(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property
`Organization
`International Bureau
`
`lllllllllll
`
`(43) International Publication Date
`30 September 2004 (30.09.2004)
`
`PCT
`
`(10) International Publication Number
`WO 2004/082822 A2
`
`(51) International Patent Classification7:
`
`B01J
`
`(21) International Application Number:
`PCT/US2004/007896
`
`(22) International Filing Date: 16 March 2004 (16.03.2004)
`
`(25) Filing Language:
`
`(26) Publication Language:
`
`English
`
`English
`
`(30) Priority Data:
`10/390,530
`
`17 March 2003 (17.03.2003)
`
`US
`
`(71) Applicants
`except US):
`States
`(for all designated
`PHARMION CORPORATION
`[US/US]; 2525 28th
`Street, Boulder, CO 80301 (US). ASH STEVENS INC
`[US/US]; 5861 John C Lodge Freeway, Detroit, MI 48202
`(US).
`
`(72) Inventors; and
`IONESCU, Du-
`(75) Inventors/Applicants (for US only):
`mitru [RO/US]; 2828 Grant Drive, Ann Arbor, MI 48108
`(US). BLUMBERGS, Peter [US/US]; 4105 Springer,
`Royal Oak, MI 48073 (US). SILVEY, Gary, L. [US/US];
`10139 Switzer Circle, Overland Park, KS 66212 (US).
`
`(81) Designated States (unless otherwise
`indicated, for every
`kind of national protection available): AE, AG, AL, AM,
`AT, AU, AZ, BA, BB, BG, BR, BW, BY, BZ, CA, CH, CN,
`CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, EG, ES, FI,
`GB, GD, GE, GH, GM, HR, HU, ID, IL, IN, IS, IP, KE,
`KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD,
`MG, MK, MN, MW, MX, MZ, NA, NI, NO, NZ, OM, PG,
`PH, PL, PT, RO, RU, SC, SD, SE, SG, SK, SL, SY, TJ, TM,
`TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, YU, ZA, ZM,
`ZW.
`
`(84) Designated States (unless otherwise
`indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LS, MW, MZ, SD, SL, SZ, TZ, UG, ZM, ZW),
`Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), Euro(cid:173)
`pean (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, Fl, FR,
`GB, GR, HU, IE, IT, LU, MC, NL, PL, PT, RO, SE, SI, SK,
`TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ, GW,
`ML, MR, NE, SN, TD, TG).
`
`Published:
`— without international search report and to be republished
`upon receipt of that report
`
`(74) Agents: SWANSON & BRATSCHUN LLC et a l; 1745
`Shea Center Drive, Suite 330, Highlands Ranch, CO 80129
`(US).
`
`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.
`
`(54) Title: METHODS FOR ISOLATING CRYSTALLINE FORM I OF 5-AZACYTIDINE
`
`(57) Abstract: The invention includes methods for isolating crystalline Form I of 5-aza-
`cytidine substantially free of other forms, wherein 5-azacytidine is represented by the
`formula: The invention also includes pharmaceutical compositions comprising Form I of
`5-azacytidine.
`
`Apotex v. Cellgene - IPR2023-00512
`Petitioner Apotex Exhibit 1009-0001
`
`

`

`WO 2004/082822
`
`PCT7US2004/007896
`
`5
`
`METHODS FOR ISOLATING CRYSTALLINE FORM I OF
`5-AZACYTIDINE
`
`Field of the Invention
`
`The invention relates to the isolation of crystalline polymorphic Form I of 5-azacytidine
`
`(also known as azacitidine and 4-amino-l-P-D-ribofuranosyl-S-triazin-2(l/f)-one). 5-
`
`10
`
`azacytidine may be used in the treatment of disease, including the treatment of
`
`myelodysplastic syndromes (MDS).
`
`Background of the Invention
`
`Polymorphs exist as two or more crystalline phases that have different arrangements
`
`15
`
`and/or different conformations of the molecule in a crystal lattice. When a solvent molecule(s)
`
`is contained within the crystal lattice the resulting crystal is called a pseudopolymorph, or
`
`solvate. If the solvent molecule(s) within the crystal structure is a water molecule, then the
`
`pseudopolymorph/solvate is called a hydrate. The polymorphic and pseudopolymorphic solids
`
`display different physical properties, including those due to packing, and various
`
`20
`
`thermodynamic, spectroscopic, interfacial and mechanical properties (See H. Brittain,
`
`Polymorphism in Pharmaceutical Solids, Marcel Dekker, New York, NY, 1999, pp. 1-2).
`
`Polymorphic and pseudopolymorphic forms of the drug substance (also known as the "active
`
`pharmaceutical ingredient" (API)), as administered by itself or formulated as a drug product
`
`(also known as the final or finished dosage form, or as the pharmaceutical composition) are
`
`25 well known and may affect, for example, the solubility, stability, flowability, fractability, and
`
`compressibility of drug substances and the safety and efficacy of drug products, (see, e.g.,
`
`Knapman, K Modem Drug Discoveries, March 2000: 53).
`
`5-Azacytidine (also known as azacitidine and 4-aimno-l-P-D-ribofuranosyl-S-triazin-
`
`30
`
`2(l#)-one; Nation Service Center designation NSC-102816; CAS Registry Number 320-67-2)
`
`has undergone NCI-sponsored trials for the treatment of myelodysplastic syndromes (MDS).
`
`See Komblith et aL, J. Clin. Oncol. 20(10): 2441-2452 (2002) and Silverman et al, J. Clin.
`
`Oncol. 20(10): 2429-2440 (2002). 5-azacytidine may be defined as having a formula of
`
`C8H12N4O5, a molecular weight of 244.20 and a structure of:
`
`35
`
`- 1-
`
`Apotex v. Cellgene - IPR2023-00512
`Petitioner Apotex Exhibit 1009-0002
`
`

`

`WO 2004/082822
`
`PCT/US2004/007896
`
`NH2
`
`'N^N,
`
`HOv
`
`10
`
`15
`
`Hi
`I
`OH
`OH
`In the United States Patent Application Serial No. 10/390,578 entitled "Forms of 5-
`
`azacytidine," filed March 17, 2003 and incorporated herein by reference in its entirety, eight
`
`different polymorphic and pseudopolymorphic forms of 5-azacytidine (Forms I-VIII), in
`
`addition to an amorphous form, are described. Forms I-VIII each have characteristic X-Ray
`
`Powder Diffraction (XRPD) patterns and are easily distinguished from one another using
`
`XRPD.
`
`5-azacytidine drug substance used in the previous clinical trials has typically been
`
`20
`
`synthesized from 5-azacytosine and 1,2,3,5,-tetra-O-acetyl-P-D-ribofuranose by the method
`
`presented in Example 1. The last step of this method is a recrystallization of the crude
`
`synthesis product from a methanol/DMSO co-solvent system. Specifically, the crude
`synthesis product is dissolved in DMSO (preheated to about 90oC), and then methanol is
`added to the DMSO solution. The product is collected by vacuum filtration and allowed to air
`
`25
`
`dry.
`
`In In the United States Patent Application Serial No. 10/390,578 entitled "Forms of 5-
`
`azacytidine," filed March 17, 2003 and incorporated herein by reference in its entirety, it is
`
`demonstrated that this prior art method for the recrystallization of the crude synthesis product
`
`30
`
`does not control for the polymorphic forms of 5-azacytidine. Specifically, the prior art
`
`recrystallization procedure produces either Form I substantially free of other forms, or a Form
`
`I/II mixed phase i.e. a solid material in which 5-azacytidine is present in a mixed phase of both
`
`polymorphic Form I and polymorphic Form II. Thus, the prior art procedures do not allow
`
`one to reliably target Form I as the single polymorphic form in the drug substance. The present
`
`35
`
`invention provides methods that allow one to recrystallize 5-azacytidine as polymorphic Form
`
`I robustly and reproducibly.
`
`- 2-
`
`Apotex v. Cellgene - IPR2023-00512
`Petitioner Apotex Exhibit 1009-0003
`
`

`

`WO 2004/082822
`
`PCT7US2004/007896
`
`Summary of the Invention
`The present invention provides methods for robustly and reproducibly isolating 5-
`
`azacytidine as polymorphic Form I substantially free of other forms. The methods involve
`
`recrystallizing dissolved 5-azacytidine from a primary solvent/co-solvent mixture and then
`
`collecting the resultant crystals. The invention also provides pharmaceutical compositions
`
`10
`
`comprising Form I of 5-azacytidine together with a pharmaceutically acceptable excipient,
`
`diluent, or carrier.
`
`Detailed Description of the Preferred Embodiments
`
`15
`
`Polymorphic Form I of 5-azacytidine
`
`Form I of 5-azacytidine is described in United States Patent Application Serial No.
`
`10/390,578 entitled "Forms of 5-azacytidme," filed March 17,2003 and incorporated herein
`
`by reference in its entirety. Table 1 provides the most prominent 28 angles, d-spacing and
`
`20
`
`relative intensities for Form I observed using X-Ray Powder Diffraction (XRPD) performed
`
`according the method of Example 4:
`
`20Angle (0)
`12.182
`13.024
`14.399
`16.470
`18.627
`19.049
`20.182
`21.329
`23.033
`23.872
`26.863
`27.135
`29.277
`29.591
`30.369
`32.072
`
`d-spacing (A)
`7.260
`6.792
`6.146
`5.378
`4.760
`4.655
`4.396
`4.162
`3.858
`3.724
`3.316
`3.284
`3.048
`3.016
`2.941
`2.788
`
`Relative Intensity
`39.1
`44.1
`31.5
`27.1
`16.0
`35.9
`37.0
`12.4
`100.0
`28.0
`10.8
`51.5
`25.6
`11.5
`10.8
`13.4
`
`25
`
`Table 1: 5-azacytidine Form I - the most prominent 29 angles, d-spacing and relative
`intensities (Cu Ka radiation)
`
`Apotex v. Cellgene - IPR2023-00512
`Petitioner Apotex Exhibit 1009-0004
`
`

`

`WO 2004/082822
`
`5
`
`PCT/US2004/007896
`
`Isolation of Polymorphic Form I of 5-azacvtidine by Recrystallization
`
`Form I of S-azacytidine may be reproducibly isolated substantially free of other forms by
`
`recrystallizing dissolved 5-azacytidine and collecting the resultant crystals. Specifically, 5-
`
`10
`
`azacytidine is first dissolved completely in at least one suitable primary solvent, preferably a
`
`polar solvent, more preferably a polar aprotic solvent. Suitable polar aprotic solvents include,
`
`but are not limited to, dimethylformamide (DMF), dimethylacetamide (DMA),
`
`dimethylsulfoxide (DMSO), and N-methylpyrrolidinone (NMP). The most preferred polar
`
`aprotic solvent is DMSO. Mixtures of two or more primary solvents are also contemplated for
`
`15
`
`dissolving the 5-azacytidine, for example a mixture of DMSO and DMF.
`
`The 5-azacytidine used to form the solution may be synthesized by any procedure known
`
`in the art; an exemplary prior art synthesis scheme is provided in Example 1. Any
`
`polymorphic or pseudopolymorphic fonn(s) of 5-azacytidine, including mixed phases, may be
`
`20
`
`used to form the solution. Amorphous 5-azacytidine may also be used to form the solution. It
`
`is preferred, but not required, that the primary solvent is preheated to an elevated temperature
`
`in order to ensure that the 5-azacytidine is dissolved completely. An especially preferred
`
`primary solvent is dimethyl sulfoxide, (DMSO), most preferably preheated to a temperature in
`the range of about 40oC to about 90oC.
`
`25
`
`Following solvation of the 5-azacytidine in the primary solvent, at least one co-solvent is
`added to the solution of 5-azacytidine. Suitable co-solvents include C2-C5 alcohols (which
`term hereinafter refers to C2-C5 alcohols that are independently: branched or unbranched,
`
`substituted or unsubstituted), aliphatic ketones (which term hereinafter refers to aliphatic
`
`30
`
`ketones that are indepedently: branched or unbranched, substituted or unsubstituted), and alkyl
`
`cyanides (which term hereinafter refers to alkyl cyanides that are independently: branched or
`
`unbranched, substituted or unsubstituted). Preferred C2-C5 alcohols, aliphatic ketones, and
`
`alkyl cyanides, along with other suitable solvents, are listed below as Class 2 (solvents to be
`
`limited) and Class 3 (solvents of low toxic potential) per the International Conference on
`
`35 Harmonization's (ICH) Guideline for Residual Solvents, July 1997). The use of mixtures of
`two or more of any of the aforementioned co-solvents is also included within the scope of the
`invention.
`
`Apotex v. Cellgene - IPR2023-00512
`Petitioner Apotex Exhibit 1009-0005
`
`

`

`WO 2004/082822
`
`5
`
`Class 2
`
`PCT/US2004/007896
`
`10
`
`15
`
`20
`
`Acetonitrile
`
`Chlorobenzene
`
`Cyclohexane
`
`1,2-Dichloroethene
`
`Dicliloromethane
`
`1,2-Dimethoxyethane
`
`N,N-Dimethylfomiamide
`
`N,N-Dimetliylacetamide
`
`1,4-Dioxane
`
`2-Ethoxyethanol
`
`Ethyleneglycol
`
`Formamide
`
`2-Methoxyethanol
`
`Methylbutyl ketone
`
`Methylcyclohexane
`
`Nitromethane
`
`Pyridine
`
`Sulfolane
`
`Tetralin
`
`25
`
`1,1,2-Trichloroethene
`
`30
`
`35
`
`Class 3
`
`1-Butanol
`
`1-Pentanol
`
`l-Propanol
`
`2-Butanol
`
`2-Methyl-1 -propanol
`
`2-Propanol (isopropyl alcohol)
`
`3-Methyl-1 -butanol
`
`Acetone
`
`Anisole
`
`Butyl acetate
`
`Cumene
`
`- 5-
`
`Apotex v. Cellgene - IPR2023-00512
`Petitioner Apotex Exhibit 1009-0006
`
`

`

`WO 2004/082822
`
`PCT/US2004/007896
`
`5
`
`10
`
`Ethanol
`
`Ethyl acetate
`
`Ethyl ether
`
`Ethyl formate
`
`Isobutyl acetate
`
`Isopropyl acetate
`
`Methyl acetate
`
`Methylethyl ketone
`
`Methylisobutyl ketone
`
`Propyl acetate
`
`15
`
`terz'-Butylmethyl
`
`ether
`
`Tetrahydrofuran
`
`It is preferred, but not required, that the co-solvents are preheated before mixing with the
`
`20
`
`primary solvent, preferably to a temperature below the temperature at which a substantial
`portion of the co-solvent would boil, most preferably to about 50oC. It is also preferred, but
`not required, that the co-solvent(s) is added gradually to the primary solvent(s).
`
`Following mixing, the primary solvent(s)/co-solvent(s) mixture is then equilibrated at
`
`25
`
`different temperatures in order to promote either a slow recrystallization or a fast
`
`recrystallization of Form I of 5-azacytidine, as described below.
`
`By slow recrystallization is meant that the co-solvent/DMSO solution is allowed to
`equilibrate at a temperature in the range from about 00C to about 40oC, preferably in the range
`of about 150C to about 30oC, and most preferably at about ambient temperature. Slow
`recrystallization of Form I of 5-azacytidine is preferably performed using C2-C5 alcohols,
`
`30
`
`aliphatic ketones, or alkyl cyanides as the co-solvent. More preferably, slow recrystallization
`
`is performed with Class 3 C2-C5 alcohols, Class 3 aliphatic ketones, or acetonitrile (Class 2).
`
`The most preferred Class 3 C2-C5 alchohols are ethanol, isopropyl alcohol, and 1-propanol,
`
`35
`
`and the most preferred Class 3 aliphatic ketone is methylethyl ketone.
`
`By fast recrystallization is meant that the co-solvent solution is allowed to equilibrate at a
`temperature of below 0oC, preferably below about -10oC, and most preferably at about -20oC.
`- 6-
`
`Apotex v. Cellgene - IPR2023-00512
`Petitioner Apotex Exhibit 1009-0007
`
`

`

`WO 2004/082822
`
`PCT/US2004/007896
`
`Fast recrystallization of Form I of 5-azacytidine is preferably performed with a C3 - C5 alcohol
`
`(which term hereinafter refers to C3-C5 alcohols which are independently: branched or
`
`unbranched, substituted or unsubstituted) or an alkyl cyanide as the co-solvent. More
`
`preferably the C3 - C5 alcohol is a Class 3 solvent, and the alkyl cyanide is acetonitrile. The
`
`most preferred Class 3 C3-C5 alcohols are isopropyl alcohol (2-propanol) and 1-propanol.
`
`10
`
`Non-limiting examples of protocols for the recrystallization of Form I according to the
`
`methods described herein are provided in Examples 2 (slow recrystallization with DMSO as
`
`the primary solvent and ethanol, isopropyl alcohol, acetonitrile, or methylethyl ketone as the
`
`co-solvent) and 3 (fast recrystallization with DMSO as the primary solvent, and isopropyl
`
`15
`
`alcohol or acetonitrile as the co-solvent) below.
`
`Following recrystallization, the Form I of 5-azacytidine crystals may be isolated from the
`co-solvent mixture by any suitable method known in the art. Preferably, the Form I crystals
`are isolated using vacuum filtration through a suitable filter medium or by centrifugation.
`
`20
`
`Using the novel methods provided herein, it is possible for the first time to target Form I of
`
`5-azacytidine as the drug substance reproducibly and robustly. In particular, isopropyl alcohol
`
`and acetonitrile reliably produce Form I independent of cooling rate (either slow
`
`recrystallization or fast recrystallization) and are preferred as the recrystallization co-solvents
`
`25
`
`to recover Form I. Most preferably, Form I is isolated using isopropyl alcohol as the co-
`
`solvent since isopropyl alcohol carries a Class 3 risk classification (solvent of low toxic
`
`potential), whereas acetonitrile carries a Class 2 risk classification (solvent to be limited). The
`
`use of the DMSO/isopropyl alcohol system allows Form I of 5-azacytidine to be reliably
`
`recovered for the first time from solvents of low toxic potential without requiring control over
`
`30
`
`the rate of recrystallation. In the most preferred embodiment, Form I of 5-azacytidine may be
`
`recovered simply by dissolving 5-azacytidine in DMSO (preferably heated to a temperature in
`the range of about 40oC to about 90oC prior to the addition of 5-azacytidine), adding isopropyl
`alcohol, and allowing the resulting solvent mixture to equilibrate at about ambient
`
`temperature.
`
`35
`
`In some embodiments of the invention, Form I of 5-azacytidine may be recovered from a
`
`primary solvent(s)/co-solvent(s) mixture by "seeding" with a small amount of Form I of 5-
`
`azacytidine either prior to, or during, the addition of the co-solvent(s). By seeding with Form
`
`- 7-
`
`Apotex v. Cellgene - IPR2023-00512
`Petitioner Apotex Exhibit 1009-0008
`
`

`

`WO 2004/082822
`
`PCT/US2004/007896
`
`5
`
`I, it is possible to expand the list of suitable co-solvents and co-solvent classes beyond those
`
`listed above. For example, it is known that recrystallization from the DMSO/methanol system
`
`produces either Form I, or a Form I/II mixed phase (see Example 1). If a small amount of
`
`Form I is added to the solution of 5-azacytidine in DMSO prior to addition of the methanol co-
`
`solvent, or is added during the addition of the methanol co-solvent, then Form I of 5-
`
`10
`
`azacytidine may be reliably isolated.
`
`By allowing the isolation of a single polymorphic form, one skilled in the art will
`
`appreciate that the present invention allows for the first time the production of 5-azacytidine
`
`drag substance with uniform and consistent properties from batch to batch, which properties
`
`15
`
`include but are not limited to solubility and dissolution rate. In turn, this allows one to provide
`
`5-azacytidine drug product (see below) which also has uniform and consistent properties from
`
`batch to batch.
`
`Pharmaceutical Formulations
`
`20
`
`For the most effective administration of drug substance of the present invention, it is
`
`preferred to prepare a pharmaceutical formulation (also known as the "drug product" or
`
`"pharmaceutical composition") preferably in unit dose form, comprising one or more of the 5-
`
`azacytidine polymorphs of the present invention and one or more pharmaceutically acceptable
`
`carrier, diluent, or excipient. Most preferably, Form I 5-azacytidine prepared according to the
`
`25 methods provided herein is used to prepare the pharmaceutical formulation.
`
`Such pharmaceutical formulation may, without being limited by the teachings set forth
`
`herein, include a solid form of the present invention which is blended with at least one
`
`pharmaceutically acceptable excipient, diluted by an excipient or enclosed within such a
`
`30
`
`carrier that can be in the form of a capsule, sachet, tablet, buccal, lozenge, paper, or other
`
`container. When the excipient serves as a diluent, it may be a solid, semi-solid, or liquid
`
`material which acts as a vehicle, carrier, or medium for the 5-azacytidine polymorph(s). Thus,
`
`the formulations can be in the form of tablets, pills, powders, elixirs, suspensions, emulsions,
`
`solutions, syrups, capsules (such as, for example, soft and hard gelatin capsules),
`
`35
`
`suppositories, sterile injectable solutions, and sterile packaged powders.
`
`Examples of suitable excipients include, but are not limited to, starches, gum arable,
`
`calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and
`
`- 8-
`
`Apotex v. Cellgene - IPR2023-00512
`Petitioner Apotex Exhibit 1009-0009
`
`

`

`WO 2004/082822
`
`PCT/US2004/007896
`
`5 methyl cellulose. The formulations can additionally include lubricating agents such as, for
`
`example, talc, magnesium stearate and mineral oil; wetting agents; emulsifying and
`
`suspending agents; preserving agents such as methyl- and propyl- hydroxybenzoates;
`
`sweetening agents; or flavoring agents. Polyols, buffers, and inert fillers may also be used.
`
`Examples of polyols include, but are not limited to: mannitol, sorbitol, xylitol, sucrose,
`
`10 maltose, glucose, lactose, dextrose, and the like. Suitable buffers encompass, but are not
`
`limited to, phosphate, citrate, tartrate, succinate, and the like. Other inert fillers which may be
`
`used encompass those which are known in the art and are useful in the manufacture of various
`
`dosage forms. If desired, the solid pharmaceutical compositions may include other
`
`components such as bulling agents and/or granulating agents, and the like. The compositions
`
`15
`
`of the invention can be formulated so as to provide quick, sustained, controlled, or delayed
`release of the drug substance after administration to the patient by employing procedures well
`known in the art.
`
`In certain embodiments of the invention, the 5-azacytidine polymorph(s) may made into
`
`20
`
`the form of dosage units for oral administration. The 5-azacytidine polymorph(s) may be
`
`mixed with a solid, pulverant carrier such as, for example, lactose, saccharose, sorbitol,
`
`mannitol, starch, amylopectin, cellulose derivatives or gelatin, as well as with an antifriction
`
`agent such as for example, magnesium stearate, calcium stearate, and polyethylene glycol
`
`waxes. The mixture is then pressed into tablets or filled into capsules. If coated tablets,
`
`25
`
`capsules, or pulvules are desired, such tablets, capsules, or pulvules may be coated with a
`
`concentrated solution of sugar, which may contain gum arable, gelatin, talc, titanium dioxide,
`or with a lacquer dissolved in the volatile organic solvent or mixture of solvents. To this
`coating, various dyes may be added in order to distinguish among tablets with different active
`compounds or with different amounts of the active compound present.
`
`30
`
`Soft gelatin capsules may be prepared in which capsules contain a mixture of the 5-
`
`azacytidine polymorph(s) and vegetable oil or non-aqueous, water miscible materials such as,
`
`for example, polyethylene glycol and the like. Hard gelatin capsules may contain granules or
`
`powder of the 5-azacytidine polymorph in combination with a solid, pulverulent carrier, such
`
`35
`
`as, for example, lactose, saccharose, sorbitol, mannitol, potato starch, com starch,
`
`amylopectin, cellulose derivatives, or gelatin.
`
`Apotex v. Cellgene - IPR2023-00512
`Petitioner Apotex Exhibit 1009-0010
`
`

`

`WO 2004/082822
`
`PCT/US2004/007896
`
`5
`
`Tablets for oral use are typically prepared in the following manner, although other
`
`techniques may be employed. The solid substances are gently ground or sieved to a desired
`
`particle size, and a binding agent is homogenized and suspended in a suitable solvent. The 5-
`
`azacytidine polymorph(s) and auxiliary agents are mixed with the binding agent solution. The
`
`resulting mixture is moistened to form a uniform suspension. The moistening typically causes
`
`10
`
`the particles to aggregate slightly, and the resulting mass is gently pressed through a stainless
`
`steel sieve having a desired size. The layers of the mixture are then dried in controlled drying
`
`units for a pre-determined length of time to achieve a desired particle size and consistency.
`
`The granules of the dried mixture are gently sieved to remove any powder. To this mixture,
`
`disintegrating, anti-friction, and anti-adhesive agents are added. Finally, the mixture is pressed
`
`15
`
`into tablets using a machine with the appropriate punches and dies to obtain the desired tablet
`size.
`
`In the event that the above formulations are to be used for parenteral administration,
`
`such a formulation typically comprises sterile, aqueous and non-aqueous injection solutions
`
`20
`
`comprising one or more 5-azacytidine polymorphs for which preparations are preferably
`
`isotonic with the blood of the intended recipient. These preparations may contain anti(cid:173)
`
`oxidants, buffers, bacteriostats, and solute; which render the formulation isotonic with the
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`blood of the intended recipient. Aqueous and non-aqueous suspensions may include
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`suspending agents and thickening agents. The formulations may be present in unit-dose or
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`25 multi-dose containers, for example, sealed ampules and vials. Extemporaneous injection
`
`solutions and suspensions may be prepared from sterile powders, granules, and tablets of the
`
`kind previously described.
`
`Liquid preparations for oral administration are prepared in the form of solutions, syrups, or
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`30
`
`suspensions with the latter two forms containing, for example, 5-azacytidine polymorph(s),
`
`sugar, and a mixture of ethanol, water, glycerol, and propylene glycol. If desired, such liquid
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`preparations contain coloring agents, flavoring agents, and saccharin. Thickening agents such
`
`as carboxymethylcellulose may also be used.
`
`35
`
`As such, the pharmaceutical formulations of the present invention are preferably
`
`prepared in a unit dosage form, each dosage unit containing from about 5 mg to about 200 mg,
`
`more usually about 100 mg of the 5-azacytidine polymorph(s). In liquid form, dosage unit
`
`contains from about 5 to about 200 mg, more usually about 100 mg of the 5-azacytidine
`
`- 1 0-
`
`Apotex v. Cellgene - IPR2023-00512
`Petitioner Apotex Exhibit 1009-0011
`
`

`

`WO 2004/082822
`
`PCT7US2004/007896
`
`polymorph(s). The term "unit dosage form" refers to physically discrete units suitable as
`unitary dosages for human subjects/patients or other mammals, each unit containing a
`predetermined quantity of the 5-azacytidine polymorph calculated to produce the desired
`therapeutic effect, in association with preferably, at least one pharmaceutically acceptable
`canier, diluent, or excipient.
`
`10
`
`The following examples are provided for illustrative purposes only, and are not to be
`construed as limiting the scope of the claims in any way.
`
`-11
`
`Apotex v. Cellgene - IPR2023-00512
`Petitioner Apotex Exhibit 1009-0012
`
`

`

`WO 2004/082822
`
`Examples
`Example 1
`
`PCT/US2004/007896
`
`Prior Art Procedure for Synthesis and Recrystallization of 5-azacvtidine Drug Substance
`
`5-azacytidine may be synthesized using commercially available 5-azacytosine and 1,2,3,5-
`
`tetra-O-acetyl-P-D-ribofuranose (RTA) according to the following pathway:
`
`NH,
`
`NHSKCHjJs
`
`A..J
`
`"N
`
`HO'
`
`HN(Si(CH3)3)2
`
`N -'
`
`"N
`
`4>
`
`(NH2)2S04,Heat
`
`(HaChSiO
`
`(1)
`
`NHSKCHaJj
`
`(HaOaSiO'
`
`"N
`
`AcO^
`
`+
`
`OAo
`
`H
`
`H
`
`|
`OAo
`
`H;
`
`I H
`OAo
`
`(2)
`
`(1) SnCl^CHjCN
`
`(2) NaOCHs, CH3OH
`
`NH2
`
`N
`
`^N
`
`HCk
`
`N
`
`^O
`
`.H
`
`H
`
`I
`OH
`
`H:
`
`I H
`OH
`
`(3)
`
`The crude synthesis product is dissolved in DMSO (preheated to about 90oC)3 and then
`methanol is added to the DMSO solution. The co-solvent mixture is equilibrated at
`approximately -20oC to allow 5-azacytidine crystal formation. The product is collected by
`vacuum filtration and allowed to air dry.
`
`Example 2
`
`Form I of 5-azacvtidine: Slow Recrystallization of 5-azacvtidine from Co-Solvent Systems
`
`Approximately 250 mg of 5-azacytidine was dissolved with approximately 5 ml of
`dimethyl sulfoxide (DMSO), preheated to approximately 90 0C, in separate 100-mL beakers.
`The solids were allowed to dissolve to a clear solution. Approximately 45 mL of ethanol,
`
`isopropyl alcohol, acetonitrile, or methyl ethyl ketone co-solvent, preheated to approximately
`50 0C, was added to the solution and the resultant solution was mixed. The solution was
`covered and allowed to equilibrate at ambient conditions. The product was collected by
`
`vacuum filtration using a Buchner funnel.
`
`-12
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`Apotex v. Cellgene - IPR2023-00512
`Petitioner Apotex Exhibit 1009-0013
`
`

`

`WO 2004/082822
`
`PCT/US2004/007896
`
`5
`
`10
`
`Example 3
`
`Form I of 5-azacvtidine: Fast Recrystallization of 5-azacvtidine from Co-Solvent Systems
`
`Approximately 250 mg of 5-azacytidine was dissolved with approximately 5 mL of
`DMSO, preheated to approximately 90 0C, in separate 100-ml beakers. The solids were
`allowed to dissolve to a clear solution. Approximately 45 mL of isopropyl alcohol or
`acetonitrile co-solvent, preheated to approximately 50 0C, was added to the solution and the
`resultant solution was mixed. The solution was covered and placed in a freezer to equilibrate
`at approximately -20oC to allow crystal formation. Solutions were removed from the freezer
`after crystal formation. The product was collected by vacuum filtration using a Buchner
`
`15
`
`funnel.
`
`Example 4
`
`X-Ray Powder Diffraction of Recrystallized 5-azacvtidine
`
`X-ray powder diffraction (XRPD) patterns for each sample were obtained on a Scintag
`
`20 XDS 2000 or a Scintag Xa 9/9 diffractometer operating with copper radiation at 45 kV and 40
`
`mA using a Kevex Psi Peltier-cooled silicon detector or a Thermo ART Peltier-cooled solid
`
`state detector. Source slits of 2 or 4 mm and detector slits of 0.5 or 0.3 mm were used for data
`
`collection. Recrystallized material was gently milled for approximately one minute using an
`
`agate mortar and pestle. Samples were placed in a stainless steel or silicon sample holder and
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`25
`
`leveled using a glass microscope slide. Powder diffraction patterns of the samples were
`obtained from 2 to 42° 29 at l0/minute. Calibration of the X2 diffractometer is verified
`annually using a silicon powder standard.
`
`XRPD performed according to this method revealed that the Form I of 5-azacytidine was
`
`30
`
`isolated in Example 2 by slow recrystallization using either ethanol, isopropyl alcohol,
`
`acetonitrile, or methyl ethyl ketone as the co-solvent, and in Example 3 by fast
`
`recrystallization using isopropyl alcohol or acetonitrile as the co-solvent. The results indicate
`
`that Form I of 5-azacytidine may be reliably recovered from the DMSO/isopropyl alcohol and
`
`DMSO/acetonitrile solvent systems without control of the rate of recrystallization.
`
`35
`
`-13
`
`Apotex v. Cellgene - IPR2023-00512
`Petitioner Apotex Exhibit 1009-0014
`
`

`

`WO 2004/082822
`
`What is claimed is:
`
`PCT/US2004/007896
`
`1. A method for isolating crystalline Form I of 5-azacytidine substantially free of other forms,
`
`the method comprising:
`
`recrystallizing 5-azacytidine from a solvent mixture comprising at least one primary
`
`solvent and at least one co-solvent selected from the group consisting of:
`
`10
`
`15
`
`1,1,2-Trichloroethene
`
`1,2-Dichloroethene
`152-Dimethoxyethane
`1,4-Dioxane
`
`1-Butanol
`
`1-Pentanol
`
`1-Propanol
`
`2-Butanol
`
`2-Ethoxyethanol
`
`20
`
`2-Methoxyethanol
`
`2-Methyl-1 -propanol
`
`2-Propanol (isopropyl alcohol)
`
`3 -Methyl-1 -butanol
`
`25
`
`Acetone
`
`Acetonitrile
`
`Anisole
`
`Butyl acetate
`
`Chlorobenzene
`
`Cumene
`
`30
`
`Cyclohexane
`
`35
`
`Dichloromethane
`
`Ethanol
`
`Ethyl acetate
`
`Ethyl ether
`
`Ethyl formate
`
`Ethyleneglycol
`
`Formamide
`
`Isobutyl acetate
`
`14-
`
`Apotex v. Cellgene - IPR2023-00512
`Petitioner Apotex Exhibit 1009-0015
`
`

`

`PCT/US2004/007896
`
`WO 2004/082822
`
`5
`
`Isopropyl acetate
`
`Methyl acetate
`
`Methylbutyl ketone
`
`Methylcyclohexane
`
`Methylethyl ketone
`
`10
`
`Methylisobutyl ketone
`
`N,N-Dimethylacetamide
`
`N,N-Dimetliylforaiamide
`
`Nitromethane
`
`Propyl acetate
`
`Pyridine
`
`Sulfolane
`
`terr-Butylmethyl ether
`
`Tetrahydrofuran, and
`
`Tetralin
`
`15
`
`20
`
`by cooling said solvent mixture from a temperature selected to allow said 5-azacytidine to
`
`dissolve completely to about ambient temperature; and
`
`isolating the recrystallized 5-azacytidine.
`
`25
`
`2.
`
`The method of claim 1 wherein said primary solvent is a polar solvent.
`
`3.
`
`The method of claim 2 wherein said polar solvent is a polar aprotic solvent.
`
`4.
`
`The method of claim 3 wherein said polar aprotic solvent is selected from the
`
`30
`
`group consisting of dimethylsulfoxide, dimethylformamide, dimethylacetamide, andN-
`
`methylpyrrolidinone.
`
`5.
`
`The method of claim 4 wherein said polar aprotic solvent is dimethylsulfoxide.
`
`35
`
`6.
`
`The method of claim 1 wherein said co-solvent is acetonitrile.
`
`7.
`
`The method of claim 1 wherein said co-solvent is ethanol.
`
`- 1 5-
`
`Apotex v. Cellgene - IPR2023-00512
`Petitioner Apotex Exhibit 1009-0016
`
`

`

`WO 2004/082822
`
`PCT/US2004/007896
`
`5
`
`8.
`
`The method of claim 1 wherein said co-solvent is methylethyl ketone.
`
`9.
`
`The method of claim 1 wherein said co-solvent is 2-propanol (isopropyl alcohol).
`
`10.
`
`The method of claim 1 wherein said co-solvent is 1-propanol.
`
`10
`
`11. A method for isolating crystalline Form I of 5-azac3'tidme substantially free of other
`
`forms, the method comprising:
`
`recrystallizing 5-azacytidine from a solvent mixture comprising at least one primary
`
`solvent and at least one co-solvent selected from the group consisting of C2-C5 alcohols,
`
`15
`
`aliphatic ketones, and