HK1185263A

HK1185263A - Antineoplastic combinations with mtor inhibitor, herceptin, and/or hki-272

Info

Publication number: HK1185263A
Application number: HK13112663.6A
Authority: HK
Inventors: Moore Laurence; Zacharchuk Charles; Krishna Rabindran Sridhar
Original assignee: Wyeth
Priority date: 2005-11-04
Filing date: 2013-11-12
Publication date: 2014-02-14

Description

Antitumor combinations of mTOR inhibitors, herceptins and/or HKI-272

The application is a divisional application of an invention patent application entitled "antitumor combination of mTOR inhibitor, herceptin and/or HKI-272" with an international application PCT/US2006/042915 entering China at the 2006, 11/2/2006 and an application number of 200680041247.1.

Technical field and background

The present invention relates to the use of herceptin (herceptin) in combination with an mTOR inhibitor and/or HKI-272 for the treatment of neoplasms associated with overexpression or amplification of HER 2.

CCI-779 is rapamycin 42-ester with 3-hydroxy-2- (hydroxymethyl) -2-methylpropionic acid, which has been shown to have significant inhibitory effects on tumor growth in both in vitro and in vivo models. This compound is currently commonly referred to as temsirolimus (temsirolimus). The preparation and use of hydroxy esters of rapamycin (including temsirolimus) is described in U.S. Pat. Nos. 5,362,718 and 6,277,983.

Temsirolimus is cytostatic, a property that, in contrast to cytotoxicity, delays the time to progression or recurrence of the tumor. The mechanism of action of temsirolimus is believed to be similar to that of sirolimus. Temsirolimus binds to the cytoplasmic protein FKBP forming a complex that inhibits the enzyme mTOR (a mammalian target of rapamycin, also known as FKBP 12-rapamycin associated protein FRAP). Inhibition of mTOR kinase activity blocks a variety of signal transduction pathways, including cytokine-stimulated cell proliferation, messenger RNA translation of some key proteins that regulate the G1 phase of the cell cycle, and IL-2-induced transcription, resulting in inhibition of cell cycle progression from the G1 phase to the S phase. The mechanism of action of temsirolimus which results in the arrest of the G1-S phase is novel for anticancer drugs.

Metastatic Breast Cancer (MBC) is essentially untreatable with standard therapy, and the mean survival time of MBC patients after transplantation is documented to be about 2 years. Thus, the goal of treatment is to improve the patient's symptoms while trying to maintain (or in some cases improve) quality of life. Prolonged survival remains a clear goal, particularly in breast cancers where the her-2 oncogene is overexpressed or amplified.

Herceptin(Trastuzumab) is an FDA approved therapeutic monoclonal antibody for metastatic breast cancer with overexpression of the HER2 protein. Murine monoclonal antibodies are described [ see U.S. Pat. No. 5,705,151]. The murine MAb4D5 molecule described in this document has been subjected to humanization in an attempt to improve its clinical efficacy by reducing immunogenicity and allowing it to support human effector functions. WO 92/22653. The development of lyophilized formulations comprising the full length humanized antibody huMAb4D5-8 is described in later document WO 92/22653. Herceptin is currently FDA approved for the treatment of metastatic breast cancer that overexpresses HER2, (1) as a single drug after previous treatment of metastatic breast cancer with one or more chemotherapeutic regimens, and (2) in combination with paclitaxel in patients that have not previously undergone metastatic breast cancer chemotherapy. In addition, there is evidence that addition of herceptin to chemotherapy with taxane adjuvant or neoadjuvant (neoadjuvant) improves patients in the pre-mammary stage.

HKI-272, (E) -N- {4- [ 3-chloro-4- (2-pyridylmethoxy) anilino ] -3-cyano-7-ethoxy-6-quinolinyl } -4- (dimethylamino) -2-butenamide, is described as a promising candidate anticancer drug for the treatment of breast cancer and other HER-2 dependent cancers. Because HKI-272 also inhibits EGFR kinase with similar potency, HKI-272 can also be used to treat tumors that overexpress HER-2 and EGFR, and is more effective than a specific EGFR or HER-2 antagonist. Rabindran et al, "anticancer Activity of HKI-272, an oraly Active, Irreversible Inhibitor of the HER-2 Tyrosine Kinase", Cancer Research 64, 3958-. See U.S. Pat. nos. 6,288,082; U.S. Pat. No. 6,297,258.

There remains a need for improved anti-tumor therapies.

Drawings

FIG. 1 shows the results obtained in BT474[ HER-2+ (amplified); in ATCC HTB-20] cells, a three-dimensional contour plot between herceptin and HKI-272, the horizontal plane at 0% indicates additive interaction, and the peaks and troughs indicate synergistic or antagonistic zones, respectively.

FIG. 2 shows a schematic representation of MCF-7[ HER-2 ]^-EGFR-; adenocarcinoma; ATCC HTB22]In the cells, in the three-dimensional contour plot between herceptin and HKI-272, the horizontal plane at 0% represents the additive interaction, and the peaks and valleys represent the synergistic or antagonistic zones, respectively.

FIG. 3 shows the expression in MDA-MB-361[ HER-2 ]⁺(unamplified); adenocarcinoma; ATCC HTB27]In the cells, in the three-dimensional contour plot between herceptin and HKI-272, the horizontal plane at 0% represents the additive interaction, and the peaks and valleys represent the synergistic or antagonistic zones, respectively.

Disclosure of Invention

The present invention provides the use of a combination comprising herceptin, an mTOR inhibitor and/or HKI-272 in the treatment of neoplasia. Accordingly, the present invention provides the use of a herceptin in combination with an mTOR inhibitor, the use of a herceptin in combination with an HKI-272, the use of an mTOR inhibitor in combination with an HKI-272, or the use of a herceptin in combination with an mTOR inhibitor and an HKI-272. The invention further provides a product containing a herceptin in combination with an mTOR inhibitor and/or HKI-272 formulated for simultaneous, separate or sequential use in the treatment of neoplasia in a mammal. The present invention may also be used as an adjuvant and/or neoadjuvant treatment for breast cancer. The following detailed description describes temsirolimus. However, other mTOR inhibitors may be substituted for temsirolimus in the methods, combinations, and products described herein.

These methods, combinations and products are useful for treating a variety of tumors associated with overexpression or amplification of HER2, including, for example, lung cancer (including bronchioloalveolar carcinoma and non-small cell lung cancer), breast cancer, prostate cancer, myeloma, head and neck cancer, or transitional cell carcinoma; small cell and large cell neuroendocrine carcinoma of the cervix.

In one embodiment, the combination of temsirolimus and herceptin is particularly useful for treating metastatic breast cancer. In further embodiments, the combination of herceptin and an mTOR inhibitor and/or HKI-272 is well suited for the treatment of breast, kidney, bladder, mouth, throat, esophageal, gastric, colon, ovarian, and lung cancers), as well as polycystic kidney disease.

The term mTOR inhibitor as used herein refers to a compound or ligand, or a pharmaceutically acceptable salt thereof, that prevents cell replication by blocking progression of the cell cycle from G1 phase to S phase. The term includes the neutral tricyclic compound rapamycin (sirolimus) and other rapamycin compounds, including, for example, rapamycin derivatives, rapamycin analogues, other macrolide compounds that inhibit mTOR activity, and all compounds included under the definition of the term "rapamycin". These include compounds having structural similarity to "rapamycins", e.g., compounds having similar macrocyclic structures modified to enhance therapeutic benefit. FK-506 can also be used in the method of the present invention.

The term rapamycin as used herein defines a class of immunosuppressive compounds that contain a rapamycin-based nucleus as shown below.

The rapamycins of the present invention include compounds that have been chemically or biologically modified to be derivatives of the rapamycin nucleus and yet retain immunosuppressive properties. Thus, the term rapamycin includes rapamycin, as well as esters, ethers, carbamates, oximes, hydrazones, and hydroxylamines of rapamycin, and rapamycins in which functional groups on the rapamycin nucleus have been modified (e.g., reduced or oxidized). The term rapamycin also includes pharmaceutically acceptable salts of rapamycins.

The term rapamycin also includes the 42-and/or 31-esters and ethers of rapamycin described in the following patents, which are incorporated herein by reference in their entirety: alkyl esters (U.S. Pat. No. 4,316,885); aminoalkyl esters (U.S. Pat. No. 4,650,803); fluorinated esters (U.S. patent 5,100,883); amide esters (U.S. patent 5,118,677); carbamates (U.S. patent 5,118,678); silyl esters (U.S. patent 5,120,842); aminodiesters (U.S. Pat. No. 5,162,333); sulfonates and sulfates (U.S. patent 5,177,203); esters (U.S. patent 5,221,670); alkoxy esters (U.S. patent 5,233,036); o-aryl, -alkyl, -alkenyl, and-alkynyl ethers (U.S. patent 5,258,389); carbonates (U.S. patent 5,260,300); arylcarbonyl and alkoxycarbonyl carbamates (U.S. patent 5,262,423); carbamates (U.S. patent 5,302,584); hydroxy esters (U.S. Pat. No. 5,362,718); hindered esters (U.S. patent 5,385,908); heterocyclic esters (U.S. Pat. No. 5,385,909); gem-disubstituted esters (U.S. patent 5,385,910); aminoalkanoic acid esters (U.S. patent 5,389,639); phosphoryl carbamates (U.S. Pat. No. 5,391,730); carbamates (U.S. patent 5,411,967); carbamates (U.S. patent 5,434,260); amidinocarbamates (U.S. patent 5,463,048); carbamates (U.S. patent 5,480,988); carbamates (U.S. patent 5,480,989); carbamates (U.S. patent 5,489,680); sterically hindered N-oxide esters (U.S. patent 5,491,231); biotin esters (U.S. patent 5,504,091); o-alkyl ethers (U.S. Pat. No. 5,665,772); and PEG esters of rapamycin (us patent 5,780,462). The preparation of these esters and ethers is disclosed in the above patents.

The term rapamycin class definitions also includes 27-esters and ethers of rapamycin, which is disclosed in U.S. patent 5,256,790. Also described are C-27 keto rapamycins, which are reduced to the corresponding alcohol and then converted back to the corresponding ester or ether. The preparation of these esters and ethers is disclosed in the above patents. Also included are oximes, hydrazones, and hydroxylamines of rapamycin, disclosed in U.S. Pat. Nos. 5,373,014, 5,378,836, 5,023,264, and 5,563,145. The preparation of these oximes, hydrazones and hydroxylamines is disclosed in the above patents and the preparation of 42-oxorapamycin is disclosed in 5,023,263.

The term CCI-779 as used herein refers to the rapamycin 42-ester with 3-hydroxy-2- (hydroxymethyl) -2-methylpropionic acid (temsirolimus) and includes prodrugs, derivatives, pharmaceutically acceptable salts, or analogs thereof.

Examples of rapamycins include, for example, 32-deoxorapamycin, 16-pent-2-ynyloxy-32 (S) -dihydro-rapamycin, 16-pent-2-ynyloxy-32 (S) -dihydro-40-O- (2-hydroxyethyl) -rapamycin, rapamycin 42-ester with 3-hydroxy-2- (hydroxymethyl) -2-methylpropionic acid (CCI-779), 40- [ 3-hydroxy-2- (hydroxymethyl) -2-methylpropionic acid ester.]-rapamycin, or a pharmaceutically acceptable salt thereof, as disclosed in U.S. Pat. No. 5,362,718, ABT578, or 40- (tetrazolyl) -rapamycin, 40-epi- (tetrazolyl) -rapamycin, for example as disclosed in International patent publication No. WO99/15530, or rapamycin analogues as disclosed in International patent publication No. WO 98/02441 and WO01/14387, such as AP 23573. In further embodiments, the compound is Certican^TM(everolimus, 2-O- (2-hydroxy) ethyl rapamycin, Novartis, U.S. Pat. No. 5,665,772).

As used herein, "HKI-272" refers to a compound having the following nucleus:

or a derivative or pharmaceutically acceptable salt thereof. Suitable derivatives may include, for example, esters, ethers, or carbamates. The chemical name of core structure HKI-272 is (E) -N- {4- [ 3-chloro-4- (2-pyridylmethoxy) anilino ] -3-cyano-7-ethoxy-6-quinolinyl } -4- (dimethylamino) -2-butenamide.

In one embodiment, the invention also provides the use of substituted 3-cyanoquinolines having the structure:

wherein R is₁Is halogen;

R₂is pyridyl, thiophene, pyrimidine, thiazole, or phenyl optionally substituted with up to three substituents;

R₃is-O-or-S-;

R₄is methyl or CH₂CH₂OCH₃；

R₅Is ethyl or methyl; and

n is 0 and 1.

These compounds, including the specific compound HKI-272, are characterized as being capable of acting as potent HER-2 inhibitors. See, for example, U.S. Pat. No. 6,288,082 and U.S. Pat. No. 6,297,258. These compounds and their preparation are described in detail in U.S. published patent application No. 2005/0059678. For convenience, HKI-272 has been used throughout this specification. However, it is understood that compounds having the above structure may be substituted for HKI-272 in combination with mTOR inhibitors and/or herceptin, as described in detail below.

The following standard pharmacological test procedures may be used to determine whether a compound is an mTOR inhibitor as defined herein. Treatment of growth factor-stimulated cells with an mTOR inhibitor, such as rapamycin, completely blocks phosphorylation of serine 389, as demonstrated by western blotting, and thus constitutes a good assay for mTOR inhibition. Thus, whole cell lysates produced by cells stimulated in culture with growth factors (e.g., IGF1) in the presence of mTOR inhibitors do not show bands on acrylamide gels labeled with p70s6K serine 389 specific antibodies.

The mTOR inhibitor used in the antineoplastic combination of the present invention is preferably a rapamycin, more preferably the mTOR inhibitor is rapamycin, temsirolimus or 42-O- (2-hydroxy) ethyl rapamycin. The preparation of 42-O- (2-hydroxy) ethyl rapamycin is described in U.S. Pat. No. 5,665,772.

The preparation of temsirolimus is described in U.S. Pat. No. 5,362,718. A regiospecific synthesis of temsirolimus is described in U.S. patent 6,277,983, which is incorporated herein by reference. Another regiospecific method for the synthesis of temsirolimus is described in U.S. patent publication No.2005-0033046-A1 (application No. 10/903,062, filed on 7/30/2004), published on 8/7/2005, and its corresponding International patent publication No. WO 2005/016935, published on 10/2/2005.

Herceptin and methods of preparation and formulation thereof are described in, for example, us patents 6,821,515; U.S. Pat. No. 6,399,063 and U.S. Pat. No. 6,387,371. Herceptin is commercially available from Genentech. The term "herceptin" as used herein includes trastuzumab and variants and derivatives of trastuzumab. The term "herceptin" includes drugs that target the same epitope on the Her-2 receptor as that targeted by trastuzumab. Epitopes are known in H.S. Cho et al, Structure of the extracellular region of HER2 alone and in complex with the said receptor Fab, Nature 421(2003), pp.756-760.

HKI-272 and methods for its preparation and formulation are described, for example, in U.S. published patent application No. 2005/0059678; U.S. Pat. No. 6,002,008, which is incorporated herein by reference, is also useful for preparing substituted 3-quinolines for use in the present invention. In addition to the methods described in these documents, the methods described in WO-9633978 and WO-9633980 can be used to prepare these compounds. Although these methods describe the preparation of certain quinazolines, they are also suitable for the preparation of the corresponding substituted 3-cyanoquinolines and are incorporated herein by reference.

The term "treatment" as used herein refers to the treatment of a mammal suffering from a neoplasm by providing to said mammal an effective amount of a combination of two or three drugs selected from the group consisting of mTOR inhibitors, herceptin and/or components of HKI-272, to inhibit the progression of the neoplastic disease, inhibit the growth of the tumor in the mammal, eradicate the neoplastic disease, prolong the survival of the mammal and/or alleviate the mammal.

The term "providing," with respect to providing an mTOR inhibitor with a herceptin and/or HKI-272, as used herein, refers to the administration of the mTOR inhibitor directly, or a prodrug, derivative, or analog that forms an effective amount of the mTOR inhibitor in vivo, and the administration of the herceptin and/or HKI-272 directly, or a prodrug, derivative, or analog that forms an effective amount of the herceptin or HKI-272 in vivo.

The use of a combination of an mTOR inhibitor (e.g., temsirolimus), herceptin and/or HKI-272 also provides for the combination of each of the drugs wherein one, two or all three of the drugs are used at subtherapeutically effective dosages. Subtherapeutically effective dosages can be readily determined by those skilled in the art in view of the teachings herein. In one embodiment, a subtherapeutically effective dose refers to a dose that is effective at a lower dose when used in the combination therapy of the invention compared to a dose that is effective when used alone. The present invention further provides for the use of one or more active agents in a combination of the invention in a supratherapeutic amount, i.e. in a higher dose in the combination than the dose when used alone. In this embodiment, the additional one or more active agents may be used in a therapeutic or sub-therapeutic amount.

The combination of the invention may take the form of a kit of parts. Thus, the present invention includes products comprising an mTOR inhibitor, herceptin, and/or HKI-272 as a combined preparation for simultaneous, separate, or sequential delivery to treat a neoplasm in a mammal in need thereof. In one embodiment, the product comprises temsirolimus and herceptin as a combined preparation for simultaneous, separate or sequential use in treating a neoplasm in a mammal in need thereof. Optionally, the product additionally comprises HKI-272. HKI-272 can be formulated separately, for example, for oral delivery. In another embodiment, the product comprises temsirolimus and HKI-272 as a combined preparation for simultaneous, separate or sequential use in treating a neoplasm in a mammal in need thereof. Optionally, the product additionally comprises herceptin. In yet another embodiment, the product comprises herceptin and HKI-272. Optionally, the product additionally contains an mTOR inhibitor. In one embodiment, the neoplasm is metastatic breast cancer.

In one embodiment, the pharmaceutical pack comprises a course of treatment of a neoplasm in one mammalian subject, wherein the pack contains units of an mTOR inhibitor in unit dosage form, and units of a herceptin in unit dosage form, optionally in additional combination with units of HKI-272 in unit dosage form. In a further embodiment, the pharmaceutical pack comprises a course of treatment of a neoplasm in one mammalian subject, wherein the pack contains mTOR inhibitor units in unit dosage form and HKI-272 units in unit dosage form, optionally in further combination with herceptin units in unit dosage form. In yet another embodiment, the pharmaceutical pack comprises a course of treatment of a neoplasm in one mammalian subject, wherein the pack contains units of herceptin in unit dosage form, and units of HKI-272 in unit dosage form, optionally in additional combination with units of an mTOR inhibitor in unit dosage form. In one embodiment, the pharmaceutical pack described herein contains a course of treatment for metastatic breast cancer in a mammalian subject.

The route of administration of the composition may be oral, intravenous, respiratory (e.g., nasal or intrabronchial), infusion, parenteral (other than intravenous, such as intralesional (intraperitoneal), intraperitoneal, and subcutaneous injection), intraperitoneal, transdermal (all modes of administration including lining through body surfaces and body passages including epithelial and mucosal tissues), and vaginal (including intrauterine) administration. Other routes of administration are also possible, such as by liposome-mediated delivery; topical, nasal, sublingual, urethral, intrathecal, ocular or otic, implant, rectal, intranasal.

Although the components of the invention may be administered by the same route, the product or kit of the invention may contain a rapamycin such as temsirolimus which is delivered by a route different from that of herceptin or HKI-272, e.g., one or more of the components may be delivered orally while one or more of the other components are administered intravenously. In one embodiment, temsirolimus is for oral delivery, HKI-272 is for oral delivery, and herceptin is for intravenous delivery. In further embodiments, both temsirolimus and herceptin are used for intravenous delivery. In other embodiments, all components are for oral delivery. Optionally, the route of delivery of the other active ingredient may be the same as or different from the route of delivery of the mTOR inhibitor (e.g., temsirolimus) or herceptin. Other variations will be apparent to those skilled in the art and are contemplated within the scope of the invention.

The combination of an mTOR inhibitor and herceptin may be administered in the absence of HKI-272. In one embodiment, these drugs are the only active antineoplastic agents used in the dosing regimen. In a further embodiment, the combination of mTOR inhibitor/herceptin is used in combination with HKI-272.

The combination of an mTOR inhibitor and HKI-272 can be administered in the absence of herceptin. In another embodiment, the mTOR inhibitor/HKI-272 combination is administered in combination with herceptin. In one embodiment, the combination of the two and three is the only active antineoplastic agent used in the dosing regimen. In further embodiments, combinations of these two and three may be used in further combinations with other active agents.

The combination of herceptin and HKI-272 may be administered in the absence of an mTOR inhibitor. In another embodiment, the herceptin/HKI-272 combination is administered in combination with an mTOR inhibitor. In one embodiment, the combination of the two and three is the only active antineoplastic agent used in the dosing regimen. In further embodiments, combinations of these two and three may be used in further combinations with other active agents.

According to what is commonly used for oncology treatments, the dosing regimen is closely monitored by the treating physician in light of various factors including the severity of the disease, the response to the disease, any treatment-related toxicities, the age and health of the patient. It is contemplated that the dosage regimen will vary depending upon the route of administration.

When administered on a weekly dosing schedule, the initial intravenous infusion dose of the mTOR inhibitor (e.g., temsirolimus) is about 5 to about 175 milligrams, or about 5 to about 25 milligrams. Oral doses of mTOR inhibitors useful in the present invention are scheduled from 10 mg/week to 250 mg/week, from about 20 mg/week to about 150 mg/week, from about 25 mg/week to about 100 mg/week, or from about 30 mg/week to about 75 mg/week. For rapamycin, the planned oral dose is from 0.1 mg/day to 25 mg/day. The precise dosage will be determined by the administering physician based on experience with the individual undergoing treatment.

Other dosing regimens and variations are foreseeable and can be determined by physician guidance. Preferably the mTOR inhibitor is administered by intravenous infusion or orally, preferably in the form of a tablet or capsule.

For herceptin, single and multiple doses are contemplated. In one embodiment, a single loading dose of herceptin is administered as a 90 minute intravenous infusion, about 4-5 mg/kg on the first day, followed by about 2 mg/kg weekly starting on day eight. Typically, 3 weeks is 1 cycle. There may be 1,2 or 3 weeks between cycles. Herceptin may also be administered in a dose of 6 mg/kg once every 3-4 weeks. In addition, herceptin may also be administered after completion of chemotherapy as a maintenance therapy.

For HKI-272, it is desirable that the compounds of the present invention be in unit dosage form. Suitable unit dosage forms include tablets, capsules and powders in sachets or vials. The unit dosage form may contain from 0.1 to 300 mg of a compound of the invention, preferably from 2 to 100 mg. A further preferred unit dosage form comprises 5 to 50 mg of a compound of the invention. The compounds of the invention are administered in a dosage range of about 0.01 to 100 mg/kg, or preferably 0.1 to 10 mg/kg. In one embodiment, the compound is administered orally 1-6 times per day, typically 1-4 times per day. Alternatively, the compounds may be administered by another suitable route, such as intravenous. In another embodiment, the compound is administered once a week. In some cases, administration of HKI-272 may be delayed or discontinued for a short period of time (e.g., 1,2, or 3 weeks) during the course of treatment. Such delay or interruption may occur one or more times during the course of treatment. The effective amount is known to those skilled in the art and may also depend on the form of the compound. One skilled in the art can routinely conduct experimental activity assays to determine the biological activity of a compound in a biological assay and thus determine the dosage administered.

These schemes may be repeated or alternated as desired. Other dosing regimens and variations are foreseeable and are determined by physician guidance.

For example, in one embodiment, the dosing regimen further comprises administering a taxane such as docetaxel and paclitaxel [ e.g., a suspension of paclitaxel bound to albumin nanoparticles, which is obtained as Abraxane ]. Paclitaxel may also be administered on a weekly schedule, with a dose of 60-100 mg/m administered over 1 hour, once per week, or a weekly dose of 2-3 weeks followed by a one week rest. In one embodiment, paclitaxel is administered intravenously at a dose of 175 mg/m over 3 hours, optionally followed by cisplatin at a dose of 75 mg/m; alternatively, paclitaxel is administered intravenously at a dose of 135 mg/m over 24 hours, optionally followed by 75 mg/m cisplatin. Paclitaxel can be injected in several doses and schedules in patients previously treated with tumor therapy. However, optimal treatment regimens are not known. The recommended treatment regimen is to administer 135 mg/m or 175 mg/m of paclitaxel intravenously over 3 hours every 3 weeks. Wherein these dosages may be varied as needed or desired.

Other active agents may be included in the combination of the mTOR inhibitor and herceptin, including, for example, chemotherapeutic agents such as alkylating agents; hormonal agents (i.e., estramustine, tamoxifen, toremifene, anastrozole, or letrozole); antibiotics (i.e., plicamycin, bleomycin, mitoxantrone, idarubicin, actinomycin D, mitomycin, or daunorubicin); antimitotic agents (i.e., vinblastine, vincristine, teniposide, or vinorelbine, available as Navelbine); topoisomerase inhibitors (i.e., topotecan, irinotecan, etoposide, or doxorubicin, such as CAELYX or Doxil, polyethylene glycol conjugated (pegylated) liposomal doxorubicin hydrochloride); and other drugs (i.e., hydroxyurea, hexamethylmelamine, rituximab, paclitaxel, docetaxel, levoasparaginase, or gemtuzumab ozogamicin); a biochemical modulator, imatinib (imatib), an EGFR inhibitor such as EKB-569 or other multi-kinase inhibitors such as those targeting serine/threonine and receptor tyrosine kinases in both tumor cells and tumor vasculature, or an immunomodulatory agent (i.e., interferon, IL-2, or BCG). Examples of suitable interferons include interferon alpha, interferon beta, interferon gamma, and mixtures thereof.

In one embodiment, the combination of an mTOR inhibitor and a herceptin may be further combined with an antineoplastic alkylating agent (such as those described in US 2002-. Antineoplastic alkylating agents are roughly divided into several classes based on their structural or reactive moieties, including nitrogen mustards such as mustagen (nitrogen mustard), cyclophosphamide, ifosfamide, melphalan, and chlorambucil; azides (azidines) and epoxides, such as thiotepa, mitomycin C, dianhydrogalactitol, and dibromodulcitol; alkyl sulfinates, such as busulfan; nitrosoureas such as dichloroethyl nitrosourea (BCNU), Cyclohexyl Chloroethyl Nitrosourea (CCNU), and methylcyclohexyl chloroethyl nitrosourea (MeCCNU); hydrazine and triazine derivatives, such as procarbazine, dacarbazine and temozolomide; streptazolin, melphalan, chlorambucil, carmustine, methcloethamine, lomustine) and platinum compounds. Platinum compounds are platinum-containing reagents that preferentially react at the N7 position of guanine and adenine residues to form a variety of monofunctional and difunctional adducts. (Johnson S W, Stevenson J P, O' Dwyer P J. Cisplatin and Its analogs, in cancer principles & Practice of Oncology, 6 th edition, editors DeVita V T, Hellman S, Rosenberg S A. Lippincott Williams & Wilkins. Philadelphia 2001. p.378.). These compounds include cisplatin, carboplatin, platinum IV compounds, and multinuclear platinum complexes.

The following are typical examples of alkylating agents of the present invention. Nitrogen mustards are commercially available as injections (mustagen). Cyclophosphamide is commercially available as an injection (cyclophophamide, lyophilized CYTOXAN, or NEOSAR) and oral tablet (cyclophophamide or CYTOXAN). Ifosfamide is commercially available as an Injection (IFEX). Melphalan is commercially available as an injection (alkenr) and oral tablet (alkenr). Chlorambucil is commercially available as an oral tablet (LEUKERAN). Thiotepa is commercially available as an injection (thiotepa or THIOPLEX). Mitomycin is commercially available as an injection (mitomycin or MUTAMYCIN). Busulfan is commercially available as an injection (BUSULFEX) and oral tablet (myrenr). Lomustine (CCNU) is commercially available as an oral Capsule (CEENU). Carmustine (BCNU) is commercially available as intracranial implant (GLIADEL) and injection (BICNU). Procarbazine is commercially available as oral capsules (MATULANE). Temozolomide is commercially available as an oral capsule (TEMODAR). Cisplatin is commercially available as an injection (cissplatin, placanol or placanol-AQ). Carboplatin is commercially available as an injection (parapeltin). Oxiplatin is commercially available as ELOXATIN.

In another embodiment, the combination of the invention may additionally comprise treatment with an anti-tumor anti-metabolite such as described in US patent publication No. US 2005-0187184A1 or US 2002-0183239A 1. The term "antimetabolite" as used herein refers to a substance that is structurally similar to a natural intermediate (metabolite) that is critical in the biochemical pathway leading to DNA or RNA synthesis, which is used by the subject in that pathway, but serves to inhibit completion of that pathway (i.e., synthesis of DNA or RNA). More specifically, antimetabolites typically function by the following mechanisms: (1) compete with metabolites for catalytic or regulatory sites of key enzymes in DNA or RNA synthesis, or (2) replace metabolites that normally bind into DNA or RNA, thereby producing DNA or RNA that cannot support replication. The main classification of antimetabolites includes (1) folic acid analogs, which are inhibitors of dihydrofolate reductase (DHFR); (2) purine analogs that mimic natural purines (adenine or guanine) but have different structures such that they competitively or irreversibly inhibit nuclear processing of DNA or RNA; and (3) pyrimidine analogs that mimic natural pyrimidines (cytosine, thymidine, and uracil) but have different structures such that they competitively or irreversibly inhibit nuclear processing of DNA or RNA.

The following are typical examples of the antimetabolite of the present invention. 5-Fluorouracil (5-FU; 5-fluoro-2, 4(1H, 3H) -pyrimidinedione) is commercially available as a topical cream (FLUOROPLEX or EFUDEX), a topical solution (FLUOROPLEX or EFUDEX), and an injection (ADRUCIL or Fluuroacil) containing 50 mg/ml of 5-fluorouracil. Floxuridine (2' -deoxy-5-fluorouracil) is commercially available as an injection (FUDR or floxuridine) containing 500 mg/vial of floxuridine. Thioguanine (2-amino-1, 7-dihydro-6-H-purine-6-thione) is commercially available as a 40 mg oral tablet (thioguanine). Cytarabine (4-amino-1- (. beta. -D-arabinofuranosyl-2 (1H) -pyrimidinone) is commercially available as a liposome injection containing 10mg/mL cytarabine (DEPOCYT) or a liquid injection containing 1 mg-1 g/vial or 20mg/mL (cytarabine or CyTOSAR-U). Fludarabine (9-H-purin-6-amine, 2-fluoro-9- (5-O-phosphono- (. beta. -D-arabinofuranosyl) as a solution containing 50 mg/vialLiquid injection (FLUDARA) is commercially available. 6-mercaptopurine (1, 7-dihydro-6H-purine-6-thione) is commercially available as a 50 mg oral tablet (PURINETHOL). Methotrexate (MTX; N- [4- [ [ (2, 4-diamino-6-pteridinyl) methyl ] methyl]Methylamino radical]Benzoyl radical]-L-glutamic acid) are commercially available as liquid injection (methotrexate sodi um or FOLEX) and 2.5 mg oral tablet (methotrexate) containing 2.5-25mg/mL and 20 mg-1 g/vial. Gemcitabine (2 ' -deoxy-2 ', 2 ' -difluorocytidine monohydrochloride ((β) -isomer)) is commercially available as a liquid injection (GEMZAR) containing 200 mg-1 g/vial. Capecitabine (5' -deoxy-5-fluoro-N- [ (pentyloxy) carbonyl)]-cytidine) is commercially available as oral tablets (XELODA) of 150 or 500 mg. Pentostatin ((R) -3- (2-deoxy- (. beta. -D-erythro-pentofuranosyl) -3, 6,7, -8-tetrahydroimidazo [4, 5-D)][1，3]Diaza derivatives-8-ol) is commercially available as a liquid injection (NIPENT) containing 10 mg/vial. Trimethotrexate (2, 4-diamino-5-methyl-6- [ (3, 4, 5-trimethoxyanilino) methyl group)]Quinazoline mono-D-glucuronic acid) is commercially available as a liquid injection (netrexin) containing 25-200 mg/vial. Cladribine (2-chloro-6-amino-9- (2-deoxy- (. beta. -D-erythro-pentofuranosyl) purine) is commercially available as a liquid injection containing 1mg/mL (LEUSTATIN).

The term "biochemical modulator" is well known and understood by those skilled in the art and is administered as an adjunct to anticancer therapy for enhancing the antitumor activity of the anticancer therapy and for counteracting the side effects of active agents such as antimetabolites. Folinic acid and levofolinic acid (levofolinate) are commonly used as biochemical modulators of methotrexate and 5-fluorouracil treatment. Folinic acid (5-formyl-5, 6,7, 8-tetrahydrofolic acid) is commercially available as an injection solution (leucovorincicum or WELLCOVORIN) and 5-25mg oral tablets (leucovorin calcum) containing 5-10mg/mL or 50-350 mg/vial. Levofolinic acid (a pharmacologically active isomer of 5-formyltetrahydrofolic acid) is commercially available as an injection (ISOVORIN) or 2.5-7.5 mg oral tablet (ISOVORIN) containing 25-75 mg levofolinic acid.

In another embodiment, the combination of the invention additionally comprises an active agent selected from kinase inhibitors. What is particularly needed are multi-kinase inhibitors that target serine/threonine and receptor tyrosine kinases in both tumor cells and tumor vasculature. An example of a suitable kinase inhibitor is sorafenib (BAY 43-9006, Bayer, commercially available as NEXAVAR), which is approved for metastatic renal cell carcinoma by the FDA's rapid approval channel. Another suitable farnesyltransferase inhibitor is Zarnestra (R115777, tipifarnib). Another compound is Sunitinib (SUTENT). Other suitable compounds targeting Ras/Raf/MEK and/or MAP kinases include, for example, avastin, ISIS 5132, and MEK inhibitors such as CI-1040 or PD 0325901.

As used herein, subtherapeutically effective amounts of herceptin and temsirolimus, when administered in combination, may be used to achieve a therapeutic effect. For example, herceptin, when provided with temsirolimus, may be provided in a dosage of 5 to less than 50%, 10 to less than 25%, or 15 to less than 20%. For example, the final herceptin dose may be about 8 to 40 mg, or about 8 to 30 mg, or 8 to 25 mg. A subtherapeutically effective amount of herceptin is expected to reduce the side effects of herceptin treatment. The present invention further provides one or more active agents for use in a combination of the invention in a supratherapeutic amount, i.e. a supratherapeutic amount is a higher dose when used in combination than when used alone. In this embodiment, the additional one or more active agents may be used in a therapeutic or sub-therapeutic amount.

The mTOR inhibitor, herceptin, HKI-272 or other active compounds used in the combinations and products of the present invention may be formulated in any suitable manner. For example, oral formulations containing an mTOR inhibitor (and optionally other active compounds) useful in the combinations and products of the present invention may include any of the commonly used oral dosage forms, including tablets, capsules, buccal forms, lozenges and oral liquids, suspensions or solutions. Capsules may contain mixtures of the active compound with inert fillers and/or diluents such as the pharmaceutically acceptable starches (e.g., corn, potato or tapioca starch), sugars, artificial sweeteners, cellulose powders such as crystalline and microcrystalline cellulose powders, flours, gelatins, gums, and the like. Useful tablets may be prepared by conventional tableting, wet or dry granulation processes using pharmaceutically acceptable diluents, binders, lubricants, disintegrants, surface modifying agents (including surfactants), suspending or stabilizing agents including, but not limited to, magnesium stearate, stearic acid, talc, sodium lauryl sulfate, microcrystalline cellulose, carboxymethylcellulose calcium, polyvinylpyrrolidone, gelatin, alginic acid, gum arabic, xanthan gum, sodium citrate, complex silicates, calcium carbonate, glycine, dextrin, sucrose, sorbitol, calcium hydrogen phosphate, calcium sulfate, lactose, kaolin, mannitol, sodium chloride, talc, dry starch and powdered sugar. Preferred surface modifying agents include nonionic and anionic surface modifying agents. Typical examples of surface modifying agents include, but are not limited to, poloxamer 188, benzalkonium chloride, calcium stearate, cetostearyl alcohol, cetomacrogol emulsifying wax, sorbitan esters, colloidal silicon dioxide, phosphates, sodium dodecylsulfate, magnesium aluminum silicate, and triethanolamine. Oral formulations as used herein may employ standard delayed release or timed release formulations to modify the absorption of the active compound. Oral formulations may also include the administration of the active ingredient in water or fruit juices, containing appropriate solubilizers or emulsifiers as required. A preferred oral formulation for rapamycin 42-ester with 3-hydroxy-2- (hydroxymethyl) -2-methylpropionic acid is described in U.S. patent publication No.2004/0077677A1, published 4 months and 22 days 2004.

In some cases, it may be desirable to administer the compound directly into the airways in the form of an aerosol.

The compounds may also be administered parenterally or intraperitoneally. The preparation of solutions or suspensions of these active compounds in the form of the free base or pharmacologically acceptable salts is carried out by suitably mixing in water with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof in oils. Under ordinary storage conditions and use conditions, these preparations contain a preservative to prevent the growth of microorganisms.

Pharmaceutical forms suitable for injection include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and must be fluid to the extent that easy syringability is achieved. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), suitable mixtures thereof, and vegetable oils. A preferred injection of rapamycin 42-ester with 3-hydroxy-2- (hydroxymethyl) -2-methylpropionic acid is described in U.S. patent publication No.2004/0167152A1, 8/26/2004.

For purposes of this disclosure, transdermal administration is understood to include all administration across the body surface and lining of body passages, including epithelial and mucosal tissues. Such administration may be carried out using the compounds of the invention or pharmaceutically acceptable salts thereof in the form of lotions, creams, foams, patches, suspensions, solutions and suppositories (rectal and vaginal).

Transdermal administration can be accomplished by the use of a transdermal patch containing the active compound and a carrier which is inert to the active compound, is non-toxic to the skin, and allows the drug to be delivered through the skin into the bloodstream for systemic absorption. The carrier may take a wide variety of forms such as creams and ointments, pastes, gels, and occlusive devices. Creams and ointments may be viscous liquid or semisolid emulsions of the oil-in-water or water-in-oil type. Pastes comprised of absorbent powders containing active ingredients dispersed in petroleum or hydrophilic petroleum are also suitable. Various occlusion devices can be used to release the active ingredient into the bloodstream, such as a reservoir containing the active ingredient and either a carrier or non-carrier, covered by a semi-permeable membrane, or a matrix containing the active ingredient. Other occlusion means are known in the literature.

Suppositories can be prepared from conventional materials including cocoa butter, with or without the addition of waxes to alter the suppository's melting point, and glycerin. Water-soluble suppository bases such as polyethylene glycols having various molecular weights may also be used.

The combination regimen used in the present invention may be administered simultaneously or may be administered in a staggered regimen with the mTOR inhibitor administered at a different time than the herceptin during the course of the chemotherapy. The time between administration of the at least two drugs may differ by minutes, hours, days, weeks, or longer. Thus, the term combination does not necessarily mean that each of the components are administered simultaneously or as a single dose, but rather within the desired treatment period. These drugs may also be administered by different routes.

Kit/kit:

the invention includes a pharmaceutical pack containing a course of anti-tumour therapy for one mammalian subject comprising one or more containers containing 1, 1 to 4, or more units of an mTOR inhibitor (e.g. temsirolimus), and optionally 1, 1 to 4, or more units of herceptin, in unit dosage form, and optionally an additional active agent.

In another embodiment, a pharmaceutical pack contains a course of anti-neoplastic therapy for one mammalian subject comprising a container containing units of the rapamycin, in unit dosage form, a container containing units of herceptin, and optionally a container containing an additional active agent. In other embodiments, the rapamycin is rapamycin, an ester of rapamycin (including 42-esters, ethers (including 42-ethers), oximes, hydrazones, or hydroxylamines in another embodiment, the rapamycin is 42-O- (2-hydroxy) ethyl rapamycin.

In another embodiment, the rapamycin is temsirolimus and the kit comprises one or more containers comprising 1, 1-4, or more units of temsirolimus and the components described herein.

In some embodiments, the compositions of the present invention are packaged in a form ready for administration. In other embodiments, the compositions of the present invention are in the form of packaged concentrates, optionally containing diluents necessary to prepare the final solution for administration. In other embodiments, the product contains a compound useful in the present invention in solid form, and optionally a separate container containing a suitable solvent or carrier for the compound of the present invention.

In other embodiments, the kits/kits described above include other components, e.g., instructions for product dilution, mixing, and/or administration, other containers, syringes, needles, etc. Other such kit/kit components will be apparent to those skilled in the art.

The following examples illustrate the use of the combinations of the present invention. It is understood that changes or modifications, such as changes and modifications to the formulation of the composition, the route of delivery, and the dosing, may be reasonably made by one skilled in the art.

Detailed Description

Example 1: combination regimen of temsirolimus (CCI-779) and herceptin in the treatment of neoplasms

Temsirolimus (IV) and herceptin (IV) were administered intravenously weekly starting on the first day of the first month at the doses provided below.

Temsirolimus and herceptin may be administered simultaneously, sequentially, or on alternating days.

Temsirolimus was administered IV weekly over 30 minutes using an in-line filter and an automatic dispensing pump. Optionally, the antihistamine (diphenhydramine, 25 to 50 mg IV, or equivalent amount) is administered about 30 minutes prior to the infusion of temsirolimus.

The loading dose of herceptin was given IV weekly over 90 minutes. The weekly dose administered is typically half the amount of the loading dose. For example, a loading dose of 4 mg/kg is typically followed by a weekly dose of 2 mg/kg. These amounts can be adjusted. In one embodiment, no loading dose is required, and the same dose is administered throughout the course of treatment.

Allowing for adjustment and/or delay of the dosage of temsirolimus and/or herceptin. For example, treatment may be for 6 months as described herein, with temsirolimus dosing weekly. Herceptin may be administered on a weekly basis for one cycle (e.g., three weeks). Typically, between cycles, 2 to 3 weeks are provided.

In certain instances, dosing of temsirolimus may be delayed or discontinued for a short period of time (e.g., 1,2, or 3 weeks) in a treatment regimen. Likewise, the cycle of treatment with herceptin may be shortened by one or more weeks, extended by one or more weeks, or the period of the week period may be extended or eliminated. Such delay or interruption may occur one or more times during the course of treatment.

Example 2: use of a combination regimen of HKI-272 and temsirolimus (CCI-779) for the treatment of neoplasia

HKI-272 was administered daily and temsirolimus was administered weekly Intravenously (IV) beginning on the first day of the first month at the doses provided below.

HKI-272 was administered orally on the first month, day one, prior to temsirolimus. Preference is given to temsirolimus after HKI-272 within 30 minutes.

Temsirolimus was administered IV weekly over 30 minutes using an in-line filter and an automated dispensing pump. Optionally, the antihistamine (diphenhydramine, 25 to 50 mg IV, or equivalent amount) is administered within about 30 minutes prior to the infusion of temsirolimus.

Therefore, HKI-272 is preferably administered orally with food once a day in the morning.

Allowing the adjustment and/or delay of the dosages of HKI-272 and temsirolimus. For example, treatment may be continued for 6 months, with HKI-272 administered daily and temsirolimus administered weekly as described herein. However, in some cases, administration of one or both of the agents may be delayed or discontinued for a short period of time (e.g., 1,2, or 3 weeks) during the course of treatment. Such delay or interruption may occur one or more times during the course of treatment.

Example 3: use of a combination regimen of HKI-272, temsirolimus (CCI-779) and herceptin in the treatment of neoplasia

HKI-272 was administered daily and temsirolimus and herceptin (IV) was administered weekly, beginning on the first day of the first month at the doses provided below.

HKI-272 was administered orally on the first month, day one, prior to temsirolimus. Temsirolimus and herceptin are administered preferably within 30 minutes after HKI-272.

The loading dose of herceptin was given IV weekly over 90 minutes. The weekly dose administered is typically half the amount of the loading dose. For example, a 4 mg/kg loading dose is usually followed by a weekly dose of 2 mg/kg. These amounts can be adjusted. In one embodiment, no loading dose is required and the same dose can be administered throughout the treatment.

Allowing the adjustment and/or delay of the dosage of HKI-272, temsirolimus and/or herceptin. For example, treatment may be for 6 months, with HKI-272 administered daily and temsirolimus administered weekly as described herein. Herceptin may be administered on a weekly basis for one cycle (e.g., three weeks). Typically, between cycles, 2 to 3 weeks are provided. However, in certain instances, the dosing of HKI-272 and/or temsirolimus may be delayed or discontinued for a short period of time (e.g., 1,2, or 3 weeks) during the course of a treatment regimen or procedure. Such delay or interruption may occur one or more times during the course of treatment.

Likewise, the cycle of treatment with herceptin may be shortened by one or more weeks, extended by one or more weeks, or the period of the week period may be extended or eliminated. Such delay or interruption may occur one or more times during the course of treatment.

Example 4: use of a combination regimen of HKI-272 and herceptin in the treatment of neoplasia

The antitumor activity of the HKI-272 and herceptin combination was demonstrated by standard pharmacological in vitro testing procedures. The procedure used and the results obtained are briefly described below.

This combination was determined in three breast cancer cell lines of different genotypes. More specifically, BT474[ HER-2+ (amplified); ATCC HTB-20]Is highly sensitive to both HKI-272 and herceptin. MDA-MB-361[ HER-2 ]⁺(non-amplified); adenocarcinoma; ATCC HTB27]Has low levels of non-amplified HER-2 and has less sensitivity to both herceptin and HKI-272. MCF-7[ HER-2 ]^-EGFR-; adenocarcinoma; ATCC HTB22]Has no HER-2 and is resistant to herceptin and HKI-272.

Cells selected from each of these cell lines were cultured in the presence of a certain concentration (0.0041, 0.012, 0.037, 0.11, 0.33, 0.1, 3 μ g/mg) of each drug.

At 37 ℃ and7％CO₂the cells were then maintained in RPMI 1640 medium (Life Technologies, inc., Gaithersburg, Md.) supplemented with 10% fetal bovine serum (FBS, Life Technologies) and 50 μ g/ml gentamicin (Life Technologies). Cells were plated in 96-well microtiter dishes (12,000 cells/well for BT474 cells, 6000 cells/well for MCF-7 cells, 10,000 cells/well for MDA-MB-361 cells) in 100. mu.l RPMI 1640 medium with 5% FBS and 50. mu.g/ml gentamicin and incubated overnight at 37 ℃. Compounds were diluted in the same medium at 2X final concentration and 100 μ Ι of drug dilution was added to the wells containing cells.

Serial dilutions of one compound were prepared in the presence of a fixed dose of the second compound. Alternatively, a checkerboard dilution series is used. Cells were cultured for 3 days in the presence of the drug. Untreated cells were included as controls. The percentage of surviving cells was determined using sulforhodamine (sulforhodamine) B (SRB, Sigma-Aldrich, St Louis, Mo.), a protein binding dye. The cellular proteins in each well were precipitated by adding 50 μ l of 50% cold trichloroacetic acid. After 1 hour, the plates were washed thoroughly in water and dried. SRB dye reagent (0.4% SRB in 1% acetic acid, 80 μ Ι per well) was added and the plate was held at room temperature for 10 minutes. The plates were then washed thoroughly in 1% acetic acid and dried. The cell-bound dye was dissolved in 10mM Tris (150. mu.l) and the absorbance read at 540nm in a microtiter plate reader. The concentration of compound that caused a fixed percentage of growth inhibition was determined by plotting the compound dose-cell viability (relative to untreated cells).

Models to study drug interactions have been developed by Prichard and Shipman in [ antiviral research.14: 181-206 (1990); prichard, MN et al, 1993, MacSynergy II.version 1.0. User's manual. university of Michigan, Ann Arbor. This is a three-dimensional model: the first two dimensions are used for each drug, and the third for biological effects. Theoretical additive interactions were calculated from each dose-response curve based on different positional models of additivity (Bliss independent model). The calculated addition plane (representing predicted cytotoxicity) was subtracted from the experimental plane, showing areas of enhanced toxicity (synergy) or reduced toxicity (antagonism). If the interaction is additive, the resulting surface appears as a 0% suppressed level above the calculated additive surface. The peaks and troughs from this plane represent synergy and antagonism, respectively. All calculations were performed automatically using MacSynergy II (Microsoft Excel-based software). This spreadsheet program calculates the theoretical additive interactions and locates and quantifies synergistic or antagonistic interactions that have significance at the 95% confidence level. These results are plotted as three-dimensional curves, or contour plots, with a plane at 0% representing the additive interaction, and peaks and troughs representing regions of synergy or antagonism, respectively, between the two drugs.

For this study, the Pritchad and Shipman methods were modified so that the combined effect at different levels of statistical significance (p values of 0.05, 0.01, 0.001) could be determined. A p value of 0.05 was considered significant. The method of assessing statistical variability in each experiment was also improved. Variability was assessed for all compound combinations, whereas in the original plate variability was assessed individually for each compound combination. It is believed that a better variability estimate is obtained using the improved method. Generally, a single point of synergy or antagonism is not considered to represent synergistic or antagonistic activity. Thus, single point peaks and troughs were not counted in the analysis. In addition, if no synergy or antagonism is observed at adjacent side concentrations, peaks or troughs occurring only along a single concentration of one of the compounds are not counted. Finally, all experiments were repeated at least twice and synergy and antagonism were determined by examining all data.

Figures 1-3 provide results from a set of experiments. In MDA-MB-361 cells, for HKI-272 at a concentration of 0.012 μ g/mL, the antagonistic region was present at 0.11-3 μ g/mL herceptin, with a 95% confidence level. In MCF7 cells, for HKI-272 at a concentration of 0.11 μ g/mL, there was a synergistic region at 0.037-0.33 μ g/mL herceptin, 95% confidence level. In BT474 cells, for HKI-272 at a concentration of 0.11. mu.g/mL, an antagonistic region was present at 0.33-1. mu.g/mL herceptin, at a 95% confidence level. When repeated at 99% confidence levels, no statistically significant antagonism or synergy zones were found. Based on the above, it is believed that the combination of herceptin and HKI-272 has an additive effect at all concentrations.

The results of these standard pharmacological test procedures from multiple independent experiments indicate that the combination of HKI-272 is not significantly antagonistic or synergistic, but has an additive effect over a range of concentrations. These data support the use of the combination in the treatment of HER2+ cancer. Because these combinations contain at least two active antineoplastic agents, the use of these combinations also provides for the use of a combination of each agent in which one or both agents are used in a supra-therapeutically effective amount, thereby reducing the toxicity associated with the use of a single chemotherapeutic agent.

All patents, patent publications, articles, and other documents referred to in this specification are incorporated herein by reference. It will be apparent to those skilled in the art that various modifications can be made to the specific embodiments described herein without departing from the scope of the specific invention.

Claims

1. A method of treating a neoplasm associated with overexpression or amplification of HER2 in a mammal in need thereof, which comprises providing to said mammal an effective amount of a combination of active ingredients comprising:

i) an mTOR inhibitor, and

ii) HKI-272 comprising (E) -N- {4- [ 3-chloro-4- (2-pyridylmethoxy) anilino ] -3-cyano-7-ethoxy-6-quinolinyl } -4- (dimethylamino) -2-butenamide, or a derivative or a pharmaceutically acceptable salt thereof.

2. The method of claim 1, wherein HKI-272 is (E) -N- {4- [ 3-chloro-4- (2-pyridylmethoxy) anilino ] -3-cyano-7-ethoxy-6-quinolinyl } -4- (dimethylamino) -2-butenamide or a pharmaceutically acceptable salt thereof.

3. The method of claim 1, wherein the mTOR inhibitor comprises:

i) rapamycin or a derivative or analogue thereof, or

ii) CCI-779 (temsirolimus) or a prodrug, derivative, pharmaceutically acceptable salt, or analog thereof.

4. The method of claim 1, wherein said mTOR inhibitor comprises rapamycin (sirolimus), 42-O- (2-hydroxy) ethyl rapamycin, or CCI-779 (temsirolimus).

5. The method of claim 2, wherein said mTOR inhibitor is CCI-779 (temsirolimus).

6. The method of claim 5, wherein the combination of active ingredients further comprises herceptin (trastuzumab).

7. The method of claim 5, wherein the combination further comprises one or more antineoplastic alkylating agents, one or more antimetabolite antineoplastic agents, one or more biochemical immunomodulators, imatinib, one or more EGFR inhibitors, multi-kinase inhibitors targeting serine/threonine and receptor tyrosine kinases in both tumor cells and tumor vasculature, or an interferon.

8. The method of claim 7, wherein:

i) the one or more antineoplastic agents comprise nitrogen mustard, cyclophosphamide, ifosfamide, melphalan, chlorambucil, thiotepa, mitomycin, busulfan, lomustine, carmustine, procarbazine, temozolomide, oxaliplatin, cisplatin, or carboplatin;

ii) the one or more antimetabolite antineoplastic agents comprise 5-fluorouracil, floxuridine, thioguanine, cytarabine, fludarabine, 6-mercaptopurine, methotrexate, gemcitabine, capecitabine, taxanes, pentostatin, trimetrexate, or cladribine; or

iii) the one or more biochemical modulators comprise folinic acid or levofolinic acid.

9. The method of claim 7, wherein the one or more antimetabolite antineoplastic agents are taxanes.

10. The method of claim 5, wherein the one or more active ingredients are provided in subtherapeutically effective amounts.

11. The method of claim 5, wherein the neoplasm comprises lung cancer, including bronchioloalveolar carcinoma and non-small cell lung cancer, breast cancer, myeloma, prostate cancer, head and neck cancer, transitional cell carcinoma, small cell and large cell neuroendocrine carcinoma of the cervix.

12. The method of claim 11, wherein the breast cancer is metastatic breast cancer.

13. The method of claim 5, wherein the active components of the combination are provided to the mammal simultaneously, separately, sequentially or in a staggered schedule.

14. The method of claim 5, wherein the active components of the combination are provided to the mammal in a formulation suitable for one or more of the following routes of administration: orally, as an aerosol, parenterally, intraperitoneally, transdermally, or as a suppository.

15. The method of claim 5, wherein the active ingredients provided are administered to the mammal using the same route of administration or using different routes of administration.

16. The method of claim 5, wherein said active ingredient is contained in a pharmaceutical pack or kit.

17. A method of treating a neoplasm associated with overexpression or amplification of HER2 in a mammal in need thereof, which comprises providing to said mammal an effective amount of a combination of active ingredients comprising:

i) herceptin (trastuzumab); and

18. The method of claim 17, wherein HKI-272 is (E) -N- {4- [ 3-chloro-4- (2-pyridylmethoxy) anilino ] -3-cyano-7-ethoxy-6-quinolinyl } -4- (dimethylamino) -2-butenamide or a pharmaceutically acceptable salt thereof.

19. The method of claim 18, wherein the combination further comprises one or more antineoplastic alkylating agents, one or more antimetabolite antineoplastic agents, one or more biochemical immunomodulators, imatinib, one or more EGFR inhibitors, multi-kinase inhibitors targeting serine/threonine and receptor tyrosine kinases in both tumor cells and tumor vasculature, or an interferon.

20. The method of claim 19, wherein:

21. The method of claim 19, wherein the one or more antimetabolite antineoplastic agents are taxanes.

22. The method of claim 21, wherein the one or more active ingredients are provided in subtherapeutically effective amounts.

23. The method of claim 21, wherein the neoplasm comprises lung cancer, including bronchioloalveolar carcinoma and non-small cell lung cancer, breast cancer, myeloma, prostate cancer, head and neck cancer, transitional cell carcinoma, small cell and large cell neuroendocrine carcinoma of the cervix.

24. The method of claim 21, wherein the breast cancer is metastatic breast cancer.

25. The method of claim 21, wherein the active components of the combination are provided to the mammal simultaneously, separately, sequentially or in a staggered schedule.

26. The method of claim 21, wherein the active components of the combination are provided to the mammal in a formulation suitable for one or more of the following routes of administration: orally, as an aerosol, parenterally, intraperitoneally, transdermally, or as a suppository.

27. The method of claim 21, wherein the active ingredients provided are administered to the mammal using the same route of administration or using different routes of administration.

28. The method of claim 21, wherein said active ingredient is contained in a pharmaceutical pack or kit.

29. A pharmaceutical composition for treating a neoplasm in a mammal, said composition comprising:

i) one or more units of an mTOR inhibitor;

ii) one or more units of HKI-272 comprising (E) -N- {4- [ 3-chloro-4- (2-pyridylmethoxy) anilino ] -3-cyano-7-ethoxy-6-quinolinyl } -4- (dimethylamino) -2-butenamide, or a derivative or pharmaceutically acceptable salt thereof; and

iii) at least one pharmaceutically acceptable carrier.

30. The pharmaceutical composition of claim 29, wherein HKI-272 is (E) -N- {4- [ 3-chloro-4- (2-pyridylmethoxy) anilino ] -3-cyano-7-ethoxy-6-quinolinyl } -4- (dimethylamino) -2-butenamide or a pharmaceutically acceptable salt thereof.

31. The pharmaceutical composition of claim 30, wherein the mTOR inhibitor comprises:

i) rapamycin or a derivative or analogue thereof, or

32. The pharmaceutical composition of claim 30, wherein said mTOR inhibitor comprises rapamycin (sirolimus), 42-O- (2-hydroxy) ethyl rapamycin, or CCI-779 (temsirolimus).

33. The pharmaceutical composition of claim 30, wherein said mTOR inhibitor is CCI-779 (temsirolimus).

34. The pharmaceutical composition of claim 33, wherein the pharmaceutical composition is contained in one or more pharmaceutical packs or one or more kits.

35. The pharmaceutical composition of claim 33, wherein the pharmaceutical composition further comprises one or more units of herceptin (trastuzumab).

36. The pharmaceutical composition of claim 33, wherein the combination further comprises one or more antineoplastic alkylating agents, one or more antimetabolite antineoplastic agents, one or more biochemical immunomodulators, imatinib, one or more EGFR inhibitors, a multi-kinase inhibitor targeting serine/threonine and receptor tyrosine kinases in both tumor cells and tumor vasculature, or an interferon.

37. The pharmaceutical composition of claim 36, wherein:

38. The pharmaceutical composition of claim 36, wherein the one or more antimetabolite antineoplastic agents are taxanes.

39. The pharmaceutical composition of claim 38, wherein the pharmaceutical composition is contained in one or more pharmaceutical packs or one or more pharmaceutical kits.

40. A pharmaceutical composition for treating a neoplasm in a mammal, said composition comprising:

i) one or more units of herceptin (trastuzumab);

iii) at least one pharmaceutically acceptable carrier.

41. The pharmaceutical composition of claim 40, wherein HKI-272 is (E) -N- {4- [ 3-chloro-4- (2-pyridylmethoxy) anilino ] -3-cyano-7-ethoxy-6-quinolinyl } -4- (dimethylamino) -2-butenamide or a pharmaceutically acceptable salt thereof.

42. The pharmaceutical composition of claim 41, wherein the pharmaceutical composition is contained in one or more pharmaceutical packs or one or more pharmaceutical kits.

43. The pharmaceutical composition of claim 41, wherein the combination further comprises one or more antineoplastic alkylating agents, one or more antimetabolite antineoplastic agents, one or more biochemical immunomodulators, imatinib, one or more EGFR inhibitors, multi-kinase inhibitors targeting serine/threonine and receptor tyrosine kinases in both tumor cells and tumor vasculature, or an interferon.

44. The pharmaceutical composition of claim 43, wherein:

45. The pharmaceutical composition of claim 43, wherein the one or more antimetabolite antineoplastic agents are taxanes.

46. The pharmaceutical composition of claim 45, wherein the pharmaceutical composition is contained in one or more pharmaceutical packs or one or more pharmaceutical kits.

47. A product, comprising:

i) an mTOR inhibitor; and

ii) HKI-272 comprising (E) -N- {4- [ 3-chloro-4- (2-pyridylmethoxy) anilino ] -3-cyano-7-ethoxy-6-quinolinyl } -4- (dimethylamino) -2-butenamide, or a derivative or pharmaceutically acceptable salt thereof

As a combined preparation for simultaneous, separate or sequential use in the treatment of a neoplasm in a mammal.

48. The product of claim 47, wherein HKI-272 is (E) -N- {4- [ 3-chloro-4- (2-pyridylmethoxy) anilino ] -3-cyano-7-ethoxy-6-quinolinyl } -4- (dimethylamino) -2-butenamide or a pharmaceutically acceptable salt thereof.

49. The article of manufacture of claim 48, wherein said mTOR inhibitor comprises:

i) rapamycin or a derivative or analogue thereof, or

50. The product of claim 48, wherein said mTOR inhibitor comprises rapamycin (sirolimus), 42-O- (2-hydroxy) ethyl rapamycin, or CCI-779 (temsirolimus).

51. The product of claim 48, wherein said mTOR inhibitor is CCI-779 (temsirolimus).

52. The product of claim 51, further comprising herceptin (trastuzumab).

53. The product of claim 51, wherein the product further comprises one or more antineoplastic alkylating agents, one or more antimetabolite antineoplastic agents, one or more biochemical immunomodulators, imatinib, one or more EGFR inhibitors, multi-kinase inhibitors targeting serine/threonine and receptor tyrosine kinases in both tumor cells and tumor vasculature, or interferons.

54. The product of claim 53, wherein the one or more antimetabolite antineoplastic agents are taxanes.

55. The product of claim 54, further comprising herceptin (trastuzumab).

56. A product, comprising:

i) herceptin (trastuzumab); and

57. The product of claim 56, wherein HKI-272 is (E) -N- {4- [ 3-chloro-4- (2-pyridylmethoxy) anilino ] -3-cyano-7-ethoxy-6-quinolinyl } -4- (dimethylamino) -2-butenamide or a pharmaceutically acceptable salt thereof.

58. The product of claim 57, wherein the product additionally comprises one or more antineoplastic alkylating agents, one or more antimetabolite antineoplastic agents, one or more biochemical immunomodulators, imatinib, one or more EGFR inhibitors, multi-kinase inhibitors targeting serine/threonine and receptor tyrosine kinases in both tumor cells and tumor vasculature, or interferons.

59. The product of claim 58, wherein said one or more antimetabolite antineoplastic agents are taxanes.

60. A pharmaceutical pack containing a course of anti-tumour therapy for a mammalian subject, wherein the pack contains:

i) at least one unit of an mTOR inhibitor in unit dosage form; and

ii) at least one unit of HKI-272 in unit dosage form comprising (E) -N- {4- [ 3-chloro-4- (2-pyridylmethoxy) anilino ] -3-cyano-7-ethoxy-6-quinolinyl } -4- (dimethylamino) -2-butenamide, or a derivative or pharmaceutically acceptable salt thereof.

61. The pharmaceutical pack according to claim 60, wherein HKI-272 is (E) -N- {4- [ 3-chloro-4- (2-pyridylmethoxy) anilino ] -3-cyano-7-ethoxy-6-quinolinyl } -4- (dimethylamino) -2-butenamide or a pharmaceutically acceptable salt thereof.

62. The pharmaceutical pack of claim 61, wherein the mTOR inhibitor comprises:

i) rapamycin or a derivative or analogue thereof, or

63. The pharmaceutical pack of claim 61, wherein the mTOR inhibitor comprises rapamycin (sirolimus), 42-O- (2-hydroxy) ethyl rapamycin, or CCI-779 (temsirolimus).

64. The pharmaceutical pack of claim 63, further comprising (c) at least one unit of herceptin (trastuzumab).

65. The pharmaceutical pack of claim 61, wherein the mTOR inhibitor is CCI-779 (temsirolimus).

66. The pharmaceutical pack of claim 65, further comprising (c) at least one unit of herceptin (trastuzumab).

67. A pharmaceutical pack containing a course of anti-tumour therapy for a mammalian subject, wherein the pack contains:

i) at least one unit of herceptin (trastuzumab) in unit dosage form; and

68. The pharmaceutical pack according to claim 67, wherein HKI-272 is (E) -N- {4- [ 3-chloro-4- (2-pyridylmethoxy) anilino ] -3-cyano-7-ethoxy-6-quinolinyl } -4- (dimethylamino) -2-butenamide or a pharmaceutically acceptable salt thereof.

69. The pharmaceutical pack of claim 68, wherein said product additionally comprises one or more antineoplastic alkylating agents, one or more antimetabolite antineoplastic agents, one or more biochemical immunomodulators, imatinib, one or more EGFR inhibitors, multi-kinase inhibitors targeting serine/threonine and receptor tyrosine kinases in both tumor cells and tumor vasculature, or interferons.

70. The pharmaceutical pack of claim 69, wherein the one or more antimetabolite antineoplastic agents are taxanes.