(12) United States Patent
`Zhu et al.
`
`I lllll llllllll Ill lllll lllll lllll lllll lllll 111111111111111111111111111111111
`US006432973Bl
`US 6,432,973 Bl
`Aug.13, 2002
`
`(10) Patent No.:
`(45) Date of Patent:
`
`(54) WATER SOLUBLE RAPAMYCIN ESTERS
`
`(75)
`
`Inventors: Tianmin Zhu, Monroe, NY (US);
`Mahdi Fawzi, Morristown, NJ (US)
`
`(73) Assignee: Wyeth, Madison, NJ (US)
`
`( *) Notice:
`
`Subject to any disclaimer, the term of this
`patent is extended or adjusted under 35
`U.S.C. 154(b) by 0 days.
`
`(21) Appl. No.: 09/954,782
`
`(22) Filed:
`
`Sep. 18, 2001
`
`Related U.S. Application Data
`(60) Provisional application No. 60/233,776, filed on Sep. 19,
`2000.
`Int. Cl.7 .................... C07D 498/16; C07D 498/18;
`C07D 491/06; A61K 31/395; A61K 31/445
`(52) U.S. Cl. ........................................ 514/291; 540/456
`(58) Field of Search ........................... 540/456; 514/291
`
`(51)
`
`(56)
`
`References Cited
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`................... 514/183
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`
`FOREIGN PATENT DOCUMENTS
`
`EP
`EP
`WO
`
`0 525 960 Al
`781 776
`WO 94/25072
`
`2/1993
`* 7 /1997
`* 11/1994
`
`OTHER PUBLICATIONS
`
`C.V. Vezina et al., J. Anitbiot., 1975, 721,28.
`H.A. Baker et al., J. Antibiot., 1978, 539,31.
`FASEB, 1989, 3411,3.
`FASEB, 1989, 5256,3.
`R.Y. Caine et al., Lancet, 1978, 1183.
`R. Martel et al., Can. J. Physiol. Pharmacol., 1977, 48, 55.
`T. Matsumoto et al., Atheroschlerosis, 1998, 95, 139.
`S.E. Roselaar et al., J. Clin. Invest., 1995, 1906, 96.
`K.B. Lemstrom et al., Arterioscler. Thomb. Vase. Biol. 1996,
`553, 16( 4).
`T. Quaschning et al., Kidney Int., 1999, S235, 56(71).
`E.E. Emeson et al., Am. J. Pathol., 1993, 1906, 142(6).
`J. Gibbons et al., Proc. Am. Assoc. Can. Res., 1999, 301, 40.
`B. Geoerger et al., Proc. Am. Assoc. Can. Res., 1999, 603,
`40.
`J. Alexandre, Bull. Cancer, 1999, 808-811, 86.
`J. Alexandre et al., Clin. Cancer Res., 1999, Abstract 7, Nov.
`Suppl.
`* cited by examiner
`
`Primary Examiner-Bruck Kifie
`(74) Attorney, Agent, or Firm-Arnold S. Milowsky
`
`(57)
`
`ABSTRACT
`
`This invention provide pegylated hydroxyesters of rapamy(cid:173)
`cin which are useful in inducing immunosuppression and in
`the treatment of transplantation rejection, autoimmune
`diseases, solid tumors, fungal infections, and vascular dis(cid:173)
`ease.
`
`18 Claims, No Drawings
`
`West-Ward Exhibit 1092
`Zhu USP '973
`Page 001
`
`

`

`1
`WATER SOLUBLE RAPAMYCIN ESTERS
`
`US 6,432,973 Bl
`
`2
`soluble in water and most organic solvents. At molecular
`weights less than 1000 are the viscous, colorless liquids;
`higher molecular weight PEGs are waxy, white solids. The
`melting point of the solid is proportional to the molecular
`5 weight, approaching a plateau at 67° C. Molecular weight
`range from a few hundred to approximately 20,000 are
`commonly used in biological and biotechnological applica(cid:173)
`tions. Of much interest in the biomedical areas is the fact that
`PEG is nontoxic and was approved by FDA for internal
`10 consumption. Peglyated rapamycin is disclosed in US Pat.
`No. 5,780,462.
`
`DESCRIPTION OF THE INVENTION
`
`This invention provides methoxypoly( ethylene glycol)
`esters of hydroxyesters of rapamycin having the structure
`
`15
`
`20
`
`This application claims priority from provisional appli(cid:173)
`cation Ser. No. 60/233,776, filed Sep. 19, 2000, the entire
`disclosure of which is hereby incorporated by reference.
`BACKGROUND OF THE INVENTION
`This invention relates to water soluble rapamycin esters
`which are useful in inducing immunosuppression and in the
`treatment of transplantation rejection, autoimmune diseases,
`solid tumors, fungal infections, and vascular disease. More
`particularly, this invention concerns methoxypoly( ethylene
`glycol) esters of hydroxyesters of rapamycin and methods
`for using them for inducing immunosuppression, and in the
`treatment of transplantation rejection, graft vs. host disease,
`autoimmune diseases, diseases of inflammation, adult T-cell
`leukemia/lymphoma, solid tumors, fungal infections, car(cid:173)
`diovascular disease, cerebral vascular disease, peripheral
`vascular disease or hyperproliferative vascular disorders.
`Rapamycin is a macrocyclic triene antibiotic produced by
`Streptomyces hygproscopicus, 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. Pat. No. 25
`3,929,992; and U.S. Pat. No. 3,993,749]. Additionally, rapa(cid:173)
`mycin 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.
`The immunosuppressive effects of rapamycin have been 30
`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 use-
`ful in preventing transplant rejection [FASEB 3, 3411
`(1989); FASEB 3, 5256 (1989); R. Y. Caine et al., Lancet 35
`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; 40
`and effectively inhibited the formation of IgE-like antibod-
`ies.
`Rapamycin is also useful in preventing or treating sys(cid:173)
`temic lupus erythematosus [U.S. Pat. No. 5,078,999], pul(cid:173)
`monary inflammation [U.S. Pat. No. 5,080,899], insulin 45
`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 50
`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].
`A rapamycin ester, rapamycin 42-ester with 3-hydroxy-
`2-(hydroxymethyl)-2-methylpropionic acid [disclosed in
`U.S. Pat. No. 5,362,718], also known as CCl-779, has been
`shown to have antitumor activity against a variety of tumor
`cell lines, in in vivo animal tumor models, and in Phase I 60
`clinical trials. [Gibbons, J., Proc. Am. Assoc. Can. Res. 40:
`301 (1999); Geoerger, B., Proc. Am. Assoc. Can. Res. 40:
`603 (1999); Alexandre, J., Proc. Am. Assoc. Can. Res. 40:
`613 (1999); and Alexandre, J., Clin. Cancer. Res. 5
`(November Supp.): Abstr. 7 (1999)].
`Polyethylene glycol (PEG) is a linear or branched, neutral
`polymer available in a variety of molecular weights and is
`
`;
`
`wherein
`R1 and R2 are each, independently, hydrogen or -CO
`(CR3R4h(CR5R 6)aCR7R8 R9
`R3 and R4 are each, independently, hydrogen, alkyl of 1-6
`carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of
`2-7 carbon atoms, trifluoromethyl, or -F;
`R5 and R6 are each, independently, hydrogen, alkyl of 1-6
`carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of
`2-7 carbon atoms, ---(CR3R4)pR10
`, -CF3, -F, or
`-C02R11;
`R 7 is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of 2-7
`carbon atoms, alkynyl of 2-7 carbon atoms,
`, -CF3, -F, or -C02R11;
`---(CR3R4)pR10
`R8 and R9 are each, independently, hydrogen, alkyl of 1-6
`carbon atoms, alkenyl of 2-7 carbon atoms, alkynyl of
`2-7 carbon atoms, ---(CR3R4)pR10
`, -CF3, -F, or
`-C02R11;
`R 10 is hydrogen or -COCH2-S-CH2CH2-(0-
`CH2-CH2)n -OCH3;
`R11 is hydrogen, alkyl of 1-6 carbon atoms, alkenyl of
`2-7 carbon atoms, alkynyl of 2-7 carbon atoms, or
`phenylalkyl of 7-10 carbon atoms;
`b=0-6;
`d=0-6;
`f=0--6;
`n=5-450;
`with the proviso that R1 and R2 are both not hydrogen and
`further provided that either R 1 or R 2 contains at least one
`65 ---(CR3R4)pR10 group in which R10 is -COCH2-S(cid:173)
`CH2CH2---(0-CH2-CH2)n-OCH3, or a pharmaceuti(cid:173)
`cally acceptable salt thereof which are useful for inducing
`
`55
`
`West-Ward Exhibit 1092
`Zhu USP '973
`Page 002
`
`

`

`US 6,432,973 Bl
`
`3
`immunosuppression, and in the treatment of transplantation
`rejection, graft vs. host disease, autoimmune diseases, dis(cid:173)
`eases of inflammation, adult T-cell leukemia/lymphoma,
`solid tumors, fungal infections, cardiovascular disease, cere(cid:173)
`bral vascular disease, peripheral vascular disease or hyper(cid:173)
`proliferative vascular disorders. The compounds of this
`invention can also be referred to as pegylated hydroxyesters
`of rapamycin.
`When applicable, pharmaceutically acceptable salts can
`be formed from organic and inorganic bases (i.e., when a
`compound contains a free hydroxyl group), 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 or antiestrogen con(cid:173)
`tains a suitable acidic moiety.
`As used in accordance with this invention, the term
`"providing," with respect to providing a compound or sub(cid:173)
`stance covered by this invention, means either directly
`administering such a compound or substance, or adminis(cid:173)
`tering a prodrug, derivative, or analog which will form the
`equivalent amount of the compound or substance within the
`body.
`Of the pegylated hydroxyesters of rapamycin covered by
`this invention, it is preferred that the hydroxyester of rapa(cid:173)
`mycin is CCl-779, in which one or both of the hydroxyl
`groups of the 3-hydroxy-2-(hydroxymethyl)-2-
`methylpropionic acid moiety are pegylated. Of the com(cid:173)
`pounds of this invention, it is preferred that n=5-200; more
`preferred that n=8-135. Most preferred members are those
`in which n=8-20 and those in which n=90-120. The values 30
`of n refer to the range of repeating ethoxy units in the PEG
`side chain. For example, when a compound is described as
`having n=5-200, it means that such compound consists of a
`mixture of compounds having a normal distribution between
`n=5 and n=200, with approximately n=lOO having the 35
`greatest frequency. With compounds III and IV, the average
`n was 108, and 99% of n being between 65 and 155. The
`compounds of this invention may also be described and
`understood based upon the average molecular weight of the
`polyethylene glycol chains used to produce their ester
`chains. For instance, an CCl-779-PEG 5000 ester refers to a
`compound of the general formula above in which one side
`chain PEG ester is formed utilizing a polyethylene glycol
`derivative having an average molecular weight range at or
`near 5,000; and CCl-779-(PEG 5000)2 ester refers to a
`compound of the general formula above in two side chain
`PEG esters are formed utilizing a polyethylene glycol
`derivative having an average molecular weight range at or
`near 5,000.
`The reagents used in the preparation of the compounds of
`this invention can be either commercially obtained or can be
`prepared by standard procedures described in the literature.
`The preparation of the hydroxyesters of rapamycin, from
`which the pegylated hydroxyesters are made from, are
`described in U.S. Pat. No. 5,362,718, which is hereby
`incorporated by reference. One or more of the hydroxyesters
`may be acylated with a acylating agent having the general
`structure X-CH2C02 H, where Xis a suitable leaving group,
`such as bromine or iodine, in the presence of a coupling
`agent such as dicyclohexylcarbodiimide (DCC) in the pres(cid:173)
`ence of a base catalyst, such as dimethylaminopyridine
`(DMAP). Accordingly, rapamycin 42-ester with 3-hydroxy- 60
`2-(2-iodo-acetoxymethy 1)-2-methy 1-propionic acid
`(Compound I) and rapamycin 42-ester with 3-(2-Iodo(cid:173)
`ace to xy )-2-( 2-iodo-ace to xyme thy 1)-2-me thy 1-propionic
`acid (Compound II) are intermediates prepared using this
`methodology, that are useful in the production of the corre- 65
`sponding pegylated hydroxyesters of rapamycin
`(Compounds III and IV, respectively).
`
`40
`
`4
`The pegylated esters may be produced utilizing the poly(cid:173)
`ethylene glycols known in the art, such as those described on
`pages 355 to 361 of the Handbook of Pharmaceutical
`Excipients, Second Edition, 1994. The preferred compounds
`5 of this invention may also be described as those of the
`formula esterified using polyethylene glycols having an
`average molecular weight of from about 200 to about
`200,000. A preferred range of the PEG esters of this inven(cid:173)
`tion includes those in which the molecular weight of the
`polyethylene glycol portion of the ester chain has a molecu-
`10 lar weight in the range of from about 300 to about 20,000,
`more preferably between about 350 and about 6,000. Reac(cid:173)
`tion of the hydroxyester of rapamycin which has been
`acylated as described above, with a suitable polyethyleneg(cid:173)
`lycol thiol in the presence of a base, such as sodium
`bicarbonate provides the desired pegylated hydroxyester of
`15 rapamycin.
`The antitumor activity of the compounds of this invention
`were confirmed in a standard pharmacological test proce(cid:173)
`dure which measures the inhibition of U87MG human
`glioblastoma cell growth (as a function of 3 H-thymidine
`20 incorporation), using CCl-779-PEG 5000 ester (Compound
`III) and CCl-779-(PEG 5000)2 ester (Compound IV) as
`representative compounds of this invention. The following
`briefly describes the procedure used and results obtained.
`U87MG human glioblastoma cells (ATCC #HTB-14; avail-
`25 able from the American Type Culture Collection; 10801
`University Boulevard; Manassas, Va. 20110;), were grown
`in the following media.
`Growth Medium: ERL Minimum Essential Medium with
`Earle Salts (500 mL)
`+5 mL ERL MEM Non-Essential Amino Acids (10 mM)
`+5 mL ERL Penicillin-Streptomycin (100000 u/mL,
`10000 µg/mL)
`+5 mL ERL Na Pyruvate Solution (100 mM)
`+5 mL ERL L-Glutamine 200 mM
`+50 mL ERL Fetal Bovine Serum (Qualified)
`Test Procedure:
`1. Cells were trypsinized and plated at a concentration of
`104 cells/well in a final volume of 200 µL growth medium
`in 96-well fiat bottom plates and allowed to adhere for 24
`hours at 37° C.
`2. The media was removed by aspiration with care to not
`disturb the cell monolayer. 200 µL of fresh growth media
`was added per well, allowing enough wells for samples to be
`run in triplicate. Test compounds were added in 10 µL
`phosphate buffer solution (PBS) and incubated for another
`45 48 hours at 37° C.
`3. During the last 5 hours of incubation, plates were
`labeled with 1 µCi 3 H thymidine per well. (New England
`Nuclear thymidine, catalog #NET-027, 6.7 Ci/mmole). The
`1 µCi was added in 10 µL of PBS (on the day of harvest). The
`50 plates were returned to the incubator for the last 5 hours.
`4. The radioactive media was removed by aspiration, with
`care not to disturb the cell monolayer. Then 50 µL of ERL
`lOX Trypsin was added to each well, followed by incubation
`at 37° C. for 10 minutes or until the monolayer was loosened
`55 from the well bottom. Samples were harvested on a glass
`fiber filter mat using a Skatron 96 well harvester. Mats were
`counted in a Wallac Betaplate counter.
`
`Results:
`
`Compound
`
`CCI-779
`Compound III
`Compound IV
`
`0.6 ng/mL
`1.0 ng/mL*
`4.0 ng/mL*
`
`*ng/mL equivalent of CCI-779
`
`West-Ward Exhibit 1092
`Zhu USP '973
`Page 003
`
`

`

`US 6,432,973 Bl
`
`10
`
`5
`The results obtained in this standard pharmacological test
`procedure show that the compounds of this invention inhibit
`tumor cell growth and are therefore useful as antineoplastic
`agents. In particular, the compounds of this invention are
`useful in treating or inhibiting the growth of solid tumors, 5
`including sarcomas and carcinomas, such as astrocytomas,
`prostate cancer, breast cancer, small cell lung cancer, and
`ovanan cancer.
`The compounds of this invention are also useful treatment
`or inhibition of transplantation rejection such as kidney,
`heart, liver, lung, bone marrow, pancreas (islet cells), cornea,
`small bowel, and skin allografts, and heart valve xenografts;
`in the treatment or inhibition of graft vs. host disease; in the
`treatment or inhibition of autoimmune diseases such as
`lupus, rheumatoid arthritis, diabetes mellitus, myasthenia
`gravis, and multiple sclerosis; and diseases of inflammation 15
`such as psoriasis, dermatitis, eczema, seborrhea, inflamma(cid:173)
`tory bowel disease, pulmonary inflammation (including
`asthma, chronic obstructive pulmonary disease,
`emphysema, acute respiratory distress syndrome, bronchitis,
`and the like) and ocular uveitis; adult T-cell leukemia/ 20
`lymphoma; fungal infections; hyperproliferative vascular
`diseases such as restenosis; graft vascular atherosclerosis;
`and cardiovascular disease, cerebral vascular disease, and
`peripheral vascular disease, such as coronary artery disease,
`cerebrovascular disease, arteriosclerosis, atherosclerosis, 25
`nonatheromatous arteriosclerosis, or vascular wall damage
`from cellular events leading toward immune mediated vas(cid:173)
`cular damage, and inhibiting stroke or multiinfarct dementia.
`When used for restenosis, it is preferred that the com(cid:173)
`pounds of this invention are used to treat restenosis that 30
`occurs following an angioplasty procedure. When used for
`this treating restenosis following an angioplasty, the com(cid:173)
`pounds of this invention can be administered prior to the
`procedure, during the procedure, subsequent to the
`procedure, or any combination of the above.
`This invention also covers analogous pegylated hydrox(cid:173)
`yesters of other rapamycins known in the art such as, but not
`limited to, 29-demethoxyrapamycin, [U.S. Pat. No. 4,375,
`464, 32-demethoxyrapamycin under C. A nomenclature];
`rapamycin derivatives in which the double bonds in the 1-, 40
`3-, and/or 5-positions have been reduced [U.S. Pat. No.
`5,023,262]; 29-desmethylrapamycin [U.S. Pat. No. 5,093,
`339, 32-desmethylrapamycin under C. A nomenclature];
`7,29-bisdesmethylrapamycin [U.S. Pat. No. 5,093,338,
`7,32-desmethylrapamycin under C. A nomenclature]; 45
`27-hydroxyrapamycin [U.S. Pat. No. 5,256,790] and
`15-hydroxyrapamycin [U.S. Pat. No. 5,102,876]. This
`invention also covers esters at the 31-position of
`42-oxorapamycin [U.S. Pat. No. 5,023,263]. The disclosures
`in the above cited U.S. Pat. Nos. are hereby incorporated by 50
`reference.
`When administered for the treatment or inhibition of a
`particular disease state or disorder, it is understood that the
`effective dosage of the pegylated hydroxyester of rapamycin
`may vary depending upon the particular compound utilized,
`the mode of administration, the condition, and severity
`thereof, of the condition being treated, as well as the various
`physical factors related to the individual being treated. As
`used in accordance with invention, satisfactory results may
`be obtained when the pegylated hydroxyester of rapamycin
`is administered in a daily oral dosage of from about pro(cid:173)
`jected daily dosages of active compound would be 0.1
`µg/kg-100 mg/kg, preferably between 0.001-25 mg/kg, and
`more preferably between 0.01-5 mg/kg. The projected daily
`dosages are expected to vary with route of administration.
`Such doses may be administered in any manner useful in
`directing the active compounds herein to the recipient's
`
`6
`bloodstream, including orally, via implants, parenterally
`(including intravenous, intraperitoneal and subcutaneous
`injections), rectally, intranasally, vaginally, and transder-
`mally. The pegylated esters of this invention provide an
`advantage in ease of formulation and administration over the
`non-pegylated esters of rapamycin, in that they are signifi-
`cantly more water soluble than corresponding non-pegylated
`esters. For example, both compounds 3 and 4 had a water
`solubility of > 100 mg/mL, whereas CCl-779 had a water
`solubility of <1.1 µg/mL. This advantage is particularly
`important for oral and parenteral administration.
`Oral formulations containing the active compounds of this
`invention may comprise any conventionally used oral forms,
`including tablets, capsules, buccal forms, troches, lozenges
`and oral liquids, suspensions or solutions. Capsules may
`contain mixtures of the active compound(s) with inert fillers
`and/or diluents such as the pharmaceutically acceptable
`starches (e.g. corn, potato or tapioca starch), sugars, artificial
`sweetening agents, powdered celluloses, such as crystalline
`and microcrystalline celluloses, flours, gelatins, gums, etc.
`Useful tablet formulations may be made by conventional
`compression, wet granulation or dry granulation methods
`and utilize pharmaceutically acceptable diluents, binding
`agents, lubricants, disintegrants, surface modifying agents
`(including surfactants), suspending or stabilizing agents,
`including, but not limited to, magnesium stearate, stearic
`acid, talc, sodium lauryl sulfate, microcrystalline cellulose,
`carboxymethylcellulose calcium, polyvinylpyrrolidone,
`gelatin, alginic acid, acacia gum, xanthan gum, sodium
`citrate, complex silicates, calcium carbonate, glycine,
`dextrin, sucrose, sorbitol, dicalcium phosphate, calcium
`sulfate, lactose, kaolin, mannitol, sodium chloride, talc, dry
`starches and powdered sugar. Preferred surface modifying
`agents include nonionic and anionic surface modifying
`agents. Representative examples of surface modifying
`35 agents include, but are not limited to, poloxamer 188,
`benzalkonium chloride, calcium stearate, cetostearl alcohol,
`cetomacrogol emulsifying wax, sorbitan esters, colloidal
`silicon dioxide, phosphates, sodium dodecylsulfate, magne-
`sium aluminum silicate, and triethanolamine. Oral formula(cid:173)
`tions herein may utilize standard delay or time release
`formulations to alter the absorption of the active compound
`(s). The oral formulation may also consist of administering
`the active ingredient in water or a fruit juice, containing
`appropriate solubilizers or emulsifiers as needed.
`In some cases it may be desirable to administer the
`compounds directly to the airways in the form of an aerosol.
`The compounds of this invention may also be adminis-
`tered parenterally or intraperitoneally. Solutions or suspen(cid:173)
`sions of these active compounds as a free base or pharma(cid:173)
`cologically acceptable salt can be prepared in water suitably
`mixed with a surfactant such as hydroxy-propylcellulose.
`Dispersions can also be prepared in glycerol, liquid poly(cid:173)
`ethylene glycols and mixtures thereof in oils. Under ordi(cid:173)
`nary conditions of storage and use, these preparation contain
`55 a preservative to prevent the growth of microorganisms.
`The pharmaceutical forms suitable for injectable use
`include sterile aqueous solutions or dispersions and sterile
`powders for the extemporaneous preparation of sterile
`injectable solutions or dispersions. In all cases, the form
`60 must be sterile and must be fluid to the extent that easy
`syringability exists. It must be stable under the conditions of
`manufacture and storage and must be preserved against the
`contaminating action of microorganisms such as bacteria
`and fungi. The carrier can be a solvent or dispersion medium
`65 containing, for example, water, ethanol, polyol (e.g.,
`glycerol, propylene glycol and liquid polyethylene glycol),
`suitable mixtures thereof, and vegetable oils.
`
`West-Ward Exhibit 1092
`Zhu USP '973
`Page 004
`
`

`

`8
`-continued
`
`Compound II
`
`O=<I
`o~
`, .... ''
`(j-o
`~
`
`o
`
`o
`
`···V··""'ocH3
`
`)
`
`'"""" [''''
`
`''
`
`I
`
`0
`
`N
`
`0
`
`US 6,432,973 Bl
`
`7
`For the purposes of this disclosure, transdermal adminis(cid:173)
`trations are understood to include all administrations across
`the surface of the body and the inner linings of bodily
`passages including epithelial and mucosal tissues. Such
`administrations may be carried out using the present 5
`compounds, or pharmaceutically acceptable salts thereof, in
`lotions, creams, foams, patches, suspensions, solutions, and
`suppositories (rectal and vaginal).
`Transdermal administration may be accomplished
`through the use of a transdermal patch containing the active
`compound and a carrier that is inert to the active compound,
`is non toxic to the skin, and allows delivery of the agent for
`systemic. absorption into the blood stream via the skin. The
`carrier may take any number of forms such as creams and
`ointments, pastes, gels, and occlusive devices. The creams
`and ointments may be viscous liquid or semisolid emulsions 15
`of either the oil-in-water or water-in-oil type. Pastes com(cid:173)
`prised of absorptive powders dispersed in petroleum or
`hydrophilic petroleum containing the active ingredient may
`also be suitable. A variety of occlusive devices may be used
`to release the active ingredient into the blood stream such as 20
`a semi-permeable membrane covering a reservoir containing
`the active ingredient with or without a carrier, or a matrix
`containing the active ingredient. Other occlusive devices are
`known in the literature.
`Suppository formulations may be made from traditional 25
`materials, including cocoa butter, with or without the addi(cid:173)
`tion of waxes to alter the suppository's melting point, and
`glycerin. Water soluble suppository bases, such as polyeth(cid:173)
`ylene glycols of various molecular weights, may also be
`used.
`The preparation of representative examples of this inven-
`tion is described below.
`EXAMPLE 1
`
`10
`
`30
`
`OH
`
`l.Ox10- 3 mole),
`g
`(1.03
`CCl-779
`4-Dimethylaminopyridine (3.0 mg) and 1,3-
`Dicyclohexylcarbodiimide (0.136 g, 6.6x10- 4 mole) were
`dissolved in 50 mL anhydrous methylene chloride in a 250
`Preparation of Rapamycin 42-Ester with 3-
`35 mL round-bottom flask. Iodoacetic acid (0.185 g, 1.0x10-3
`Hydroxy-2-(2-iodo-acetoxymethyl)-2-methyl(cid:173)
`propionic Acid (Compound I) and Rapamycin 42-
`mole) was dissolved in 10 mL anhydrous methylene chlo(cid:173)
`Ester with 3-(2-Iodo-acetoxy)-2-(2-iodo(cid:173)
`ride. The iodoacetic acid solution was added into reaction
`acetoxymethyl)-2-methyl-propionic Acid
`mixture over a period of 10 min with stirring by a magnetic
`(Compound II)
`Compound I 40 bar. Then the reaction mixture was stirred at room tempera(cid:173)
`ture for another 2.5 h. The solution was then filtered through
`a filter paper. The filtrate was transferred to a separatory
`funnel, washed 50 mL of sodium bicarbonate solution (5.5
`g/100 mL) and then washed with 2x50 mL of water. The
`45 methylene chloride layer was dried with 10 g anhydrous
`sodium sulfate for 2 h. Then sodium sulfate was filtered out
`and methylene chloride was removed by rotary evaporation.
`A total of 0. 93 g yellow solid was obtained. Isolation of pure
`50 compound I and compound II was performed by preparative
`HPLC on a Prep Nova-pak HR C18 (300x19 mm) column
`from Waters. Compound I eluted at 18.4 min and compound
`11 eluted at 24.4 min using a gradient (30% A, 70% B for
`5 min. then to 100% B in 30 min.). A is 90% water, 10%
`55 acetonitrile; B is 10% water, 90% acetonitrile. The fraction
`was collected and extracted by 2x100 ml methylene chlo(cid:173)
`ride. The organic layer was combined and dried with anhy(cid:173)
`drous sodium sulfate for 4 h. The organic solvent was
`removed by rotary evaporation to dryness. Compound I, a
`yellowish solid was obtained (0.14 g). 1 H NMR (CDCL3 ,
`400 MHz) ll 3.68 (s, 2H, I-CH2-C02 - ) , 4.28 (dd, 2H,
`-C02-CH2 - ) . MS m/z 1215.8 (M+NH4t. Compound
`II, a yellowish solid was obtained (0.08 g). 1H NMR
`65 (CDCL3 , 400 MHz) ll 3.72 (s, 4H, 2xl-CH2-C02 - ) ,
`4.28 (dd, 4H, 2x-C0 2-CH2 - ) . MS m/z 1383.6
`(M+NH4t.
`
`O=<I
`o~
`0:0
`~
`"""" [''''
`I"""' 0
`
`...
`
`OCH3
`
`,,
`
`N
`
`0
`
`60
`
`West-Ward Exhibit 1092
`Zhu USP '973
`Page 005
`
`

`

`9
`EXAMPLE 2
`Preparation of CCl-779-PEG 5000-Rapamycin 42-
`Ester with 3-Hydroxy-2-(hydroxymethyl)-2-
`methylpropionic Acid Methoxypoly( ethylene
`glycol)thiol 5000 Conjugate (Compound III)
`
`(Compound III)
`
`o~s~o0ocH3 10
`
`o~
`
`( ) :0 OH
`
`""" ('"
`
`''
`
`;CH3
`
`"'f'
`
`0
`
`N
`
`~~:en~ ,p
`
`with average n = 108
`
`CCl-779-PEG 5000
`
`US 6,432,973 Bl
`
`10
`HPLC analysis. Then mPEG-SH 5000 (450 mg, 9.lxl0-5
`mole) was added to the reaction solution and the reaction
`mixture was stirred at room temperature for another 45 min.
`The reaction was checked again by taking 10 µL sample for
`5 HPLC analysis. The chromatogram showed that compound
`I was 100% converted to Compound III. The reaction
`mixture was extracted with 2x100 mL methylene chloride.
`The organic layer was dried with anhydrous sodium sulfate
`then filtered. The filtrate was concentrated to a volume of 20
`mL by rotaty evaporation. The crude product was precipi(cid:173)
`tated o