A PROCESS FOR THE PREPARATION OF ANTINEOPLASTIC COMBINATIONS
This application is divided out of Indian Patent Application No. 1475/KOLNP/2003 filed on
12th November 2003.
This invention relates to the use of combinations of an mTOR inhibitor (e.g
rapamycin 42-ester with 3-hydroxy-2-(hydroxymethyl)-2-methylpropionic add
(CCI-779)) and an antineoplastic alkylating agent in the treatment of neoplasms, to
the use of an mTOR inhibitor and an antineoplastic alkylating agent in the preparation
of a medicament for the treatment of a neoplasm, to a product comprising an mTOR
inhibitor and an antineoplastic alkylating agent as a combined preparation for
simultaneous, separate or sequential use in the treatment of a neoplasm, and to
pharmaceutical compositions comprising an mTOR inhibitor, an antineoplastic
alkylating agent and a pharmaceutically acceptable carrier.
BACKGROUND OF THE INVENTION
Rapamycin is a macrocyclic triene antibiotic produced by Streptomvces
hygroscopicus. which was found to have antifungal activity, particularly against
Candida albicans, both in vitro and in vivo [C. Vezina et al., J. Antibiot. 28,721 (1975);
S.N. Sehgal et al., J. Antibiot. 28, 727 (1975); H. A. Baker et al., J. Antibiot 31, 539
(1978); U.S. Patent 3,929,992; and U.S. Patent 3,993,749]. Additionally, rapamycin
alone (U.S. Patent 4,885,171) or in combination with picibanil (U.S. Patent 4,401,653)
has been shown to have antitumor activity.
The immunosuppressive effects of rapamycin have been disclosed in FASEB
3, 3411 (1989). Cyclosporin A and FK-506, other macrocyclic molecules, also have
been shown to be effective as immunosuppressive agents, therefore useful in
preventing transplant rejection [FASEB 3, 3411 (1989); FASEB 3, 5256 (1989); R. Y.
Calne et al., Lancet 1183 (1978); and U.S. Patent 5,100,899]. R. Mattel et al. [Can. J.
Physiol. Pharmacol. 55, 48 (1977)] disclosed that rapamycin is effective in the
experimental allergic encephalomyelitis model, a model for multiple sclerosis; in the
adjuvant arthritis model, a model for rheumatoid arthritis; and effectively inhibited the
formation of IgE-like antibodies.
Rapamycin is also useful in preventing or treating systemic lupus
erythematosus [U.S. Patent 5,078,999], pulmonary inflammation [U.S. Patent
5,080,899], insulin dependent diabetes mellitus [U.S. Patent 5,321,009], skin
disorders, such as psoriasis [U.S. Patent 5,286,730], bowel disorders [U.S. Patent
5,286,731], smooth muscle cell proliferation and intimal thickening following vascular
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injury [U.S. Patents 5,288,711 and 5,516,781], adult T-cell leukemia/lymphoma
[European Patent Application 525,960 A1], ocular inflammation [U.S. Patent
5,387,589], malignant carcinomas [U.S. Patent 5,206,018], cardiac inflammatory
disease [U.S. Patent 5,496,832], and anemia [U.S. Patent 5,561,138].
Rapamycin 42-ester with 3-hydroxy-2-(hydroxymethyl)-2-methylpropionic acid
(CCI-779) is ester of rapamycin which has demonstrated significant inhibitory effects
on tumor growth in both in vitro and in vivo models. The preparation and use of
hydroxyesters of rapamycin, including CCI-779, are disclosed in U.S. Patent
5,362,718.
CCI-779 exhibits cytostatic, as opposed to cytotoxic properties, and may delay
the time to progression of tumors or time to tumor recurrence. CCI-779 is considered
to have a mechanism of action that is similar to that of sirolimus. CCI-779 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 kinase activity inhibits a variety of
signal transduction pathways, including cytokine-stimulated cell proliferation,
translation of mRNAs for several key proteins that regulate the G1 phase of the cell
cycle, and IL-2-induced transcription, leading to inhibition of progression of the cell
cycle from GI to S. The mechanism of action of CCI-779 that results in the Gl S
phase block is novel for an anticancer drug.
In vitro, CCI-779 has been shown to inhibit the growth of a number of
histologically diverse tumor cells. Central nervous system (CNS) cancer, leukemia
(T-cell), breast cancer, prostate cancer, and melanoma lines were among the most
sensitive to CCI-779. The compound arrested cells in the G1 phase of the cell cycle.
In vivo studies in nude mice have demonstrated that CCI-779 has activity
against human tumor xenografts of diverse histological types. Gliomas were
particularly sensitive to CCI-779 and the compound was active in an orthotopic glioma
model in nude mice. Growth factor (platelet-derived)-induced stimulation of a human
glioblastoma cell line in vitro was markedly suppressed by CCI-779. The growth of
several human pancreatic tumors in nude mice as well as one of two breast cancer
lines studied in vivo also was inhibited by CCI-779.
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DESCRIPTION OF THE INVENTION
This invention provides the use of combinations of an mTOR inhibitor and an
antineoplastic alkylating agent as antineoplastic combination chemotherapy. In
particular, these combinations are useful in the treatment of renal cancer, soft tissue
cancer, breast cancer, neuroendocrine tumor of the lung, cervical cancer, uterine
cancer, head and neck cancer, glioma, non-small lung cell cancer, prostate cancer,
pancreatic cancer, lymphoma, melanoma, small cell lung cancer, ovarian cancer,
colon cancer, esophageal cancer, gastric cancer, leukemia, colorectal cancer, and
unknown primary cancer. This invention also provides combinations of an mTOR
inhibitor and an antineoplastic alkylating agent for use as antineoplastic combination
chemotherapy, in which the dosage of either the mTOR inhibitor or the antineoplastic
alkylating agent or both are used in subtherapeutically effective dosages.
In another aspect, the invention provides the use of combinations of an mTOR
inhibitor and an antineoplastic alkylating agent in the preparation of a medicament for
the treatment of a neoplasm. In a further aspect, the invention provides a product
comprising an mTOR inhibitor and an antineoplastic alkylating agent as a combined
preparation for simultaneous, separate or sequential use in the treatment of a
neoplasm in a mammal. In a still further aspect, the invention provides a
pharmaceutical composition comprising an mTOR inhibitor, an antineoplastic
alkylating agent and a pharmaceutically acceptable carrier.
As used in accordance with this invention, the term "treatment" means treating
a mammal having a neoplastic disease by providing said mammal an effective amount
of a combination of an mTOR inhibitor and an antineoplastic alkylating agent with the
purpose of inhibiting growth of the neoplasm in such mammal, eradication of the
neoplasm, or palliation of the mammal.
As used in accordance with this invention, the term "providing," with respect to
providing the combination, means either directly administering the combination, or
administering a prodrug, derivative, or analog of one or both of the components of the
combination which will form an effective amount of the combination within the body.
mTOR is the mammalian target of rapamycin, also known as FKBP12-
rapamycin associated protein [FRAP]. Inhibition of mTOR's kinase activity inhibits a
variety of signal transduction pathways, including cytokine-stimulated cell proliferation,
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translation of mRNAs for several key proteins that regulate the G1 phase of the cell
cycle, and IL-2-induced transcription, leading to inhibition of progression of the cell
cycle from G1 to S.
mTOR regulates the activity of at least two proteins involved in the translation
of specific cell cycle regulatory proteins (Burnett, P.E., PNAS 95: 1432 (1998) and
Isotani, S., J. Biol. Chem. 274: 33493 (1999)). One of these proteins p70s6 kinase is
phosphorylated by mTOR on serine 389 as well as threonine 412. This
phosphorylation can be observed in growth factor treated cells by Western blotting of
whole cell extracts of these cells with antibody specific for the phosphoserine 389
residue.
As used in accordance with this invention, an "mTOR inhibitor" means a
compound or ligand which inhibits cell replication by blocking progression of the cell
cycle from G1 to S by inhibiting the phosphorylation of serine 389 of p70s6 kinase by
mTOR.
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 (eg. 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.
Materials:
NuPAGE LDS Sample Buffer (Novex Cat # NP0007)
NuPAGE Sample Reducing Agent (NovexCat#NP0004)
NuPAGE 4-12% Bis-Tris Gel (Novex Cat #NP0321)
NuPAGE MOPS SDS Running Buffer (Novex Cat #NP0001)
Nitrocellulose (Novex Cat #LC2001)
NuPAGE Transfer Buffer (Novex Cat #NP0006)
Hyperfilm ECL (Amersham Cat # RPN3114H)
ECL Western Blotting Detection Reagent (Amersham Cat # RPN2134)
Primary antibody: Phospho-p70 S6 Kinase (Thr389) (Cell Signaling Cat #9205)
Secondary antibody: Goat anti-rabbit IgG-HRP conjugate (Santa Cruz Cat #sc-2004)
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Methods:
A. Preparation of Cell Lysates
Cell lines were grown in optimal basal medium supplemented with 10% fetal
bovine serum and penicillin/treptomycin. For phosphorylation studies, cells were
subcultured in 6-well plates. After the cells have completely attached, they were
either serum-starved. Treatment with mTOR inhibitors ranged from 2 to 16
hours. After drug treatment, the cells were rinsed once with PBS (phosphate
buffered saline without Mg++ and Ca++) and then lysed in 150-200 u.l NuPAGE
LDS sample buffer per well. The lysates were briefly sonicated and then
centrifuged for 15 minutes at 14000 rpm. Lysates were stored at minus -80°C until
use.
The test procedure can also be run by incubating the cells in growth medium
overnight after they have completely attached. The results under both sets of
conditions should be the same for an mTOR inhibitor.
B. Western Blot Analysis
1) Prepare total protein samples by placing 22.5 p.l of lysate per tube and then
add 2.5 u.l NuPAGE sample reducing agent. Heat samples at 70°C for 10
minutes. Electrophoresed using NuPAGE gels and NuPAGE SDS buffers.
2) Transfer the gel to a nitrocellulose membrane with NuPAGE transfer buffer.
The membrane are blocked for 1 hour with blocking buffer (Tris buffered
saline with 0.1%-Tween and 5% nonfat-milk). Rinse membranes 2x with
washing buffer (Tris buffered saline with 0.1%-Tween).
3) Blots/membrane are incubated with the P-p70 S6K (T389) primary antibody
(1:1000) in blocking buffer overnight at 40C in a rotating platform.
4) Blots are rinsed 3x for 10 minutes each with washing buffer, and incubated
with secondary antibody (1:2000) in blocking buffer for 1 hour at room
temperature.
5) After the secondary antibody binding, blots are washed 3x for 10 minutes
each with washing buffer, and 2x for 1 minute each with Tris-buffered saline,
followed by chemiluminescent (ECL) detection and then exposed to
chemiluminescence films.
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As used in accordance with this invention, the term "a rapamycin" defines a
class of immunosuppressive compounds which contain the basic rapamycin nucleus
(shown below). The rapamycins of this invention include compounds which may be
chemically or biologically modified as derivatives of the rapamycin nucleus, while still
retaining immunosuppressive properties. Accordingly, the term "a rapamycin"
includes esters, ethers, 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. The term "a rapamycin" also
includes pharmaceutically acceptable salts of rapamycins, which are capable of
forming such salts, either by virtue of containing an acidic or basic moiety.

It is preferred that the esters and ethers of rapamycin are of the hydroxyl
groups at the 42- and/or 31-positions of the rapamycin nucleus, esters and ethers of a
hydroxyl group at the 27-position (following chemical reduction of the 27-ketone), and
that the oximes, hydrazones, and hydroxylamines are of a ketone at the 42-position
(following oxidation of the 42-hydroxyl group) and of 27-ketone of the rapamycin
nucleus.
Preferred 42- and/or 31-esters and ethers of rapamycin are disclosed in the
following patents, which are all hereby incorporated by reference: alkyl esters (U.S.
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Patent 4,316,885); aminoalkyl esters (U.S. Patent 4,650,803); fiuorinated esters (U.S.
Patent 5,100,883); amide esters (U.S. Patent 5,118,677); carbamate esters (U.S.
Patent 5,118,678); silyl ethers (U.S. Patent 5,120,842); aminoesters (U.S. Patent
5,130,307); acetals (U.S. Patent 5,51,413); aminodiesters (U.S. Patent 5,162,333);
sulfonate and sulfate esters (U.S. Patent 5,177,203); esters (U.S. Patent 5,221,670);
alkoxyesters (U.S. Patent 5,233,036); O-aryl, -alkyl, -alkenyl, and -alkynyl ethers (U.S.
Patent 5,258,389); carbonate esters (U.S. Patent 5,260,300); arylcarbonyl and
alkoxycarbonyl carbamates (U.S. Patent 5,262,423); carbamates (U.S. Patent
5,302,584); hydroxyesters (U.S. Patent 5,362,718); hindered esters (U.S. Patent
5,385,908); heterocydic esters (U.S. Patent 5,385,909); gem-disubstituted esters
(U.S. Patent 5,385,910); amino alkanoic esters (U.S. Patent 5,389,639);
phosphorylcarbamate esters (U.S. Patent 5,391,730); carbamate esters (U.S. Patent
5,411,967); carbamate esters (U.S. Patent 5,434,260); amidino carbamate esters
(U.S. Patent 5,463,048); carbamate esters (U.S. Patent 5,480,988); carbamate esters
(U.S. Patent 5,480,989); carbamate esters (U.S. Patent 5,489,680); hindered N-oxide
esters (U.S. Patent 5,491,231); biotin esters (U.S. Patent 5,504,091); O-alkyl ethers
(U.S. Patent 5,665,772); and PEG esters of rapamycin (U.S. Patent 5,780,462). The
preparation of these esters and ethers are disclosed in the patents listed above.
Preferred 27-esters and ethers of rapamycin are disdosed in U.S. Patent
5,256,790, which is hereby incorporated by reference. The preparation of these esters
and ethers are disclosed in the patents listed above.
Preferred oximes, hydrazones, and hydroxylamines of rapamycin are
disclosed in U.S. Patents 5,373,014, 5,378,836, 5,023,264, and 5,563,145, which are .
hereby incorporated by reference. The preparation of these oximes, hydrazones, and
hydroxylamines are disclosed in the above listed patents. The preparation of 42-
oxorapamycin is disclosed in 5,023,263, which is hereby incorporated by reference.
Particularly preferred rapamycins include rapamycin [U.S. Patent 3-,929,992],
CCI-779 [rapamycin 42-ester with 3-hydroxy-2-(hydroxymethyl)-2-methylpropionic
acid; U.S. Patent 5,362,718], and 42-O-(2-hydroxy)ethyl rapamycin [U.S. Patent
5,665,772].
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When applicable, pharmaceutically acceptable salts of the rapamycin can be
formed from organic and inorganic acids, for example, acetic, propionic, lactic, citric,
tartaric, succinic, fumaric, maleic, malonic, mandelic, malic, phthalic, hydrochloric,
hydrobromic, phosphoric, nitric, sulfuric, methanesulfonic, napthalenesulfonic,
benzenesulfonic, toluenesulfonic, camphorsulfonic, and similarly known acceptable
aids when the rapamycin contains a suitable basic moiety. Salts may also be formed
from organic and inorganic bases, such as alkali metal salts (for example, sodium,
lithium, or potassium) alkaline earth metal salts, ammonium salts, alkylammonium
salts containing 1-6 carbon atoms or dialkylammonium salts containing 1-6 carbon
atoms in each alkyl group, and trialkylammonium salts containing 1-6 carbon atoms in
each alkyl group, when the rapamycin contains a suitable acidic moiety.
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, CCI-779, or 42-O-(2-hydroxy)ethyl rapamycin.
As described herein, CCI-779 was evaluated as a representative mTOR
inhibitor in the mTOR inhibitor plus antimetabolite combinations of this invention.
The preparation of CCI-779 is described in U.S. Patent 5,362,718, which is
hereby incorporated by reference. When CCI-779 is used as an antineoplastic agent,
it is projected that initial i.v. infusion dosages will be between about 0.1 and 100
mg/m2 when administered on a daily dosage regimen (daily for 5 days, every 2-3
weeks), and between about 0.1 and 1000 mg/m2 when administered on a once
weekly dosage regimen. Oral or intravenous infusion are the preferred routes of
administration, with intravenous being more preferred.
As used in accordance with this invention, the term "antineoplastic alkylating
agent" means a substance which reacts with (or "alkylates") many electron-rich atoms
in cells to form covalent bonds. The most important reactions with regard to their
antitumor activities are reactions with DNA bases. Some alkylating agents are
monofunctional and react with only one strand of DNA. Others are bifunctional and
react with an atom on each of the two strands of DNA to produce a "cross-link" that
covalently links the two strands of the DNA double helix. Unless repaired, this lesion
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will prevent the cell from replicating effectively. The lethality of the monofunctional
alkylating agents results from the recognition of the DNA lesion by the cell and the
response of the cell to that lesion. (Colvin OM. Antitumor Alkylating Agents. In
Cancer Principles & Practice of Oncology 6th Edition, ed. DeVita VT, Hellman S,
Rosenberg SA. Lippincott Williams & Wilkins. Philadelphia 2001. p. 363.)
Antineoplastic alkylating agents are roughly classified, according to their
structure or reactive moiety, into several categories which include nitrogen mustards,
such as mustargen, cyclophosphamide, ifosfamide, melphalan, and chlorambucil;
azidines and epoxides, such as thiotepa, mitomycin C, dianhydrogalactitol, and
dibromodulcitol; alkyl sulfonates, such as busulfan; nitrosoureas, such as
bischloroethylnitrosourea (BCNU), cyclohexyl-chloroethylnitrosourea (CCNU), and
methylcyclohexylchloroethylnitrosourea (MeCCNU); hydrazine and triazine derivatives,
such as procarbazine, dacarbazine, and temozolomide; 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 SW, Stevenson JP, O'Dwyer PJ. Cisplatin and Its
Analogues. In Cancer Principles & Practice of Oncology 6th Edition, ed. DeVita VT,
Hellman S, Rosenberg SA. 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 antineoplastic 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 (IFEX).
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).
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Busulfan is commercially available as an injectable (BUSULFEX) and in oral
tablets (MYLERAN).
Lomustine (CCNU) is commercially avaitable 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, PLATINOL, or
PLAT1NOL-AQ).
Carboplatin is commercially available as an injectable (PARAPLATIN).
The following table briefly summarizes some of the recommended dosages for
the antineoplastic alkylating agents listed above.

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Preferred mTOR inhibitor plus antineoplastic alkylating agent combinations of
this invention include CCI-779 plus cisplatin; CCI-779 plus cyclophosphamide; CCI-
779 plus carboplatin; and CCI-779 plus BCNU.
The antineoplastic activity of the mTOR inhibitor plus antineoplastic alkylating
agent combinations were confirmed using CCI-779 as a representative mTOR
inhibitor in in vitro and in vivo standard pharmacological test procedures using
combinations of CCI-779 plus cisplatin; CCI-779 plus cyclophosphamide; and CCI-779
plus BCNU as representative combinations of this invention. The following briefly
describes the procedures used and the results obtained.
Human rhabdomyosarcoma lines Rh30 and Rh1 and the human glioblastoma
line SJ-GBM2 were used for in vitro combination studies with CCI-779 and alkylating
agents. In vivo studies used a human neuroblastoma (NB1643) and human colon line
GC3.
Dose response curves were determined for each of the drugs of interest. The
cell lines Rh30, Rh1 and SJ-G2 were plated in six-well cluster plates at 6x103, 5x103
and 2.5x104 cells/well respectively. After a 24 hour incubation period, drugs were
added in either 10%FBS+RPMI 1640 for Rh30 and Rh1 or 15%FBS+DME for SJ-G2.
After seven days exposure to drug containing media, the nuclei were released by
treating the cells with a hypotonic solution followed by a detergent. The nuclei were
then counted with a Coulter Counter. The results of the experiments were graphed
and the IC50 (drug concentration producing 50% inhibition of growth) for each drug
was determined by extrapolation. Because the IC50s varied slightly from experiment
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to experiment, two values that bracketed the IC50 of each drug were used in the
interaction studies. The point of maximum interaction between two drugs occurs
when they are present in a 1:1 ratio if the isobole is of standard shape. Therefore,
each of the three approximate IC50 concentrations of CCI-779 was mixed in a 1:1
ratio with each of three approximated IC50s of cisplatin, BCNU, and melphanan. This
resulted in nine 1:1 combinations of drugs in each experiment plus three IC50
concentrations for CCI-779 and the other drug. This protocol usually resulted in at
least one combination for each drug containing an IC50 value. The 1:1 combination of
IC50 concentrations for CCI-779 and each chemotherapy drug was then used to
calculate additivity, synergism, or antagonism using Berenbaum's formula:
x/X50+y/y 50'=:1><1>>1- If the three concentrations of CCI-779 tested alone didn't
produce an IC that matched any of the three ICs of the other compound tested alone,
all the 1:1 combinations were checked to see if their ICs fell between the appropriate
ICs of drugs tested singly. If they did, the effect was considered additive.
The results obtained in the in vitro standard pharmacological test procedure
showed when tested against Rh30 tumor line, the combination of CCI-779 plus
cisplatin was synergistic; the combination was greater than additive but did not reach
levels of being mathematically synergystic against the Rh1 tumor cell line, and was
additive against the SJ-G2 tumor cell line. A combination of CCI-779 plus BCNU was
synergistic against the SJ-G2 tumor cell line and greater than additive but did not
reach levels of being mathematically synergystic against the Rh30 cell line, and
additive against the Rh1 cell line. The combination of CCI-779 plus melphanan was
additive against each of the cell lines.
Female CBA/CaJ mice (Jackson Laboratories, Bar Harbor, ME), 4 weeks of
age, were immune-deprived by thymectomy, followed 3 weeks later by whole-body
irradiation (1200 cGy) using a 137Cs source. Mice received 3 x 10* nucleated bone
marrow cells within 6-8 h of irradiation. Tumor pieces of approximately 3 mm3 were
implanted in the space of the dorsal lateral flanks of the mice to initiate tumorgrowth.
Tumor-bearing mice were randomized into groups of seven prior to initiating therapy.
Mice bearing tumors each received drug when tumors were approximately 0.20-1 cm
in diameter. Tumor size was determined at 7-day intervals using digital Vernier
calipers interfaced with a computer. Tumor volumes were calculated assuming tumors
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to be spherical using the formula [(TT/6) X d 3 ], where d is the mean diameter. CCI-
779 was given on a schedule of 5 consecutive days for 2 weeks with this cycle
repeated every 21 days for 3 cycles. This resulted in CCI-779 being given on days 1-
5, 8-12 (cycle 1); 21-25, 28-32 (cycle 2); and 42-46,49-53 (cycle 3). The schedule of
the other chemotherapy drug for each study was as follows:
Cyclophosphamide on days 1 and 8 every 21 days for 3 cycles
The combination of CCI-779 and cyclophosphamide was evaluated using a
human rhabdosarcoma (Rh18) using the mouse xenograft test procedure described
above. In this test procedure, the effect of CCI-779 with cyclophosphamide (44
mg/kg) was additive. When combined as suboptimum dosages, CCI-779 plus
cyclophosphamide was equivalent to cyclophosphamide given at an optimum dosage.
Based on the results of these standard pharmacological test procedures,
combinations of an mTOR inhibitor plus an antineoplastic alkylating agent are useful
as antineopfastic therapy. More particularly, these combinations are useful in the
treatment of renal carcinoma, soft tissue sarcoma, breast cancer, neuroendocrine
tumor of the lung, cervical cancer, uterine cancer, head and neck cancer, glioma, non-
small cell lung cancer, prostate cancer, pancreatic cancer, lymphoma, melanoma,
small cell lung cancer, ovarian cancer, colon cancer, esophageal cancer, gastric
cancer, leukemia, colorectal cancer, and unknown primary cancer. 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
In providing chemotherapy, multiple agents having different modalities of
action are typically used as part of a chemotherapy "cocktail." It is anticipated that the
combinations of this invention will be used as part of a chemotherapy cocktail that
may contain one or more additional antineoplastic agents depending on the nature of
the neoplasia to be treated. For example, this invention also covers the use of the
mTOR inhibitor/alkylating agent combination used in conjunction with other
chemotherapeutic agents, such as antimetabolites (i.e., 5-fluorouracil, floxuradine,
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thioguanine, cytarabine, fludarabine, 6-mercaptopurine, methotrexate, gemcitabine,
capecitabine, pentostatin, trimetrexate, or cladribine); hormonal agents (i.e.,
estramustine, tamoxifen, toremifene, anastrozole, or letrozole); antibiotics (i.e.,
plicamycin, bleomycin, mitoxantrone, idarubicin, dactinomycin, mitomycin, or
daunorubicin); immunomodulators (i.e., interferons, IL-2, or BCG); antimitotic agents
(i.e., vinblastine, vincristine, teniposide, or vinorelbine); topoisomerase inhibitors (i.e.,
topotecan, irinotecan, etoposide, or doxorubicin); and other agents (i.e., hydroxyurea,
trastuzumab, altretamine, retuximab, paclitaxel, docetaxel, L-asparaginase, or
gemtuzumab ozogamicin).
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 aikylating agent.
This time differential may range from several minutes, hours, days, weeks, or longer
between administration of the two agents. Therefore, the term combination 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. For example, in the combination of an
mTOR inhibitor plus an aikylating agent, it is anticipated that the mTOR inhibitor will
be administered orally or parenterally, with parenterally being preferred, while the
aikylating agent may be administered parenterally, orally, or by other acceptable
means. These combination can be administered daily, weekly, or even once monthly.
As typical for chemotherapeutic regimens, a course of chemotherapy may be
repeated several weeks later, and may follow the same timeframe for administration
of the two agents, or may be modified based on patient response.
As typical with chemotherapy, 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, health of the patient,
and other concomitant disorders or treatments.
Based on the results obtained with the CCI-779 plus aikylating agent
combinations, it is projected that the initial i.v. infusion dosage of the mTOR inhibitor
will be between about 0.1 and 100 mg/m2, with between about 2.5 and 70 mg/m2
being preferred. It is also preferred that the mTOR inhbitor be administered by i.v.,
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typically over a 30 minute period, and administered about once per week. The initial
dosages of the alkylating agent component will depend on the component used, and
will be based initially on physician experience with the agents chosen. After one or
more treatment cycles, the dosages can be adjusted upwards or downwards
depending on the results obtained and the side effects observed.
For commercially available alkylating agents, the existing dosage form can be
used, with the dosages divided as need be. Alternatively, such agents or alkylating
agents that are not commercially available can be formulated according to standard
pharmaceutical practice. Oral formulations containing the active compounds 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. com, 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.
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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-propylceliulose. Dispersions can also be prepared in
glycerol, liquid polyethylene glycols and mixtures thereof in oils. Under ordinary
conditions of storage and use, these preparation 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.
For the purposes of this disclosure, transderma! 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
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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.
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AM100661/100662-01 IN
WE .CLAIM :
1. A process of manufacture of a antineoplastic combination which comprises
bringing an mTOR inhibitor and an antineoplastic alkylating agent into association in
an effective amount.
2. A process according to claim 1, wherein the antineoplastic alkylating agent is
selected from the group consisting of meclorethamine, cyclophosphamide, ifosfamide,
melphalan, chlorambucil, thiotepa, mitomycin, busulfan, lomustine, carmustine,
procarbazine, temozolomide, cisplatin, and carboplatin.
3. A process according to claim 1 or 2, wherein the effective amount of the
combination includes the mTOR inhibitor in a subtherapeutically effective amount, the
alkylating agent in a subtherapeutically effective amount or both in subtherapeutically
effective amounts.
4. The process according to claim 3 in which the mTOR inhibitor is provided in a
subtherapeutically effective amount.
5. The process according to claim 3 in which the alkylating agent is provided in a
subtherapeutically effective amount.
6. The process according to claim 3 in which both the mTOR inhibitor and the
alkylating agent are provided in subtherapeutically effective amounts.
7. The process according to any one of claims 1 to 6, wherein the mTOR inhibitor
is a rapamycin.
8. The process according to claim 7, wherein the rapamycin is rapamycin.
9. The process according to claim 7, wherein the rapamycin is 42-O-(2-
hydroxy)ethyl rapamycin.
10. The process according to claim 7 wherein the rapamycin is CCI-779.
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AM100661/100662-01 IN
11. A process for the preparation of a product comprising an mTOR inhibitor and
an antineoplastic alkylating agent as a combined preparation for simultaneous,
separate or sequential use in the treatment of a neoplasm in a mammal, which
comprises bringing an mTOR inhibitor and an antineoplastic alkylating agent into
combination in an effective amount.
12. A process according to claim 11, wherein the antineoplastic alkylating agent is
selected from the group consisting of meclorethamine, cyclophosphamide, ifosfamide,
melphalan, chlorambucil, thiotepa, mitomycin, busulfan, lomustine, carmustine,
procarbazine, temozolomide, cisplatin, and carboplatin.
13. A process according to claim 11 or 12, wherein the effective amount of a
combination includes the mTOR inhibitor in a subtherapeutically effective amount, the
alkylating agent in a subtherapeutically effective amount, or both in subtherapeutically
effective amounts.
14. The process according to claim 13 in which the mTOR inhibitor is provided in a
subtherapeutically effective amount.
15. The process according to claim 13 in which the alkylating agent is provided in
a subtherapeutically effective amount.
16. The process according to claim 13 in which both the mTOR inhibitor and the
alkylating agent are provided in subtherapeutically effective amounts.
17. The process according to any one of claims 11 to 16, wherein the mTOR
inhibitor is a rapamycin.
18. The process according to claim 17, wherein the rapamycin is rapamycin.
19. The process according to claim 17, wherein the rapamycin is 42-O-(2-
hydroxy)ethyl rapamycin.
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AM100661/100662-01 IN
20. The process according to claim 17 wherein the rapamycin is CCI-779.
21. A process for the preparation of a pharmaceutical composition, which
comprises bringing an mTOR inhibitor, an antineoplastic alkylating agent and a
pharmaceutically acceptable carrier into association or combination.
22. A process according to claim 21 in which the antineoplastic alkylating agent is
selected from the group consisting of meclorethamine, cyclophosphamide, ifosfamide,
melphalan, chlorambucil, thiotepa, mitomycin, busulfan, lomustine, carmustine,
procarbazine, temozolomide, cisplatin, and carboplatin
23. A process according to claim 21 or 22, wherein the mTOR inhibitor is CCI-779.
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24. A process of manufacture of an antineoplastic
combination, substantially as herein described.
25. A process for the preparation of a product
comprising an mTOR inhibitor and an antineoplastic
alkylating agent,substantially as herein
described.
25. A process for the preparation of a pharmaceutical
composition, substantially as herein described.
21

This invention provides the use of a combination of an mTOR inhibitor and
an antineoplastic alkylating agent in the treatment of neoplasms.