I lllll llllllll II llllll lllll lllll lllll lllll lllll lllll lllll 111111111111111111111111111111111
`US 20030008923Al
`
`(19) United States
`(12) Patent Application Publication
`Dukart et al.
`
`(10) Pub. No.: US 2003/0008923 Al
`Jan. 9, 2003
`(43) Pub. Date:
`
`(54) ANTINEOPLASTIC COMBINATIONS
`
`Related U.S. Application Data
`
`(75)
`
`Inventors: Gary Dukart, Ambler, PA (US); James
`Joseph Gibbons JR., Westwood, NJ
`(US)
`
`Correspondence Address:
`Arnold S. Milowsky
`5 Giralda Farms
`Madison, NJ 07940 (US)
`
`(60) Provisional application No. 60/295,190, filed on Jun.
`1, 2001.
`
`Publication Classification
`
`(51)
`Int. Cl.7 .......................... A61K 31/44; A61K 31/13
`(52) U.S. Cl. .............................................................. 514/672
`
`(73) Assignee: Wyeth, Madison, NJ (US)
`
`(57)
`
`ABSTRACT
`
`(21) Appl. No.:
`
`10/157,656
`
`(22) Filed:
`
`May 29, 2002
`
`This invention provides the use of a combination of an
`mTOR inhibitor and an antineoplastic alkylating agent in the
`treatment of neoplasms.
`
`West-Ward Exhibit 1024
`Dukart USPA '923
`Page 001
`
`

`

`US 2003/0008923 Al
`
`Jan.9,2003
`
`1
`
`ANTINEOPLASTIC COMBINATIONS
`
`BACKGROUND OF THE INVENTION
`
`[0001] This application claims priority from copending
`provisional application Serial No. 60/295,190, filed Jun. 1,
`2001, the entire disclosure of which is hereby incorporated
`by reference.
`
`[0002] This invention relates to the use of combinations of
`an mTOR inhibitor and an alkylating agent in the treatment
`of neoplasms.
`
`[0003] Rapamycin is a macrocyclic triene antibiotic pro(cid:173)
`duced by Streptomyces hygroscopicus, which was found to
`have antifungal activity, particularly against Candida albi(cid:173)
`cans, 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.
`Pat. Nos. 3,929,992; and 3,993,749]. Additionally, rapamy(cid:173)
`cin alone (U.S. Pat. No. 4,885,171) or in combination with
`picibanil (U.S. Pat. No. 4,401,653) has been shown to have
`antitumor activity.
`
`[0004] The immunosuppressive effects of rapamycin have
`been disclosed in FASEB a* 3, 3411 (1989). CyclosporinA
`and FK-506, other macrocyclic molecules, also have been
`shown to be effective as immunosuppressive agents, there(cid:173)
`fore useful in preventing transplant rejection [FASEB 3,
`3411 (1989); FASEB 3, 5256 (1989); R. Y. Calne et al.,
`Lancet 1183 (1978); and U.S. Pat. No. 5,100,899]. R. Martel
`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 arthri(cid:173)
`tis; and effectively inhibited the formation of IgE-like anti(cid:173)
`bodies.
`
`[0005] Rapamycin is also useful in preventing or treating
`systemic lupus erythematosus [U.S. Pat. No. 5,078,999],
`pulmonary inflammation [U.S. Pat. No. 5,080,899], insulin
`dependent diabetes mellitus [U.S. Pat. No. 5,321,009], skin
`disorders, such as psoriasis [U.S. Pat. No. 5,286,730], bowel
`disorders [U.S. Pat. No. 5,286,731], smooth muscle cell
`proliferation and intimal thickening following vascular
`injury [U.S. Pat. Nos. 5,288,711and5,516,781], adult T-cell
`leukemia/lymphoma [European Patent Application 525,960
`Al], ocular inflammation [U.S. Pat. No. 5,387,589], malig(cid:173)
`nant carcinomas [U.S. Pat. No. 5,206,018], cardiac inflam(cid:173)
`matory disease [U.S. Pat. No. 5,496,832], and anemia [U.S.
`Pat. No. 5,561,138].
`
`[0006] Rapamycin 42-ester with 3-hydroxy-2-(hydroxym(cid:173)
`ethyl)-2-methylpropionic acid (CCI-779) is ester of rapa(cid:173)
`mycin 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, includ(cid:173)
`ing CCI-779, are disclosed in U.S. Pat. No. 5,362,718.
`
`[0007] CCI-779 exhibits cytostatic, as opposed to cyto(cid:173)
`toxic 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 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
`CCI-779 that results in the G 1 S phase block is novel for an
`anticancer drug.
`
`[0008]
`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 G 1 phase of the cell cycle.
`
`[0009]
`In vivo studies in nude mice have demonstrated
`that CCI-779 has activity against human tumor xenografts of
`diverse histological types. Gliomas were particularly sensi(cid:173)
`tive to CCI-779 and the compound was active in an ortho(cid:173)
`topic glioma model in nude mice. Growth factor (platelet(cid:173)
`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.
`
`DESCRIPTION OF THE INVENTION
`
`[0010] 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 can(cid:173)
`cer, gastric cancer,
`leukemia, colorectal cancer, and
`unknown primary cancer. This invention also provides com(cid:173)
`binations of an mTOR inhibitor and an antineoplastic alky(cid:173)
`lating agent for use as antineoplastic combination chemo(cid:173)
`therapy, in which the dosage of either the mTOR inhibitor or
`the antineoplastic alkylating agent or both are used in
`subtherapeutically effective dosages.
`
`[0011] 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.
`
`[0012] 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.
`
`[0013] 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
`
`West-Ward Exhibit 1024
`Dukart USPA '923
`Page 002
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`

`US 2003/0008923 Al
`
`Jan.9,2003
`
`2
`
`cell proliferation, translation of mRNAs for several key
`proteins that regulate the G 1 phase of the cell cycle, and
`IL-2-induced transcription, leading to inhibition of progres(cid:173)
`sion of the cell cycle from Gl to S.
`
`[0014] 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.
`
`[0015] 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 Gl to S by inhibiting the phosphorylation of
`serine 389 of p70s6 kinase by mTOR.
`
`[0016] The following standard pharmacological test pro(cid:173)
`cedure 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 rapa(cid:173)
`mycin 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. IGFl) 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.
`
`pletely attached. The results under both sets of conditions
`should be the same for an mTOR inhibitor.
`
`[0021] B. Western Blot Analysis
`
`[0022] 1) Prepare total protein samples by placing
`22.5 µl of lysate per tube and then add 2.5 µl
`NuPAGE sample reducing agent. Heat samples at
`70° C. for 10 minutes. Electrophoresed using
`NuPAGE gels and NuPAGE SDS buffers.
`
`[0023] 2) Transfer the gel to a nitrocellulose mem(cid:173)
`brane with NuPAGE transfer buffer. The membrane
`are blocked for 1 hour with blocking buffer (Tris
`buffered saline with 0.1 %-Tween and 5% nonfat(cid:173)
`milk). Rinse membranes 2x with washing buffer
`(Tris buffered saline with 0.1%-Tween).
`
`[0024] 3) Blots/membrane are incubated with the
`P-p70 S6K (T389) primary antibody (1:1000) in
`blocking buffer overnight at 4 ° C. in a rotating
`platform.
`
`[0025] 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.
`
`[0026] 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) detec(cid:173)
`tion and then exposed to chemiluminescence films.
`
`Materials:
`
`(Novex Cat # NP0007)
`NuPAGE LOS Sample Buffer
`(Novex Cat # NP0004)
`NuPAGE Sample Reducing Agent
`(Novex Cat# NP0321)
`NuPAGE 4--12% Bis-Tris Gel
`(Novex Cat# NPOOOl)
`NuPAGE MOPS SOS Running Buffer
`(Novex Cat# LC2001)
`Nitrocellulose
`(Novex Cat # NP0006)
`NuPAGE Transfer Buffer
`(Amersham Cat# RPN3114H)
`Hyperfilm ECL
`ECL Western Blotting Detection Reagent
`(Amersham Cat # RPN2134)
`(Cell Signaling Cat # 9205)
`Primary antibody: Phospho-p70 S6 Kinase (Thr389)
`Secondary antibody: Goat anti-rabbit IgG-HRP conjugate (Santa Cruz Cat# sc-2004)
`
`[0017] Methods
`[0018] A Preparation of Cell Lysates
`[0019] Cell lines were grown in optimal basal medium
`supplemented with 10% fetal bovine serum and penicillin/
`treptomycin. For phosphorylation studies, cells were sub(cid:173)
`cultured 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 µl 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.
`[0020] The test procedure can also be run by incubating
`the cells in growth medium overnigh, after they have com-
`
`[0027] As used in accordance with this invention, the term
`"a rapamycin" defines a class of immunosuppressive com(cid:173)
`pounds which contain the basic rapamycin nucleus (shown
`below). The rapamycins of this invention include com(cid:173)
`pounds 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 form(cid:173)
`ing such salts, either by virtue of containing an acidic or
`basic moiety.
`
`West-Ward Exhibit 1024
`Dukart USPA '923
`Page 003
`
`

`

`US 2003/0008923 Al
`
`Jan.9,2003
`
`3
`
`2
`
`-a OH
`w---n·
`
`, [ '
`
`O'Me
`
`0
`
`0
`
`OMe
`
`RAPAMYCIN
`
`[0028]
`It is preferred that the esters and ethers of rapamy(cid:173)
`cin 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.
`
`[0029] Preferred 42- and/or 31-esters and ethers of rapa(cid:173)
`mycin are disclosed in the following patents, which are all
`hereby incorporated by reference: alkyl esters (U.S. Pat. No.
`4,316,885); aminoalkyl esters (U.S. Pat. No. 4,650,803);
`fluorinated esters (U.S. Pat. No. 5,100,883); amide esters
`(U.S. Pat. No. 5,118,677); carbamate esters (U.S. Pat. No.
`5,118,678); silyl ethers (U.S. Pat. No. 5,120,842); ami(cid:173)
`noesters (U.S. Pat. No. 5,130,307); acetals (U.S. Pat. No.
`5,51,413); aminodiesters (U.S. Pat. No. 5,162,333); sul(cid:173)
`fonate and sulfate esters (U.S. Pat. No. 5,177,203); esters
`(U.S. Pat. No. 5,221,670); alkoxyesters (U.S. Pat. No.
`5,233,036); 0-aryl, -alkyl, -alkenyl, and -alkynyl ethers
`(U.S. Pat. No. 5,258,389); carbonate esters (U.S. Pat. No.
`5,260,300); arylcarbonyl and alkoxycarbonyl carbamates
`(U.S. Pat. No. 5,262,423); carbamates (U.S. Pat. No. 5,302,
`584); hydroxyesters (U.S. Pat. No. 5,362,718); hindered
`esters (U.S. Pat. No. 5,385,908); heterocyclic esters (U.S.
`Pat. No. 5,385,909); gem-disubstituted esters (U.S. Pat. No.
`5,385,910); amino alkanoic esters (U.S. Pat. No. 5,389,639);
`phosphorylcarbamate esters (U.S. Pat. No. 5,391,730); car(cid:173)
`bamate esters (U.S. Pat. No. 5,411,967); carbamate esters
`(U.S. Pat. No. 5,434,260); amidino carbamate esters (U.S.
`Pat. No. 5,463,048); carbamate esters (U.S. Pat. No. 5,480,
`988); carbamate esters (U.S. Pat. No. 5,480,989); carbamate
`esters (U.S. Pat. No. 5,489,680); hindered N-oxide esters
`(U.S. Pat. No. 5,491,231); biotin esters (U.S. Pat. No.
`5,504,091); 0-alkyl ethers (U.S. Pat. No. 5,665,772); and
`PEG esters of rapamycin (U.S. Pat. No. 5,780,462). The
`preparation of these esters and ethers are disclosed in the
`patents listed above.
`
`[0030] Preferred 27-esters and ethers of rapamycin are
`disclosed in U.S. Pat. No. 5,256,790, which is hereby
`incorporated by reference. The preparation of these esters
`and ethers are disclosed in the patents listed above.
`
`[0031] Preferred oximes, hydrazones, and hydroxy(cid:173)
`lamines of rapamycin are disclosed in U.S. Pat. Nos. 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 dis(cid:173)
`closed in U.S. Pat. No. 5,023,263, which is hereby incor(cid:173)
`porated by reference.
`
`[0032] Particularly preferred rapamycins include rapamy(cid:173)
`cin [U.S. Pat. No. 3,929,992], CCI-779 [rapamycin 42-ester
`with
`3-hydroxy-2-(hydroxymethyl)-2-methylpropionic
`acid; U.S. Pat. No. 5,362,718], and 42-0-(2-hydroxy)ethyl
`rapamycin [U.S. Pat. No. 5,665,772].
`
`[0033] 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, sul(cid:173)
`furic, methanesulfonic,
`napthalenesulfonic,
`benzene(cid:173)
`sulfonic, 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 car(cid:173)
`bon 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.
`
`[0034]
`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 42-0-(2-hydroxy)ethyl rapamycin.
`
`[0035] As described herein, CCI-779 was evaluated as a
`representative mTOR inhibitor in the mTOR inhibitor plus
`antimetabolite combinations of this invention.
`
`[0036] The preparation of CCI-779 is described in U.S.
`Pat. No. 5,362,718, which is hereby incorporated by refer(cid:173)
`ence. 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.
`
`[0037] 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 will
`prevent the cell from replicating effectively. The lethality of
`
`West-Ward Exhibit 1024
`Dukart USPA '923
`Page 004
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`US 2003/0008923 Al
`
`Jan.9,2003
`
`4
`
`the monofunctional alkylating agents results from the rec(cid:173)
`ognition of the DNA lesion by the cell and the response of
`the cell to that lesion. (Colvin 0 M. Antitumor Alkylating
`Agents. In Cancer Principles & Practice of Oncology 6th
`Edition. ed. De Vita V T, Hellman S, Rosenberg S A
`Lippincoft Williams & Wilkins. Philadelphia 2001. p. 363.)
`
`[0038] Antineoplastic alkylating agents are roughly clas(cid:173)
`sified, 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, mito(cid:173)
`mycin C, dianhydrogalactitol, and dibromodulcitol; alkyl
`sulfonates, such as busulfan; nitrosoureas, such as bischlo(cid:173)
`roethylnitrosourea
`(BCNU),
`cyclohexyl-chloroethylni(cid:173)
`trosourea
`(CCNU),
`and methylcyclohexylchloroeth(cid:173)
`ylnitrosourea
`(MeCCNU);
`hydrazine
`and
`triazine
`derivatives, such as procarbazine, dacarbazine, and temozo(cid:173)
`lomide; 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 JP, O'Dwyer P J. Cisplatin and Its Analogues. In
`Cancer Principles & Practice of Oncology 6th Edition. ed.
`De Vita VT, Hellman S, Rosenberg SA Lippincott Williams
`& Wilkins. Philadelphia 2001. p. 378.) These compounds
`include cisplatin, carboplatin, platinum IV compounds, and
`multinuclear platinum complexes.
`
`[0039] The following are representative examples of alky(cid:173)
`lating agents of this invention.
`
`[0040] Meclorethamine is commercially available as an
`injectable (MUSTARGEN).
`
`[0041] Cyclophosphamide is commercially available as
`an
`injectable
`( cyclophosphamide,
`lyophilized
`CYTOXAN, or NEOSAR) and in oral tablets (cyclo(cid:173)
`phosphamide or CYTOXAN).
`
`[0042]
`Ifosfamide is commercially available as an
`injectable (IFEX).
`
`[0043] Melphalan is commercially available as an
`injectable (ALKERAN) and in oral tablets (ALKE(cid:173)
`RAN).
`
`[0044] Chlorambucil is commercially available in oral
`tablets (LEUKERAN).
`
`[0045] Thiotepa is commercially available as an inject(cid:173)
`able (thiotepa or THIOPLEX).
`
`[0046] Mitomycin is commercially available as an
`injectable (mitomycin or MUTAMYCIN).
`
`[0047] Busulfan is commercially available as an inject(cid:173)
`able (BUSULFEX) and in oral tablets (MYLERAN).
`
`[0048] Lomustine (CCNU) is commercially available in
`oral capsules (CEENU).
`
`[0049] Carmustine (BCNU) is commercially available
`as an intracranial implant (GLIADEL) and as an inject(cid:173)
`able (BICNU).
`
`[0050] Procarbazine is commercially available in oral
`capsules (MATULANE).
`
`[0051] Temozolomide is commercially available in oral
`capsules (TEMODAR).
`
`[0052] Cisplatin is commercially available as an inject(cid:173)
`able (cisplatin, PLATINOL, or PLATINOL-AQ).
`
`[0053] Carboplatin is commercially available as an
`injectable (PARAPLATIN).
`
`[0054] The following table briefly summarizes some of the
`recommended dosages for the antineoplastic alkylating
`agents listed above.
`
`TABLE 1
`
`Recommended Dosages of Antineoplastic Alkylating Agents
`
`Drug
`
`Mustargen
`
`Cyclophosphamide
`
`Ifosfamide
`
`Melphalan
`
`Chlorambucil
`Thiotepa
`Mitomycin
`Busulfan
`
`Dosage
`
`Regimen
`
`0.4 mg/kg
`
`40-50 mg/kg i.v.
`10-15 mg/kg i.v.
`3-5 mg/kg i.v.
`1-5 mg/kg oral
`1.2 g/m2 i. v.
`
`each course given as a singe dose or in
`divided doses of 0.1 to 0.2 mg/kg/day.
`in divided doses over a period of 2-5 days
`every 7-10 days
`twice weekly
`daily
`daily for 5 consecutive days; repeated
`every 3 weeks or after recovery from
`hematologic toxicity.
`daily for 2-3 weeks followed by 4 weeks
`rest, then 2 mg daily maintenance dosage
`daily for 7-10 days followed by 2 mg daily
`maintenance after white blood cell count
`has recovered.
`0.15 mg/kg orally daily for 7 days, followed by a rest period
`of at least 14 days, then 0.005 mg/kg
`daily maintenance.
`single infusion over 15-20 minutes every
`2 weeks for 4 doses, followed by a rest
`period, then administered at 4 week
`intervals for maintenance.
`0.1-0.2 mg/kg orally daily for 3-6 weeks
`0.3-0.4 mg/kg i.v.
`every 1-4 weeks
`20 mg!m2 i.v.
`every 6--8 weeks
`1.8 mg!m2 orally daily
`
`6 mg orally
`
`10 mg orally
`
`16 mg!m2 i.v.
`
`West-Ward Exhibit 1024
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`Page 005
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`

`US 2003/0008923 Al
`
`Jan.9,2003
`
`5
`
`TABLE 1-continued
`
`Recommended Dosages of Antineoplastic Alkylating Agents
`
`Drug
`
`Lorn us tine
`Carmustine
`Procarbazine
`
`Temozolomide
`
`Cisplatin
`
`Carboplatin
`
`Dosage
`
`Regimen
`
`130 mg/m2 orally every 6 weeks
`150-200 mg/m2 i.v.
`every 6 weeks
`2-4 mg/kg orally daily for first week, then 4-6 mg/kg until
`maximum response is achieved
`1-2 mg/kg orally mainentance
`150 mg!m2 orally once daily for 5 days per 28-day
`treatment cycle
`daily for 5 days per cycle
`once every 4 week cycle
`once every 4 week cycle
`
`20 mg!m2 i.v.
`75-100 mg!m2 i.v.
`360 mg!m2 i.v.
`
`4
`
`[0055] Preferred mTOR inhibitor plus antineoplastic alky(cid:173)
`lating agent combinations of this invention include CCI-779
`plus cisplatin; CCI-779 plus cyclophosphamide; CCI-779
`plus carboplatin; and CCI-779 plus BCNU.
`[0056] The antineoplastic activity of the mTOR inhibitor
`plus antineoplastic alkylating agent combinations were con(cid:173)
`firmed using CCI-779 as a representative mTOR inhibitor in
`in vitro and in vivo standard pharmacological test proce(cid:173)
`dures using combinations of CCI-779 plus cisplatin; CCI-
`779 plus cyclophosphamide; and CCI-779 plus BCNU as
`representative combinations of this invention. The following
`briefi y describes the procedures used and the results
`obtained.
`[0057] Human rhabdomyosarcoma lines Rh30 and Rhl
`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.
`[0058] Dose response curves were determined for each of
`the drugs of interest. The cell lines Rh30, Rhl and SJ-G2
`, 5xl03 and
`were plated in six-well cluster plates at 6xl03
`2.5xl0
`cells/well respectively. After a 24 hour incubation
`period, drugs were added in either 10%FBS+RPMI 1640 for
`Rh30 and Rhl 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 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
`IC concentrations of CCI-779 was mixed in a 1: 1 ratio with
`ea~h 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/Y50,=l,<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 I Cs of drugs tested singly. If they
`did, the effect was considered additive.
`[0059] The results obtained in the in vitro standard phar(cid:173)
`macological 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 Rhl 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 Rhl cell line. The combination of
`CCI-779 plus melphanan was additive against each of the
`cell lines.
`[0060] Female CBNCaJ mice (Jackson Laboratories, Bar
`Harbor, Me.), 4 weeks of age, were immune-deprived by
`thymectomy, followed 3 weeks later by whole-body irradia(cid:173)
`137
`Cs source. Mice received 3xl06
`tion (1200 cGy) using a
`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 tumor
`growth. 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 inter(cid:173)
`vals using digital Vernier calipers interfaced with a com(cid:173)
`puter. Tumor volumes were calculated assuming tumors to
`be spherical using the formula [(it/6)xd3
`], 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 chemo(cid:173)
`therapy drug for each study was as follows:
`[0061] Cyclophosphamide on days 1 and 8 every 21
`days for 3 cycles
`[0062] The combination of CCI-779 and cyclophospha(cid:173)
`mide 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 cyclophos(cid:173)
`phamide ( 44 mg/kg) was additive. When combined as
`suboptimum dosages, CCI-779 plus cyclophosphamide was
`equivalent to cyclophosphamide given at an optimum dos(cid:173)
`age.
`
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`Dukart USPA '923
`Page 006
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`US 2003/0008923 Al
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`Jan.9,2003
`
`6
`
`[0063] Based on the results of these standard pharmaco(cid:173)
`logical test procedures, combinations of an mTOR inhibitor
`plus an antineoplastic alkylating agent are useful as antine(cid:173)
`oplastic 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, pancre(cid:173)
`atic 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.
`[0064]
`In providing chemotherapy, multiple agents having
`different modalities of action are typically used as part of a
`chemotherapy "cocktail." It is anticipated that the combina(cid:173)
`tions of this invention will be used as part of a chemotherapy
`cocktail that may contain one or more additional antine(cid:173)
`oplastic 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-fiuorouracil, fioxuradine, thiogua(cid:173)
`nine, cytarabine, fiudarabine, 6-mercaptopurine, methotrex(cid:173)
`ate, gemcitabine, capecitabine, pentostatin, trimetrexate, or
`cladribine ); hormonal agents (i.e., estramustine, tamoxifen,
`toremifene, anastrozole, or letrozole ); antibiotics (i.e., pli(cid:173)
`camycin, bleomycin, mitoxantrone, idarubicin, dactinomy(cid:173)
`cin, mitomycin, or daunorubicin); immunomodulators (i.e.,
`interferons, IL-2, or ECG); antimitotic agents (i.e., vinblas(cid:173)
`tine, vincristine, teniposide, or vinorelbine ); topoisomerase
`inhibitors (i.e., topotecan, irinotecan, etoposide, or doxoru(cid:173)
`bicin); and other agents (i.e., hydroxyurea, trastuzumab,
`altretamine, retuximab, paclitaxel, docetaxel, L-asparagi(cid:173)
`nase, or gemtuzumab ozogamicin).
`[0065] 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 alkylating
`agent. This time differential may range from several min(cid:173)
`utes, 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 adminis(cid:173)
`tered 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 alkylating agent,
`it is anticipated that the mTOR inhibitor will be administered
`orally or parenterally, with parenterally being preferred,
`while the alkylating agent may be administered parenterally,
`orally,