ANTINEOPLASTIC COMBINATIONS WITH MTOR INHIBITOR,
HERCEPTIN, AND/OR HKI-272
BACKGROUND OF THE INVENTION
This invention relates to the use of combinations of herceptin with an mTOR
inhibitor and/or HKI-272, for the treatment of neoplasms associated with
overexpression or amplification of HER2.
CCI-779, is rapamycin 42-ester with 3-hydroxy-2-(hydroxymethyl)-2-
methylpropionic acid, an ester of rapamycin which has demonstrated significant
inhibitory effects on tumor growth in both in vitro and in vivo models. This
compound is now known generically under the name temsirolirnus. The preparation
and use of hydroxyesters of rapamycin, including temsirolimus, are described in US
Patents 5,362,718 and 6,277,983.
Temsirolimus exhibits cytostatic, as opposed to cytotoxic properties, and may
delay the time to progression of tumors or time to tumor recurrence. Temsirolimus is
considered to have a mechanism of action that is similar to that of sirolimus.
Temsirolimus binds to and forms a complex with the cytoplasmic protein FKBP,
which inhibits an enzyme, mTOR (mammalian target of rapamycin, also known as
FKBP12-rapamycin associated protein [FRAP]). Inhibition of mTOR's lrinase
activity inhibits a variety of signal transduction pathways, including cytokine-
stimulated cell proliferation, translation of mRNAs for several key proteins that
regulate the Gl phase of the cell cycle, and IL-2-induced transcription, leading to
inhibition of progression of the cell cycle from Gl to S. The mechanism of action of
temsirolimus that results in the Gl-S phase block is novel for an anticancer drag.
Metastatic breast cancer (MBC) is essentially incurable with standard therapy,
and patients with MBC have a median survival of about 2 years after documentation
of metastasis. As a consequence, the goals of treatment are to improve patients'
symptoms while trying to maintain (or improve, in certain cases) quality of life.
Prolonging survival remains a clear goal, particularly in breast cancer that has
overexpression or amplification of the her-2 oncogene.

Herceptin® (Trastuzumab) is an FDA-approved therapeutic monoclonal
antibody for HER2 protein overexpressing metastatic breast cancer. A murine
monoclonal antibody was described [see, US Patent 5,705,151]. The murine
MAb4D5 molecule described in that document has been humanized in an attempt to
improve its clinical efficacy by reducing immunogenicity and allowing it to support
human effector functions. WO 92/22653. Later documents describe the development
of a lyophilized formulation comprising full length humanized antibody huMAb4D5-8
described in WO 92/22653. Herceptin is currently approved by the FDA for the
treatment of metastatic breast cancer that overexpresses HER2, (1) as a single agent
after previous treatment of the metastatic breast cancer with one or more
chemotherapy regimens and (2) in combination with paclitaxel in such patients
without prior chemotherapy for their metastatic breast cancer. Moreover, there is
evidence that the addition of herceptin to taxane adjuvant or neoadjuvant
chemotherapy improves to patients with earlier stage breast cancer.
HKI-272, (E)-N-{4-[3-chloro-4-(2-pyridinylmethoxy) anilino]-3-cyano-7-
etioxy-6-qumolmyl}-4-(dimethylamino)-2-butenamide, has been described as a
promising anticancer drug candidate for the treatment of breast cancers and other
HER-2-dependent cancers. Because it also inhibits the EGFR kinase with similar
potency, HKI-272 may be useful to treat tumors that overexpress both HER-2 and
EGFR and be more efficacious than a specific EGFR or HER-2 antagonist. S. K.
Rabindran et al, "Antitumor Activity of HKI-272, an Orally Active, Irreversible
Inhibitor of the HER-2 Tyrosine Kinase", Cancer Research 64,3958-3965, June 1,
2004. See, US Patent No. 6,288,082; US Patent No. 6,297,258.
What is needed is an improved antineoplastic therapy.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 is a 3-dimensional contour plot with the plane at 0 % representing
additive interaction, and peaks and valleys representing areas of synergy or
antagonism, respectively, between the herceptin and HKI-272 in BT474 [HER-2+
(amplified); ATCC HTB-20] cells.

Fig. 2 is a 3-dimensional contour plot with the plane at 0 % representing
additive interaction, and peaks and valleys representing areas of synergy or
antagonism between the herceptin and HKI-272 in MCF-7 [HER-2", EGFR-;
adenocarcinoma; ATCC HTB22] cells.
Fig. 3 is a 3-dimensional contour plot with the plane at 0 % representing
additive interaction, and peaks and valleys representing areas of synergy or
antagonism between the herceptin and HKI-272 in MDA-MB-361 [HER-2+ (non-
amplified); adenocarcinoma; ATCC HTB 27] cells.
DETAILED DESCRIPTION OF THE INVENTION
This invention provides the use of combinations comprising herceptin, an
mTOR inhibitor and/or HKI-272 in the treatment of neoplasms. Thus, the invention
provides for the combined use of a herceptin with an mTOR inhibitor, the combined
use of herceptin with an HKI-272, the combined use of an mTOR inhibitor with HKI-
272, or the combined use of a herceptin with mTOR inhibitor and an HKI-272. The
invention further provides products containing a herceptin in combination with an
mTOR inhibitor and/or HKI-272 formulated for simultaneous, separate or sequential
use in treating neoplasms in a mammal. The invention is also useful as an adjuvant
and/or neoadjuvant therapy of earlier stages of breast cancer. The following detailed
description illustrates 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 in the treatment of a
variety of neoplasms associated with overexpression or amplification of HER2,
including, for example, lung cancers, including bronchioalveolar carcinoma and non
small cell lung cancer, breast cancers, prostate cancers, myeloma, head and neck
cancer, or transitional cell carcinoma; small cell and large cell neuroendocrine
carcinoma of the uterine cervix.
In one embodiment, the combination of temsirolimus and herceptin is
particularly well suited for treatment of metastatic breast cancer. In another
embodiment, the combination of herceptin and a mTOR inhibitor and/or an HKI-272,

are well suited for treatment of breast, kidney, bladder, mouth, larynx, esophagus,
stomach, colon, ovary, and lung), and polycystic kidney disease.
As used herein, the term mTOR inhibitor means a compound or ligand, or a
pharmaceutically acceptable salt thereof, that inhibits cell replication by blocking the
progression of the cell cycle from G1 to S. The term includes the neutral tricyclic
compound rapamycin (sirolimus) and other rapamycin compounds, including, e.g.,
rapamycin derivatives, rapamycin analogues, other macrolide compounds that inhibit
mTOR activity, and all compounds included within the definition below of the term "a
rapamycin". These include compounds with a structural similarity to "a rapamycin",
e.g., compounds with a similar macrocyclic structure that have been modified to
enhance therapeutic benefit. FK.-506 can also be used in the method of the invention.
As used herein, the term a rapamycin defines a class of immunosuppressive
compounds that contain the basic rapamycin nucleus as shown below.

The rapamycins of this invention include compounds that are chemically or
biologically modified as derivatives of the rapamycin nucleus, while still retaining
immunosuppressive properties. Accordingly, the term a rapamycin includes
rapamycin, and esters, ethers, carbamates, oximes, hydrazones, and hydroxylamines of
rapamycin, as well as rapamycins in which functional groups on the rapamycin
nucleus have been modified, for example through reduction or oxidation. Also
included in the term a rapamycin are pharmaceutically acceptable salts of
rapamycins.
The term a rapamycin also includes 42- and/or 31-esters and ethers of
rapamycin as described in the following patents, which are all hereby incorporated by

reference: alkyl esters (U.S. Patent No. 4,316,885); aminoalkyl esters (U.S. Patent No.
4,650,803); fluorinated esters (U.S. Patent No. 5,100,883); amide esters (U.S. Patent
No. 5,118,677); carbamate esters (U.S. Patent No. 5,118,678); silyl esters (U.S.
Patent No. 5,120,842); aminodiesters (U.S. Patent No. 5,162,333); sulfonate and
sulfate esters (U.S. Patent No. 5,177,203); esters (U.S. Patent No. 5,221,670);
alkoxyesters (U.S. Patent No. 5,233,036); O-aryl, -alkyl, -alkenyl, and -alkynyl ethers
(U.S. Patent No. 5,258,389); carbonate esters (U.S. Patent No. 5,260,300);
arylcarbonyl and alkoxycarbonyl carbamates (U.S. Patent No. 5,262,423); carbamates
(U.S. Patent No. 5,302,584); hydroxyesters (U.S. Patent No. 5,362,718); hindered
esters (U.S. Patent No. 5,385,908); heterocyclic esters (U.S. Patent No. 5,385,909);
gem-disubstituted esters (U.S. Patent No. 5,385,910); amino alkanoic esters (U.S.
Patent No. 5,389,639); phosphorylcarbamate esters (U.S. Patent No. 5,391,730);
carbamate esters (U.S. Patent No. 5,411,967); carbamate esters (U.S. Patent No.
5,434,260); amidino carbamate esters (U.S. Patent No. 5,463,048); carbamate esters
(U.S. Patent No. 5,480,988); carbamate esters (U.S. Patent No. 5,480,989); carbamate
esters (U.S. Patent No. 5,489,680); hindered N-oxide esters (U.S. Patent No.
5,491,231); biotin esters (U.S. Patent No. 5,504,091); O-alkyl ethers (U.S. Patent No.
5,665,772); and PEG esters of rapamycin (U.S. Patent No. 5,780,462). The
preparation of these esters and ethers is disclosed in the patents listed above.
Further included within the definition of the term a rapamycin are 27-esters
and ethers of rapamycin, which are disclosed in U.S. Patent No. 5,256,790. Also
described are C-27 ketone rapamycins which are reduced to the corresponding
alcohol, which is in turn converted to the corresponding ester or ether. The preparation
of these esters and ethers is disclosed in the patent listed above. Also included are
oximes, hydrazones, and hydroxylamines of rapamycin are disclosed in U.S. Patent
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-listed patents. The
preparation of 42-oxorapamycin is disclosed in 5,023,263.
As used herein, the term a CCI-779 means rapamycin 42-ester with 3-
hydroxy-2-(hydroxymethyl)-2-methylpropionic acid (temsirolimus), and encompasses
prodrugs, derivatives, pharmaceutically acceptable salts, or analogs thereof.

Examples of arapatnycin include, e.g., rapamycin, 32-deoxorapamycin, 16-
pent-2-ynyloxy-32-deoxorapamycin, 16-pent-2-ylyloxy-32(S)-dihydro-rapamycin, 16-
pent-2-ylyloxy-32(S)-dihydr-o-40-O-(2-hydroxyethyl)-rapamycin, 40-O-(2-
hydroxyethyl)-rapamycin, rapamycin 42-ester with 3-hydroxy-2-(hydroxymethyl)-2-
methylpropionic acid (CCI-779), 40-[3-hydroxy-2-(hydroxymethyl)-2-meth-
ylpropanoate]-rapamycin, or apharmaceutically acceptable salt thereof, as disclosed
in U.S. Patent No. 5,362,718, ABT578, or 40-(tetrazolyl)-rapamycin, 40-epi-
(tetrazolyl)-rapamycin, e.g., as disclosed in International Patent Publication No. WO
99/15530, or rapamycin analogs as disclosed in International Patent Publication No.
WO 98/02441 and WO 01/14387, e.g., AP23573. In another embodiment, the
compound is Certican™ (everolimus, 2-O-(2-hydroxy)ethyl rapamycin, Novartis, U.S.
Patent No. 5,665,772).
As used herein, "an HKI-272" refers to a compound having the following

or a derivative or pharmaceutically acceptable salt thereof. Suitable derivatives may
include, e.g., an ester, ether, or carbamate. The core structure, HKI-272, has the
chemical name (E)-N-{4-[3-chloro-4-(2-pyridinyhnethoxy) anilino] -3-cyano-7-
ethoxy-6-quinolinyl}-4-(dimethylamino)-2- -butenamide.
In one embodiment, the invention also provides for use of substituted 3-cyano
quinolines having structure:


where R1 is halogen;
R2 is pyridinyl, thiophene, pyrimidine, thiazole, or phenyl optionally
substituted with up to three substituents;
R3is-O-or-S-;
R4 is methyl or CH2CH2OCH3;
R5 is ethyl or methyl; and
n is O or l.
These compounds, of which HKI-272 is a species, are characterized by the ability to
act as potent HER-2 inhibitors. See, e.g., US Patent 6,288,082 and US Patent
6,297,258. These compounds and their preparation are described in detail in US
Published Patent Application No. 2005/0059678. For convenience, HKI-272 is used
throughout this specification. However, it will be understood that the compound of
the structure provided above can be substituted for HKI-272 in the combinations with
an mTOR inhibitor and/or herceptin which are described in detail below.
The following standard pharmacological test procedure can be used to
determine whether a compound is an mTOR inhibitor, as defined herein. Treatment of
growth factor stimulated cells with an mTOR inhibitor like rapamycin completely
blocks phosphorylation of serine 389 as evidenced by Western blot and as such
constitutes a good assay for mTOR inhibition. Thus, whole cell lysates from cells
stimulated by a growth factor (e.g-. IGF1) in culture in the presence of an mTOR
inhibitor should fail to show a band on an acrylamide gel capable of being labeled
with an antibody specific for serine 389 of p70s6K.

It is preferred that the mTOR inhibitor used in the antineoplastic combinations
of this invention is a rapamycin, and more preferred that 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. Patent 5,665,772.
The preparation of temsirolimus is described in U.S. Patent 5,362,718. A
regiospecific synthesis of temsirolimus is described in US Patent 6,277,983, which is
hereby incorporated by reference. Still another regiospecific method for synthesis of
temsirolimus is described in US Patent Publication No. 2005-0033046-A1, published
Feb 10, 2005 (Application No. 10/903,062, filed July 30, 2004), and its counterpart,
International Patent Publication No. WO 2005/016935, published Apr 7, 2005.
Herceptin, and methods of making and formulating same have been described.
See, e.g., US Patent 6,821,515; US Patent No. 6,399,063 and US Patent No.
6,387,371. Herceptin is available commercially from Genentech. As used herein, the
term "a herceptin" includes includes trastuzumab and altered forms of, and derivatives
of, trastuzumab. The term "a herceptin" includes agents that target the same epitope
on the Her-2 receptor as targeted by trastuzumab. The epitope is known from H.S.
Cho et ah, Structure of the extracellular region of HER2 alone and in complex with
the Herceptin Fab, Nature 421 (2003), pp. 756-760.
HKI-272 and methods of making and formulating same have been described.
See, e.g., US Published Patent Application No. 2005/0059678; US Patent No.
6,002,008, can also be used to prepare the substituted 3-quinoline compounds used
this invention and are hereby incorporated by reference. In addition to the methods
described in these documents, WO-9633978 and WO-9633980 describe methods that
are useful for the preparation of these compounds. Although these methods describe
the preparation of certain quinazolines, they are also applicable to the preparation of
correspondingly substituted 3-cyanoquinolines and are hereby incorporated by
reference.
As used in accordance with this invention, the term "treatment" means treating
a mammal having a neoplasm by providing said mammal an effective amount of a
combination of a two or three-way combination of the components selected from an
mTOR inhibitor, a herceptin and/or HKI-272 with the purpose of inhibiting

progression of the neoplastic disease, growth of a tumor in such mammal, eradication
of the neoplastic disease, prolonging survival of the mammal and/or palliation of the
mammal.
As used in accordance with this invention, the term "providing," with respect
to providing an mTOR inhibitor with herceptin and/or HKI-272, means either directly
administering the mTOR inhibitor, or administering a prodrug, derivative, or analog
which will form an effective amount of the mTOR inhibitor within the body, along
with herceptin and/or HKI-272 directly, or administering a prodrug, derivative, or
analog which will form an effective amount of herceptin or HKI-272 in the body.
Use of a combination of an mTOR inhibitor (e.g., temsirolimus), a herceptin
and/or HKI-272 also provides for the use of combinations of each of the agents in
which one, two, or all three agents is used at subtherapeutically effective dosages.
Subtherapeutically effective dosages may be readily determined by one of skill in the
art, in view of the teachings herein. In one embodiment, the subtherapeutically
effective dosage is a dosage which is effective at a lower dosage when used in the
combination regimen of the invention, as compared to the dosage that is effective
when used alone. The invention further provides for one or more of the active agents
in the combination of the invention to be used in a supratherapeutic amount, i.e., at a
higher dosage in the combination than when used alone. In this embodiment, the
other active agent(s) may be used in a therapeutic or subtherapeutic amount.
The combinations of the invention may be in the form of a kit of parts. The
invention therefore includes a product containing an mTOR inhibitor, a herceptin
and/or HKI-272 as a combined preparation for simultaneous, separate or sequential
delivery for the treatment of a neoplasm in a mammal in need thereof. In one
embodiment, a product contains temsirolimus and a herceptin as a combined
preparation for simultaneous, separate or sequential use in treating a neoplasm in a
mammal in need thereof. Optionally, the product further contains an HKI-272. HKI-
272 may be separately formulated, e.g., for oral delivery. In another embodiment, a
product contains temsirolimus and an HKI-272 as a combined preparation for
simultaneous, separate or sequential use in a neoplasm in a mammal in need thereof.
Optionally, the product further contains herceptin. In yet another embodiment, the

product contains a herceptin and an HKI-272. Optionally, the product further contains
an mTOR inhibitor. In one embodiment, the neoplasm is metastatic breast cancer.
In one embodiment, a pharmaceutical pack contains a course of treatment of a
neoplasm for one individual mammal, wherein the pack contains units of an mTOR
inhibitor in unit dosage form and units of herceptin in unit dosage form, optionally
further in combination with units of an HKI-272 in unit dosage form. In another
embodiment, a pharmaceutical pack contains a course of treatment of a neoplasm for
one individual mammal, wherein the pack contains-units of an mTOR inhibitor in unit
dosage form and units of HKI-272 in unit dosage form, optionally further in
combination with units of herceptin in unit dosage form. In yet another embodiment,
a pharmaceutical pack contains a course of treatment of a neoplasm for one individual
mammal, wherein the pack contains units of a herceptin in unit dosage form and units
of HKI-272 in unit dosage form, optionally further in combination with units of an
mTOR inhibitor in unit dosage form. la one embodiment, a pharmaceutical pack as
described herein contains a course of treatment of metastatic breast cancer for one
individual mammal.
Administration of the compositions may be oral, intravenous, respiratory (e.g.,
nasal or intrabronchial), infusion, parenteral (besides i.v., such as intralesional,
intraperitoneal and subcutaneous injections), intraperitoneal, transdermal (including
all administration across the surface of the body and the inner linings of bodily
passages including epithelial and mucosal tissues), and vaginal (including intrauterine
administration). Other routes of administration are also feasible, such as via liposome-
mediated delivery; topical, nasal, sublingual, uretheral, intrathecal, ocular or otic delivery,
implants, rectally, intranasally.
While the components of the invention may be delivered via the same route, a
product or pack according to the invention may contain a rapamycin, such as
temsirolimus, for delivery by a different route than that of the herceptin or the HKI-
272, e.g., one or more of the components may be delivered orally, while one or more
of the others are administered intravenously. In one embodiment, temsirolimus is
prepared for oral delivery, HKI-272 is prepared for oral delivery and herceptin is
prepared for intravenous delivery. In another embodiment, both temsirolimus and

herceptin are prepared for intravenous delivery. In still another embodiment, all of the
components are prepared for oral delivery. Optionally, other active components may
be delivered by the same or different routes as the mTOR inhibitor (e.g.,
temsirolimus) or herceptin. Other variations would be apparent to one skilled in the
art and are contemplated within the scope of the invention.
The mTOR inhibitor plus herceptin combination may be administered in the
absence of HKI-272. In one embodiment, these are the sole active antineoplastic
agents utilized in the regimen. In another embodiment, the mTOR inhibitor/herceptin
combination is administered in combination with HKI-272.
The mTOR inhibitor plus HKI-272 combination may 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,
these two and three-way combinations are the sole active antineoplastic agents utilized
in the regimen. In another embodiment, these two and three-way combinations may
be utilized in further combination with other active agents.
The herceptin plus HKI-272 combination 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, these two
and three-way combinations are the sole active antineoplastic agents utilized in the
regimen. In another embodiment, these two and three-way combinations may be
utilized in further combination with other active agents.
As is typical with oncology treatments, dosage regimens are closely monitored
by the treating physician, based on numerous factors including the severity of the
disease, response to the disease, any treatment related toxicities, age, and health of the
patient. Dosage regimens are expected to vary according to the route of
administration.
It is projected that initial i.v. infusion dosages of the mTOR inhibitor (e.g.,
temsirolimus) will be from about 5 to about 175 mg, or about 5 to about 25 mg, when
administered on a weekly dosage regimen. It is projected that the oral dosage of an
mTOR useful in the invention will be 10 mg/ week to 250 mg/week, about 20
mg/week to about 150 mg/week, about 25 mg/week to about 100 mg/week, or about

30 mg/week to about 75 mg/week. For rapamycin, the projected oral dosage will be
between 0.1 mg/day to 25 mg/day. Precise dosages will be determined by the
administering physician based on experience with the individual subject to be treated.
Other dosage regimens and variations are foreseeable, and will be determined
through physician guidance. It is preferred that the mTOR inhibitor is administered
by i.v. infusion or orally, preferably in the form of tablets or capsules.
For herceptin, single doses and multiple doses are contemplated. In one
embodiment, a single loading dose of herceptin is administered as a 90-minute
intravenous infusion in a range of about 4- 5 mg/kg on day 1, followed by about 2
mg/kg per week starting on day 8. Typically, 3 weeks is 1 cycle. From 1, to 2 to 3,
weeks may be provided between cycles. Herceptin may also be given at a dose of 6
mg/kg once every 3-4 weeks. In addition, herceptin may also be given after
completion of chemotherapy as maintenance therapy.
For an HKI-272, it is desired that a compound of the invention is in the form
of a unit dose. Suitable unit dose forms include tablets, capsules and powders in
sachets or vials. Such unit dose forms may contain from 0.1 to 300 mg of a compound
of the invention and preferably from 2 to 100 mg. Still further preferred unit dosage
forms contain 5 to 50 mg of a compound of the present invention. The compounds of
the present invention can be administered at a dose range of about 0.01 to 100 mg/kg
or preferably at a dose range of 0.1 to 10 mg/kg. In one embodiment, the compounds
are administered orally from 1 to 6 times a day, more usually from 1 to 4 times a day.
Alternatively, the compounds may be administered through another suitable route,
e.g., intravenous. In still another embodiment, the compounds are administered once
a week. In certain situations, dosing with the HKI-272 may be delayed or
discontinued for a brief period (e.g., 1,2 or three weeks) during the course of
treatment. Such a delay or discontinuation may occur once, or more, during the
course of treatment. The effective amount will be known to one of skill in the art; it
will also be dependent upon the form of the compound. One of skill in the art could
routinely perform empirical activity tests to determine the bioactivity of the compound
in bioassays and thus determine what dosage to administer.

These regimens may be repeated, or alternated, as desired. Other dosage
regimens and variations are foreseeable, and will be determined through physician
guidance.
For example, in one embodiment, the regimen further comprises
administration of a taxane, e.g., docetaxel and paclitaxel [e.g., a suspension of
paclitaxel bound to albumen nanoparticles, which is available as Abraxane].
Paclitaxel may also be administered on a weekly schedule, at doses 60 - 100 mg/m2
administered over 1 hour, weekly, or 2 - 3 weekly doses followed by a one week rest.
In one embodiment, paclitaxel is administered intravenously over 3 hours at a dose of
175 mg/m2, optionally followed by cisplatin at a dose of 75 mg/m2; or paclitaxel
administered intravenously over 24 hours at a dose of 135 mg/m2, optionally followed
by cisplatin at a dose of 75 mg/m2. In patients previously treated with therapy for
carcinoma, paclitaxel can be injected at several doses and schedules. However, the
optimal regimen is not yet clear. The recommended regimen is paclitaxel 135 mg/m2
or 175 mg/m2 administered intravenously over 3 hours every 3 weeks. These doses
may be altered as needed or desired.
Still other active agents may be included in a combination with an mTOR
inhibitor and herceptin, including, e.g., chemotherapeutic agents, such as alkylating
agents; hormonal agents (i.e., estramustine, tamoxifen, toremifene, anastrozole, or
letrozole); antibiotics (i.e., plicamycin, bleomycin, mitoxantrone, idarubicin,
dactinomycin, mitomycin, or daunorubicin); antimitotic agents {i.e., vinblastine,
vincristine, teniposide, or vinorelbine, available as Navelbine); topoisomerase
inhibitors (i.e., topotecan, irinotecan, etoposide, or doxorubicin, e.g., CAELYX or
Doxil, pegylated liposomal doxorubicin hydrochloride); and other agents (i.e.,
hydroxyurea, altretamine, rituximab, paclitaxel, docetaxel, L-asparaginase, or
gemtuzumab ozogamicin); biochemical modulating agents, imatib, EGFR inhibitors
such as EKB-569 or other multi-kinase inhibitors, e.g., those that targets
serine/threonine and receptor tyrosine kinases in both the tumor cell and tumor
vasculature, or immunomodulators (i.e., interferons, IL-2, or BCG). Examples of
suitable interferons include interferon α, interferon β, interferon λ, and mixtures
thereof.

In one embodiment, the combination of an mTOR inhibitor and herceptin may
be further combined with antineoplastic alkylating agents, e.g., those described in US
2002-0198137A1. Antineoplastic alkylating agents are roughly classified, according
to their structure or reactive moiety, into several categories which include nitrogen
mustards, such as MUSTARGEN (meclorethamine), cyclophosphamide, ifosfamide,
melphalan, and chlorambucil; azidines and epoxides, such as thiotepa, mitomycin C,
dianhydrogalactitol, and dibromodulcitol; alkyl sulfinates, such as busulfan;
nitrosoureas, such as bischloroethylnitrosourea (BCNU), cyclohexyl-
chloroethylnitrosourea (CCNU), and methylcyclohexylchloroethyhiitrosourea
(MeCCNU); hydrazine and triazine derivatives, such as procarbazine, dacarbazine,
and temozolomide; streptazoin, melphalan, chlorambucil, carmustine,
methclorethamine, lomustine)and platinum compounds. Platinum compounds are
platinum containing agents that react preferentially at the N7 position of guanine and
adenine residues to form a variety of monofunctional and bifunctional adducts.
(Johnson S W, Stevenson J P, O'Dwyer P J. Cisplatin and Its Analogues, hi Cancer
Principles & Practice of Oncology 6th Edition, ed. 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 representative examples of alkylating agents of this
invention. Meclorethamine is commercially available as an injectable
(MUSTARGEN). Cyclophosphamide is commercially available as an injectable
(cyclophosphamide, lyophilized CYTOXAN, or NEOSAR) and in oral tablets
(cyclophosphamide or CYTOXAN). Ifosfamide is commercially available as an
injectable (EFEX). Melphalan is commercially available as an injectable (ALKERAN)
and in oral tablets (ALKERAN). Chlorambucil is commercially available in oral
tablets (LEUKERAN). Thiotepa is commercially available as an injectable (thiotepa
or THIOPLEX). Mitomycin is commercially available as an injectable (mitomycin or
MUTAMYCIN). Busulfan is commercially available as an injectable (BUSULFEX)
and in oral tablets (MYLERAN). Lomustine (CCNU) is commercially available in
oral capsules (CEENU). Carmustine (BCNU) is commercially available as an

intracranial implant (GLIADEL) and as an injectable (BICNU). Procarbazine is
commercially available in oral capsules (MATULANE). Temozolomide is
commercially available in oral capsules (TEMODAR). Cisplatin is commercially
available as an injectable (cisplatin, PLATENOL, or PLATTNOL-AQ). Carboplatin is
commercially available as an injectable (PARAPLATIN). Oxiplatin is commercially
available as ELOXATIN.
In another embodiment, a combination of the invention may further include
treatment with an antineoplastic antimetabolite, such as is described in US Patent
Publication No. US 2005-0187184A1 or US 2002-0183239 Al. As used in
accordance with this invention, the term "antimetabolite" means a substance which is
structurally similar to a critical natural intermediate (metabolite) in a biochemical
pathway leading to DNA or RNA synthesis which is used by the host in that pathway,
but acts to inhibit the completion of that pathway (i.e., synthesis of DNA or RNA).
More specifically, antimetabolites typically function by (1) competing with
metabolites for the catalytic or regulatory site of a key enzyme in DNA or RNA
synthesis, or (2) substitute for a metabolite that is normally incorporated into DNA or
RNA, and thereby producing a DNA or RNA that cannot support replication. Major
categories of antimetabolites include (1) folic acid analogs, which are inhibitors of
dihydrofolate reductase (DHFR); (2) purine analogs, which mimic the natural purines
(adenine or guanine) but are structurally different so they competitively or irreversibly
inhibit nuclear processing of DNA or RNA; and (3) pyrimidine analogs, which mimic
the natural pyrhnidines (cytosine, thymidine, and uracil), but are structurally different
so thy competitively or irreversibly inhibit nuclear processing of DNA or RNA.
The following are representative examples of antimetabolites of this invention.
5-Fluorouracil (5-FU; 5-fluoro-2,4(lH,3H)-pyrimidinedione) is commercially
available in a topical cream (FLUOROPLEX or EFUDEX), a topical solution
(FLUOROPLEX or EFUDEX), and as an injectable containing 50 mg/mL 5-
fluorouracil (ADRUCIL or flurouracil). Floxuradine (2'-deoxy-5-fluorouridine) is
commercially available as an injectable containing 500 mg/vial of floxuradine (FUDR
or floxuradine). Thioguanine (2-arnino-l,7-dihydro-6-H-purine-6-thione) is
commercially available in 40 mg oral tablets (thioguanine). Cytarabine (4-amino-l-

(beta)-D-arabinofuranosyl-2(lH)-pyrimidinoiie) is commercially available as a
liposomal injectable containing 10 mg/mL cytarabine (DEPOCYT) or as a liquid
injectable containing between 1 mg -1 g/vial or 20 mg/mL (cytarabine or CYTOSAR-
U). Fludarabine (9-H-Purin-6-amine,2-fluoro-9-(5-O-phosphono-(beta)-D-a-
rabinofuranosyl) is commercially available as a liquid injectable containing 50 mg/vial
(FLUDARA). 6-Mercaptopurine (l,7-dihydro-6H-purine-6-thione) is commercially
available in 50 mg oral tablets (PURMETHOL). Methotrexate (MTX; N-[4-[[(2,4-
diarnmo-6-pteridmyl)methyl]memylaniino]benzoyl]-L-glutamic acid) is commercially
available as a liquid injectable containing between 2.5 - 25 mg/mL and 20 mg -1
g/vial (methotrexate sodium or FOLEX) and in 2.5 mg oral tablets (methotrexate
sodium). Gemcitabine (2l-deoxy~2',2l-difluorocytidine monohydrochloride ((beta)-
isomer)), is commercially available as a liquid injectable containing between 200 mg -
1 g/vial (GEMZAR). Capecitabine (5'-deoxy-5-fluoro-N-[(pentyloxy)carbonyl]-
cytidine) is commercially available as a 150 or 500 mg oral tablet (XELODA).
Pentostatin ((R)-3-(2-deoxy-(beta)-D-erythro-pentofuranosyl)-3,6,7,- 8-
tetrahydroimidazo[4,5-d][l,3]diazepin-8-ol) is commercially available as a liquid
injectable containing 10 mg/vial (NIPENT). Trimetrexate (2,4-diamino-5-methyl-6-
[(3,4,5-trimethoxyanilino)methyl]quinazoline mono-D-glucuronate) is commercially
available as a liquid injectable containing between 25 - 200 mg/vial (NEUTREXIN).
Cladribine (2-chloro-6-ammo-9-(2-deoxy-(beta)-D-erythropento-furanosyl) purine) is
commercially available as a liquid injectable containing 1 mg/mL (LEUSTATIN).
The term "biochemical modulating agent" is well known and understood to
those skilled in the art as an agent given as an adjunct to anti-cancer therapy, which
serves to potentate its antineoplastic activity, as well as counteract the side effects of
the active agent, e.g., an antimetabolite. Leucovorin and levofolinate are typically
used as biochemical modulating agents for methotrexate and 5-FU therapy.
Leucovorin (5-formyl-5,6,7,8-tetrahydrofolic acid) is commercially available as an
injectable liquid containing between 5-10 mg/mL or 50 - 350 mg/vial (leucovorin
calcium or WELLCOVORIN) and as 5 - 25 mg oral tablets (leucovorin calcium).
Levofolinate (pharmacologically active isomer of 5-formyltetrahydrofolic acid) is

commercially available as an injectable containing 25 - 75 mg levofolinate
(ISOVORIN) or as 2.5 - 7.5 mg oral tablets (ISOVORIN).
la another embodiment, the combination of the invention further includes an
active agent selected from among akinase inhibitor. Particularly desirable are multi-
kinase inhibitors target serine/threonine and receptor tyrosine kinases in both the
tumor cell and tumor vasculature. Examples of suitable kinase inhibitors are
Sorafenib (BAY 43-9006, Bayer, commercially available as NEXAVAR), which has
been granted Fast Track status by the FDA for metastatic renal cell cancer. Another
suitable famesyltransferase inhibitor is Zarnestra (Rl15777, tipifarnib). Yet another
compound is suntinib (SUTENT). Still other suitable compounds that target
Ras/Raf/MEK and/or MAP kinases include, e.g., avastin, ISIS 5132, and MEK
inhibitors such as CI-1040 or PD 0325901.
As described herein, subtherapeutically effective amounts of herceptin and
temsirolimus may be used to achieve a therapeutic effect when administered in
combination. For example, herceptin may be provided at dosages of 5 to 50% lower,
10 to 25% lower, or 15 to 20% lower, when provided along with temsirolimus. For
example, a resulting herceptin dosage can be from about 8 to 40 mg, or about 8 to 30
mg, or 8 to 25 mg. Subtherapeutically effective amounts of herceptin are expected to
reduce the side-effects of herceptin treatment. The invention further provides for one
or more of the active agents in the combination of the invention to be used in a
supratherapeutic amount, i.e., at a higher dosage in the combination than when used
alone. In this embodiment, the other active agent(s) may be used in a therapeutic or
subtherapeutic amount.
The mTOR inhibitor, herceptin, HKI-272 or other active compounds used in
the combination and products of the invention may be formulated in any suitable
manner. For example, oral formulations containing the mTOR inhibitor (and
optionally, other active compounds) useful in combination and products of this
invention may comprise any conventionally used oral forms, including tablets,
capsules, buccal forms, troches, lozenges and oral liquids, suspensions or solutions.
Capsules may contain mixtures of the active compound(s) with inert fillers and/or
diluents such as the pharmaceutically acceptable starches (e.g. corn, potato or tapioca

starch), sugars, artificial sweetening agents, powdered celluloses, such as crystalline
and microcrystalline celluloses, flours, gelatins, gums, etc. Useful tablet formulations
may be made by conventional compression, wet granulation or dry granulation
methods and utilize pharmaceutically acceptable diluents, binding agents, 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, acacia gum, xanthan gum, sodium citrate,
complex silicates, calcium carbonate, glycine, dextrin, sucrose, sorbitol, dicalcium
phosphate, calcium sulfate, lactose, kaolin, mannitol, sodium chloride, talc, dry
starches and powdered sugar. Preferred surface modifying agents include nonionic
and anionic surface modifying agents. Representative 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 herein may utilize standard delay or time
release formulations to alter the absorption of the active compound(s). The oral
formulation may also consist of administering the active ingredient in water or a fruit ■
juice, containing appropriate solubilizers or emulsifiers as needed. Preferred oral
formulations for rapamycin 42-ester with 3-hydroxy-2-(hydroxymethyl)-2-
methylpropionic acid are described in US Patent Publication No. 2004/0077677 Al,
published April 22, 2004.
In some cases it may be desirable to administer the compounds directly to the
airways in the form of an aerosol.
The compounds may also be administered parenterally or intraperitoneally.
Solutions or suspensions of these active compounds as a free base or
pharmacologically acceptable salt can be prepared in water suitably mixed with a
surfactant such as hydroxy-propylcellulose. Dispersions can also be prepared in
glycerol, liquid polyethylene glycols and mixtures thereof in oils. Under ordinary
conditions of storage and use, these preparations contain a preservative to prevent the
growth of microorganisms.

The pharmaceutical forms suitable for injectable use 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 exists. 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
(e.g., glycerol, propylene glycol and liquid polyethylene glycol), suitable mixtures
thereof, and vegetable oils. Preferred injectable formulations for rapamycin 42-ester
with 3-hydroxy-2-(hydroxymethyl)-2-methyIpropionic acid are described in US Patent
Publication No. 2004/0167152 A1, published August 26, 2004.
For the purposes of this disclosure, transdermal administrations are understood
to include all administrations across the surface of the body and the inner linings of
bodily passages including epithelial and mucosal tissues. Such administrations may
be carried out using the present compounds, or pharmaceutically acceptable salts
thereof, in lotions, creams, foams, patches, suspensions, solutions, and suppositories
(rectal and vaginal).
Transdermal administration may be accomplished through the use of a
transdermal patch containing the active compound and a carrier that is inert to the
active compound, is non toxic to the skin, and allows delivery of the agent for
systemic absorption into the blood stream via the skin. The carrier may take any
number of forms such as creams and ointments, pastes, gels, and occlusive devices.
The creams and ointments may be viscous liquid or semisolid emulsions of either the
oil-in-water or water-in-oil type. Pastes comprised of absorptive powders dispersed in
petroleum or hydrophilic petroleum containing the active ingredient may also be
suitable. A variety of occlusive devices may be used to release the active ingredient
into the blood stream such as a semi-permeable membrane covering a reservoir
containing the active ingredient with or without a carrier, or a matrix containing the
active ingredient. Other occlusive devices are known in the literature.
Suppository formulations may be made from traditional 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 of
various molecular weights, may also be used.
As used in this invention, the combination regimen can be given
simultaneously or can be given in a staggered regimen, with the mTOR inhibitor being
given at a different time during the course of chemotherapy than the herceptin. This
time differential may range from several minutes, hours, days, weeks, or longer
between administration of the at least two agents. Therefore, the term combination (or
combined) does not necessarily mean administered at the same time or as a unitary
dose, but that each of the components are administered during a desired treatment
period. The agents may also be administered by different routes.
Pharmaceutical Packs/Kits:
The invention includes a product or pharmaceutical pack containing a course
of an anti-neoplastic treatment for one individual mammal comprising one or more
containers) having one, one to four, or more unit(s) of an mTOR inhibitor (e.g.,
temsirolimus) in unit dosage form and, optionally, one, one to four, or more unit(s) of
herceptin, and optionally, another active agent.
In another embodiment, pharmaceutical packs contain a course of anti-
neoplastic treatment for one individual mammal comprising a container having a unit
of a rapamycin in unit dosage form, a containing having a unit of herceptin, and
optionally, a container with another active agent, In other embodiments, the
rapamycin is rapamycin, an ester (including a 42-ester, ether (including a 42-ether),
oxime, hydrazone, or hydroxylamine of rapamycin. In another embodiment, the
rapamycin is 42-O-(2-hydroxy)ethyl rapamycin.
In another embodiment, the rapamycin is temsirolimus, and the pack contains
one or more containers) comprising one, one to four, or more unit(s) of temsirolimus
with the components described herein.
In some embodiments, the compositions of the invention are in packs in a form
ready for administration. In other embodiments, the compositions of the invention are
in concentrated form in packs, optionally with the diluent required to make a final
solution for administration, In still other embodiments, the product contains a

compound useful in the invention in solid form and, optionally, a separate container
with a suitable solvent or carrier for the compound useful in the invention.
In still other embodiments, the above packs/kits include other components,
e.g., instructions for dilution, mixing and/or administration of the product, other
containers, syringes, needles, etc. Other such pack/kit components will be readily
apparent to one of skill in the art.
The following examples illustrate of the uses of the combinations of the
invention. It will be readily understood that alterations or modifications, e.g., in the
formulation of the components, the routes of delivery, and the dosing, can be made for
reasons known to those of skill in the art.
EXAMPLE 1: COMBINATION REGIMEN OF TEMSIROLIMUS (CCI-779) and
HERCEPTIN IN TREATMENT OF NEOPLASMS
Dosing begins at month 1, day 1 with weekly intravenous (TV) temsirolimus
and herceptin (TV) at the dosages provided below.
Temsirolimus and herceptin can be administered simultaneously,
consecutively, or on alternative days.
Temsirolimus is administered IV weekly over a 30-minute period using an in-
line filter and an automatic dispensing pump. Optionally, antihistamine
(diphenhydramine, 25 to 50 mg IV or the equivalent) is administered about 30
minutes prior to temsirolimus infusion.
A herceptin loading dose is administered IV weekly over a 90 minute period.
Weekly doses are administered, which are typically half the amount of the loading
dose. For example, a 4 mg/kg loading dose is typically followed by 2 mg/kg weekly
doses. These amounts may be adjusted. In one embodiment, no loading dose is
required and the same dose is administered throughout the course of treatment.


Dose adjustments and/or delays for temsirolimus, and/or herceptin are
permitted. For example, treatment may continue as described herein for six months,
with weekly doses of temsirolimus. The herceptin may be provided on a weekly basis
for a cycle, e.g., three weeks. Typically, 2 to 3 weeks is provided between cycles.
In certain situations, dosing with the temsirolimus may be delayed or
discontinued for a brief period (e.g., 1,2 or three weeks) during the regimen.
Similarly, a cycle of treatment with herceptin may be shortened by one or more weeks,
lengthened by one or more weeks, or the period between cycles delayed or eliminated.
Such a delay or discontinuation may occur once, or more, during the course of
treatment.
. EXAMPLE 2: USE OF A COMBINATION REGIMEN OF HKI-272 AND
TEMSIROLIMUS (CCI-779) IN TREATMENT OF NEOPLASMS
Dosing begins at month 1, day 1 with daily HKI-272 and weekly intravenous
(TV) temsirolimus at the dosages provided below.
On month 1, day 1, HKI-272 is administered orally prior to temsirolimus.
Temsirolimus is administered following HKI-272, preferably within 30 minutes.
Temsirolimus is administered IV weekly over a 3 0-minute period using an in-
line filter and an automatic dispensing pump. Optionally, antihistamine
(diphenhydramine, 25 to 50 mg IV or the equivalent) is administered about 30
minutes prior to temsirolimus infusion.
Thereafter, HKI-272 is taken orally once daily with food, preferably in the
morning.


Dose adjustments and/or delays for HKI-272 and temsirolimus are permitted.
For example, treatment may continue as described herein for six months, with daily
doses of HKI-272 and weekly doses of temsirolimus. However, in certain situations,
dosing with one or both drugs may be delayed or discontinued for a brief period (e.g.,
1,2 or three weeks) during the regimen course of treatment. Such a delay or
discontinuation may occur once, or more, during the course of treatment.
EXAMPLE 3: USE OF A COMBINATION REGIMEN OF HKI-272,
TEMSIROLIMUS (CCI-779), and HERCEPTIN IN TREATMENT OF
NEOPLASMS
Dosing begins at month 1, day 1 with daily HKI-272 and weekly intravenous
(IV) temsirolimus and herceptin (TV) at the dosages provided below.
On month 1, day 1, HKI-272 is administered orallyprior to temsirolimus.
Temsirolimus and herceptin are administered following HKI-272, preferably within
30 minutes.
Temsirolimus is administered IV weekly over a 30-minute period using an in-
line filter and an automatic dispensing pump. Optionally, antihistamine
(diphenhydramine, 25 to 50 mg IV or the equivalent) is administered about 30
minutes prior to temsirolimus infusion.
A herceptin loading dose is administered IV weekly over a 90 minute period.
Weekly doses are administered, which are typically half the amount of the loading
dose. For example, a 4 mg/kg loading dose is typically followed by 2 mg/kg weekly
doses. These amounts may be adjusted. In one embodiment, no loading dose is
required and the same dose is administered throughout the course of treatment.


Dose adjustments and/or delays for HKI-272, temsirolimus, and/or herceptin
are permitted. For example, treatment may continue as described herein for six
months, with daily doses of HKI-272 and a weekly dose of temsirolimus. The
herceptin may be provided on a weekly basis for a cycle, e.g., three weeks. Typically,
2 to 3 weeks is provided between cycles. However, in certain situations, dosing with
the HKI-272 and/or temsirolimus may be delayed or discontinued for a brief period
(e.g., 1, 2 or three weeks) during the regimen or course of treatment. Such a delay or
discontinuation may occur once, or more, during the course of treatment.
Similarly, a cycle of treatment with herceptin may be shortened by one or more
weeks, lengthened by one or more weeks, or the period between cycles delayed or
eliminated. Such a delay or discontinuation may occur once, or more, during the
course of treatment.
EXAMPLE 4: USE OF A COMBINATION REGIMEN OF HKI-272 and
HERCEPTIN IN TREATMENT OF NEOPLASMS
The antineoplastic activity of the HKI-272 plus herceptin combination was
confirmed in in vitro standard pharmacological test procedure. The following briefly
describes the procedure used and the results obtained.
The combination was tested in three breast cancer cell lines of differing
genotypes. More particularly, BT474 [HER-2+ (amplified); ATCC HTB-20] and is
highly sensitive to both HKI-272 and Herceptin. MDA-MB-361 [HER-2+ (non-

amplified); adenocarcinoma; ATCC HTB 27] has lower levels of HER-2 without
amplification and less sensitive to both herceptin and HKI-272. MCF-7 [HER-2",
EGFR-; adenocarcinoma; ATCC HTB22] has no HER-2 and is resistant to both
Herceptin and HKI-272.
Cells from each of these cell lines were incubated in the presence of a range of
concentrations (0.0041, 0.012, 0.037, 0.11, 0.33, 0.1, 3 jtig/mg) for each drug.
The cells were maintained in RPMI 1640 medium (Life Technologies, Inc.,
Gaithersburg, Md.) supplemented with 10% fetal bovine serum (FBS, Life
Technologies) and 50 g/ml gentamicin (Life Technologies) under 7% CO2 at 37 °C.
Cells were plated in 96-well microtiter dishes (12,000 cells/well for BT474 Cells,
6000 cells/well MCF-7 Cells and 10,000 cells/well for MDA-MB-361 Cells) in 100 l
RPMI 1640 medium containing 5% FBS and 50 g/ml gentamicin and incubated
overnight at 37 °C. Compound dilutions were prepared in the same medium, at 2X
final concentration, and 100 l of the drug dilution was added to the cell-containing
wells.
Serial dilutions of one compound were prepared in the presence of a fixed
dose of a second compound. Alternatively, a checkerboard dilution series was
employed. Cells were cultured for three days in the presence of the drugs. Untreated
cells were included as controls. The percentage of surviving cells was determined
using sulforhodamine B (SRB, Sigma-Aldrich, St Louis, Mo.), a protein binding dye.
Cellular protein was precipitated in each well by the addition of 50 l 50% cold
trichloroacetic acid. After 1 hour, the plates were washed extensively in water and
dried. SRB dye reagent (0.4% SRB in 1% acetic acid, 80 l per well) was added and
plates were kept at room temperature for ten minutes. Plates were then washed
thoroughly in 1% acetic acid and dried. Cell-associated dye was dissolved in 10 mM
Tris (150 l) and the absorbance was read at 540 nm in a microtiter plate reader. The
concentration of compound that caused a fixed percentage inhibition of growth was
determined by plotting cell survival (relative to untreated cells) against the compound
dose.

A model for studying drug interactions has been described by Prichard and
Shipman [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 3-dimensional model: one for each drug and the third for the biological
effect. Theoretical additive interactions are calculated from the individual dose-
response curves, based on a dissimilar sites model of additivity (Bliss independence).
The calculated additive surface, representing predicted cytotoxicity is subtracted from
the experimental surface to reveal areas of enhanced toxicity (synergy) or reduced
toxicity (antagonism). The resulting surface appears as a horizontal plane at 0%
inhibition above the calculated additive surface, if the interaction is additive. Peaks
and valleys deviating from this plane are indicative of synergy and antagonism,
respectively. MacSynergy H, a Microsoft .Excel-based software was used to perform
all calculations automatically. This spreadsheet calculates the theoretical additive
interactions, and locates and quantifies synergistic or antagonistic interactions that are
significant at the 95% confidence levels. The results were plotted as a 3-dimensional
plot, or as a contour plot with the plane at 0% representing additive interaction, and
peaks and valleys representing areas of synergy or antagonism, respectively, between
the two drugs.
For purposes of this study, the Pritchard and Shipman method was modified to
allow determination of the combination effects at different levels of statistical
significance (p-values 0.05, 0.01, 0.001). Ap-value of 0.05 is considered significant.
The method of estimating statistical variability within each experiment was also
modified. Variability was determined across all compound combinations, whereas in
the original version, variability was estimated separately for each compound
combination. It is believed that better estimates of the variability are obtained with
the modified approach. In general, single points of synergy or antagonism are not
considered representative of either synergistic or antagonistic activity. Thus, single
point peaks or valleys are disregarded in the analysis. Furthermore, peaks or valleys
that occur only along single concentration of one of the compounds are also
disregarded, if no synergy or antagonism is observed at the adjacent, flanking

concentrations. Finally, all experiments are repeated at least twice and determinations
of synergy and antagonism are made by examination of all the data.
Figs. 1-3 provide the results from a single set of experiments. In MDA-MB-
361 cells, there is an area of antagonism at 0.11 — 3 g/mL herceptin for at a
concentration of 0.012 g/mL HKI-272 at the 95% confidence level. In MCF7 cells,
there is an area of synergy at 0.037 - 0.33 /Ag/mL herceptin for at a concentration of
0.11 g/mL HKI-272 at the 95% confidence level. For the BT474 cells, there is an
area of antagonism at 0.33 -1 /ig/mL herceptin for at a concentration of 0.11 g/mL
HKI-272 at the 95% confidence level. When repeated at the 99% confidence level, no
statistically significant areas of antagonism or synergy were found. Based on the
above criteria, the combination of herceptin and HKI-272 is considered additive
across all concentrations.
The results of these standard pharmacological test procedures derived from
multiple independent experiments, indicate that combinations of HKI-272 are not
significantly antagonistic or synergistic, but are additive over a range of
concentrations. These data support the use of the combinations in the treatment of
HER2+ cancers. As these combinations contain at least two active antineoplastic
agents, the use of such combinations also provides for the use of combinations of each
of the agents in which one or both of the agents is used at subtherapeutically effective
dosages, thereby lessening toxicity associated with the individual chemotherapeutic
agent.
All patents, patent publications, articles, and other documents referenced
herein are incorporated by reference. It will be clear to one of skill in the art that
modifications can be made to the specific embodiments described herein without
departing from the scope of the 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 components comprising a
herceptin and an mTOR inhibitor and/or HKI-272.
2. The method according to claim 1, wherein the combination comprises
HKI-272.
3. The method according to claim 1 or 2, wherein one or more of the
active components is provided in subtherapeutically effective amounts.
4. 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 comprising a rapamycin and
an HKI-272.
5. The method according to any one of claims 1 to 4, wherein the
neoplasm is selected from the group consisting of lung cancers, including
bronchioalveolar carcinoma and non small cell lung cancer, breast cancers, myeloma,
prostate cancers, head and neck cancer, or transitional cell carcinoma; small cell and
large cell neuroendocrine carcinoma of the uterine cervix.
6. The method according to any one of claims 1 to 5, wherein said
combination further comprises another active component selected from the group
consisting of one or more antineoplastic alkylating agent, one or more antimetabolite
antineoplastic agents, one or more biochemical immune modulators, imatinib, one or
more EGFR inhibitors, a multi-kinase inhibitor that targets serine/threonine and
receptor tyrosine kinases in both the tumor cell and tumor vasculature or an interferon.

7. The method according to claim 6, wherein said antineoplastic agents
are selected from the group consisting of meclorethamine, cyclophosphamide,
ifosfamide, melphalan, chlorambucil, thiotepa, mitomycin, busulfan, lomustine,
carmustine, procarbazine, temozolomide, oxaliplatin, cisplatin, and carboplatin.
8. The method according to claim 7, wherein the antimetabolite
antineoplastic agent is selected from the group consisting of: 5-fluorouracil;
floxuradine; thioguanine; cytarabine; fludarabine; 6-mercaptopurine; methotrexate;
gemcitabine; capecitabine; taxanes; pentostatin; trimetrexatel; and cladribine.
9. The method according to claim 7, wherein the biochemical modulating
agent is selected from the group consisting of leucovorin and levofolinate.
10. The method according to claim 9, wherein the combination further comprises
a taxane.
11. The method according to any one of claims 1 to 10, wherein the
rapamycin is rapamycin.
12. The method according to any one of claims 1 to 10, wherein the
rapamycin is 42-O-(2-hydroxy)ethyl rapamycin.
13. The method according to any one of claims 1 to 12, wherein the
neoplasm is metastatic breast cancer.
14. Use of a rapamycin in the preparation of an antineoplastic medicament
administrable in a regimen with a herceptin, or use of a herceptin in the preparation of
a medicament administrable in a regimen with a rapamycin, said regimen optionally
further comprising an HKI-272.

15. Use of a rapamycin in the preparation of an antineoplastic medicament
administrable in a regimen with an HKI-272, or use of an HKI-272 in the preparation
of a medicament administrable in a regimen with a rapamycin, said regimen optionally
further comprising a herceptin.
16. Use of a herceptin in the preparation of an antineoplastic medicament
administrable in a regimen with an HKI-272, or use of an herceptin in the preparation
of a medicament administrable in a regimen with an HKI-272, said regimen optionally
further comprising an mTOR inhibitor.
17. Use according to any one of claims 14 to 16, wherein the medicament
is for the treatment of a neoplasm selected from the group consisting of lung cancers,
including bronchioalveolar carcinoma and non small cell lung cancer, breast cancers,
myeloma, prostate cancers, head and neck cancer, or transitional cell carcinoma; small
cell and large cell neuroendocrine carcinoma of the uterine cervix.
18. Use according to any one of claims 14 to 17, wherein the rapamycin is
rapamycin.
19. Use according to any one of claims 14 to 17, wherein the rapamycin is
42-O-(2-hydroxy)ethyl rapamycin.
20. Use according to any one of claims 14 to 19, wherein the medicament is
for the treatment of metastatic breast cancer.
21. A regimen for treatment of breast cancer associated with overexpression
or amplification of HER2, said method comprising:
delivering a dosage amount amount of a herceptin; and
delivering a dose of at least one additional compound selected from the
group consisting of an mTOR inhibitor and a HKI-272.

22. The regimen according to claim 21, wherein the rapamycin mTOR
inhibitor is delivered intravenously.
23. The regimen according to claim 21, wherein the rapamycin is delivered
weekly.
24. The regimen according to claim 21, wherein the rapamycin is delivered
orally.
25. The regimen according to claim 21, wherein the rapamycin is delivered
weekly.
26. The regimen according to claim 21, wherein the herceptin is delivered
intravenously.
27. The regimen according to claim 21, wherein the herceptin is delivered
for at least two weeks following at least one week off.
28. The regimen according to claim 21, wherein the herceptin is delivered
for a period of four weeks followed by two weeks off.
29. The regimen according to claim 21, wherein the herceptin is delivered
once every three to four weeks.
30. The regimen according to claim 21, wherein the rapamycin is
rapamycin.
31. The regimen according to claim 30, wherein the rapamycin is
temsirolimus.

32. The regimen according to claim 21, wherein the HKI-272 is delivered
orally.
33. A product containing temsirolimus and a herceptin as a combined
preparation for simultaneous, separate or sequential use in treating a neoplasm in a
mammal.
34. A product containing a rapamycin and an HKI-272 as a combined
preparation for simultaneous, separate or sequential use in treating a neoplasm in a
mammal.
35. A product containing a herceptin and an HKI-272 as a combined
preparation for simultaneous, separate or sequential use in treating a neoplasm in a
mammal.
36. A pharmaceutical pack containing a course of an anti-neoplastic
treatment for one individual mammal, wherein the pack contains (a) at least one unit of
temsirolimus and (b) at least one unit of herceptin in unit dosage form.
37. A pharmaceutical pack containing a course of an anti-neoplastic
treatment for one individual mammal, wherein the pack contains (a) at least one unit of
a rapamycin and (b) at least one unit of HKI-272 in unit dosage form.
38. A pharmaceutical pack containing a course of an anti-neoplastic
treatment for one individual mammal, wherein the pack contains (a) at least one unit of
herceptin and (b) at least one unit of HKI-272 in unit dosage form.
39. A pharmaceutical composition useful in treating a neoplasm in a
mammal, the composition comprising (a) at least one unit of temsirolimus and (b) at
least one unit of a herceptin in unit dosage form, and at least one pharmaceutically
acceptable carrier.

40. A pharmaceutical composition useful in treating a neoplasm in a
mammal, the composition comprising (a) at least one unit of a rapamycin and (b) at
least one unit of an HKI-272 in unit dosage form, and at least one phannaceutically
acceptable carrier.
41. A pharmaceutical composition useful in treating a neoplasm in a
mammal, the composition comprising (a) at least one unit of a herceptin and (b) at least
one unit of an HKI-272 in unit dosage form, and at least one pharmaceutically
acceptable carrier.
42. Use of a rapamycin in the preparation of an antineoplastic medicament
administrable in a regimen with a herceptin, or use of a herceptin in the preparation of
a medicament administrable in a regimen with a rapamycin, said regimen optionally
further comprising a compound having the formula A

where R1 is halogen;
R2 is pyridinyl, thiophene, pyrimidine, thiazole, or phenyl optionally
substituted with up to three substituents;
R3is-O-or-S-;
R4 is methyl or CH2CH2OCH3;
R5 is ethyl or methyl; and
n is O or 1 or a pharmaceutically acceptable salt thereof.

43. Use of a rapamycin in the preparation of an antineoplastic medicament
administrable in a regimen with a compound having the formula A (as illustrated and
defined in claim 14) or a pharmaceutically acceptable salt thereof, or use of a
compound having the formula A (as illustrated and defined in claim 14) or a
pharmaceutically acceptable salt thereof in the preparation of a medicament
administrable in a regimen with a rapamycin, said regimen optionally further
comprising a herceptin.
44. Use of a herceptin in the preparation of an antineoplastic medicament
administrable in a regimen with a compound having the formula A (as illustrated and
defined in claim 14) or a pharmaceutically acceptable salt thereof, or use of an
herceptin in the preparation of a medicament administrable in a regimen with a
compound having the formula A (as illustrated and defined in claim 14) or a
pharmaceutically acceptable salt thereof, said regimen optionally further comprising an
mTOR inhibitor.

A combination of temsirolimus and herceptin in the treatment of cancer is provided. A combination of temsirolimus
and HKI-272 is provided. A combination of herceptin and HKI-272 is also provided. Regimens and kits for treatment of metastatic
breast cancer, containing herceptin, temsirolimus and/or HKI-272, optionally in combination with other anti-neoplastic agents, or
immune modulators are described.