I lllll llllllll II llllll lllll lllll lllll lllll lllll lllll lllll 111111111111111111111111111111111
`US 20020183239Al
`
`(19) United States
`(12) Patent Application Publication
`Gibbons, JR. et al.
`
`(10) Pub. No.: US 2002/0183239 Al
`Dec. 5, 2002
`( 43) Pub. Date:
`
`(54) ANTINEOPLASTIC COMBINATIONS
`
`Related U.S. Application Data
`
`(75)
`
`Inventors: James J. Gibbons JR., Westwood, NJ
`(US); Gary Dukart, Ambler, PA (US);
`Jurgen Frisch, Marburg (DE)
`
`Correspondence Address:
`Arnold S. Milowsky
`5 Giralda Farms
`Madison, NJ 07940 (US)
`
`(73) Assignee: Wyeth, Madison, NJ
`
`(21) Appl. No.:
`
`10/116,902
`
`(22) Filed:
`
`Apr. 5, 2002
`
`(60) Provisional application No. 60/282,388, filed on Apr.
`6, 2001.
`
`Publication Classification
`
`Int. Cl.7 ..................................................... A61K 31/00
`(51)
`(52) U.S. Cl. .................................................................. 514/1
`
`(57)
`
`ABSTRACT
`
`This invention provides the use of a combination of an
`mTOR inhibitor and an antimetabolite antineoplastic agent
`in the treatment of neoplasms.
`
`West-Ward Exhibit 1023
`Gibbons USPA '239
`Page 001
`
`

`

`US 2002/0183239 Al
`
`Dec. 5, 2002
`
`1
`
`ANTINEOPLASTIC COMBINATIONS
`
`[0001] This application claims priority from provisional
`application Serial No. 60/282,388, filed Apr. 6, 2001, the
`entire disclosure of which is hereby incorporated by refer(cid:173)
`ence.
`
`BACKGROUND OF THE INVENTION
`
`[0002] This invention relates to the use of combinations of
`an mTOR inhibitor and an antimetabolite antineoplastic
`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 3, 3411 (1989). CyclosporinAand
`FK-506, other macrocyclic molecules, also have been shown
`to be effective as immunosuppressive agents, therefore use(cid:173)
`ful 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 encepha(cid:173)
`lomyelitis model, a model for multiple sclerosis; in the
`adjuvant arthritis model, a model for rheumatoid arthritis;
`and effectively inhibited the formation of IgE-like antibod(cid:173)
`ies.
`
`[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 antimetabolite antineoplastic
`agent as antineoplastic combination chemotherapy. In par(cid:173)
`ticular, these combinations are useful in the treatment of
`renal cancer, soft tissue cancer, breast cancer, neuroendo(cid:173)
`crine 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 antimetabolite anti(cid:173)
`neoplastic agent for use as antineoplastic combination che(cid:173)
`motherapy, in which the dosage of either the mTOR
`inhibitor or the antimetabolite antineoplastic 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 antimetabolite
`antineoplastic 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
`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.
`
`West-Ward Exhibit 1023
`Gibbons USPA '239
`Page 002
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`

`US 2002/0183239 Al
`
`Dec. 5, 2002
`
`2
`
`[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.
`
`Materials:
`
`NuPAGE LOS Sample Buffer
`NuPAGE Sample Reducing Agent
`NuPAGE 4-12% Bis-Tris Gel
`NuPAGE MOPS SOS Running Buffer
`Nitrocellulose
`NuPAGE Transfer Buffer
`Hyperfilm ECL
`ECL Western Blotting Detection
`Reagent
`Primary antibody: Phospho-p70 S6
`Kinase (Thr389)
`Secondary antibody: Goat anti-rabbit
`IgG-HRP conjugate
`
`(Novex Cat # NP0007)
`(Novex Cat # NP0004)
`(Novex Cat# NP0321)
`(Novex Cat# NPOOOl)
`(Novex Cat# LC2001)
`(Novex Cat # NP0006)
`(Amersham Cat# RPN3114H)
`(Amersham Cat # RPN2134)
`
`(Cell Signaling Cat # 9205)
`
`(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(cid:173)
`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.
`[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.
`
`RAPAMYCIN
`
`2
`
`CXOH
`~--- "~r'
`
`'
`
`0
`
`0
`
`OMe
`
`West-Ward Exhibit 1023
`Gibbons USPA '239
`Page 003
`
`

`

`US 2002/0183239 Al
`
`Dec. 5, 2002
`
`3
`
`[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
`"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 incorpo(cid:173)
`rated 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 gua(cid:173)
`nine) but are structurally different so thy competitively or
`irreversibly inhibit nuclear processing of DNA or RNA; and
`(3) pyrimidine analogs. which mimic the natural pyrim(cid:173)
`idines (cytosine, thymidine, and uracil) but are structurally
`different so thy competitively or irreversibly inhibit nuclear
`processing of DNA or RNA
`
`[0038] The following are representative examples of anti(cid:173)
`metabolites of this invention.
`
`[0039] 5-Fluorouracil (5-FU; 5-fiuoro-2,4(1H,3H)-pyrim(cid:173)
`idinedione) is commercially available in a topical cream
`(FLUOROPLEX or EFUDEX) a topical solution (FLUO(cid:173)
`ROPLEX or EFUDEX), and as an injectable containing 50
`mg/mL 5-fiuorouracil (ADRUCIL or fiurouracil).
`
`[0040] Floxuradine (2'-deoxy-5-fiuorouridine) is commer(cid:173)
`cially available as an injectable containing 500 mg/vial of
`fioxuradine (FUDR or fioxuradine).
`
`[0041] Thioguanine (2-amino-1,7-dihydro-6-H-purine-6-
`thione) is commercially available in 40 mg oral tablets
`(thioguanine ).
`
`West-Ward Exhibit 1023
`Gibbons USPA '239
`Page 004
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`

`US 2002/0183239 Al
`
`Dec. 5, 2002
`
`4
`
`-continued
`
`Drug
`
`Dosage
`
`Regimen
`
`[0042] Cytarabine ( 4-amino-l-(beta)-D-arabinofuranosyl-
`2(1H)-pyrimidinone) is commercially available as a liposo(cid:173)
`mal injectable containing 10 mg/mL cytarabine (DEPO(cid:173)
`CYT) or as a liquid injectable containing between 1 mg-1
`g/vial or 20 mg/mL (cytarabine or CYTOSAR-U).
`
`[0043] Fludarabine (9-H-Purin-6-amine,2-fluoro-9-(5-0-
`phosphono-(beta)-D-arabinofuranosyl)
`is
`commercially
`available as a liquid injectable containing 50 mg/vial (FLU(cid:173)
`DARA).
`
`[0044] 6-Mercaptopurine
`(1,7-dihydro-6H-purine-6-
`thione) is commercially available in 50 mg oral tablets
`(PURINETHOL).
`
`[0045] Methotrexate (MTX; N-[ 4-[[(2,4-diamino-6-pte(cid:173)
`ridinyl)methyl]methylamino ]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).
`
`[0046] Gemcitabine
`(2'-deoxy-2',2'-difluorocytidine
`monohydrochloride ((beta)-isomer)), is commercially avail(cid:173)
`able as a liquid injectable containing between 200 mg-1
`g/vial (GEMZAR).
`
`[0047] Capecitabine
`(5'-deoxy-5-fluoro-N-[(pentyloxy(cid:173)
`)carbonyl]-cytidine) is commercially available as a 150 or
`500 mg oral tablet (XELODA).
`
`[0048] Pentostatin
`((R)-3-(2-deoxy-(beta)-D-erythro(cid:173)
`pentofuranosy 1)-3,6, 7,8-tetrahydroimidazo[ 4,5-d ][ 1,3 ]diaz(cid:173)
`epin-8-ol) is commercially available as a liquid injectable
`containing 10 mg/vial (NIPENT).
`
`[0049] Trimetrexate (2,4-diamino-5-methyl-6-[ (3,4,5-tri(cid:173)
`methoxyanilino )methyl]quinazoline mono-D-glucuronate)
`is commercially available as a liquid injectable containing
`between 25-200 mg/vial (NEUTREXIN).
`
`[0050] Cladribine
`(2-chloro-6-amino-9-(2-deoxy-(beta)(cid:173)
`D-erythropento-furanosyl) purine) is commercially avail(cid:173)
`able as a liquid injectable containing 1 mg/mL (LEUSTA(cid:173)
`TIN).
`
`[0051] The following table briefly summarizes some of the
`recommended dosages for the antimetabolites listed above.
`
`Drug
`
`Dosage
`
`Regimen
`
`5-Fluorouracil
`
`12 mg/kg oral
`6 mg/kg oral
`
`Floxuradine
`(FUDR)
`Cytarabine
`(DEPOCYT)
`
`Cytarabine
`(injectable)
`Fludarabine
`(FLUDARA)
`6-Mercaptopurine
`(PURINETHOL)
`
`370-600 mg/m2
`i.v.
`0.1-0.6 mg/kg
`
`50 mg
`
`100 mg/m2
`2-3 g/m2
`25 mg/m2
`
`2.5-5 mg/kg
`1. 5-2.5 mg/kg
`
`daily for 4 days
`days 6, 8, 10, 12
`no drug on days 5, 7, 9, and 11;
`doses cut in half if toxicity
`observed
`daily for 5 days, every 3-4
`weeks
`daily by arterial infusion
`
`every 14 days for 5 doses during
`induction period; followed by
`every 28 days for maintenance
`daily for 7 days
`twice daily for 2-6 days
`30 min infusion for 5 consecu-
`tive days; every 28 days
`daily for induction
`daily for maintenance
`
`Methotrexate
`
`Gemcitabine
`(GEMZAR)
`
`Capecitabine
`(XELODA)
`Pentostatin
`(NIP ENT)
`Trimetrexate
`(NEUTREXIN)
`Cladribine
`(LEUSTATIN)
`
`15-30 mg oral
`
`1000-1250 mg/m2
`/
`30 min
`
`2500 mg/m2
`
`daily for 5 day course; repeated
`3-5 times
`1000 mg/m2/30 min single agent: once weekly for 7
`weeks, followed by 1 week rest,
`then once weekly for 3 out of
`every 4 weeks
`combination therapy: days 1, 8,
`15 per 28 day cycle, or days 1
`and 8 per 21 day cycle
`daily for 2 weeks followed by 1
`week rest period
`as bolus injection or diluted as
`i.v. infusion; every other week
`i.v. infusion once daily for 21
`days
`continuous infusion for 7
`consecutive days
`
`4 mg/m2
`
`45 mg/m2
`
`0.09 mg/kg/day
`
`[0052] This invention also covers the use of an mTOR
`inhibitor plus an antimetabolite in which a biochemical
`modifying agent is part of the chemotherapeutic regimen.
`The term "biochemical modifying agent" is well known and
`understood to those skilled in the art as an agent given as an
`adjunct to antimetabolite therapy, which serves to potentate
`its antineoplastic activity, as well as counteract the side
`effects of the antimetabolite. Leucovorin and levofolinate
`are typically used as biochemical modifying agents for
`methotrexate and 5-FU therapy.
`
`[0053] 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 (leucov(cid:173)
`orin calcium).
`
`[0054] Levofolinate (pharmacologically active isomer of
`5-formyltetrahydrofolic acid) is commercially available as
`an injectable containing 25-75 mg levofolinate (ISO(cid:173)
`VORIN) or as 2.5-7.5 mg oral tablets (ISOVORIN).
`
`[0055] Preferred mTOR
`inhibitor plus antimetabolite
`combinations of this invention include CCI-779 plus gem(cid:173)
`citabine; CCI-779 plus 5-fluorouracil; and CCI-779 plus
`5-fluorouracil plus leucovorin. It is preferred that the CCI-
`779 plus gemcitabine combination be used in treating pan(cid:173)
`creatic cancer and that the CCI-779 plus 5-fluorouracil
`combination (with or without leucovorin) be used in treating
`colorectal cancer.
`
`[0056] The antineoplastic activity of the CCI-779 plus
`antimetabolite combination was confirmed in in vitro and in
`vivo standard pharmacological test procedures using com(cid:173)
`binations of CCI-779 plus gemcitabine; and CCI-779 plus
`5-fluorouracil as representative combinations of this inven(cid:173)
`tion. The following briefly 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 antimetabolite
`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
`
`West-Ward Exhibit 1023
`Gibbons USPA '239
`Page 005
`
`

`

`US 2002/0183239 Al
`
`Dec. 5, 2002
`
`5
`
`4
`
`, 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 gemcitabine or 5-FU.
`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 I C50 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,=1,<1,>1. If the three concentrations of CCI-
`779 tested alone didn't produce an IC that matched any of
`the three I Cs of the other compound tested alone, all the 1: 1
`combinations were checked to see if their I Cs fell between
`the appropriate ICs 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 that in no case did the
`combinations yield less than a 50% inhibition of growth
`indicating that the combinations were at least additive and
`produced no evidence of antagonism.
`
`[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)
`tion (1200 cGy) using a 137Cs source. Mice received 3xl06
`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] Gemcitabine on days 1, 4, 8 in cycle 1 only
`
`[0062] The combination of CCI-779 and gemcitabine was
`evaluated in a human colon (GC3) mouse xenograft test
`procedure. In this test procedure, CCI-779 was given daily
`5 for 2 consecutive weeks every 21 days for 3 cycles and
`gemcitabine given on days 1, 4, and 8 in the first cycle only.
`
`The presence of CCI-779 did not enhance tumor regression
`seen in the first cycle with gemcitabine treatment. However,
`groups treated with CCI-779 were delayed in the time
`required to reach 2-3x the original pretreatment tumor
`volume (versus gemcitabine alone), indicating that there was
`at least an additive benefit derived from the combination
`treatment.
`
`[0063] Based on the results of these standard pharmaco(cid:173)
`logical test procedures, combinations of an mTOR inhibitor
`plus an antimetabolite chemotherapeutic agent are useful as
`antineoplastic therapy. More particularly, these combina(cid:173)
`tions useful in treating 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/antimetabolite combination used in
`conjunction with other chemotherapeutic agents, such as
`alkylating agents (i.e., cisplatin, carboplatin, streptazoin,
`melphalan, chlorambucil, carmustine, methclorethamine,
`lomustine, bisulfan, thiotepa, ifofamide, or cyclophospha(cid:173)
`mide ); 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, doxorubicin, or daunorubicin); immuno(cid:173)
`modulators (i.e., interferons, IL-2, or ECG); antimitotic
`agents (i.e., vinblastine, vincristine, teniposide, or vinorel(cid:173)
`bine ); topoisomerase inhibitors (i.e., topotecan, irinotecan,
`or etoposide); and other agents (i.e., hydroxyurea, trastu(cid:173)
`zumab, altretamine, retuximab, paclitaxel, docetaxel, L-as(cid:173)
`paraginase, 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