(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property Organization
`International Bureau
`
`I lllll llllllll II llllll lllll llll I II Ill lllll lllll lllll 111111111111111111111111111111111
`
`(43) International Publication Date
`17 October 2002 (17.10.2002)
`
`PCT
`
`(10) International Publication Number
`WO 02/080975 Al
`
`A61K 45/06,
`(51) International Patent Classification7:
`31/436, 3117068, 311513, 311519, A61P 35100 II (A61K
`3117068, 31:436) (A61K 3117068, 31:519, 31:436) (A61K
`31/513, 31:436) (A61K 311519, 31:513, 31:436)
`
`(21) International Application Number: PCT/US02/10912
`
`(22) International Filing Date:
`
`5 April 2002 (05.04.2002)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`601282,385
`601282,388
`
`6 April 2001 (06.04.2001) US
`6 April 2001 (06.04.2001) US
`
`(71) Applicant: WYETH [US/US]; Five Giralda Farms, Madi(cid:173)
`son, NJ 07940-0874 (US).
`
`(72) Inventors: GIBBONS, James, Joseph, Jr.; 33 Terrace
`Drive, Westwood, NJ 07675 (US). DUKART, Gary; 1714
`Benjamin Drive, Ambler, PA 19002 (US). FRISCH, Jur(cid:173)
`gen, Hermann, Ernst; Johannes-Acker-Strasse 15, 35041
`Marburg (DE).
`
`(74) Agents: MILOWSKY, Arnold, S. et al.; Wyeth, Patent
`Law Department, Five Giralda Farms, Madison, NJ 07940-
`0874 (US).
`
`(81) Designated States (national): AE, AG, AL, AM, AT, AU,
`AZ, BA, BB, BG, BR, BY, BZ, CA, CH, CN, CO, CR, CU,
`CZ, DE, DK, DM, DZ, EC, EE, ES, Fl, GB, GD, GE, GH,
`GM, HR, HU, ID, IL, IN, IS, JP, KE, KG, KP, KR, KZ, LC,
`LK, LR, LS, LT, LU, LV, MA, MD, MG, MK, MN, MW,
`MX, MZ, NO, NZ, OM, PH, PL, PT, RO, RU, SD, SE, SG,
`SI, SK, SL, TJ, TM, TN, TR, TT, TZ, UA, UG, UZ, VN,
`YU, ZA, ZM, ZW.
`
`(84) Designated States (regional): ARIPO patent (GH, GM,
`KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZM, ZW),
`Eurasian patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
`European patent (AT, BE, CH, CY, DE, DK, ES, Fl, FR,
`GB, GR, IE, IT, LU, MC, NL, PT, SE, TR), OAPI patent
`(BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW, ML, MR,
`NE, SN, TD, TG).
`
`Published:
`with international search report
`before the expiration of the time limit for amending the
`claims and to be republished in the event of receipt of
`amendments
`
`For two-letter codes and other abbreviations, refer to the "Guid(cid:173)
`ance Notes on Codes and Abbreviations" appearing at the begin(cid:173)
`ning of each regular issue of the PCT Gazette.
`
`---iiiiiiii
`iiiiiiii --!!!!!!!!
`
`iiiiiiii
`
`--
`!!!!!!!! -iiiiiiii
`iiiiiiii ----
`
`ln
`
`l" °" = QO
`= ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
`= ROURACIL
`0 > (57) Abstract: The invention provides the use of a combination of an mTOR inhibitor such as a rapamycin and an antimetabolite
`
`...........
`M (54) Title: ANTINEOPLASTIC COMBINATIONS SUCH AS RAPAMYCIN TOGETHER WITH GEMCITABINE OR FLUO-
`
`~ antineoplastic agent such as gemsitabine or fluorouracil in the treatment of neoplasms.
`
`West-Ward Exhibit 1037
`Gibbons WO '975
`Page 001
`
`

`

`WO 02/080975
`
`PCT/US02/10912
`
`ANTINEOPLASTIC COMBINATIONS SUCH AS RAPAMYCIN TOGETHER WITH GEMCITABINE OR
`FLUOROURACIL
`
`5
`
`10
`
`This invention relates to antineoplastic combinations, more particularly to the
`use of combinations of an mTOR inhibitor (e.g. rapamycin 42-ester with 3-hydroxy-2-
`(hydroxymethyl)-2-methylpropionic
`acid
`(CCl-779))
`and
`an
`antimetabolite
`antineoplastic agent in the treatment of neoplasms.
`
`BACKGROUND OF THE INVENTION
`
`Rapamycin is a macrocyclic triene antibiotic produced by Streptomyces
`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,
`
`15
`
`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.
`
`20
`
`25
`
`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.
`Caine et al., Lancet 1183 (1978); and U.S. Patent 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 arthritis; and effectively inhibited the
`
`formation of lgE-like antibodies.
`lupus
`treating systemic
`in preventing or
`is also useful
`Rapamycin
`erythematosus
`[U.S. Patent 5,078,999], pulmonary
`inflammation
`[U.S. Patent
`
`[U.S. Patent 5,321,009], skin
`insulin dependent diabetes mellitus
`5,080,899],
`disorders, such as psoriasis [U.S. Patent 5,286,730], bowel disorders [U.S. Patent
`
`30
`
`5,286,731], smooth muscle cell proliferation and intimal thickening following vascular
`
`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].
`
`- 1 -
`
`West-Ward Exhibit 1037
`Gibbons WO '975
`Page 002
`
`

`

`WO 02/080975
`
`PCT/US02/10912
`
`Rapamycin 42-ester with 3-hydroxy-2-(hydroxymethyl)-2-methylpropionic acid
`
`(CCl-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 CCl-779, are disclosed
`in U.S. Patent
`5,362,718.
`
`5
`
`CCl-779 exhibits cytostatic, as opposed to cytotoxic properties, and may delay
`
`the time to progression of tumors or time to tumor recurrence. CCl-779 is considered
`to have a mechanism of action that is similar to that of sirolimus. CCl-779 binds to
`and forms a complex with the cytoplasmic protein FKBP, which inhibits an enzyme,
`
`target of rapamycin, also known as FKBP12-rapamycin
`
`1 O mTOR (mammalian
`Inhibition of mTOR's kinase activity inhibits a variety of
`associated protein [FRAP]).
`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
`
`15
`
`cycle from 01 to S. The mechanism of action of CCl-779 that results in the Gl S
`
`phase block is novel for an anticancer drug.
`In vitro, CCl-779 has been shown to inhibit the growth of a number of
`
`histologically diverse tumor cells. Central nervous system (CNS) cancer, leukemia (T(cid:173)
`
`eel!), breast cancer, prostate cancer, and melanoma lines were among the most
`sensitive to CCl-779. The compound arrested cells in the G1 phase of the cell cycle.
`
`20
`
`In vivo studies in nude mice have demonstrated that CCl-779 has activity
`against human tumor xenografts of diverse histological types. Gliomas were
`
`particularly sensitive to CCl-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 CCl-779. The growth of
`
`25
`
`several human pancreatic tumors in nude mice as well as one of two breast cancer
`lines studied in vivo also was inhibited by CCl-779.
`
`DESCRIPTION OF THE INVENTION
`
`30
`
`This invention provides the use of combinations of an mTOR inhibitor and an
`antimetabolite antineoplastic 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,
`
`- 2 -
`
`West-Ward Exhibit 1037
`Gibbons WO '975
`Page 003
`
`

`

`WO 02/080975
`
`PCT/US02/10912
`
`5
`
`1 O
`
`15
`
`20
`
`25
`
`30
`
`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 antimetabolite antineoplastic agent for use as antineoplastic
`combination chemotherapy, in which the dosage of either the mTOR inhibitor or the
`antimetabolite antineoplastic agent or both are used in subtherapeutically effective
`dosages.
`
`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.
`
`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,
`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
`lsotani, 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
`
`- 3 -
`
`West-Ward Exhibit 1037
`Gibbons WO '975
`Page 004
`
`

`

`WO 02/080975
`
`PCT/US02/10912
`
`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 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
`
`(Novex Cat # NP0007)
`(Novex Cat# NP0004)
`(Novex Cat# NP0321)
`(Novex Cat# NP0001)
`(Novex Cat # LC2001)
`(Novex Cat# NP0006)
`(Amersham Cat# RPN3114H)
`(Amersham Cat# RPN2134)
`
`(Cell Signaling Cat# 9205)
`Phospho-p70 S6 Kinase (Thr389)
`Primary antibody:
`Secondary antibody: Goat anti-rabbit lgG-HRP conjugate (Santa Cruz Cat # sc-2004)
`
`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 µI NuP.A:GE
`
`LOS sample buffer per well. The lysates were briefly
`sonicated and then
`centrifuged for 15 minutes at 14000 rpm. Lysates were stored at minus -8o0c until
`use.
`
`5
`
`1 O
`
`15
`
`20
`
`25
`
`30
`
`35
`
`-4-
`
`West-Ward Exhibit 1037
`Gibbons WO '975
`Page 005
`
`

`

`WO 02/080975
`
`PCT/US02/10912
`
`The test procedure can also be run by incubating the cells in growth medium
`overnigh, after they have completely attached. The results under both sets of
`conditions should be the same for an mTOR inhibitor.
`
`5
`
`B. Western Blot Analysis
`
`1) Prepare total protein samples by placing 22.5 µI of lysate per tube and then
`
`add 2.5 µI NuPAGE sample reducing agent. Heat samples at 10°c for 1 O
`
`minutes. Electrophoresed using NuPAGE gels and NuPAGE SDS buffers.
`2) Transfer the gel to a nitrocellulose membrane with NuPAGE transfer buffer.
`
`10
`
`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 4 C in a rotating platform.
`
`0
`
`15
`
`4) Blots are rinsed 3x for 10 minutes each with washing buffer, and incubated
`
`20
`
`25
`
`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
`chemiluminescence films.
`
`(ECL) detection and
`
`then exposed
`
`to
`
`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
`
`30 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 by virtue of containing either an acidic or basic moiety.
`
`- 5 -
`
`West-Ward Exhibit 1037
`Gibbons WO '975
`Page 006
`
`

`

`WO 02/080975
`
`PCT/US02/10912
`
`0
`
`OMe
`I
`
`RAPAMYCIN
`
`5
`
`10
`
`15
`
`20
`
`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.
`Patent 4,316,885); aminoalkyl esters (U.S. Patent 4,650,803); fluorinated 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); 0-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); heterocyclic 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
`
`- 6 -
`
`West-Ward Exhibit 1037
`Gibbons WO '975
`Page 007
`
`

`

`WO 02/080975
`
`PCT/US02/10912
`
`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 ); 0-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 disclosed 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],
`CCl-779
`[rapamycin 42-ester with 3-hydroxy-2-(hydroxymethyl)-2-methylpropionic
`acid; U.S. Patent 5,362,718], and 42-0-(2-hydroxy)ethyl rapamycin [U.S. Patent
`
`5,665,772].
`
`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.
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`- 7 -
`
`West-Ward Exhibit 1037
`Gibbons WO '975
`Page 008
`
`

`

`WO 02/080975
`
`PCT/US02/10912
`
`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, CCl-779, or 42-0-(2-hydroxy)ethyl rapamycin.
`
`5
`
`As described herein, CCl-779 was evaluated as a representative mTOR
`inhibitor in the mTOR inhibitor plus antimetabolite combinations of this invention.
`The preparation of CCl-779 is described in U.S. Patent 5,362,718, which is
`hereby incorporated by reference. When CCl-779 is used as an antineoplastic agent,
`it is projected that initial i.v. infusion dosages will be between about 0.1 and 100
`
`10 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.
`
`15
`
`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
`20 metabolites for the catalytic or regulatory site of a key enzyme in DNA or RNA
`synthesis, or (2) substitute for a metabolite that is normally incorporated into DNA or
`RNA, and thereby producing a DNA or RNA that cannot support replication. Major
`categories of antimetabolites include (1) folic acid analogs, which are inhibitors of
`dihydrofolate reductase (DHFR); (2) purine analogs, which mimic the natural purines
`(adenine or guanine) but are structurally different so they competitively or irreversibly
`inhibit nuclear processing of DNA or RNA; and (3) pyrimidine analogs. which mimic
`the natural pyrimidines (cytosine, thymidine, and uracil) but are structurally different so
`they competitively or irreversibly inhibit nuclear processing of DNA or RNA.
`The following are representative examples of antimetabolites of this invention.
`5-Fluorouracil (5-FU; 5-fluoro-2,4(1 H,3H)-pyrimidinedione)
`is commercially
`available
`in a topical cream (FLUOROPLEX or EFUDEX) a
`topical solution
`(FLUOROPLEX or EFUDEX}, and as an injectable containing 50 mg/ml 5-fluorouracil
`(ADRUCIL or flurouracil).
`
`25
`
`30
`
`- 8 -
`
`West-Ward Exhibit 1037
`Gibbons WO '975
`Page 009
`
`

`

`WO 02/080975
`
`PCT/US02/10912
`
`commercially
`
`is commercially available as an
`(2'-deoxy-5-fluorouridine)
`Floxuradine
`injectable containing 500 mg/vial of floxuradine (FUDR or floxuradine).
`Thioguanine
`(2-amino-1, 7-dihydro-6-H-purine-6-thione)
`is
`available in 40 mg oral tablets {thioguanine).
`Cytarabine
`(4-amino-1-(beta)-D-arabinofuranosyl-2(1 H)-pyrimidinone)
`is
`commercially available as a liposomal injectable containing 10 mg/ml cytarabine
`(DEPOCYT) or as a liquid injectable containing between 1 mg - 1 g/vial or 20 mg/ml
`(cytarabine or CYTOSAR-U).
`Fludarabine (9-H-Purin-6-amine,2-fluoro-9-(5-0-phosphono-(beta)-D-arabino-
`furanosyl) is commercially available as a liquid injectable containing 50 mg/vial
`(FlUDARA).
`6-Mercaptopurine (1,7-dihydro-6H-purine-6-thione) is commercially available in
`50 mg oral tablets (PURINETHOl).
`Methotrexate
`{MTX; N-[4-[[(2,4-diamino-6-pteridinyl)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).
`((beta)-
`Gemcitabine
`(2'-deoxy-2',2'-difluorocytidine monohydrochloride
`isomer)), is commercially available as a liquid injectable containing between 200 mg -
`1g/vial (GEMZAR).
`(5'-deoxy-5-fluoro-N-[(pentyloxy)carbonyl]-cytidine)
`Capecitabine
`commercially available as a 150 or 500 mg oral tablet (XElODA).
`Pentostatin
`( (R )-3-(2-deoxy-(beta )-D-erythro-pentofuranosyl)-3, 6, 7 ,8-tetra-
`hyd roimidazo[4,5-d][1, 3]d iazepin-8-ol) is commercially available as a liquid injectable
`containing 10 mg/vial (NIPENT).
`Trimetrexate
`{2,4-diamino-5-methyl-6-((3,4,5-trimethoxyanilino)methyl]-
`quinazoline mono-D-glucuronate) is commercially available as a liquid injectable
`containing between 25 - 200 mg/vial (NEUTREXIN).
`Cladribine
`(2-chloro-6-amino-9-(2-deoxy-(beta)-D-erythropento-furanosyl)-
`purine)
`is commercially available as a
`liquid
`injectable containing 1 mg/ml
`(lEUSTATIN).
`
`is
`
`5
`
`1 O
`
`15
`
`20
`
`25
`
`30
`
`The following table briefly summarizes some of the recommended dosages for
`the antimetabolites listed above.
`
`- 9 -
`
`West-Ward Exhibit 1037
`Gibbons WO '975
`Page 0010
`
`

`

`WO 02/080975
`
`PCT/US02/10912
`
`Drug
`
`5-Fluorouracil
`
`Dosage
`
`12 mg/kg oral
`6 mg/kg oral
`
`Regimen
`
`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
`
`370 - 600 mgtm2 i.v.
`
`daily for 5 days, every 3-4 weeks
`
`Floxuradine (FUDR)
`Cytarabine (DEPOCYT)
`
`0.1-0.6 mg/kg
`
`50 mg
`
`Cytarabine (injectable)
`
`Fludarabine (FLUDARA)
`
`6-Mercaptopurine
`(PURINETHOL)
`
`Methotrexate
`Gemcitabine (GEMZAR)
`
`100 mgtm2
`
`2-3 gtm2
`
`25 mg/m2
`
`2.5-5 mg/kg
`1.5-2.5 mg/kg
`
`15-30 mg oral
`
`1000 mg/m2/30 min
`
`1000 -1250 mgtm2t
`30 min
`
`Capecitabine (XELODA)
`
`Pentostatin (NIPENT)
`
`Trimetrexate (NEUTREXIN)
`
`2500 mg/m2
`
`4 mg/m2
`
`45 mg/m2
`
`Cladribine (LEUSTATIN)
`
`0.09 mg/kg/day
`
`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 consecutive days;
`every 28 days
`daily for induction
`daily for maintenance
`
`daily for 5 day course; repeated 3-5 times
`
`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
`oeriod
`injection or diluted as
`as bolus
`infusion; every other week
`i.v. infusion once daily for 21 days
`
`i.v.
`
`continuous
`days
`
`infusion
`
`for 7 consecutive
`
`5
`
`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
`potentiate its antineoplastic activity, as well as counteract the side effects of the
`antimetabolite.
`Leucovorin and
`levofolinate are typically used as biochemical
`10 modifying agents for methotrexate and 5-FU therapy.
`
`- 10 -
`
`West-Ward Exhibit 1037
`Gibbons WO '975
`Page 0011
`
`

`

`WO 02/080975
`
`PCT/US02/10912
`
`Leucovorin (5-formyl-5,6,7,8-tetrahydrofolic acid) is commercially available as
`an injectable liquid containing between 5 - 10 mg/ml or 50 - 350 mg/vial (leucovorin
`calcium or WELLCOVORIN) and as 5 - 25 mg oral tablets (leucovorin calcium).
`Levofolinate (pharmacologically active isomer of 5-formyltetrahydrofolic acid)
`is commercially available as an
`injectable containing 25 - 75 mg
`levofolinate
`(ISOVORIN) or as 2.5 - 7.5 mg oral tablets (ISOVORIN).
`
`Preferred mTOR inhibitor plus antimetabolite combinations of this invention
`include CCl-779 plus gemcitabine; CCl-779 plus 5-fluorouracil; and CCl-779 plus 5-
`fluorouracil plus leucovorin.
`It is preferred that the CCl-779 plus gemcitabine
`combination be used in treating pancreatic cancer and that the CCl-779 plus 5-
`fluorouracil combination (with or without leucovorin) be used in treating colorectal
`
`cancer.
`
`The antineoplastic activity of the CCl-779 plus antimetabolite combination was
`confirmed in in vitro and in vivo standard pharmacological test procedures using
`combinations of CCl-779 plus gemcitabine; and CCl-779 plus 5-fluorouracil as
`representative combinations of this invention. The following briefly describes the
`procedures used and the results obtained.
`
`5
`
`1 O
`
`15
`
`20
`
`Human rhabdomyosarcoma lines Rh30 and Rh1 and the human glioblastoma
`for in vitro combination studies with CCl-779 and
`line SJ-GBM2 were used
`In vivo studies used a human neuroblastoma (NB1643) and
`antimetabolite agents.
`
`human colon line GC3.
`Dose response curves were determined for each of the drugs of interest. The
`
`25
`
`cell lines Rh30, Rh1 and SJ-G2 were plated in six-well cluster plates at 6x1 o3, 5x1 o3
`
`and 2.5x1Q4 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
`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
`
`30
`
`35
`
`- 11 -
`
`West-Ward Exhibit 1037
`Gibbons WO '975
`Page 0012
`
`

`

`WO 02/080975
`
`PCT/US02/10912
`
`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 CCl-779 was mixed in a 1 :1
`ratio with each 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
`5 CCl-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 CCl-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 CCl-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 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.
`
`10
`
`15
`
`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
`
`20
`
`irradiation (1200 cGy) using a 137Cs source. Mice received 3 x 106 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 intervals using digital Vernier
`calipers interfaced with a computer. Tumor volumes were calculated assuming tumors
`
`to be spherical using the formula [(n/6) x d 3 ], where d is the mean diameter. CCl-
`
`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 CCl-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:
`
`25
`
`30
`
`Gemcitabine on days 1, 4, 8 in cycle 1 only
`
`- 12 -
`
`West-Ward Exhibit 1037
`Gibbons WO '975
`Page 0013
`
`

`

`WO 02/080975
`
`PCT/US02/10912
`
`The combination of CCl-779 and gemcitabine was evaluated in a human colon
`(GC3) mouse xenograft test procedure.
`In this test procedure, CCl-779 was given
`daily x 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 CCl-779 did not enhance
`tumor regression seen in the first cycle with gemcitabine treatment. However, groups
`treated with CCl-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.
`
`Based on the results of these standard pharmacological test procedures,
`combinations of an mTOR inhibitor plus an antimetabolite chemotherapeutic agent
`are useful as antineoplastic therapy. More particularly, these combinations 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, 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 indivi