small
molecule kinase pan inhibitor of p110 isoforms (WO 2007/129161),
potentiated the activity of trastuzumab-DM1.
[0109] T-DM1 combined with the PI3K inhibitor GDC-0941 enhanced
anti-tumor activity of, in HER2-amplified breast cancer lines with
mutated PI3K: BT-474 (K111N), MDA-361.1 (E545K), and KPL4 (H1047R).
Combination treatment in vitro resulted in additive or synergistic
inhibition of cell proliferation, as well as increased apoptosis.
Similarly, in vivo efficacy was augmented with combined drug
treatment compared to single agent activity in the MDA-MB-361.1 and
Fo5 HER2-amplified xenograft models. Biochemical analyses of
biomarkers for each agent showed inhibition of phospho-Akt and
phospho-ERK by both T-DM1 and GDC-0941, decreased phosphorylation
of Rb and PRAS40 by GDC-0941, and increased levels of the mitotic
markers phospho-histone H3 and cyclin B1 after treatment with T-DM
1. In addition, T-DM 1 treatment resulted in apoptosis in these
breast cancer models as determined by appearance of the 23 kDa
PARP-cleavage fragment, decreased levels of Bcl-XL, as well as
activation of caspases 3 and 7. Addition of GDC-0941 to T-DM1
further enhanced apoptosis induction. These studies provide
evidence for the use of rational drug combinations in
HER2-amplified breast cancer and offer additional therapeutic
approaches for patients whose disease progresses on trastuzumab or
lapatinib-based therapy.
[0110] In Vitro Cell Proliferation Assays
[0111] The in vitro potency of the combinations of
trastuzumab-MCC-DM1 with chemotherapeutic agents was measured by
the cell proliferation assay of Example 2; the CellTiter-Glo.RTM.
Luminescent Cell Viability Assay, commercially available from
Promega Corp., Madison, Wis. This homogeneous assay method is based
on the recombinant expression of Coleoptera luciferase (U.S. Pat.
No. 5,583,024; U.S. Pat. No. 5,674,713; U.S. Pat. No. 5,700,670)
and determines the number of viable cells in culture based on
quantitation of the ATP present, an indicator of metabolically
active cells (Crouch et al (1993) J. Immunol. Meth. 160:81-88; U.S.
Pat. No. 6,602,677). The CellTiter-Gloms Assay was conducted in 96
or 384 well format, making it amenable to automated high-throughput
screening (HTS) (Cree et al (1995) AntiCancer Drugs 6:398-404). The
homogeneous assay procedure involves adding the single reagent
(CellTiter-Glo.RTM. Reagent) directly to cells cultured in
serum-supplemented medium. Cell washing, removal of medium and
multiple pipetting steps are not required. The system detects as
few as 15 cells/well in a 384-well format in 10 minutes after
adding reagent and mixing.
[0112] The homogeneous "add-mix-measure" format results in cell
lysis and generation of a luminescent signal proportional to the
amount of ATP present. The amount of ATP is directly proportional
to the number of cells present in culture. The CellTiter-Glo.RTM.
Assay generates a "glow-type" luminescent signal, produced by the
luciferase reaction, which has a half-life generally greater than
five hours, depending on cell type and medium used. Viable cells
are reflected in relative luminescence units (RLU). The substrate,
Beetle Luciferin, is oxidatively decarboxylated by recombinant
firefly luciferase with concomitant conversion of ATP to AMP and
generation of photons. The extended half-life eliminates the need
to use reagent injectors and provides flexibility for continuous or
batch mode processing of multiple plates. This cell proliferation
assay can be used with various multiwell formats, e.g. 96 or 384
well format. Data can be recorded by luminometer or CCD camera
imaging device. The luminescence output is presented as relative
light units (RLU), measured over time.
[0113] The anti-proliferative effects of trastuzumab-MCC-DM1 and
combinations with chemotherapeutic agents were measured by the
CellTiter-Glo.RTM. Assay (Example 2) against the tumor cell lines
in FIGS. 1-9 and 18-33.
[0114] Exemplary embodiments include a method for determining
compounds to be used in combination for the treatment of cancer
comprising: a) administering a therapeutic combination of
trastuzumab-MCC-DM1 (T-DM1) and a chemotherapeutic agent to an in
vitro tumor cell line, and b) measuring a synergistic or
non-synergistic effect. A combination index (CI) value greater than
1.3 denotes antagonism; CI values between 0.7-1.3 denote
additivity, and CI values less than 0.7 denote synergistic drug
interactions.
[0115] FIG. 1 shows the antagonistic effect of trastuzumab in
combination with trastuzumab-MCC-DM1 (T-DM1) at various
concentrations in multiples of the individual IC50 values (Table 1)
in SK-BR-3 cells which are trastuzumab-sensitive. The viable cell
number is plotted relative to the IC50 multiple values. The
combination index (CI) over IC.sub.10 to IC.sub.90 for each
combination is greater than 2, indicating antagonism in vitro.
However the combination of T-DM1+trastuzumab in vivo does not show
an antagonistic effect.
TABLE-US-00001 TABLE 1 SK-BR-3 Proliferation - 3 days IC50 multiple
trastuzumab ng/ml T-DM1 ng/ml Effect (%) CI 0.5 X.sup. 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
[0116] FIG. 2 shows the antagonistic effect of trastuzumab in
combination with trastuzumab-MCC-DM1 (T-DM1) at various
concentrations in multiples of the individual IC50 values (Table 2)
in BT-474 EEI cells which are trastuzumab-resistant. The viable
cell number is plotted relative to the IC50 multiple values. The
combination index (CI) over IC.sub.10 to IC.sub.90 for each
combination is great than 2, indicating antagonism.
TABLE-US-00002 TABLE 2 BT-474-EEI Proliferation - 3 days IC50
multiple 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.sup. 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 4
X 370.4 370.4 11.6 >2 8 X 1111.1 1111.1 18.4 >2
[0117] FIG. 3 shows the synergistic effect of pertuzumab in
combination with trastuzumab-MCC-DM1 (T-DM1) at various
concentrations in multiples of the individual IC50 values (Table 3)
in MDA-MB-175 cells. The viable cell number is plotted relative to
the IC50 multiple values. The combination index (CI) over IC.sub.10
to IC.sub.90 for each combination is under 1, with the average
CI=0.387, indicating synergism (Table 3).
TABLE-US-00003 TABLE 3 MDA-MB-175 Proliferation - 5 days IC50
multiple 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.sup. 312.5 250 33.6 0.597 1 X 625 500 50.7 0.391 2 X
1250 1000 67.7 0.259
[0118] FIG. 3a shows a plot of MDA-MB-175 in vitro cell viability
at 5 days versus IC50 multiple concentrations of pertuzumab,
trastuzumab-MCC-DM1 (T-DM1), and the combination of pertuzumab and
T-DM 1. The viable cell number is plotted relative to the IC50
multiple values. The combination index (CI) over IC.sub.10 to
IC.sub.90 for each combination is under 1, with the average
CI=0.096, indicating synergism (Table 3a).
TABLE-US-00004 TABLE 3a MDA-MB-175 Proliferation - 5 days IC50
multiple Effect (%) CI 0.0625x 21.3 0.093 0.125x 37.5 0.037 0.25x
40.1 0.060 0.5x.sup. 50.3 0.052 1x 53.9 0.078 2x 57.0 0.120 4x 65.5
0.117 8x 66.8 0.208
[0119] FIG. 4 shows a plot of BT-474 in vitro cell viability at 5
days versus various fixed doses of pertuzumab in combination with
dose response of trastuzumab-MCC-DM1 (T-DM1), and various doses of
T-DM1 alone. FIG. 4 shows the effects of fixed doses of T-DM1 in
combination with various dosages of pertuzumab. Addition of
pertuzumab to T-DM1 results in slightly greater anti-proliferative
activity than T-DM 1 alone.
[0120] FIG. 5 shows a plot of BT-474 in vitro cell viability at 5
days versus various fixed doses of trastuzumab-MCC-DM1 (T-DM1) in
combination with dose response of pertuzumab, and various doses of
pertuzumab alone. FIG. 5 shows the effects of fixed doses of
pertuzumab in combination with various dosages of T-DM1 on BT-474
cell proliferation. Addition of T-DM1 to pertuzumab enhances the
effect of pertuzumab alone.
[0121] FIG. 6 shows the synergistic effect of pertuzumab in
combination with trastuzumab-MCC-DM1 (T-DM1) at various
concentrations in multiples of the individual IC50 values (Table 4)
in BT-474 cells. The viable cell number is plotted relative to the
IC50 multiple values. Combination index (CI) values from IC.sub.10
to IC.sub.90 range from 0.198 to 1.328. The average CI for this
range=0.658 indicating synergy.
TABLE-US-00005 TABLE 4 BT-474 Proliferation - 5 days IC50 multiple
pertuzumab ng/ml T-DM1 ng/ml Effect (%) CI 0.25 X 34.29 11.43 3.9
>2 0.5 X.sup. 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
[0122] FIG. 7 shows a plot of SK-BR-3 in vitro cell viability at 3
days versus varying doses of T-DM1 in combination with fixed doses
of lapatinib (4.5 nM, 14 nM, 41 nM, 123 nM), and varying doses of
T-DM1 alone (0-1000 ng/ml). Addition of lapatinib to T-DM1 results
in slightly greater anti-proliferative activity than T-DM 1
alone.
[0123] FIG. 7a shows a plot of SK-BR-3 in vitro cell viability at 3
days versus T-DM1, lapatinib, and fixed dose ratio combinations of
T-DM1 and lapatinib as shown in Table 7a. The average CI value
between the IC10 and IC90=0.793, indicating additivity.
TABLE-US-00006 TABLE 7a SK-BR-3 Proliferation - 3 days IC50
multiple lapatinib nM T-DM1 ng/ml Effect (%) CI 0.25x 4.57 1.52 3.5
>2 0.5x.sup. 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
[0124] FIG. 8a shows a plot of BT-474 in vitro cell viability at 3
days versus T-DM1, lapatinib, and fixed dose ratio combinations of
T-DM1 and lapatinib as shown in Table 8a. The average CI value
between the IC10 and IC90=0.403, indicating synergy.
TABLE-US-00007 TABLE 8a BT-474 Proliferation - 3 days IC50 multiple
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.sup. 4.57 13.72 26.8 0.493 1x 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
[0125] FIG. 8 shows a plot of BT-474 in vitro cell viability at 3
days versus varying doses of T-DM1 in combination with fixed doses
of lapatinib (1.5 nM, 4.5 nM, 14 nM, 41 nM, 123 nM), and varying
doses of T-DM1 alone (0-1000 ng/ml). Addition of lapatinib to T-DM1
results in greater anti-proliferative activity compared to either
drug alone.
[0126] FIG. 9 shows a plot of BT-474-EEI in vitro cell viability at
3 days versus varying doses of T-DM1 in combination with fixed
doses of lapatinib (14 nM, 41 nM, 123 nM, 370 nM, 1111 nM), and
varying doses of T-DM1 alone (0-1000 ng/ml). Addition of lapatinib
to T-DM1 results in greater anti-proliferative activity compared to
either drug alone.
[0127] FIG. 18 shows a plot of SK-BR-3 in vitro cell viability at 3
days versus IC50 multiple concentrations of 5-FU,
trastuzumab-MCC-DM1 (T-DM1), and fixed dose ratio combinations of
5-FU and T-DM1 (Table 18). The combination of 5-FU and T-DM1 is
additive on SK-BR-3 cells, with the average CI between the IC10 and
IC90=0.952.
TABLE-US-00008 TABLE 18 5-FU + T-DM1: SK-BR-3 Proliferation - 3
days IC50 multiple 5-FU (.mu.M) T-DM1 ng/ml Effect (%) CI 0.5x.sup.
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
[0128] FIG. 19 shows a plot of BT-474 in vitro cell viability at 3
days versus IC50 multiple concentrations of 5-FU,
trastuzumab-MCC-DM1 (T-DM1), and fixed dose ratio combinations of
5-FU and T-DM1 (Table 19). The combination of 5-FU and T-DM1 is
synergistic on BT-474 cells, with average CI value=0.623.
TABLE-US-00009 TABLE 19 5-FU + T-DM1: BT-474 Proliferation - 3 days
IC50 multiple 5-FU (.mu.M) T-DM1 ng/ml Effect (%) CI 0.25x 0.488
3.90 17.1 0.508 0.5x.sup. 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
[0129] FIG. 20 shows a plot of SK-BR-3 in vitro cell viability at 3
days versus IC50 multiple concentrations of gemcitabine,
trastuzumab-MCC-DM1 (T-DM1), and fixed dose ratio combinations of
gemcitabine and T-DM1 (Table 20). Gemcitabine combined with T-DM1
results in an antagonistic drug interaction, with CI values>1.3
at all combinations tested.
TABLE-US-00010 TABLE 20 gemcitabine (GEM) + T-DM1: SK-BR-3
Proliferation - 3 days IC50 multiple GEM (nM) T-DM1 ng/ml Effect
(%) CI 0.5x.sup. 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 50 72.2 4.957
[0130] FIG. 21 shows a plot of MDA-MD-361 in vitro cell viability
at 3 days versus IC50 multiple concentrations of gemcitabine,
trastuzumab-MCC-DM1 (T-DM1), and fixed dose ratio combinations of
gemcitabine and T-DM1 (Table 21). The drug combination gives an
antagonistic effect with the average CI=1.706.
TABLE-US-00011 TABLE 21 gemcitabine (GEM) + T-DM1: MDA-MD- 361
Proliferation - 3 days IC50 multiple 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.sup. 1.56 12.5 30.7 1.336 1x 3.12 25 59.2 1.280 2x 6.25 50
76.3 1.581 4x 12.5 100 80.3 2.747
[0131] FIG. 22 shows a plot of KPL4 in vitro cell viability
(proliferation) at 3 days after treatment with T-DM1, GDC-0941, and
fixed dose ratio combinations of T-DM1 (6.25 to 100 ng/ml) and
GDC-0941 (62.5 nM to 1 .mu.M) at IC50 multiple concentrations from
0.25.times. to 4.times.. Table 22 shows the effect in the 10-90%
inhibition range with calculated CI values and average CI of
1.111.
The Bliss prediction of additivity is plotted as the dotted line in
FIG. 22. The Bliss independence plot shows the calculated
additivity response from combination of two single compounds.
TABLE-US-00012 TABLE 22 GDC-0941 + T-DM1: KPL4 Proliferation - 3
days IC50 multiple GDC-0941 (nM) T-DM1 ng/ml Effect (%) CI 0.25x
62.5 6.25 1.0 6.319 0.5x.sup. 125 12.5 33.9 1.229 1x 250 25 71.8
1.053 2x 500 50 91.1 1.051 4x 1000 100 93.7 1.753
[0132] FIG. 23 shows a plot of KPL4 in vitro cell viability
(proliferation) at 3 days after treatment with T-DM1, GDC-0941, and
fixed dose ratio combinations of T-DM1 (1.25 to 80 ng/ml) and
GDC-0941 (31.25 nM to 2 .mu.M) at IC50 multiple concentrations from
0.0625.times. to 16.times.. The Bliss prediction of additivity is
plotted as the dotted line. Table 23 shows the effect in the 10-90%
inhibition range with calculated CI values and average CI of 0.802.
The combination of T-DM1 and GDC-0941 is additive in the KPL4 cell
line
TABLE-US-00013 TABLE 23 GDC-0941 + T-DM1: KPL4 Proliferation - 3
days IC50 multiple 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.sup. 125 5
39.2 1.263 1x 250 10 84.5 0.452 2x 500 20 94.9 0.350 4x 1000 40
97.1 0.440 8x 2000 80 97.9 0.668
[0133] FIG. 24 shows a plot of Her2 amplified, HERCEPTIN.RTM.
resistant, PIK3CA (H1047R) mutant, KPL-4 cells in vitro cell
viability (proliferation) after treatment with T-DM1, PI103,
GDC-0941, and fixed dose ratio combinations of T-DM1+PI103, and
T-DM1+GDC-0941, at IC50 multiple concentrations from 0 to
16.times.. Table 24 shows the Combination Index values. The results
suggest moderate in vitro synergy between T-DM-1 and GDC-0941 since
the CI values are between 0.5 and 1, and additivity between T-DM-1
and PI103 since CI values are near 1.
TABLE-US-00014 TABLE 24 Combinations: KPL4 Proliferation CI at:
T-DM1 + GDC-0941 T-DM1 + PI103 ED50 0.74303 1.04069 ED75 0.63448
0.9721 ED90 0.54179 0.91094
[0134] The PI3K selective inhibitor, PI103 (Hayakawa et al (2007)
Bioorg. Med. Chem. Lett. 17:2438-2442; Raynaud et al (2007) Cancer
Res. 67:5840-5850; Fan et al (2006) Cancer Cell 9:341-349; U.S.
Pat. No. 6,608,053), and has the structure:
##STR00009##
[0135] FIG. 25 shows a plot of KPL4 Caspase 3/7 in vitro cell
apoptosis (programmed cell death) at 24 hours after treatment with
T-DM1, GDC-0941, and fixed dose ratio combinations of T-DM1 and
GDC-0941. The combination of T-DM1 and GDC-0941 results in greatly
enhanced apoptosis compared to either agent alone.
[0136] FIG. 26 shows a plot of KPL4 in vitro cell apoptosis
(programmed cell death) at 3 days after treatment with T-DM1,
GDC-0941, and fixed dose ratio combinations of T-DM1 and GDC-0941.
The combination of T-DM1 and GDC-0941 results in greatly enhanced
apoptosis compared to either agent alone.
[0137] FIG. 27 shows a plot of MDA-MB-361 in vitro cell viability
(proliferation) at 3 days after treatment with T-DM1, GDC-0941, and
fixed dose ratio combinations of T-DM1 (3.125 to 50 ng/ml) and
GDC-0941 (62.5 nM to 1 .mu.M) at IC50 multiple concentrations from
0.125.times. to 8.times.. The Bliss prediction of additivity is
plotted as the dotted line. Table 27 shows the effect in the 10-90%
inhibition range with calculated CI values and average CI of 0.888.
T-DM1 combined with GDC-0941 results in additive anti-proliferative
activity in the MDA-MB-361 cells, with the average CI=0.889.
TABLE-US-00015 TABLE 27 GDC-0941 + T-DM1: MDA-MB-361 Proliferation
- 3 days IC50 multiple GDC-0941 (nM) T-DM1 ng/ml Effect (%) CI
0.25x 62.5 3.125 21.9 1.003 0.5x.sup. 125 6.25 37.3 0.862 1x 250
12.5 51.8 0.920 2x 500 25 73.1 0.742 4x 1000 50 82.3 0.917
[0138] FIG. 28 shows a plot of MDA-MB-361 in vitro cell viability
(proliferation) at 3 days after treatment with T-DM1, GDC-0941, and
fixed dose ratio combinations of T-DM1 (3.125 to 100 ng/ml) and
GDC-0941 (62.5 nM to 2 .mu.M) at IC50 multiple concentrations from
0.125.times. to 8.times.. The Bliss prediction of additivity is
plotted as the dotted line. Table 28 shows the Effect in the 10-90%
inhibition range with calculated CI values and average CI of 0.813.
T-DM1 combined with GDC-0941 results in additive anti-proliferative
activity in the MDA-MB-361 cells, with the average CI=0.813.
TABLE-US-00016 TABLE 28 GDC-0941 + T-DM1: MDA-MB-361 Proliferation
- 3 days IC50 multiple GDC-0941 (nM) T-DM1 ng/ml Effect (%) CI
0.25x 62.5 3.125 28.6 0.785 0.5x.sup. 125 6.25 36.7 0.960 1x 250
12.5 48.5 1.026 2x 500 25 66.6 0.807 4x 1000 50 82.2 0.590 8x 2000
100 87.7 0.709
[0139] FIG. 29 shows a plot of BT-474 in vitro cell viability
(proliferation) at 3 days after treatment with T-DM1, GDC-0941, and
fixed dose ratio combinations of T-DM1 (3.125 to 100 ng/ml) and
GDC-0941 (31.25 nM to 1 .mu.M) at IC50 multiple concentrations from
0.125.times. to 4.times.. The Bliss prediction of additivity is
plotted as the dotted line. Table 29 shows the effect in the 10-90%
inhibition range with calculated CI values and average CI of
1.2122. GDC-0941 and T-DM1 do not have a combination effect on
BT-474, using these dose ratios.
TABLE-US-00017 TABLE 29 GDC-0941 + T-DM1: BT-474 Proliferation - 3
days IC50 multiple 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.sup. 125 12.5
31.4 1.178 1x 250 25 43.9 1.207 2x 500 50 53.9 1.473 4x 1000 100
71.5 1.171
[0140] FIG. 30 shows a plot of BT-474 in vitro cell viability
(proliferation) at 3 days after treatment with T-DM1, GDC-0941, and
fixed dose ratio combinations of T-DM1 (6.25 to 100 ng/ml) and
GDC-0941 (62.5 nM to 1 .mu.M) at IC50 multiple concentrations from
0.25.times. to 4.times.. The Bliss prediction of additivity is
plotted as the dotted line. Table 30 shows the effect in the 10-90%
inhibition range with calculated CI values and average CI of 0.997,
indicating additivity.
TABLE-US-00018 TABLE 30 GDC-0941 + T-DM1: BT-474 Proliferation - 3
days IC50 multiple GDC-0941 (nM) T-DM1 ng/ml Effect (%) CI 0.25x
62.5 6.25 19.7 1.338 0.5x.sup. 125 12.5 31.5 1.167 1x 250 25 49.0
0.886 2x 500 50 66.0 0.708 4x 1000 100 73.9 0.886
[0141] FIG. 31 shows a plot of Her2 amplified, non-PI3K mutant,
AU565 cells in vitro cell viability (proliferation) at 3 days after
treatment with T-DM1, PI 103, GDC-0941, and fixed dose ratio
combinations of T-DM1+PI 103, and T-DM1+GDC-0941 at IC50 multiple
concentrations from 0 to 16.times.. Table 31 shows the Combination
Index values. The results suggest in vitro antagonism between
T-DM-1 and GDC-0941 since the CI values are between >1, and
additivity or slight antagonism between T-DM-1 and PI103 since CI
values are near or slightly greater than 1.
TABLE-US-00019 TABLE 31 Combinations: AU565 Proliferation CI at:
T-DM1 + GDC-0941 T-DM1 + PI103 ED50 1.19123 1.12269 ED75 1.36342
0.97338 ED90 1.56063 0.84956
[0142] FIG. 32 shows a plot of Her2 amplified, PIK3CA (C420R)
mutant, EFM192A cells in vitro cell viability (proliferation) at 3
days after treatment with T-DM1, PI103, GDC-0941, and fixed dose
combinations of T-DM1+PI103, and T-DM1+GDC-0941, at IC50 multiple
concentrations from 0 to 16.times.. Table 32 shows the Combination
Index values. The results suggest moderate in vitro synergy between
T-DM-1 and GDC-0941 since the CI values are between 0.5 and 1, and
synergy between T-DM-1 and PI103 since CI values are near 0.5.
TABLE-US-00020 TABLE 32 Combinations: EFM192A Proliferation CI at:
T-DM1 + GDC-0941 T-DM1 + PI103 ED50 0.80379 0.53861 ED75 0.66352
0.52087 ED90 0.5485 0.52001
[0143] FIG. 33 shows a plot of Her2 amplified, HERCEPTIN.RTM.
resistant, PIK3CA (H1047R) mutant, HCC1954 cells in vitro cell
viability (proliferation) at 3 days after treatment with T-DM1,
PI103, GDC-0941, and fixed dose ratio combinations of T-DM1+PI103,
and T-DM1+GDC-0941, at IC50 multiple concentrations from 0 to
16.times.. Table 33 shows the Combination Index values. The results
suggest additivity or slight in vitro synergy between T-DM-1 and
GDC-0941 since the CI values are close to 1, and slight synergy
between T-DM-1 and PI103 since CI values are <1.
TABLE-US-00021 TABLE 33 Combinations: HCC1954 Proliferatior CI at:
T-DM1 + GDC-0941 T-DM1 + PI103 ED50 1.15864 0.78902 ED75 0.92365
0.78684 ED90 0.74198 0.80771
[0144] In Vivo Tumor Xenograft Efficacy
[0145] The efficacy of the combinations of the invention may be
measured in vivo by implanting allografts or xenografts of cancer
cells in rodents and treating the tumors with the combinations.
Variable results are to be expected depending on the cell line, the
presence or absence of certain mutations in the tumor cells, the
sequence of administration of trastuzumab-MCC-DM1 and
chemotherapeutic agent, dosing regimen, and other factors. Subject
mice were treated with drug(s) or control (Vehicle) and monitored
over several weeks or more to measure the time to tumor doubling,
log cell kill, and tumor inhibition (Example 3). FIGS. 10-17 and
34-37 show the efficacy of trastuzumab-MCC-DM1 in combinations with
chemotherapeutic agents by xenograft tumor inhibition in mice.
[0146] FIG. 10 shows a plot of the in vivo mean tumor volume change
over time on KPL-4 tumors inoculated into the mammary fat pad of
SCID beige mice after dosing with: (1) ADC buffer, (2) pertuzumab
15 mg/kg, (3) T-DM1 0.3 mg/kg, (4) T-DM1 1 mg/kg, (5) T-DM1 3
mg/kg, (6) pertuzumab 15 mg/kg+T-DM1 0.3 mg, (7) pertuzumab 15
mg/kg+T-DM1 1 mg/kg, (8) pertuzumab 15 mg/kg+T-DM1 3 mg/kg. Animals
dosed with ADC buffer (1) gave 0 PR and 0 CR. Animals dosed with
pertuzumab (2) at 15 mg/kg gave 0 PR and 0 CR. Animals dosed with
T-DM1 at 0.3 mg/kg (3) alone gave 0 PR and 0 CR. Animals dosed with
T-DM1 at 1 mg/kg (4) alone gave 1 PR and 0 CR. Animals dosed with
T-DM1 at 3 mg/kg (5) alone gave 7 PR and 0 CR. Animals dosed with
the combination of pertuzumab at 15 mg/kg and T-DM1 at 0.3 mg/kg
(6) gave 5 PR and 0 CR. Animals dosed with the combination of
pertuzumab at 15 mg/kg and T-DM1 at 1 mg/kg (7) gave 8 PR and 0 CR.
Animals dosed with the combination of pertuzumab at 15 mg/kg and
T-DM1 at 3 mg/kg (8) gave 8 PR and 0 CR. The combination of
pertuzumab and T-DM1 results in greater anti-tumor activity in KPL4
xenografts than either agent alone.
[0147] FIG. 11 shows a plot of the in vivo mean tumor volume change
over time on KPL-4 tumors inoculated into the mammary fat pad of
SCID beige mice after dosing with: (1) ADC buffer, (2) 5-FU 100
mg/kg, (3) pertuzumab, 40 mg/kg, (4) B20-4.1, 5 mg/kg, (5) T-DM1, 5
mg/kg, (6) 5-FU, 100 mg/kg+T-DM1, 5 mg, (7) pertuzumab, 40
mg/kg+T-DM1, 5 mg/kg, (8), -4.1 5 mg/kg+T-DM1, 5 mg/kg, (9)
B20-4.1, 5 mg/kg+pertuzumab, 40 mg/kg. At the end of the study, all
remaining tumors less than 50 mm.sup.3 volume were histologically
evaluated and determined that 8 samples in single agent (5) T-DM1,
5 mg/kg, 5 samples in combination group (6) 5-FU, 100 mg/kg+T-DM1,
5 mg, and 8 samples in combination group (7) pertuzumab, 40
mg/kg+T-DM1, 5 mg/kg had no evidence of viable tumor cells.
[0148] FIG. 12 shows a plot of the in vivo mean tumor volume change
over time on MMTV-HER2Fo5 transgenic mammary tumor inoculated into
the mammary fat pad of CRL nu/nu mice after dosing with: (1)
Vehicle (ADC buffer), (2) B20-4.1, 5 mg/kg, (3) T-DM1, 3 mg/kg, (4)
T-DM1, 5 mg/kg, (5) T-DM1, 10 mg/kg, (6) B20-4.1, 5 mg/kg+T-DM1, 3
mg/kg, (7) B20-4.1, 5 mg/kg+T-DM1, 5 mg/kg, (8) B20-4.1, 5
mg/kg+T-DM1, 10 mg/kg. The combination of T-DM1 and B20-4.1 results
in enhanced tumor growth inhibition with T-DM1 of 3 and 5 mg/kg,
but not 10 mg/kg.
[0149] FIG. 13 shows a plot of the in vivo mean tumor volume change
over time on MMTV-HER2Fo5 transgenic mammary tumor inoculated into
the mammary fat pad of CRL nu/nu mice after dosing with: (1)
Vehicle (ADC buffer), (2) T-DM1, 10 mg/kg, (3) 5-FU, 100 mg/kg, (4)
gemcitabine, 120 mg/kg, (5) carboplatin, 100 mg/kg, (6) 5-FU, 100
mg/kg+T-DM1, 10 mg/kg, (7) gemcitabine, 120 mg/kg+T-DM1, 10 mg/kg,
(8) carboplatin, 100 mg/kg+T-DM1, 10 mg/kg. T-DM1 combined with
either 5-FU, carboplatin or gemcitabine results in enhanced
anti-tumor efficacy compared to single agent treatment.
[0150] FIG. 14 shows a plot of the in vivo mean tumor volume change
over time on MMTV-Her2 Fo5 transgenic mammary tumor xenografts
inoculated into the mammary fat pad of Harlan athymic nude mice
after dosing with: (1) Vehicle (PBS buffer) iv, qwk.times.4, (2)
lapatinib, 101 mg/kg, po, bid.times.21, (3) pertuzumab, 40 mg/kg,
iv, qwk.times.4, (4) B20-4.1, 5 mg/kg, ip, 2.times./wk.times.4, (5)
T-DM1, 15 mg/kg, iv, q3wk to end, (6) lapatinib, 101 mg/kg, po,
bid.times.21+T-DM1, 15 mg/kg, iv, q3wk to end (7) pertuzumab, 40
mg/kg, iv, qwk.times.4+T-DM1, mg/kg, iv, q3wk to end, (8) B20-4.1,
5 mg/kg, ip, 2.times./wk.times.4+T-DM1, 15 mg/kg, iv, q3wk to
end.
[0151] The single agent T-DM1 at 15 mg/kg dose (5) is not
significantly different from the combination of T-DM1 at 15 mg/kg
and B20-4.1 at 5 mg/kg (8). Lapatinib and pertuzumab were not
different from vehicle in this study. B20-4.1 showed a trend
towards increased efficacy compared to vehicle. T-DM1 was
efficacious as a single agent (p<0.01). The combination of T-DM1
with lapatinib was significantly better than lapatinib alone
(p<0.01), but not different than T-DM1 alone. The combination of
T-DM1 with pertuzumab was significantly better than pertuzumab
alone (p<0.01), but not different than T-DM1 alone. The
combination of T-DM1 with B20-4.1 was significantly better than
B20-4.1 alone (p<0.01), but not different than T-DM1 alone.
[0152] FIG. 15 shows a plot of the in vivo efficacy by mean tumor
volume change over time on MMTV-Her2 Fo5 transgenic mammary tumor
xenografts inoculated into the mammary fat pad of Harlan athymic
nude mice after dosing with: (1) Vehicle (PBS buffer) po,
bid.times.21 (2) T-DM1, 7.5 mg/kg, iv, qd.times.1 (3) T-DM1, 15
mg/kg, iv, qd.times.1 (4) ABT-869, 5 mg/kg, po, bid.times.21 (5)
ABT-869, 15 mg/kg, po, bid.times.21 (6) T-DM1, 7.5 mg/kg, iv,
qd.times.1+ABT-869, 5 mg/kg, po, bid.times.21 (7) T-DM1 7.5 mg/kg,
iv, qd.times.1+ABT-869, 15 mg/kg, po, bid.times.21 (8) T-DM1, 15
mg/kg, iv, qd.times.1+ABT-869, 5 mg/kg, po, bid.times.21 (9) T-DM1,
15 mg/kg, iv, qd.times.1+ABT-869, 15 mg/kg, po, bid.times.21.
[0153] The combination of T-DM1 and ABT-869, 5 mg/kg showed two
partial responses (8), and is not significantly more efficacious
than single agent ABT-869, 5 mg/kg (4). The combination of T-DM 1
and ABT-869, 15 mg/kg (9) is slightly more efficacious than single
agent ABT-869, 15 mg/kg (5). ABT-869 dosed at 5 mg/kg was
significantly better than vehicle by time to endpoint (p<0.01),
but was not different than vehicle by time to tumor doubling.
ABT-869 dosed at 15 mg/kg and T-DM1 dosed at either 7.5 or 15 mg/kg
were significantly better than vehicle by both time to tumor
doubling and time to tumor endpoint (p<0.01). The combination of
7.5 mg/kg T-DM1 and 5 mg/kg ABT-869 was not different than the
single agent of 7.5 mg/kg T-DM1. Compared to single agent 5 mg/kg
ABT-869, the combination of 7.5 mg/kg T-DM1+5 mg/kg ABT-869 was
significantly better by time to tumor doubling (p<0.01), but was
not different by time to endpoint. The combination of 7.5 mg/kg
T-DM1 and 15 mg/kg ABT-869 was significantly better than either
single agent (p<0.01). The combination of 15 mg/kg T-DM1+5 mg/kg
ABT-869 was not different than 15 mg/kg T-DM1 single agent.
Compared to 5 mg/kg ABT-869 single agent, the combination of 15
mg/kg T-DM1 and 5 mg/kg ABT-869 was not different by time to
endpoint, but was significantly different by time to tumor doubling
(p<0.01). The combination of 15 mg/kg T-DM1+15 mg/kg ABT-869 was
significantly better than 15 mg/kg ABT-869 alone and was better
than 15 mg/kg T-DM1 alone by time to tumor doubling (p<0.01).
The time to endpoint of 15 mg/kg T-DM1 and 15 mg/kg T-DM1+15 mg/kg
ABT-869 was not different.
[0154] FIG. 16 shows a plot of the in vivo mean tumor volume change
over time on MMTV-Her2 Fo5 transgenic mammary tumor xenografts
inoculated into the mammary fat pad of Harlan athymic nude mice
after dosing with: (1) Vehicle. iv. qwk.times.3 (2) T-DM1, 7.5
mg/kg, iv, q3wk.times.2 (3) T-DM1, 15 mg/kg, iv, q3wk.times.2 (4)
docetaxel, 30 mg/kg, iv, qwk.times.3 (5) T-DM1, 7.5 mg/kg, iv,
q3wk.times.2+docetaxel, 30 mg/kg, iv, qwk.times.3 (6) T-DM1, 15
mg/kg, iv, q3wk.times.2+docetaxel, 30 mg/kg, iv, qwk.times.3.
[0155] Animals dosed with T-DM1 at 15 mg/kg (3) alone gave 6
partial responses (PR) and 1 complete response (CR). Animals dosed
with docetaxel alone at 30 mg/kg (4) gave 2 PR. Animals dosed with
the combination of T-DM1 at 7.5 mg/kg and docetaxel at 30 mg/kg (5)
gave 10 PR. Animals dosed with the combination of T-DM1 at 15 mg/kg
and docetaxel at 30 mg/kg (6) showed a dose response with 7 PR and
3 CR. All single agent groups were significantly different than the
vehicle group (p<0.01). The combination of 7.5 mg/kg
T-DM1+docetaxel was significantly better than either single agent
by both time to tumor doubling and time to endpoint (p<0.01).
There were no objective responses in the 7.5 mg/kg T-DM1 group and
2 partial responses (PR) in the docetaxel single agent group. The
combination of 7.5 mg/kg T-DM1 and docetaxel resulted in 9 PRs and
1 complete response (CR). The combination of 15 mg/kg
T-DM1+docetaxel was significantly better than either single agent
by time to tumor doubling and time to endpoint (p<0.01). The
single agent 15 mg/kg T-DM1 treatment resulted in 5 PRs and 2 CRs.
The combination of 15 mg/kg T-DM1+docetaxel increased the objective
response rate to 7 PRs and 3 CRs. All mice in this combination
group had an objective response to treatment.
[0156] FIG. 17 shows a plot of the in vivo mean tumor volume change
over time on MMTV-Her2 Fo5 transgenic mammary tumor xenografts
inoculated into the mammary fat pad of Harlan athymic nude mice
after dosing with: (1) Vehicle, po, qd.times.21 (2) T-DM1, 7.5
mg/kg, iv, q3wk.times.2, (3) T-DM1, 15 mg/kg, iv, q3wk.times.2 (4)
lapatinib, 100 mg/kg, po, bid.times.21, (5) T-DM1, 7.5 mg/kg, iv,
q3wk.times.2+lapatinib, 100 mg/kg, po, bid.times.21, (6) T-DM1, 15
mg/kg, iv, q3wk.times.2+lapatinib, 100 mg/kg, po, bid.times.21.
[0157] Animals dosed with T-DM1 at 15 mg/kg (3) alone gave 6
partial responses (PR) and 3 complete responses (CR). Animals dosed
with the combination of T-DM1 at 7.5 mg/kg and lapatinib at 100
mg/kg (5) gave 4 PR and 5 CR. Animals dosed with the combination of
T-DM1 at 15 mg/kg and lapatinib at 100 mg/kg (6) showed a dose
response with 8 CR. All single agent groups were significantly
different from vehicle (p<0.01) by both time to tumor doubling
and time to endpoint. T-DM1 dosed at 7.5 mg/kg in combination with
lapatinib was significantly better than either lapatinib or T-DM1
at 7.5 mg/kg as a single agent (p<0.01). T-DM1 dosed at 15 mg/kg
in combination with lapatinib was significantly better than
lapatinib single agent (p<0.01). This combination was not
different than 15 mg/kg of T-DM1 dosed as a single agent.
[0158] The time to tumor doubling was measured by Kaplan-Meier
statistical analysis as 2.times.Vo. Time to tumor doubling and
survival analysis were quantified by Log-rank-p values. Time to
progression is measured as the elapsed time for tumor volume to
reach 1000 mm.sup.3, or the survival time if 1000 mm.sup.3 tumor
volume is not reached. T-DM1 combined with lapatinib resulted in
greatly enhanced anti-tumor efficacy compared to single agent
treatment.
[0159] FIG. 34 shows a plot of the in vivo mean tumor volume change
over time on MMTV-Her2 Fo5 transgenic mammary tumor inoculated into
CRL nu/nu mice after dosing with: (1) Vehicle, po, qd.times.21 (2)
T-DM1, 10 mg/kg, iv, q3wk, (3) 5-FU, 100 mg/kg, po, qwk.times.2,
(4) (5) T-DM1, 5 mg/kg, iv, q3wk+5-FU, 100 mg/kg, po, qwk.times.2.
Animals dosed with Vehicle gave 0 partial responses (PR) and 0
complete responses (CR). Animals dosed with T-DM1 gave 1 PR and 0
CR. Animals dosed with 5-FU gave 0 PR and 0 CR. Animals dosed with
the combination of T-DM1 and 5-FU gave 3 PR and 0 CR at the 42 day
time point. Treatment with T-DM1 and 5-FU results in enhanced
anti-tumor activity compared to either agent alone.
[0160] FIG. 35 shows a plot of the in vivo mean tumor volume change
over time on MMTV-Her2 Fo5 transgenic mammary tumor inoculated into
CRL nu/nu mice after dosing with: (1) Vehicle, po, qd.times.21 (2)
T-DM1, 5 mg/kg, iv, q3wk, (3) GDC-0941, 100 mg/kg, po,
bid.times.21, (4) GDC-0152, 50 mg/kg, po, qwk.times.2, (5) T-DM1, 5
mg/kg, iv, q3wk+GDC-0941, 100 mg/kg, po, bid.times.21, (6) T-DM1, 5
mg/kg, iv, q3wk+GDC-0152, 50 mg/kg, po, qwk.times.2. Treatment with
T-DM1 and GDC-0941 results in enhanced anti-tumor activity compared
to single agent treatment, while the combination of T-DM1 and
GDC-0152 was not more efficacious than T-DM1 alone.
[0161] GDC-0152 is an inhibitor of caspases which are inhibitors of
apoptosis proteins (Call et al (2008) The Lancet Oncology,
9(10):1002-1011; Deveraux et al (1999) J Clin Immunol
19:388-398).
[0162] FIG. 36 shows a plot of the in vivo mean tumor volume change
over time on MDA-MB-361.1 mammary tumor inoculated into CRL nu/nu
mice after dosing with: (1) Vehicle, po, qd.times.21, (2) GDC-0941,
25 mg/kg, po, qd.times.21, (3) GDC-0941, 50 mg/kg, po, qd.times.21,
(4) GDC-0941, 100 mg/kg, po, qd.times.21, (5) T-DM1, 3 mg/kg, iv,
q3wk, (6) T-DM1, 10 mg/kg, iv, q3wk, (7) GDC-0941, 25 mg/kg, po,
qd.times.21+T-DM1, 3 mg/kg, iv, q3wk, (8) GDC-0941, 50 mg/kg, po,
qd.times.21+T-DM1, 3 mg/kg, iv, q3wk, (9) GDC-0941, 100 mg/kg, po,
qd.times.21+T-DM1, 3 mg/kg, iv, q3wk, (10) GDC-0941, 25 mg/kg, po,
qd.times.21+T-DM1, 10 mg/kg, iv, q3wk, (11) GDC-0941, 50 mg/kg, po,
qd.times.21+T-DM1, 10 mg/kg, iv, q3wk, (12) GDC-0941, 100 mg/kg,
po, qd.times.21+T-DM1, 10 mg/kg, iv, q3wk.
[0163] Animals dosed with Vehicle (1) gave 0 partial responses (PR)
and 0 complete response (CR). Animals dosed with GDC-0941 at 25
mg/kg alone (2) gave 0 PR and 0 CR. Animals dosed with GDC-0941 at
50 mg/kg alone (3) gave 1 PR and 0 CR. Animals dosed with GDC-0941
at 100 mg/kg alone (4) gave 0 PR and 0 CR. Animals dosed with T-DM1
at 3 mg/kg (5) alone gave 1 (PR) and 1 CR). Animals dosed with
T-DM1 at 10 mg/kg (6) alone gave 8 (PR) and 1 CR). Animals dosed
with the combination of T-DM1 at 3 mg/kg and GDC-0941 at 25 mg/kg
(7) gave 5 PR and 0 CR. Animals dosed with the combination of T-DM1
at 3 mg/kg and GDC-0941 at 50 mg/kg (8) gave 3 PR and 0 CR. Animals
dosed with the combination of T-DM1 at 3 mg/kg and GDC-0941 at 100
mg/kg (9) gave 3 PR and 1 CR. Animals dosed with the combination of
T-DM1 at 10 mg/kg and GDC-0941 at 50 mg/kg (10) gave 9 PR and 0 CR.
Animals dosed with the combination of T-DM1 at 10 mg/kg and
GDC-0941 at 50 mg/kg (11) gave 7 PR and 2 CR. Animals dosed with
the combination of T-DM1 at 10 mg/kg and GDC-0941 at 100 mg/kg (12)
gave 9 PR and 1 CR.
[0164] FIG. 37 shows a plot of the in vivo mean tumor volume change
over time on MDA-MB-361.1 mammary tumor inoculated into CRL nu/nu
mice after dosing with: (1) Vehicles [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,
qd.times.21 and qd (2) GNE-390, 1.0 mg/kg, po, qd.times.21, (3)
GNE-390, 2.5 mg/kg, po, qd.times.21, (4) T-DM1, 3 mg/kg, iv, qd,
(5) GNE-390, 1.0 mg/kg, po, qd.times.21+T-DM1, 3 mg/kg, iv, qd, (6)
GNE-390, 2.5 mg/kg, po, qd.times.21+T-DM1, 3 mg/kg, iv, qd
[0165] Animals dosed with Vehicle (1) gave 0 partial responses (PR)
and 0 complete response (CR). Animals dosed with GNE-390 at 1.0
mg/kg alone (2) gave 0 PR and 0 CR. Animals dosed with GNE-390 at
2.5 mg/kg alone (3) gave 1 PR and 0 CR. Animals dosed with T-DM1 at
3 mg/kg (5) alone gave 1 (PR) and 1 CR). Animals dosed with T-DM1
at 3 mg/kg (4) alone gave 0 PR and 0 CR. Animals dosed with the
combination of T-DM1 at 3 mg/kg and GNE-390 at 25 mg/kg (5) gave 3
PR and 0 CR. Animals dosed with the combination of T-DM1 at 3 mg/kg
and GNE-390 at 2.5 mg/kg (6) gave 5 PR and 1 CR. Combination of
GNE-390 with T-DM1 significantly increased the number of partial
and complete anti-tumor responses when compared to GNE-390 or T-DM1
alone in the MDA-MB-361.1 breast cancer xenograft model.
[0166] Pharmaceutical Compositions
[0167] Pharmaceutical compositions or formulations of the present
invention include combinations of trastuzumab-MCC-DM1, a
chemotherapeutic agent, and one or more pharmaceutically acceptable
carrier, glidant, diluent, or excipient.
[0168] Trastuzumab-MCC-DM1 and chemotherapeutic agents of the
present invention may exist in unsolvated as well as solvated forms
with pharmaceutically acceptable solvents such as water, ethanol,
and the like, and it is intended that the invention embrace both
solvated and unsolvated forms.
[0169] Trastuzumab-MCC-DM1 and chemotherapeutic agents of the
present invention may also exist in different tautomeric forms, and
all such forms are embraced within the scope of the invention. The
term "tautomer" or "tautomeric form" refers to structural isomers
of different energies which are interconvertible via a low energy
barrier. For example, proton tautomers (also known as prototropic
tautomers) include interconversions via migration of a proton, such
as keto-enol and imine-enamine isomerizations. Valence tautomers
include interconversions by reorganization of some of the bonding
electrons.
[0170] Pharmaceutical compositions encompass both the bulk
composition and individual dosage units comprised of more than one
(e.g., two) pharmaceutically active agents including
trastuzumab-MCC-DM1 and a chemotherapeutic agent selected from the
lists of the additional agents described herein, along with any
pharmaceutically inactive excipients, diluents, carriers, or
glidants. The bulk composition and each individual dosage unit can
contain fixed amounts of the aforesaid pharmaceutically active
agents. The bulk composition is material that has not yet been
formed into individual dosage units. An illustrative dosage unit is
an oral dosage unit such as tablets, pills, capsules, and the like.
Similarly, the herein-described method of treating a patient by
administering a pharmaceutical composition of the present invention
is also intended to encompass the administration of the bulk
composition and individual dosage units.
[0171] Pharmaceutical compositions also embrace
isotopically-labeled compounds of the present invention which are
identical to those recited herein, but for the fact 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 usually found
in nature. All isotopes of any particular atom or element as
specified are contemplated within the scope of the compounds of the
invention, and their uses. Exemplary isotopes that can be
incorporated into compounds of the invention include isotopes of
hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine,
chlorine and iodine, such as .sup.2H, .sup.3H, .sup.11C, .sup.13C,
.sup.14C, .sup.13N, .sup.15N, .sup.15O, .sup.17O, .sup.18O,
.sup.32P, .sup.33P, .sup.35S, .sup.18F, .sup.36Cl, .sup.123I and
.sup.125I. Certain isotopically-labeled compounds of the present
invention (e.g., those labeled with .sup.3H and .sup.14C) are
useful in compound and/or substrate tissue distribution assays.
Tritiated (.sup.3H) and carbon-14 (.sup.14C) isotopes are useful
for their ease of preparation and detectability. Further,
substitution with heavier isotopes such as deuterium (.sup.2H) may
afford certain therapeutic advantages resulting from greater
metabolic stability (e.g., increased in vivo half-life or reduced
dosage requirements) and hence may be preferred in some
circumstances. Positron emitting isotopes such as .sup.15O,
.sup.13N, .sup.11C and .sup.18F are useful for positron emission
tomography (PET) studies to examine substrate receptor occupancy.
Isotopically labeled compounds of the present invention can
generally be prepared by following procedures analogous to those
disclosed in the Schemes and/or in the Examples herein below, by
substituting an isotopically labeled reagent for a non-isotopically
labeled reagent.
[0172] Trastuzumab-MCC-DM1 and chemotherapeutic agents may be
formulated in accordance with standard pharmaceutical practice for
use in a therapeutic combination for therapeutic treatment
(including prophylactic treatment) of hyperproliferative disorders
in mammals including humans. The invention provides a
pharmaceutical composition comprising trastuzumab-MCC-DM1 in
association with one or more pharmaceutically acceptable carrier,
glidant, diluent, or excipient.
[0173] Suitable carriers, diluents and excipients are well known to
those skilled in the art and include materials such as
carbohydrates, waxes, water soluble and/or swellable polymers,
hydrophilic or hydrophobic materials, gelatin, oils, solvents,
water and the like. The particular carrier, diluent or excipient
used will depend upon the means and purpose for which the compound
of the present invention is being applied. Solvents are generally
selected based on solvents recognized by persons skilled in the art
as safe (GRAS) to be administered 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 in water. Suitable
aqueous solvents include water, ethanol, propylene glycol,
polyethylene glycols (e.g., PEG 400, PEG 300), etc. and mixtures
thereof. The formulations may also include one or more buffers,
stabilizing agents, surfactants, wetting agents, lubricating
agents, emulsifiers, suspending agents, preservatives,
antioxidants, opaquing agents, glidants, processing aids,
colorants, sweeteners, perfuming agents, flavoring agents and other
known additives to provide an elegant presentation of the drug
(i.e., a compound of the present invention or pharmaceutical
composition thereof) or aid in the manufacturing of the
pharmaceutical product (i.e., medicament).
[0174] The formulations may be prepared using conventional
dissolution and mixing procedures. For example, the bulk drug
substance (i.e., compound of the present invention or stabilized
form of the compound (e.g., complex with a cyclodextrin derivative
or other known complexation agent) is dissolved in a suitable
solvent in the presence of one or more of the excipients described
above. The compound of the present invention is typically
formulated into pharmaceutical dosage forms to provide an easily
controllable dosage of the drug and to enable patient compliance
with the prescribed regimen.
[0175] The pharmaceutical composition (or formulation) for
application may be packaged in a variety of ways depending upon the
method used for administering the drug. Generally, an article for
distribution includes a container having deposited therein the
pharmaceutical formulation in an appropriate form. Suitable
containers are well known to those skilled in the art and include
materials such as bottles (plastic and glass), sachets, ampoules,
plastic bags, metal cylinders, and the like. The container may also
include a tamper-proof assemblage to prevent indiscreet access to
the contents of the package. In addition, the container has
deposited thereon a label that describes the contents of the
container. The label may also include appropriate warnings.
[0176] Pharmaceutical formulations of the compounds of the present
invention may be prepared 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.), in the form of a
lyophilized formulation, milled powder, or an aqueous solution.
Formulation may be conducted by mixing at ambient temperature at
the appropriate pH, and at the desired degree of purity, with
physiologically acceptable carriers, i.e., carriers that are
non-toxic to recipients at the dosages and concentrations employed.
The pH of the formulation depends mainly on the particular use and
the concentration of compound, but may range from about 3 to about
8.
[0177] The pharmaceutical formulation is preferably sterile. In
particular, formulations to be used for in vivo administration must
be sterile. Such sterilization is readily accomplished by
filtration through sterile filtration membranes.
[0178] The pharmaceutical formulation ordinarily can be stored as a
solid composition, a lyophilized formulation or as an aqueous
solution.
[0179] The pharmaceutical formulations of the invention will be
dosed and administered in a fashion, i.e., amounts, concentrations,
schedules, course, vehicles and route of administration, consistent
with good medical practice. Factors for consideration in this
context include the particular disorder being treated, the
particular mammal being treated, the clinical condition of the
individual patient, the cause of the disorder, the site of delivery
of the agent, the method of administration, the scheduling of
administration, and other factors known to medical practitioners.
The "therapeutically effective amount" of the compound to be
administered will be governed by such considerations, and is the
minimum amount necessary to prevent, ameliorate, or treat the
coagulation factor mediated disorder. Such amount is preferably
below the amount that is toxic to the host or renders the host
significantly more susceptible to bleeding.
[0180] As a general proposition, the initial pharmaceutically
effective amount of trastuzumab-MCC-DM1 administered per dose will
be in the range of about 0.01-100 mg/kg, namely about 0.1 to 20
mg/kg of patient body weight per day, with the typical initial
range of compound used being 0.3 to 15 mg/kg/day.
[0181] Acceptable diluents, carriers, excipients and stabilizers
are nontoxic 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, butyl, ethanol, or benzylalcohol; alkyl parabens
such as methyl or propyl paraben; catechol; resorcinol;
cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less
than about 10 residues) polypeptides; proteins, such as serum
albumin, gelatin, or immunoglobulins; 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; chelating agents such as EDTA; sugars such as sucrose,
mannitol, trehalose or sorbitol; salt-forming counter-ions such as
sodium; metal complexes (e.g., Zn-protein complexes); and/or
non-ionic surfactants such as TWEEN.TM., including Tween 80,
PLURONICS.TM. or polyethylene glycol (PEG), including PEG400. The
active pharmaceutical ingredients may also be entrapped in
microcapsules prepared, for example, by coacervation techniques or
by interfacial polymerization, for example, hydroxymethylcellulose
or gelatin-microcapsules and poly-(methylmethacylate)
microcapsules, respectively, in colloidal drug delivery systems
(for example, liposomes, albumin microspheres, microemulsions,
nano-particles and nanocapsules) or in macroemulsions. Such
techniques are disclosed in Remington's Pharmaceutical Sciences
18th edition, (1995) Mack Publ. Co., Easton, Pa.
[0182] The pharmaceutical formulations include those suitable for
the administration routes detailed herein. The formulations may
conveniently be presented in unit dosage form and may be prepared
by any of the methods well known in the art of pharmacy. Techniques
and formulations generally are found in Remington's Pharmaceutical
Sciences 18.sup.th Ed. (1995) Mack Publishing Co., Easton, Pa. Such
methods include the step of bringing into association the active
ingredient with the carrier which constitutes one or more accessory
ingredients. In general the formulations are prepared by uniformly
and intimately bringing into association the active ingredient with
liquid carriers or finely divided solid carriers or both, and then,
if necessary, shaping the product.
[0183] Formulations of a chemotherapeutic agent suitable for oral
administration may be prepared as discrete units such as pills,
hard or soft e.g., gelatin capsules, cachets, troches, lozenges,
aqueous or oil suspensions, dispersible powders or granules,
emulsions, syrups or elixirs each containing a predetermined amount
of a compound of trastuzumab-MCC-DM1 and/or a chemotherapeutic
agent. Such formulations may be prepared according to any method
known to the art for the manufacture of pharmaceutical compositions
and such compositions may contain one or more agents including
sweetening agents, flavoring agents, coloring agents and preserving
agents, in order to provide a palatable preparation. Compressed
tablets may be prepared by compressing in a suitable machine the
active ingredient in a free-flowing form such as a powder or
granules, optionally mixed with a binder, lubricant, inert diluent,
preservative, surface active or dispersing agent. Molded tablets
may be made by molding in a suitable machine a mixture of the
powdered active ingredient moistened with an inert liquid diluent.
The tablets may optionally be coated or scored and optionally are
formulated so as to provide slow or controlled release of the
active ingredient therefrom.
[0184] Tablet excipients of a pharmaceutical formulation of the
invention may include: Filler (or diluent) to increase the bulk
volume of the powdered drug making up the tablet; Disintegrants to
encourage the tablet to break down into small fragments, ideally
individual drug particles, when it is ingested and promote the
rapid dissolution and absorption of drug; Binder to ensure that
granules and tablets can be formed with the required mechanical
strength and hold a tablet together after it has been compressed,
preventing it from breaking down into its component powders during
packaging, shipping and routine handling; Glidant to improve the
flowability of the powder making up the tablet during production;
Lubricant to ensure that the tableting powder does not adhere to
the equipment used to press the tablet during manufacture. They
improve the flow of the powder mixes through the presses and
minimize friction and breakage as the finished tablets are ejected
from the equipment; Antiadherent with function similar to that of
the glidant, reducing adhesion between the powder making up the
tablet and the machine that is used to punch out the shape of the
tablet during manufacture; Flavor incorporated into tablets to give
them a more pleasant taste or to mask an unpleasant one, and
Colorant to aid identification and patient compliance.
[0185] Tablets containing the active ingredient in admixture with
non-toxic pharmaceutically acceptable excipient which are suitable
for manufacture of tablets are acceptable. These excipients may be,
for example, inert diluents, such as calcium or sodium carbonate,
lactose, calcium or sodium phosphate; granulating and
disintegrating agents, such as maize starch, or alginic acid;
binding agents, such as starch, gelatin or acacia; and lubricating
agents, such as magnesium stearate, stearic acid or talc. Tablets
may be uncoated or may be coated by known techniques including
microencapsulation to delay disintegration and adsorption in the
gastrointestinal tract and thereby provide a sustained action over
a longer period. For example, a time delay material such as
glyceryl monostearate or glyceryl distearate alone or with a wax
may be employed.
[0186] For treatment of the eye or other external tissues, e.g.,
mouth and skin, the formulations are preferably applied as a
topical ointment or cream containing the active ingredient(s) in an
amount of, for example, 0.075 to 20% w/w. When formulated in an
ointment, the active ingredients may be employed with either a
paraffinic or a water-miscible ointment base. Alternatively, the
active ingredients may be formulated in a cream with an
oil-in-water cream base.
[0187] If desired, the aqueous phase of the cream base may include
a polyhydric alcohol, i.e., an alcohol having two or more hydroxyl
groups such as propylene glycol, butane 1,3-diol, mannitol,
sorbitol, glycerol and polyethylene glycol (including PEG 400) and
mixtures thereof. The topical formulations may desirably include a
compound which enhances absorption or penetration of the active
ingredient through the skin or other affected areas. Examples of
such dermal penetration enhancers include dimethyl sulfoxide and
related analogs.
[0188] The oily phase of the emulsions of this invention may be
constituted from known ingredients in a known manner, including a
mixture of at least one emulsifier with a fat or an oil, or with
both a fat and an oil. Preferably, a hydrophilic emulsifier is
included together with a lipophilic emulsifier which acts as a
stabilizer. Together, the emulsifier(s) with or without
stabilizer(s) make up an emulsifying wax, and the wax together with
the oil and fat comprise an emulsifying ointment base which forms
the oily dispersed phase of cream formulations. Emulsifiers and
emulsion stabilizers suitable for use in the formulation of the
invention include Tween.RTM. 60, Span.RTM. 80, cetostearyl alcohol,
benzyl alcohol, myristyl alcohol, glyceryl mono-stearate and sodium
lauryl sulfate.
[0189] Aqueous suspensions of the pharmaceutical formulations of
the invention contain the active materials 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 acacia, and dispersing
or wetting agents such as a naturally occurring phosphatide (e.g.,
lecithin), a condensation product of an alkylene oxide with a fatty
acid (e.g., polyoxyethylene stearate), a condensation product of
ethylene oxide with a long chain aliphatic alcohol (e.g.,
heptadecaethyleneoxycetanol), a condensation product of ethylene
oxide with a partial ester derived from a fatty acid and a hexitol
anhydride (e.g., polyoxyethylene sorbitan monooleate). The aqueous
suspension may also contain one or more preservatives such as ethyl
or n-propyl p-hydroxybenzoate, one or more coloring agents, one or
more flavoring agents and one or more sweetening agents, such as
sucrose or saccharin.
[0190] Pharmaceutical compositions may be in the form of a sterile
injectable preparation, such as a sterile injectable aqueous or
oleaginous suspension. This suspension may be formulated according
to the known art using those suitable dispersing or wetting agents
and suspending agents which have been mentioned above. The sterile
injectable preparation may be a solution or a 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 and solvents that may be employed are
water, Ringer's solution and isotonic sodium chloride solution. In
addition, sterile fixed oils may conventionally be employed as a
solvent or suspending medium. For this purpose any bland fixed oil
may be employed including synthetic mono- or diglycerides. In
addition, fatty acids such as oleic acid may likewise be used in
the preparation of injectables.
[0191] The amount of active ingredient that may be combined with
the carrier material to produce a single dosage form will vary
depending upon the host treated and the particular mode of
administration. For example, a time-release formulation intended
for oral administration to humans may contain approximately 1 to
1000 mg of active material compounded with an appropriate and
convenient amount of carrier material which may vary from about 5
to about 95% of the total compositions (weight:weight). The
pharmaceutical composition can be prepared to provide easily
measurable amounts for administration. For example, an aqueous
solution intended for intravenous infusion may contain from about 3
to 500 .mu.g of the active ingredient per milliliter of solution in
order that infusion of a suitable volume at a rate of about 30
mL/hr can occur.
[0192] Formulations suitable for parenteral administration include
aqueous and non-aqueous sterile injection solutions which may
contain anti-oxidants, buffers, bacteriostats and solutes which
render the formulation isotonic with the blood of the intended
recipient; and aqueous and non-aqueous sterile suspensions which
may include suspending agents and thickening agents.
[0193] Formulations suitable for topical administration to the eye
also include eye drops wherein the active ingredient is dissolved
or suspended in a suitable carrier, especially an aqueous solvent
for the active ingredient. The active ingredient is preferably
present in such formulations in a concentration of about 0.5 to 20%
w/w, for example about 0.5 to 10% w/w, for example about 1.5%
w/w.
[0194] Formulations suitable for topical administration in the
mouth include lozenges comprising the active ingredient in a
flavored basis, usually sucrose and acacia or tragacanth; pastilles
comprising the active ingredient in an inert basis such as gelatin
and glycerin, or sucrose and acacia; and mouthwashes comprising the
active ingredient in a suitable liquid carrier.
[0195] Formulations for rectal administration may be presented as a
suppository with a suitable base comprising for example cocoa
butter or a salicylate.
[0196] Formulations suitable for intrapulmonary or nasal
administration have a particle size for example in the range of 0.1
to 500 microns (including particle sizes in a range between 0.1 and
500 microns in increments microns such as 0.5, 1, 30 microns, 35
microns, etc.), which is administered by rapid inhalation through
the nasal passage or by inhalation through the mouth so as to reach
the alveolar sacs. Suitable formulations include aqueous or oily
solutions of the active ingredient. Formulations suitable for
aerosol or dry powder administration may be prepared according to
conventional methods and may be delivered with other therapeutic
agents such as compounds heretofore used in the treatment or
prophylaxis disorders as described below.
[0197] Formulations suitable for vaginal administration may be
presented as pessaries, tampons, creams, gels, pastes, foams or
spray formulations containing in addition to the active ingredient
such carriers as are known in the art to be appropriate.
[0198] The formulations may be packaged in unit-dose or multi-dose
containers, for example sealed ampoules and vials, and may be
stored in a freeze-dried (lyophilized) condition requiring only the
addition of the sterile liquid carrier, for example water, for
injection immediately prior to use. Extemporaneous injection
solutions and suspensions are prepared from sterile powders,
granules and tablets of the kind previously described. Preferred
unit dosage formulations are those containing a daily dose or unit
daily sub-dose, as herein above recited, or an appropriate fraction
thereof, of the active ingredient.
[0199] The invention further provides veterinary compositions
comprising at least one active ingredient as above defined together
with a veterinary carrier therefore. Veterinary carriers are
materials useful for the purpose of administering the composition
and may be solid, liquid or gaseous materials which are otherwise
inert or acceptable in the veterinary art and are compatible with
the active ingredient. These veterinary compositions may be
administered parenterally, orally or by any other desired
route.
[0200] Combination Therapy
[0201] Trastuzumab-MCC-DM1 may be employed in combination with
other chemotherapeutic agents for the treatment of a
hyperproliferative disease or disorder, including tumors, cancers,
and neoplastic tissue, along with pre-malignant and non-neoplastic
or non-malignant hyperproliferative disorders. In certain
embodiments, trastuzumab-MCC-DM1 is combined in a pharmaceutical
combination formulation, or dosing regimen as combination therapy,
with a second compound that has anti-hyperproliferative properties
or that is useful for treating the hyperproliferative disorder. The
second compound of the pharmaceutical combination formulation or
dosing regimen preferably has complementary activities to
trastuzumab-MCC-DM1, and such that they do not adversely affect
each other. Such compounds are suitably present in combination in
amounts that are effective for the purpose intended. In one
embodiment, a composition of this invention comprises
trastuzumab-MCC-DM1 in combination with a chemotherapeutic agent
such as described herein. Examples 4 and 5 are clinical protocols
for T-DM1+pertuzumab, and T-DM1+GDC-0941, respectively.
[0202] Therapeutic combinations of the invention include a
formulation, dosing regimen, or other course of treatment
comprising the administration of 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.
[0203] The combination therapy may be administered as a
simultaneous or sequential regimen. When administered sequentially,
the combination may be administered in two or more administrations.
The combined administration includes coadministration, using
separate formulations or a single pharmaceutical formulation, and
consecutive administration in either order, wherein preferably
there is a time period while both (or all) active agents
simultaneously exert their biological activities.
[0204] Suitable dosages for any of the above coadministered agents
are those presently used and may be lowered due to the combined
action (synergy) of the newly identified agent and other
chemotherapeutic agents or treatments.
[0205] In a particular embodiment of anti-cancer therapy,
trastuzumab-MCC-DM1 may be combined with a chemotherapeutic agent,
including hormonal or antibody agents such as those described
herein, as well as combined with surgical therapy and radiotherapy.
The amounts of trastuzumab-MCC-DM1 and the other pharmaceutically
active chemotherapeutic agent(s) and the relative timings of
administration will be selected in order to achieve the desired
combined therapeutic effect.
[0206] Administration of Pharmaceutical Compositions
[0207] The compounds of the invention may be administered by any
route appropriate to the condition to be treated. Suitable routes
include oral, parenteral (including subcutaneous, intramuscular,
intravenous, intraarterial, inhalation, intradermal, intrathecal,
epidural, and infusion techniques), transdermal, rectal, nasal,
topical (including buccal and sublingual), vaginal,
intraperitoneal, intrapulmonary and intranasal. Topical
administration can also involve the use of transdermal
administration such as transdermal patches or iontophoresis
devices. Formulation of drugs is discussed in Remington's
Pharmaceutical Sciences, 18.sup.th Ed., (1995) Mack Publishing Co.,
Easton, Pa. Other examples of drug formulations can be found in
Liberman, H. A. and Lachman, L., Eds., Pharmaceutical Dosage Forms,
Marcel Decker, Vol 3, 2.sup.nd Ed., New York, N.Y. For local
immunosuppressive treatment, the compounds may be administered by
intralesional administration, including perfusing or otherwise
contacting the graft with the inhibitor before transplantation. It
will be appreciated that the preferred route may vary with for
example the condition of the recipient. Where the compound is
administered orally, it may be formulated as a pill, capsule,
tablet, etc. with a pharmaceutically acceptable carrier, glidant,
or excipient. Where the compound is administered parenterally, it
may be formulated with a pharmaceutically acceptable parenteral
vehicle or diluent, and in a unit dosage injectable form, as
detailed below.
[0208] A dose of trastuzumab-MCC-DM1 to treat human patients may
range from about 100 mg to about 500 mg. The dose of
trastuzumab-MCC-DM1 may be administered once every six weeks, once
every three weeks, weekly, or more frequently, depending on the
pharmacokinetic (PK) and pharmacodynamic (PD) properties, including
absorption, distribution, metabolism, and excretion. A 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 may be administered once every six
weeks, once every three weeks, weekly, or more frequently, such as
once or twice per day. In addition, toxicity factors may influence
the dosage and administration regimen. When administered orally,
the pill, capsule, or tablet may be ingested daily or less
frequently for a specified period of time. The regimen may be
repeated for a number of cycles of therapy.
[0209] Methods of Treatment
[0210] Therapeutic combinations of: (1) trastuzumab-MCC-DM1 and (2)
a chemotherapeutic agent are useful for treating diseases,
conditions and/or disorders including, but not limited to, those
characterized by activation of the HER2 pathway. Accordingly,
another aspect of this invention includes methods of treating
diseases or conditions that can be treated by targeting HER2 or the
VEGFR receptor 1. Therapeutic combinations of: (1)
trastuzumab-MCC-DM1 and (2) a chemotherapeutic agent may be
employed for the treatment of a hyperproliferative disease or
disorder, including tumors, cancers, and neoplastic tissue, along
with pre-malignant and non-neoplastic or non-malignant
hyperproliferative disorders.
[0211] Cancers which can be treated according to the methods of
this invention include, but are not limited to, breast, ovary,
cervix, prostate, testis, genitourinary tract, esophagus, larynx,
glioblastoma, neuroblastoma, stomach, skin, keratoacanthoma, lung,
epidermoid carcinoma, large cell carcinoma, non-small cell lung
carcinoma (NSCLC), small cell carcinoma, lung adenocarcinoma, bone,
colon, adenoma, pancreas, adenocarcinoma, thyroid, follicular
carcinoma, undifferentiated carcinoma, papillary carcinoma,
seminoma, melanoma, sarcoma, bladder carcinoma, liver carcinoma and
biliary passages, kidney carcinoma, myeloid disorders, lymphoid
disorders, hairy cells, buccal cavity and pharynx (oral), lip,
tongue, mouth, pharynx, small intestine, colon-rectum, large
intestine, rectum, brain and central nervous system, Hodgkin's and
leukemia.
[0212] Another aspect of this invention provides a pharmaceutical
composition or therapeutic combination for use in the treatment of
the diseases or conditions described herein in a mammal, for
example, a human, suffering from such disease or condition. Also
provided is the use of a pharmaceutical composition in the
preparation of a medicament for the treatment of the diseases and
conditions described herein in a warm-blooded animal, such as a
mammal, for example a human, suffering from such disorder.
[0213] Articles of Manufacture
[0214] In another embodiment of the invention, an article of
manufacture, or "kit", containing trastuzumab-MCC-DM1 useful for
the treatment of the diseases and disorders described above is
provided. In one embodiment, the kit comprises a container
comprising 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 customarily
included in commercial packages of therapeutic products, that
contain information about the indications, usage, dosage,
administration, contraindications and/or warnings concerning the
use of such therapeutic products. Suitable containers include, for
example, bottles, vials, syringes, blister pack, etc. The container
may be formed from a variety of materials such as glass or plastic.
The container may hold trastuzumab-MCC-DM1 or a formulation thereof
which is effective for treating the condition and may have a
sterile access port (for example, the container may be an
intravenous solution bag or a vial having a stopper pierceable by a
hypodermic injection needle). At least one active agent in the
composition is trastuzumab-MCC-DM1. The label or package insert
indicates that the composition is used for treating the condition
of choice, such as cancer. In one embodiment, the label or package
inserts indicates that the composition comprising
trastuzumab-MCC-DM1 can be used to treat a disorder resulting from
abnormal cell growth. The label or package insert may also indicate
that the composition can be used to treat other disorders.
Alternatively, or additionally, the article of manufacture may
further comprise a second container comprising a pharmaceutically
acceptable buffer, such as bacteriostatic water for injection
(BWFI), phosphate-buffered saline, Ringer's solution and dextrose
solution. It may further include other materials desirable from a
commercial and user standpoint, including other buffers, diluents,
filters, needles, and syringes.
[0215] The kit may further comprise directions for the
administration of trastuzumab-MCC-DM1 and, if present, the second
pharmaceutical formulation. For example, if the kit comprises a
first composition comprising trastuzumab-MCC-DM1 and a second
pharmaceutical formulation, the kit may further comprise directions
for the simultaneous, sequential or separate administration of the
first and second pharmaceutical compositions to a patient in need
thereof.
[0216] In another embodiment, the kits are suitable for the
delivery of solid oral forms of trastuzumab-MCC-DM1, such as
tablets or capsules. Such a kit preferably includes a number of
unit dosages. Such kits can include a card having the dosages
oriented in the order of their 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 packaging pharmaceutical
unit dosage forms. If desired, a memory aid can be provided, for
example in the form of numbers, letters, or other markings or with
a calendar insert, designating the days in the treatment schedule
in which the dosages can be administered.
[0217] According to one embodiment, a kit may comprise (a) a first
container with trastuzumab-MCC-DM1 contained therein; and
optionally (b) a second container with a second pharmaceutical
formulation contained therein, wherein the second pharmaceutical
formulation comprises a second compound with
anti-hyperproliferative activity. Alternatively, or additionally,
the kit may further comprise a third container comprising a
pharmaceutically-acceptable buffer, such as bacteriostatic water
for injection (BWFI), phosphate-buffered saline, Ringer's solution
and dextrose solution. It may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, and syringes.
[0218] Where the kit comprises a composition of trastuzumab-MCC-DM1
and a second therapeutic agent, i.e. the chemotherapeutic agent,
the kit may comprise a container for containing the separate
compositions such as a divided bottle or a divided foil packet,
however, the separate compositions may also be contained within a
single, undivided container. Typically, the kit comprises
directions for the administration of the separate components. The
kit form is particularly advantageous when the separate components
are preferably administered in different dosage forms (e.g., oral
and parenteral), are administered at different dosage intervals, or
when titration of the individual components of the combination is
desired by the prescribing physician.
EXAMPLES
[0219] In order to illustrate the invention, the following examples
are included. However, it is to be understood that these examples
do not limit the invention and are only meant to suggest a method
of practicing the invention.
Example 1
Preparation of Trastuzumab-MCC-DM1
[0220] Trastuzumab was purified from HERCEPTIN.RTM. by
buffer-exchange at 20 mg/mL in 50 mM potassium phosphate/50 mM
sodium chloride/2 mM EDTA, pH 6.5 and treated with 7.5 to 10 molar
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 2005/0169933; US 2005/0276812). After stirring for 2 to 4
hours under argon at ambient temperature, the reaction mixture was
filtered through a Sephadex G25 column equilibrated with 50 mM
potassium phosphate/50 mM sodium chloride/2 mM EDTA, pH 6.5.
Alternatively, the reaction mixture was gel filtered with 30 mM
citrate and 150 mM sodium chloride at pH 6. Antibody containing
fractions were pooled and assayed. Recovery of trastuzumab-SMCC was
88%.
[0221] The drug-linker intermediate, trastuzumab-MCC from above,
was diluted with 50 mM potassium phosphate/50 mM sodium chloride/2
mM EDTA, pH 6.5, to a final concentration of 10 mg/ml, and reacted
with a 10 mM solution of DM1 (1.7 equivalents assuming 5
SMCC/trastuzumab, 7.37 mg/ml) in dimethylacetamide. DM1 may be
prepared from ansamitocin fermentation products (U.S. Pat. No.
6,790,954; U.S. Pat. No. 7,432,088) and derivatized for conjugation
(U.S. Pat. No. 6,333,410; RE 39151). The reaction was stirred at
ambient temperature under argon for 4 to about 16 hours. The
conjugation reaction mixture was filtered through a Sephadex G25
gel filtration column (1.5.times.4.9 cm) with 1.times.PBS at pH
6.5. Alternatively, the reaction mixture was gel filtered with 10
mM succinate and 150 mM sodium chloride at pH 5. The
DM1/trastuzumab ratio (p) was 3.1, as measured by the absorbance at
252 nm and at 280 nm. The drug to antibody ratio (p) may also be
measured by mass spectrometry. Conjugation may also be monitored by
SDS polyacrylamide gel electrophoresis. Aggregation may be assessed
by laser light scattering analysis.
[0222] Alternatively, trastuzumab-MCC-DM1 may be prepared by
forming an MCC-DM1 linker-drug reagent and then reacting with
trastuzumab.
[0223] Typically a conjugation reaction of trastuzumab-MCC with DM1
results in a heterogeneous mixture comprising antibodies different
numbers of attached, conjugated DM1 drugs, i.e. drug loading where
p is a distribution from 1 to about 8. An additional dimension of
heterogeneity exists with different attachment sites of SMCC to
trastuzumab where many different nucleophiles on trastuzumab, e.g.
terminal lysine amino groups, can react with SMCC. Thus,
trastuzumab-MCC-DM1 includes isolated, purified species molecules
as well as mixtures of average drug loading from 1 to 8 and where
MCC-DM1 is attached through any site of the trastuzumab
antibody.
[0224] The average number of DM1 drug moieties per trastuzumab
antibody in preparations of trastuzumab-MCC-DM1 from conjugation
reactions may be characterized by conventional means such as mass
spectroscopy, ELISA assay, electrophoresis, and HPLC. The
quantitative distribution of trastuzumab-MCC-DM1 in terms of p may
also be determined. By ELISA, the averaged value of p in a
particular preparation of ADC may be determined (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 discernible by the antibody-antigen binding
and detection limitation of ELISA. Also, ELISA assay for detection
of antibody-drug conjugates does not determine where the drug
moieties are attached to the antibody, such as the heavy chain or
light chain fragments, or the particular amino acid residues. In
some instances, separation, purification, and characterization of
homogeneous trastuzumab-MCC-DM1 where p is a certain value from
trastuzumab-MCC-DM1 with other drug loadings may be achieved by
means such as reverse phase HPLC or electrophoresis.
Example 2
In Vitro Cell Proliferation Assay
[0225] Efficacy of the combinations of the invention was measured
by a cell proliferation assay employing the following protocol
(Promega Corp. Technical Bulletin TB288; Mendoza et al (2002)
Cancer Res. 62:5485-5488). The Cell-Titer Glo assay reagents and
protocol are commercially available (Promega). The assay assesses
the ability of compounds to get into cells and affect cell
proliferation. The assay principle is the determination of the
number of viable cells present by quantitating the cellular ATP.
Cell-Titer Glo is the reagent used for this quantitation. It is a
homogenous assay where addition of the Cell-Titer Glo results in
cell lysis and generation of a luminescent signal through the
luciferase reaction. The luminescent signal is proportional to the
amount of ATP present.
[0226] DMSO and Media Plates: 96-well conical bottom polypropylene
plates from Nunc (cat.#249946)
[0227] Cell Plates: 384-well black, clear bottom (microclear), TC
plates with lid from Falcon (353962)
[0228] Cell Culture Medium: RPMI or DMEM high glucose; Ham's F-12
(50:50), 10% Fetal Bovine Serum, 2 mM L-Glutamine
[0229] Cell Titer-Glo: Promega (cat.# G7572)
[0230] Procedure:
[0231] Day 1--Seed Cell Plates, Harvest cells, Seed cells at
1000-2000 cells per 54 .mu.l per well into 384 well Cell Plates for
3 days assay. Incubate overnight (approx. 16 hr) at 37 C, 5%
CO.sub.2.
[0232] Day 2--Add Drug to Cells, Compound Dilution, DMSO Plates
(serial 1:2 for 9 points). Add 20 ul compounds (10 mM stock
solution for small molecule drugs) in the 2nd column of 96 well
plate. Perform serial 1:2 across the plate (10 .mu.l+10 .mu.l 1100%
DMSO) for a total of 9 points using Precision Media Plates (1:50
dilution). Add 147 .mu.l of Media into all wells of separate
96-well media plate. Transfer 3 .mu.l of DMSO+compound from each
well in the DMSO Plate to each corresponding well on Media Plate
using Rapidplate. For 2 drug combo studies, transfer one drug1.5
.mu.l of DMSO+compound from each well in the DMSO Plate to each
corresponding well on Media Plate using Rapidplate. Then, transfer
another drug 1.5 ul to the medium plate.
[0233] Drug Addition to Cells, Cell Plate (1:10 dilution), Add 6
.mu.l of media+compound directly to cells (54 .mu.l of media on the
cells already). Incubate 3 days at 37 C, 5% CO2 in an incubator
that will not be opened often.
[0234] Day 5--Develop Plates, Thaw Cell Titer Glo Buffer at room
temperature. Remove Cell Plates from 37.degree. C. and equilibrate
to room temperature. for about 30 minutes. Add Cell Titer Glo
Buffer to Cell Titer Glo Substrate (bottle to bottle). Add 30 .mu.A
Cell Titer Glo Reagent to each well of cells. Place on plate shaker
for about 30 minutes. Read luminescence on PerkinElmer Envision
(0.1 second per well) or Analyst HT Plate Reader (half second per
well).
[0235] Cell viability assays and combination assays: Cells were
seeded at 1000-2000 cells/well in 384-well plates for 16 h. On day
two, nine serial 1:2 compound dilutions were made in DMSO in a 96
well plate. The compounds were further diluted into growth media
using a Rapidplate robot (Zymark Corp., Hopkinton, Mass.). The
diluted compounds were then added to quadruplicate wells in
384-well cell plates and incubated at 37 C and 5% CO2. After 4
days, relative numbers of viable cells were measured by
luminescence using Cell-Titer Glo (Promega) according to the
manufacturer's instructions and read on an Envision or a Wallac
Multilabel Reader (PerkinElmer, Foster City). EC50 values were
calculated using Kaleidagraph 4.0 (Synergy Software) or Prism 4.0
software (GraphPad, San Diego). Drugs in combination assays were
dosed starting at 8.times.EC50 concentrations. In cases where the
EC50 of the drug was >2.5 .mu.M, the highest concentration used
was 10 .mu.M. Trastuzumab-MCC-DM1 and chemotherapeutic agents were
added simultaneously or separated by 4 hours (one before the other)
in all assays.
[0236] An additional exemplary in vitro cell proliferation assay
includes the following steps:
[0237] 1. An aliquot of 100 .mu.l of cell culture containing about
10.sup.4 cells (see FIG. 1 for cell lines and tumor type) in medium
was deposited in each well of a 384-well, opaque-walled plate.
[0238] 2. Control wells were prepared containing medium and without
cells.
[0239] 3. The compound was added to the experimental wells and
incubated for 3-5 days.
[0240] 4. The plates were equilibrated to room temperature for
approximately 30 minutes.
[0241] 5. A volume of CellTiter-Glo Reagent equal to the volume of
cell culture medium present in each well was added.
[0242] 6. The contents were mixed for 2 minutes on an orbital
shaker to induce cell lysis.
[0243] 7. The plate was incubated at room temperature for 10
minutes to stabilize the luminescence signal.
[0244] 8. Luminescence was recorded and reported in graphs as
RLU=relative luminescence units.
[0245] Alternatively, cells were seeded at optimal density in a 96
well plate and incubated for 4 days in the presence of test
compound. Alamar Blue.TM. was subsequently added to the assay
medium, and cells were incubated for 6 h before reading at 544 nm
excitation, 590 nm emission. EC.sub.50 values were calculated using
a sigmoidal dose response curve fit.
Example 3
In Vivo Tumor Xenograft
[0246] Animals suitable for transgenic experiments can be obtained
from standard commercial sources. Groups of female CB-17 SCID beige
mice (Charles River Laboratory) were implanted with 3 million KPL-4
(Her2 overexpressing) breast cancer cells with matrigel in the
mammary fat pad. Groups of female athymic nude mice (Charles River
Laboratory or Harlan) were implanted with 2.times.2 mm3 fragments
of MMTV-Her2 Fo5 transgenic breast tumors in the mammary fat pad.
Mouse xenografts were dosed at day 0 with drug, drug combination,
or vehicle according to the schedule specified for each tumor
model. 5-FU, gemcitabine, carboplatin and B20-4.1 were administered
intraperitoneal, pertuzumab was given either intravenously or
intraperitoneal as indicated, trastuzumab-MCC-DM1 and docetaxel
were administered intravenously, lapatinib, GDC-0941 and ABT-869
were given periorally by gavage. Tumor sizes were recorded twice
weekly over the course of the study. Mouse body weights were also
recorded twice weekly, and the mice were observed regularly. Tumor
volume was measured in two dimensions (length and width) using
Ultra Cal IV calipers (Model 54-10-111; Fred V. Fowler Co., Inc.;
Newton, Mass.) and analyzed using Excel v.11.2 (Microsoft
Corporation; Redmond, Wash.). Tumor inhibition graphs were plotted
using KaleidaGraph, Version 3.6 (Synergy Software; Reading, Pa.).
The tumor volume was calculated with formula: Tumor size
(mm.sup.3)=(longer measurement.times.shorter
measurement.sup.2).times.0.5
[0247] Animal body weights were measured using an Adventurera Pro
AV812 scale (Ohaus Corporation; Pine Brook, N.J.). Graphs were
generated using KaleidaGraph Version 3.6. Percent weight change was
calculated using formula: Group percent weight change=(1-(initial
weight/new weight)).times.100.
[0248] Mice whose tumor volume exceeded 2000 mm.sup.3 or whose body
weight loss was more than 20% of their starting weight were
promptly euthanized according to regulatory guidance.
[0249] The percent tumor growth delay (% TGD) at the end of study
(EOS) was calculated using formula: % TGD=100.times.(Median time to
endpoint for the treatment group-median time to endpoint for the
control group)/Median time to endpoint for the control group.
[0250] Tumor incidence (TI) was determined based on the number of
measurable tumors remaining in each group at the end of the study.
A partial response (PR) was defined as more than 50% but less than
100% reduction in tumor volume, compared with the starting tumor
volume, observed for three consecutive measurements. A complete
response (CR) was defined as a 100% reduction in tumor volume,
compared with the initial tumor volume, observed for three
consecutive measurements. Data were analyzed and p-values were
determined using the Dunnett's t-test with JMP statistical
software, version 5.1.2 (SAS Institute; Cary, N.C.). Individual
tumor volumes at end of study and mean tumor volume.+-.SEM values
were calculated using JMP statistical software, version 5.1.2. Body
weight data were graphed based on the mean percentage of change
from initial body weights.+-.SEM.
Example 4
Clinical Study of Trastuzumab-MCC-DM1 (T-DM1) in Combination with
Pertuzumab
[0251] A Phase 1 b/II, open-label study of the safety,
tolerability, and efficacy of trastuzumab-MCC-DM1 (T-DM1) in
combination with pertuzumab administered intravenously to patients
with HER2-positive locally advanced or metastatic breast cancer who
have progressed while receiving prior therapy was designed to
characterize the safety and tolerability of the combination. The
combination is administered every 3 weeks to patients with
HER2-positive locally advanced or metastatic breast cancer who have
previously received trastuzumab in any line of therapy, have
received chemotherapy combined with HER2-targeted therapy for
advanced disease, or have progressed while receiving their most
recent therapy. Another objective is to evaluate the
pharmacokinetics of T-DM1 when the combination of T-DM1 and
pertuzumab is administered on this schedule. Another objective is
to make a preliminary assessment of the efficacy of the combination
of T-DM1 and pertuzumab administered on this schedule, as measured
by objective response rate based on investigator assessment using
modified Response Evaluation Criteria in Solid Tumors (RECIST),
Version 1.0. Secondary objectives for this study are as follows:
(1) To estimate the progression-free survival (PFS) of patients who
receive the combination of T-DM1 and pertuzumab administered on
this schedule; (2) To assess the duration of response of the
combination of T-DM1 and pertuzumab administered on this schedule;
and (3) To assess the development of anti-therapeutic antibodies to
T-DM1.
[0252] T-DM1 will be administered by intravenous (IV) infusion in
combination with pertuzumab, also administered by intravenous (IV)
infusion, in patients with HER2-positive locally advanced or
metastatic breast cancer that have previously received trastuzumab
and have progressed following or while receiving their last
therapy. Patients will receive a combination of T-DM 1 and
pertuzumab, in repeated cycles, at a minimum interval of 3
weeks.
[0253] Patients at a given dose level will be observed for DLT
(Dose-Limiting Toxicity) during the DLT Observation Period (defined
as 21 days from the time of the first dose of T-DM1) after
receiving their first doses of study drugs prior to treatment of
any patient at a higher dose level. If no DLTs are observed in
these patients during the DLT Observation Period, dose escalation
to the next dose level may proceed.
[0254] A DLT is defined as any of the following treatment-related
toxicities occurring within the DLT Observation Period: (1)
Grade.gtoreq.3 non-hematologic adverse event that is not due to
disease progression or another clearly identifiable cause, except
for alopecia of any grade; (2) Grade 3 diarrhea that responds to
standard of care therapy: (3) Grade 3 nausea or vomiting in the
absence of premedication that responds to standard of care therapy;
(4) Grade.gtoreq.3 elevation of serum bilirubin, hepatic
transaminases (ALT or AST), or alkaline phosphatase (ALP) lasting
72 hours, with the exception of patients with Grade 2 hepatic
transaminase or ALP levels at baseline, (.ltoreq.5 the upper limit
of normal [ULN]) as a result of liver or bone metastases. A hepatic
transaminase or ALP level.gtoreq.10 ULN will be considered a DLT;
(5) Grade.gtoreq.4 thrombocytopenia lasting 24 hours; (6)
Grade.gtoreq.4 neutropenia (absolute neutrophil
count<500/cells/mm3) lasting 4 days or accompanied by a fever
(oral or tympanic temperature 100.4.degree. F. or 38.degree. C.);
(7) Any subjectively intolerable toxicity felt by the investigator
to be related to either test compound; (8) Any treatment-related
toxicity prohibiting the start of the second cycle of
treatment.
[0255] Once a decision has been made to proceed to the next highest
dose level, an intra-patient dose escalation will also be allowed;
patients enrolled in the study will initially receive a reduced
dose of T-DM1 (3.0 mg/kg) along with full-dose pertuzumab. These
patients will be allowed to escalate to full doses of both drugs
for subsequent cycles once their cohort has cleared the DLT
Observation Period. However, the safety of the 3.6 mg/kg dose level
will be based on the assessment of DLT. Patients (including those
who are enrolled in the study during the dose-escalation phase of
the study) will be considered evaluable for efficacy if they remain
on study through the first follow-up tumor assessment.
Echocardiogram (ECHO) or multigated acquisition (MUGA) scans should
be performed at the end of Cycle 1, and then every three cycles
throughout the treatment period.
[0256] T-DM1 Formulation
[0257] T-DM1 may be provided as a single-use lyophilized
formulation in a 20-mL Type I USP/European Pharmacopeia glass vial
fitted with a 20-mm fluoro resin-laminated stopper and aluminum
seal with a dark gray flip-off plastic cap. Following
reconstitution with 8.0 mL Sterile Water for Injection (SWFI), the
resulting product contains 20 mg/mL T-DM1 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 T-DM1 to allow
delivery of 160 mg T-DM1. The indicated volume of T-DM 1 solution
is removed from the vial(s) and added to the IV bag. Reconstituted
T-DM1 is diluted into PVC or latex-free PVC-free polyolefin bags
(PO) containing 0.45% or 0.9% Sodium Chloride Injection (minimum
volume of 250 mL). The use of PVC or PO bags containing 0.45%
Sodium Chloride is preferred. In cases wherein PVC or PO bags
containing 0.9% Sodium Chloride are used, the use of 0.22 .mu.m
in-line filters is recommended. The bag is gently inverted to mix
the solution. The solution of T-DM1 for infusion diluted in
polyvinyl chloride (PVC) or latex-free PVC-free polyolefin (PO)
bags containing 0.9% or 0.45% Sodium Chloride Injection, USP, may
be stored at 2.degree. C.-8.degree. C. (36.degree. F.-46.degree.
F.) for a short period of time.
[0258] Pertuzumab Formulation
[0259] Pertuzumab is provided as a single-use formulation
containing 30 mg/mL pertuzumab formulated in 20 mM L-histidine (pH
6.0), 120 mM sucrose, and 0.02% polysorbate 20. Each 20-cc vial
contains approximately 420 mg of pertuzumab (14.0 mL/vial). The
indicated volume of pertuzumab solution is withdrawn from vials and
added to a 250-cc IV bag of 0.9% sodium chloride solution for
injection. The bag is gently inverted the bag to mix the solution,
and visually inspected for particulates and discoloration prior to
administration. The solution of pertuzumab for infusion diluted in
polyethylene or non-PVC polyolefin bags containing 0.9% sodium
chloride solution may be stored at 2.degree. C.-8.degree. C.
(36.degree. F.-46.degree. F.) for a short period of time.
[0260] Safety Outcome Measures
[0261] The safety and tolerability of T-DM1 and pertuzumab will be
assessed using the following primary safety outcome measures: (1)
Incidence, nature, and severity of adverse events; (2) Adverse
events or changes in physical findings and clinical laboratory
results during and following study drug administration that result
in dose modification, dose delay, or discontinuation of T-DM1
and/or pertuzumab; and (3) Change in cardiac function (i.e., left
ventricular ejection fraction [LVEF], segmental wall
abnormalities), including ECHO or MUGA scans
[0262] Pharmacokinetic and Pharmacodynamic Outcome Measures
[0263] The following pharmacokinetic parameters of T-DM1 and
pertuzumab will be determined in all patients who receive study
treatment using either non-compartmental and/or population methods,
when appropriate, as data allow: (1) Serum concentrations of T-DM1
(conjugate), total trastuzumab (free and conjugated to DM1); (2)
Plasma concentrations of free DM1; (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)
Anti-therapeutic antibodies to T-DM1
[0264] Efficacy Outcome Measures
[0265] The objective response rate using modified RECIST, v1.0 will
be assessed as the efficacy outcome measure. The secondary efficacy
outcome measures of this study are the following: (1) PFS, defined
as the time from the study treatment initiation to the first
occurrence of disease progression or death on study (within 30 days
of the last dose of study treatment) from any cause, as determined
by investigator review of tumor assessments using modified RECIST,
v1.0; and (2) Duration of response, defined as the first occurrence
of a documented objective response until the time of disease
progression, as determined by investigator review of tumor
assessments using modified RECIST (v1.0), or death on study (within
30 days of the last dose of study treatment) from any cause.
[0266] Study Treatment
[0267] T-DM1 will be administered no more frequently than every 3
weeks at a dose of 2.4, 3.0, or 3.6 mg/kg IV. Any patient may be
de-escalated to a T-DM1 dose as low as 2.4 mg/kg. Depending on the
toxicity encountered in the cohort of patients that begin therapy
at 3.0 mg/kg, and if 3.0 mg/kg T-DM1 is confirmed to be tolerable,
patients may be escalated to a dose of 3.6 mg/kg IV every 3 weeks
in subsequent cycles. Pertuzumab will be administered at a loading
dose of 840 mg IV on Day 1, Cycle 1, followed by 420 mg IV every 3
weeks in subsequent cycles.
[0268] Statistical Methods
[0269] The primary efficacy endpoint of this study is
investigator-assessed objective response, defined as a complete or
partial response determined on two consecutive occasions.gtoreq.4
weeks apart. An estimate of the objective response rate will be
computed as well as the corresponding 95% confidence interval. For
objective response, patients without a valid post-baseline tumor
assessment will be counted as non-responders. For duration of
response and PFS, data from patients who are lost to follow-up will
be treated as censored on the last date the patient was known to be
progression-free. Data for patients without post-treatment tumor
assessment or death will be censored at the date of the treatment
initiation plus 1 day.
Example 5
Clinical Study of Trastuzumab-MCC-DM1 (T-DM1) in Combination with
GDC-0941
[0270] A phase Ib, open-label study of the combination of T-DM1
administered intravenously and GDC-0941 administered orally to
patients with HER2-positive metastatic breast cancer who have
progressed on previous trastuzumab-based therapy was designed to
characterize the safety, tolerability, pharmacokinetics, and
activity of the combination. The primary objectives of this study
are: To evaluate the safety and tolerability of GDC-0941
administered with T-DM1; To estimate the MTD of GDC-0941 when
administered with T-DM1; identify a recommended Phase II dose for
GDC-0941 administered in combination with T-DM1; and To
characterize any observed anti-tumor 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.
[0271] GDC-0941 Formulation
[0272] GDC-0941 is a dry powder intended for PO administration. The
formulated drug product will be provided in hard gelatin capsules
of two strengths (15 and 50 mg) that are encapsulated with size 0
shells and differentiated by color. Excipients included in the
capsule formulations are microcrystalline cellulose NF/EP, sodium
lauryl sulfate NF/DP (in the 50 mg strength only), 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 at refrigerated temperature between
36.degree. F. and 46.degree. F. (2.degree. C. and 8.degree. C.).
Patients will be instructed to store study drug at refrigerated
temperature between 36.degree. F. and 46.degree. F. (2.degree. C.
and 8.degree. C.).
[0273] Outcome Measures
[0274] Outcome measures for safety, pharmacokinetics,
pharmacodynamic, and efficacy will be determined and assessed,
including Statistical Methods, as in Example 4.
[0275] Study Treatment
[0276] Study treatments will be administered in 3-week cycles.
Patients receiving clinical benefit from study treatment may have
the possibility of treatment for more cycles which may occur in a
separate study, depending on the development status, drug
availability, and other factors.
[0277] In the dose escalation phase of the study, patients enrolled
will receive a single dose of GDC-0941 on Day 1 of Cycle 1 on an
empty stomach, to allow pre- and post-dose GDC-0941 PK sample
collection and to observe intra-patient variability. The starting
dose of GDC-0941 will be 60 mg qd, which is a dose that has been
determined to be safe as a single agent without any dose limiting
toxicities in a phase I study. On Day 2 of Cycle 1, full-dose T-DM1
will be administered at 3.6 mg/kg IV over 90 minutes without a
loading dose. This will be followed by a dose of GDC-0941. Patients
will be monitored for 90 minutes after the first T-DM1 infusion.
GDC-0941 will then be given once daily, for a total of 14 doses
followed by 1 week off for the first cycle.
[0278] Dose escalation of GDC-0941 in subsequent patients will
continue until progression or intolerability. Subsequent study
treatment cycles will be 3 weeks in length, with T-DM1 3.6 mg/kg IV
administered over 30 minutes first on Day 1 of each cycle and
GDC-0941 administered after the T-DM1 infusion, and continuing for
a total of 2 weeks on and 1 week off. Dosing will continue until
progression or intolerability. T-DM1 will be administered as a 30
to 90 minute (.+-.10) IV infusion, depending on how T-DM1 was
tolerated in the parent study. If the 90 minute infusion is well
tolerated, subsequent infusions may be delivered over 30 (.+-.10)
minutes.
[0279] The foregoing description is considered as illustrative only
of the principles of the invention. Further, since numerous
modifications and changes will be readily apparent to those skilled
in the art, it is not desired to limit the invention to the exact
construction and process shown as described above. Accordingly, all
suitable modifications and equivalents may be considered to fall
within the scope of the invention as defined by the claims that
follow.
* * * * *