I 1111111111111111 11111 111111111111111 IIIII IIIII 1111111111 111111111111111111
`US007772199B2
`
`c12) United States Patent
`Ionescu et al.
`
`(IO) Patent No.:
`(45) Date of Patent:
`
`US 7,772,199 B2
`Aug. 10, 2010
`
`(54) FORMS OF 5-AZACYTIDINE
`
`(75)
`
`Inventors: Dumitru Ionescu, Ann Arbor, MI (US);
`Peter Blumbergs, Royal Oak, MI (US);
`Gary L Silvey, Overland Park, KS (US)
`
`(73) Assignee: Celgene Corporation, Summit, NJ (US)
`
`( *) Notice:
`
`Subject to any disclaimer, the term ofthis
`patent is extended or adjusted under 35
`U.S.C. 154(b) by O days.
`
`(21) Appl. No.: 11/458,365
`
`(22) Filed:
`
`Jul. 18, 2006
`
`(65)
`
`Prior Publication Data
`
`US 2006/0247189 Al
`
`Nov. 2, 2006
`
`Related U.S. Application Data
`
`(60) Continuation of application No. 11/052,615, filed on
`Feb. 7, 2005, now Pat. No. 7,078,518, which is a divi(cid:173)
`sion of application No. 10/390,578, filed on Mar. 17,
`2003, now Pat. No. 6,887,855.
`
`(51)
`
`Int. Cl.
`A61K 311706
`(2006.01)
`C07H 19112
`(2006.01)
`(52) U.S. Cl. ........................................ 514/43; 536/28.3
`(58) Field of Classification Search ................ 536/28.3,
`536/124, 28.5, 23.1, 24.31, 24.33, 22.1;
`514/49, 85, 269; 424/45, 450
`See application file for complete search history.
`
`(56)
`
`References Cited
`
`U.S. PATENT DOCUMENTS
`
`10/1967 Sorm et al.
`3,350,388 A
`6/1974 Vorbriiggen et al.
`3,817,980 A
`6/1975 Vorbriiggen et al.
`3,891,623 A
`4/1978 Vorbriiggen
`4,082,911 A
`6/1980 Vorbriiggen
`4,209,613 A
`5,700,640 A * 12/1997 Voss et al. ..................... 435/6
`6,723,728 B2
`4/2004 Hu et al.
`6,753,426 B2
`6/2004 Zhang et al.
`6,887,855 B2 *
`5/2005 Ionescu et al. ................ 514/43
`6,943,249 B2
`9/2005 Ionescu et al.
`7,038,038 B2
`5/2006 Ionescu et al.
`7,078,518 B2 *
`7/2006 Ionescu et al.
`7,132,552 B2
`11/2006 Dolitzky et al.
`2004/0186284 Al
`9/2004 Ionescu et al.
`
`536/28.3
`
`FOREIGN PATENT DOCUMENTS
`
`CZ
`CZ
`DE
`DE
`FR
`GB
`GB
`
`114716
`116297
`1922702
`2012888
`2 123 632
`1 227 692
`1227691
`
`11/1964
`4/1965
`4/1971
`9/1971
`9/1972
`4/1971
`4/1971
`
`OTHER PUBLICATIONS
`
`Braga et al., "Making crystals from crystals: a green route tocrystal
`engineering and polymorphism" Chemical Communications (2005)
`pp. 3635-3645.*
`
`Pharmaceutical Dosage Forms: Tablets, vol. 2, Published by Marcel
`Dekker, Inc., ed. by Lieberman, Lachman, and Schwartz, pp. 462-
`472.*
`Dean, J., Analytical Chemistry Handbook, Published bt McGraw(cid:173)
`Hill, Inc., pp. 10.23-10.26.*
`Jain et al., "Polymorphism in Pharmacy" Indian Drugs (1986) vol.
`23, No. 6, pp. 315-329.*
`Kritz et al., "Pilot study of 5-azacytidine (5-AZA) and carboplatin
`(CBDCA) in patients with relapsed/refractory leukemia" AMerican
`Journal of Hematology (1996) vol. 51, No. 2, pp. 117-121.*
`Cabri et al., "Polymorphisms and Patent, Market, and Legal Battles:
`Cefdinir Case Study" Organic Process Research and Development
`(2007)vol. ll,pp.64-72.*
`Niedballa et al. "A General Synthesis ofN-Glycosides. V. Synthesis
`of 5-azacytidine" J. Org. Chem. (1974) vol. 39 No. 25, pp. 3672-
`3674.*
`Beisler et al., "Chemistry of Antitumor Triazine Nucleosides. An
`improved Synthesis ofDihydro-5-Azacytidine" Journal of Carbohy(cid:173)
`drates, Nucleosides, and Nucleotides (1977) vol. 4 No. 5, pp. 281-
`299.*
`Pharmaceutical Dosage Forms: Tablets, vol. 2, Published by Marcel
`Dekker, Inc., (1990) ed. by Lieberman, Lachman, and Schwartz, pp.
`462-472.*
`Dean, J., Analytical Chemistry Handbook, Published bt McGraw(cid:173)
`Hill, Inc., (1995) pp. 10.23-10.26.*
`Beisler, Journal of Medicinal Chemistry, 21(2):204 ( 1978).
`Niedballa & Vorbriiggen, Journal of Organic Chemistry, 39(25):3672
`(1974).
`Kornblith et al., J. Clin Oncol. 20: 2441 (2002).
`Piskala & Sorm, Collect. Czech. Chem. Commun. 29:2060 (1964).
`Piskala & Sorm, Nucleic Acid Chemistry 1: 435 (1978).
`
`(Continued)
`
`Primary Examiner-Eric S Olson
`(74) Attorney, Agent, or Firm-Jones Day
`
`(57)
`
`ABSTRACT
`
`The invention provides novel polymorphic and pseudopoly(cid:173)
`morphic crystalline forms of 5-azacytidine, along with meth(cid:173)
`ods for preparing said forms, wherein 5-azacytidine is repre(cid:173)
`sented by the formula:
`
`NH2 NAN
`
`HO~ lNAO
`
`H
`
`H
`
`H
`
`H
`
`OH
`
`OH
`
`The invention also includes pharmaceutical compositions
`comprising said forms.
`
`2 Claims, 8 Drawing Sheets CELGENE 2037
`APOTEX v. CELGENE
`IPR2023-00512
`
`

`

`US 7,772,199 B2
`Page 2
`
`OTHER PUBLICATIONS
`
`Piskala & Sorm, Nucleic Acids Research, Special Publication No. 1:
`sl 7 (1975).
`Silverman et al., J. Clin Oncol. 20: 2429 (2002).
`Vorbriiggen et al, Chem. Ber. 114: 1234 (1981).
`Vorbriiggen & Bennua, Chem Ber. 114: 1279 (1981).
`Vorbriiggen & Ruh-Pohlenz in Organic Reactions, vol. 55, p. 100 (L.
`A. Paquette Ed., John Wiley & Sons, New York, 2000).
`Winkley & Robins, Journal of Organic Chemistry, 35(2):491-495
`(1970).
`Wittenburg, Z. Chem. 4:303 (1964).
`Beisler et al. ( 1977) "Chemistry of antitumor triazine nucleosides. An
`improved synthesis ofDihydro-5-AZacytidine" Journal of Carbohy(cid:173)
`drates, Nucleosides Nucleotides, Marcel Dekker, Basel, CH,
`4:(5):281-299.
`Zaitseva et al. ( 199 5) "Convergent synthesis and cytostatic properties
`of 2-chloro-2'-deoxy-2'-fluoroadenosine and
`its N7-isomer."
`Bioorganic & Medicinal Chemistry Letters, 5(24), 2999-3002.
`Database HCAPLUS on CAS (Columbus, OH, USA), No.
`1995:448387, Zaitseva, et al. "Convergent synthesis and cytostatic
`
`properties of 2-chloro-2'-deoxy-2'-fluoroadenosine and its N7-iso(cid:173)
`mer," abstract, Bioorganic & Medicinal Chemistry Letters, 5(24),
`2999-3002.
`Vogler et al., "5-Azacytidine (NSC 102816): A New Drug for the
`Treatment ofMyeloblastic Leukemia," Blood, Sep. 1976, 48(3):331-
`337.
`Opposition Brief dated Sep. 3, 2007 in Chile Application No. 2267-
`2005 (with English translation).
`Hanka, L.J., et al., "Microbiological Production of 5-Azacytidine: I.
`Production and Biological Activity," Antimicrobial Agents and Che(cid:173)
`motherapy, 1966, pp. 619-624.
`Bergy and Herr, "Microbiological Production of 5-Azacytidine: II.
`Isolation and Chemical Structure," Antimicrobial Agents and Che(cid:173)
`motherapy, 1966, pp. 625-630.
`Office Action dated Aug. 1, 2007 in U.S. Appl. No. 11/198,550.
`Office Action dated May 12, 2008 in U.S. Appl. No. 11/198,550.
`Notice of Allowance dated Dec. 2, 2009 in U.S. Appl. No.
`11/198,550.
`
`* cited by examiner
`
`

`

`U.S. Patent
`
`Aug. 10, 2010
`
`Sheet 1 of 8
`
`US 7,772,199 B2
`
`Figure 1. X-ray Powder Diffraction Pattern of Azacitidine, Form I, Labeled with the
`more Prominent 28 Angles (Cu Ka Radiation)
`
`5500
`
`5000
`
`4500
`
`4000
`
`3500
`
`3000
`
`... 2500
`.e,
`l= J 2000
`
`1500
`
`1000
`
`500
`
`0
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`Two-Theta
`
`

`

`U.S. Patent
`
`Aug. 10, 2010
`
`Sheet 2 of 8
`
`US 7,772,199 B2
`
`Figure 2 X-ray Powder Diffraction Pattern of Ar.acitidine, Mixed Phase Forms I and II,
`Labeled with the more Prominent 28 Angles (Cu Ket Radiation)
`
`5000
`
`4500
`
`4000
`
`3500
`
`3000
`
`...
`.e, 2500
`19
`-
`I 2000
`1500
`
`1000
`
`500
`
`0
`
`• indicates lines distn:live lo Fonn II
`
`~
`~
`}':l
`
`~
`
`~
`~-t
`~ ~
`~~
`""
`!
`
`Azacitidine
`Mixed Forms I & II
`
`h ~ *
`el ~
`B ;::i
`~ ~
`~
`
`..;
`N
`~
`be
`~
`
`II::
`~
`
`00
`
`"'
`f:i
`
`00
`
`"'
`~
`
`~
`g.
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`Two-Theta
`
`

`

`U.S. Patent
`
`Aug. 10, 2010
`
`Sheet 3 of 8
`
`US 7,772,199 B2
`
`Figure 3 X-ray Powder Diffraction Pattern of A7.acitidine, Form III, Labeled with the
`more Prominent 20 Angles (Cu Ka Radiation)
`
`4000
`
`3500
`
`3000
`
`2500
`
`...
`~ 2000
`!l
`-
`=
`QI 1500
`
`1000
`
`500
`
`0
`
`:'o
`~
`~
`
`~
`1!l
`
`~
`~
`:::
`
`~
`"i
`<:,
`
`~
`!:;
`
`00
`
`..,
`'.'.:i
`
`~
`;;j
`
`a
`
`"1
`iEl
`
`~
`~
`
`~
`;1i
`
`~
`~ ~
`;;j
`5 1:1
`
`<:,
`
`~
`
`~
`:i; ~
`;;
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`Two-Theta
`
`

`

`U.S. Patent
`
`Aug. 10, 2010
`
`Sheet 4 of 8
`
`US 7,772,199 B2
`
`Figure 4 X-ray Powder Diffraction Pattern of Azacitidine, Form IV, Labeled with the
`more Prominent 28 Angles (Cu Ka Radiation)
`
`3000
`
`2500
`
`2000
`
`1500
`....
`~
`~
`-
`I
`
`1000
`
`500
`
`0
`
`...
`.,.,
`;::j
`
`i:,
`
`~
`
`t?,,
`~
`ti
`
`~
`~
`
`.,., ,,. .
`~
`i::ls
`... N
`
`N
`
`......
`~
`,-.N
`~d
`ON
`N
`~
`...
`
`N
`
`~
`~
`
`~! ~
`
`~
`!o
`0
`"'
`
`N
`
`.
`...
`"'
`~~
`.,,
`od
`N
`~
`
`a
`
`~
`
`~
`:g
`j
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`Two-Theta
`
`

`

`U.S. Patent
`
`Aug. 10, 2010
`
`Sheet 5 of 8
`
`US 7,772,199 B2
`
`Figure 5 X-ray Powder Diffraction Pattern of Amcitidine, Form V, Labeled with the
`more Prominent 29 Angles (Cu Ka Radiation)
`
`3000
`
`2500
`
`2000
`
`B 1500
`!= ca.I = 1000
`-
`
`500
`
`0
`
`0 ..
`
`"'!
`Fl
`
`.. ~
`
`5
`
`IO
`
`15
`
`25
`20
`Two-Theta
`
`30
`
`35
`
`40
`
`

`

`U.S. Patent
`
`Aug. 10, 2010
`
`Sheet 6 of 8
`
`US 7,772,199 B2
`
`Figure 6 X-ray Powder Diffraction Pattern of Azacitidine, Form VI, Labeled with the
`more Prominent 28 Angles (Cu Kcx Radiation)
`
`1500
`
`1000
`
`,t,
`·;
`f
`
`o-4
`
`500
`
`0
`
`:iil
`~
`
`~
`a
`~
`
`~ o
`~~
`
`~:
`
`N
`
`&
`N "'
`0
`"'
`
`~~
`~~
`"'
`
`N
`
`~
`l:i
`~
`
`,0
`N
`
`00
`
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`~
`
`~
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`
`00
`
`~ .-:
`
`<'>
`
`~
`00
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`
`~ ~
`<.-i ~
`"'
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`

`

`U.S. Patent
`
`Aug. 10, 2010
`
`Sheet 7 of 8
`
`US 7,772,199 B2
`
`Figure 7 X-ray Powder Diffraction Pattern of Amcitidine, Mixed Phase Forms I and
`VII, Labeled with the more Prominent 28 Angles (Cu Ka Radiation)
`
`3500 .............. -....-...... T-1"' ...................... T-'P'""T'""'I ............... T-'P' ......... __,,.._......,... ....... __,_......,...._...
`
`• indicates lines distinctive to Form VII
`
`3000
`
`2500
`
`2000
`
`....
`,t;>
`~ 1500
`
`i - 1000
`
`500
`
`0
`
`*
`"' r--,-.
`.,.;
`
`5
`
`10
`
`15
`
`20
`
`25
`
`30
`
`35
`
`40
`
`Two-Theta
`
`

`

`U.S. Patent
`
`Aug. 10, 2010
`
`Sheet 8 of 8
`
`US 7,772,199 B2
`
`Figure 8 X-ray Powder Diffraction Pattern of Azacitidine, Form VIII, Labeled with the
`more Prominent 29 Angles (Cu Ka; Radiation)
`
`3500
`
`3000
`
`2500
`
`2000
`
`1500
`
`1000
`
`500
`
`0
`
`;;...
`;t::
`~
`-
`i
`
`Si;
`"' ~
`
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`
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`~
`
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`~
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`
`g.,
`~
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`
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`:g
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`
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`
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`.,;
`"'
`
`8
`~
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`
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`
`10
`
`15
`
`20
`25
`Two-Theta
`
`30
`
`35
`
`40
`
`

`

`US 7,772,199 B2
`
`1
`FORMS OF 5-AZACYTIDINE
`
`RELATED APPLICATIONS
`
`2
`The polymorphic form of 5-azacytidine drug substance and
`drug product has never been characterized. It is an object of
`the present invention to characterize the polymorphic forms
`of 5-azacytidine.
`
`SUMMARY OF THE INVENTION
`
`This application is a continuation of U.S. application Ser. 5
`No. 11/052,615, now U.S. Pat. No. 7,078,518, filed Feb. 7,
`2005, entitled, "Forms of 5-Azacytidine", which is a divi(cid:173)
`sional of U.S. application Ser. No. 10/390,578, now U.S. Pat.
`No. 6,887,855, filed Mar. 17, 2003, entitled "Forms of5-Aza(cid:173)
`cytidine". All of the above listed references are hereby incor(cid:173)
`porated in their entirety.
`
`FIELD OF THE INVENTION
`
`The invention relates to the isolation of crystalline poly(cid:173)
`morphic and pseudopolymorphic forms of 5-azacytidine
`(also known as azacitidine and 4-amino-l-~-D-ribofurano(cid:173)
`syl-S-triazin-2(1H)-one). 5-azacytidine may be used in the
`treatment of disease, including the treatment of myelodys(cid:173)
`plastic syndromes (MDS).
`
`BACKGROUND OF THE INVENTION
`
`20
`
`It has been unexpectedly found that 5-azacytidine exists in
`10 at least eight different polymorphic and pseudopolymorphic
`crystalline forms (Forms I-VIII), in addition to an amorphous
`form. Form I is a polymorph found in prior art retained
`samples of 5-azacytidine drug substance. Form II is a poly-
`15 morph found in some prior art retained samples of the 5-aza(cid:173)
`cytidine drug substance; in those samples, Form II is always
`found in mixed phase with Form I. Form III is a hydrate, and
`is formed when prior art retained and current samples of the
`drug product are reconstituted with water to form a "slurry"
`prior to administration to the patient. Form VI is found in prior
`art retained samples of the 5-azacytidine drug product, either
`substantially free of other polymorphs, or in mixed phase
`with Form I.
`The invention provides novel crystalline forms referred to
`as Form IV, Form V, Form VII and Form VIII. Forms I-VIII
`each have characteristic X-ray power diffraction (XRPD)
`patterns and are easily distinguished from one another using
`30 XRPD.
`Also included in the present invention are methods for
`robustly and reproducibly synthesizing 5-azacytidine drug
`substance substantially as Form IV, Form V, or Form VIII.
`Also provided are methods for robustly and reproducibly
`synthesizing a Form INII mixed phase. The invention also
`provides pharmaceutical compositions comprising the vari(cid:173)
`ous forms of 5-azacytidine together with one or more phar-
`40 maceutically acceptable excipients, diluents, or carriers.
`
`Polymorphs exist as two or more crystalline phases that
`have different arrangements and/or different conformations
`of the molecule in a crystal lattice. When a solvent 25
`molecule(s) is contained within the crystal lattice the result(cid:173)
`ing 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 thermodynamic, spectroscopic, interfa(cid:173)
`cial and mechanical properties (See H. Brittain, Polymor(cid:173)
`phism in Pharmaceutical Solids, Marcel Dekker, New York,
`N.Y., 1999, pp. 1-2). Polymorphic and pseudopolymorphic 35
`forms of the drug substance (also known as the "active phar(cid:173)
`maceutical 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 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 Modern Drug Discoveries, March 2000:
`53).
`5-azacytidine (also known as azacitidine and 4-amino-l(cid:173)
`~-D-ribofuranosyl-1,3,5-triazin-2(1H)-one; Nation Service
`Center designation NSC-102816; CAS Registry Number
`320-67-2) has undergone NCI-sponsored clinical trials for
`the treatment of myelodysplastic syndromes (MDS). See
`Komblithet al., J. Clin. Oneal. 20(10): 2441-2452 (2002) and
`Silverman et al., J. Clin. Oneal. 20(10): 2429-2440 (2002).
`5-azacytidine may be defined as having a formula of
`C 8H 12N4 0 5 , a molecular weight of244.20 and a structure of:
`
`BRIEF DESCRIPTION OF THE FIGURES
`
`FIG. 1 presents the X-Ray Powder Diffraction (XRPD)
`45 pattern of 5-azacytidine, Form I, labeled with the most promi(cid:173)
`nent 28 angles (Cu Ka radiation).
`FIG. 2 presents the XRPD pattern of 5-azacytidine, mixed
`phase Form I and Form II, labeled with the most prominent 28
`50 angles (Cu Ka radiation).
`FIG. 3 presents the XRPD pattern of 5-azacytidine, Form
`III, labeled with the most prominent 28 angles (Cu Ka radia(cid:173)
`tion).
`FIG. 4 presents the XRPD pattern of 5-azacytidine, Form
`IV, labeled with the most prominent 28 angles (Cu Ka radia(cid:173)
`tion).
`FIG. 5 presents the XRPD pattern of 5-azacytidine, Form
`V, labeled with the most prominent 28 angles (Cu Ka radia-
`60 tion).
`FIG. 6 presents the XRPD pattern of 5-azacytidine, Form
`VI, labeled with the most prominent 28 angles (Cu Ka radia(cid:173)
`tion).
`FIG. 7 presents the XRPD pattern of 5-azacytidine, mixed
`phase Form I and Form VII, labeled with the most prominent
`28 angles (Cu Ka radiation).
`
`55
`
`65
`
`NH2
`NAN
`HOlAlNAO
`
`H
`
`H
`
`OH
`
`H
`
`H
`
`OH
`
`

`

`US 7,772,199 B2
`
`3
`FIG. 8 presents the XRPD pattern of 5-azacytidine, Form
`VIII, labeled with the most prominent 28 angles (Cu Ka
`radiation).
`
`DETAILED DESCRIPTION OF THE PREFERRED
`EMBODIMENTS
`
`5-azacytidine Crystalline Farms I-VIII
`
`It has been discovered that 5-azacytidine exists in at least 10
`eight different polymorphic and pseudopolymorphic crystal(cid:173)
`line forms, and also in an amorphous form.
`Form I
`A single sample of the 5-azacytidine drug substance was
`synthesized from 5-azacytosine and 1,2,3,5,-Tetra-O-acetyl- 15
`~-D-ribofuranose according to the prior art method provided
`in Example 1. The last step of this method is a recrystalliza(cid:173)
`tion of the crude synthesis product from a DMSO/methanol
`co-solvent system. Specifically, the crude synthesis product is
`dissolved in DMSO (preheated to about 90° C.), and then 20
`methanol is added to the DMSO solution. The co-solvent
`mixture is equilibrated at approximately -20° C. to allow
`5-azacytidine crystal formation. The product is collected by
`vacuum filtration and allowed to air dry.
`The X-Ray Powder Diffraction (XRPD; see Example 5) 25
`pattern of the resulting 5-azacytidine is shown in FIG. 1 along
`with some of the 28 values. Table 1 provides the most promi(cid:173)
`nent 28 angles, d-spacing and relative intensities for this
`material, which is designated as Form I.
`
`30
`
`4
`
`TABLE2
`
`5-azacytidine, Mixed Phase Forms I and II - the most prominent
`28 angles cl-spacing and relative intensities (Cu Ka radiation)
`
`20Angle ( 0
`
`)
`
`cl-spacing (A)
`
`Relative Intensity
`
`12.244
`13.082
`13.458*
`14.452
`16.521
`17.648*
`18.677
`19.093
`20.231
`21.353
`22.309*
`23.070
`23.909
`26.641
`26.813
`27.158
`29.309
`29.609
`30.384
`32.074
`
`7.223
`6.762
`6.574
`6.124
`5.361
`5.022
`4.747
`4.645
`4.386
`4.158
`3.982
`3.852
`3.719
`3.343
`3.322
`3.281
`3.045
`3.015
`2.939
`2.788
`
`34.8
`37.0
`29.2
`25.4
`19.0
`12.1
`12.7
`41.3
`42.1
`15.5
`35.1
`100.0
`18.9
`18.2
`12.6
`46.0
`27.3
`12.7
`10.5
`12.0
`
`These results indicate that the prior art 5-azacytidine syn(cid:173)
`thesis procedures for the drug substance produce 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 Form I and Form II.
`Thermal analysis of mixed phase Form I/II is presented in
`Example 6.
`Form III
`An additional crystalline form of 5-azacytidine, designated
`Form III, is found in slurries of 5-azacytidine. See Example 8.
`35 Moreover, it has been found that all forms of 5-azacytidine
`(including the 5-azacytidine in the prior art drug product)
`convert to Form III in water. See Example 8. Thus, reconsti(cid:173)
`tution of the drug product used in the aforementioned NCI
`trials would have led to the formation of a saturated solution
`( or "slurry") in which the remaining solid 5-azacytidine was
`Form III. The XRPD powder pattern of Form III is shown in
`FIG. 3 along with some of the 28 values. Table 3 provides the
`most prominent 28 angles, d-spacing and relative intensities
`for this crystalline material. The XRPD powder pattern for
`45 Form III is distinctly different from that of all of the other
`forms of 5-azacytidine.
`
`TABLE 1
`
`5-azacytidine Form I - the most prominent 20 angles, cl-spacing
`and relative intensities (Cu Ka radiation)
`
`20Angle ( 0
`
`)
`
`cl-spacing (A)
`
`Relative Intensity
`
`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
`
`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
`
`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
`
`40
`
`50
`
`Thermal analysis of Form I indicates that this form of
`5-azacytidine is anhydrous. See Example 6.
`Form II
`Retained samples of the drug substance previously used to 55
`the formulate the drug product in the NCI-sponsored Cancer
`and Leukemia Group B (CALGB) investigations (Phase 2
`trial 8291 and Phase 3 trial 9221) for the treatment of MDS
`(Investigational New Drug (IND) 7574) were also analyzed
`by XRPD. The retained drug substance samples comprised 60
`either Form I, or a mixed phase of Form I and another poly(cid:173)
`morph: Form II. See Example 5.
`The XRPD powder pattern of mixed phase Forms I and II
`is shown in FIG. 2 along with some of the 28 values. Peaks
`distinctive to Form II are observed at 13.5, 17.6 and 22.3° 28. 65
`Table 2 provides the most prominent 28 angles, d-spacing and
`relative intensities for this mixed phase.
`
`TABLE3
`
`5-azacytidine, Form III - the most prominent 20 angles, cl-spacing
`and relative intensities (Cu Ka radiation)
`
`20Angle ( 0
`
`)
`
`cl-spacing (A)
`
`Relative Intensity
`
`6.566
`11.983
`13.089
`15.138
`17.446
`20.762
`21.049
`22.776
`24.363
`25.743
`26.305
`28.741
`31.393
`32.806
`33.043
`33.536
`
`13.450
`7.380
`6.758
`5.848
`5.079
`4.275
`4.147
`3.901
`3.651
`3.458
`3.385
`3.104
`2.847
`2.728
`2.709
`2.670
`
`32.9
`52.5
`71.0
`38.9
`48.2
`10.8
`34.8
`89.5
`13.7
`22.8
`39.9
`100.0
`22.5
`11.8
`10.1
`15.1
`
`

`

`US 7,772,199 B2
`
`5
`
`TABLE 3-continued
`
`6
`
`TABLES
`
`5-azacyticline, Form III - the most prominent 20 angles, cl-spacing
`and relative intensities (Cu Ka radiation)
`
`5-azacytidine Form V - the most prominent 28 angles, cl-spacing
`and relative intensities (Cu Ka radiation)
`
`20Angle ( 0
`
`)
`
`cl-spacing (A)
`
`Relative Intensity
`
`20Angle ( 0
`
`)
`
`cl-spacing (A)
`
`Relative Intensity
`
`36.371
`39.157
`41.643
`
`2.468
`2.299
`2.167
`
`11.0
`19.3
`12.1
`
`10
`
`15
`
`Thermal analysis and proton (1 H) NMR spectroscopy indi(cid:173)
`cate that Form III is a pseudopolymorphic form of 5-azacy(cid:173)
`tidine, specifically a monohydrate. See Examples 6-7.
`Form IV
`Form IV is a novel crystalline form of 5-azacytidine. Form
`IV was recovered by slow recrystallization from a DMSO/
`toluene co-solvent system (see Example 2) or by fast recrys(cid:173)
`tallization from the DMSO/chloroform co-solvent system 20
`(see Example 3). The XRPD powder pattern of Form IV is
`shown in FIG. 4 along with some of the 28 values. Table 4
`provides the most prominent 28 angles, d-spacing and rela(cid:173)
`tive intensities for this crystalline material. The XRPD pow(cid:173)
`der pattern for Form IV is distinctly different from that of any 25
`other form.
`
`11.018
`12.351
`13.176
`13.747
`14.548
`15.542
`16.556
`17.978
`18.549
`19.202
`19.819
`20.329
`21.518
`21.970
`22.521
`23.179
`24.018
`24.569
`27.224
`28.469
`29.041
`29.429
`30.924
`31.133
`37.938
`
`8.024
`7.160
`6.714
`6.436
`6.084
`5.697
`5.350
`4.930
`4.780
`4.618
`4.476
`4.365
`4.126
`4.042
`3.948
`3.834
`3.702
`3.620
`3.273
`3.133
`3.072
`3.033
`2.889
`2.870
`2.370
`
`40.0
`29.6
`28.3
`42.9
`18.3
`14.2
`47.8
`18.1
`83.9
`25.0
`12.1
`28.6
`100.0
`65.6
`11.5
`66.5
`13.0
`40.7
`50.2
`24.2
`24.8
`15.0
`15.6
`22.6
`10.7
`
`30
`
`Thermal analysis indicates that Form V is a solvate. See
`Example 6.
`Form VI
`The drug product used in the aforementioned NCI investi(cid:173)
`gation was typically prepared by lyophilizing a solution of
`35 5-azacytidine and mannitol (1:1 w/w). The resultant drug
`product comprised 100 mg of 5-azacytidine and 100 mg
`mannitol as a lyophilized cake in a vial and was administered
`by subcutaneous
`injection as an aqueous suspension
`("slurry"). XRPD analysis of retained samples of the drug
`40 product used in the NCI investigation revealed the existence
`of another polymorph, Form VI. The retained drug product
`samples comprised either Form VI alone, or a Form I/VI
`mixed phase. Table 6 provides the most prominent 28 angles,
`d-spacing and relative intensities for Form VI.
`
`45
`
`TABLE6
`
`5-azacyticline Form VI - the most prominent 20 angles, cl-spacing
`and relative intensities (Cu Ka radiation)
`
`50
`
`20Angle ( 0
`
`)
`
`cl-spacing (A)
`
`Relative Intensity
`
`TABLE4
`
`5-azacyticline Form IV - the most prominent 20 angles, cl-spacing
`and relative intensities (Cu Ka radiation)
`
`20Angle ( 0
`
`)
`
`cl-spacing (A)
`
`Relative Intensity
`
`5.704
`11.571
`12.563
`14.070
`15.943
`16.993
`18.066
`20.377
`20.729
`21.484
`21.803
`22.452
`22.709
`23.646
`24.068
`25.346
`25.346
`26.900
`27.991
`28.527
`28.723
`30.124
`30.673
`31.059
`35.059
`38.195
`38.403
`
`15.408
`7.642
`7.040
`6.289
`5.555
`5.213
`4.906
`4.355
`4.281
`4.132
`4.073
`3.957
`3.913
`3.760
`3.695
`3.526
`3.511
`3.312
`3.185
`3.126
`3.106
`2.964
`2.912
`2.877
`2.557
`2.354
`2.342
`
`24.9
`97.8
`22.2
`100.0
`67.4
`51.0
`20.1
`44.7
`49.0
`36.30
`11.2
`66.7
`64.0
`17.3
`19.4
`12.0
`12.5
`11.0
`11.4
`25.7
`34.1
`14.7
`53.6
`15.7
`18.1
`15.0
`12.6
`
`Thermal analysis of Form IV is presented in Example 6.
`FormV
`Form Vis a novel crystalline form of 5-azacytidine. Form 60
`V was recovered by fast recrystallization of 5-azacytidine
`from a DMSO/toluene co-solvent system (see Example 3).
`The XRPD powder pattern of Form Vis shown in FIG. 5
`along with some of the 28 values. Table 5 provides the most
`prominent 28 angles, d-spacing and relative intensities for 65
`this crystalline material. The XRPD powder pattern for Form
`V is distinctly different from that of any other form.
`
`55
`
`12.533
`12.963
`13.801
`18.929
`20.920
`21.108
`21.527
`22.623
`22.970
`24.054
`26.668
`27.210
`28.519
`29.548
`30.458
`33.810
`35.079
`37.528
`
`7.057
`6.824
`6.411
`4.6843
`4.243
`4.205
`4.125
`3.922
`3.869
`3.697
`3.340
`3.275
`3.127
`3.021
`2.932
`2.649
`2.556
`2.411
`
`10.1
`10.2
`100.0
`10.0
`34.2
`49.4
`47.0
`10.7
`13.8
`77.8
`23.0
`33.7
`12.9
`27.2
`50.3
`11.6
`12.6
`24.7
`
`

`

`US 7,772,199 B2
`
`7
`Thermal analysis and proton (1 H) NMR spectroscopy of
`Form VI is presented in Examples 6-7.
`Form VII
`Form VII is a novel crystalline form of 5-azacytidine. Form
`VII was produced by fast recrystallization from a DMSO/
`methanol co-solvent system (see Example 3). Form VII was
`always isolated by this recrystallization method as a mixed
`phase with Form I. The XRPD powder pattern of mixed phase
`Forms I and VII is shown in FIG. 7 along with some of the 28
`values and the Form VII distinctive peaks indicated with
`asterisks. Table 7 provides the most prominent 28 angles,
`d-spacing and relative intensities for this mixed phase. Form
`VII exhibits distinctive peaks at 5.8, 11.5, 12.8, 22.4 and
`26.6° 28 in addition to peaks displayed in the Form I XRPD
`powder pattern. The XRPD pattern for mixed phase Forms I 15
`and VII is distinctly different from that of any other form.
`
`10
`
`8
`
`TABLE 8-continued
`
`5-azacyticline, Form VIII - the most prominent 20 angles,
`cl-spacing and relative intensities (Cu Ka radiation)
`
`20Angle ( 0
`
`)
`
`cl-spacing (A)
`
`Relative Intensity
`
`25.740
`29.293
`32.148
`35.074
`38.306
`
`3.458
`3.046
`2.782
`2.556
`2.348
`
`7.8
`3.8
`8.8
`7.4
`2.5
`
`TABLE 7
`
`5-azacytidine, mixed Forms I and VII - the most prominent
`28 angles cl-spacing and relative intensities (Cu Ka radiation)
`
`20Angle ( 0
`
`)
`
`cl-spacing (A)
`
`Relative Intensity
`
`5.779
`11.537
`12.208
`12.759
`13.048
`14.418
`16.489
`18.649
`19.101
`20.200
`20.769
`21.355
`22.365
`23.049
`23.884
`26.628
`27.145
`29.296
`29.582
`32.078
`
`15.281
`7.664
`7.244
`6.932
`6.780
`6.138
`5.372
`4.754
`4.643
`4.392
`4.273
`4.157
`3.972
`3.856
`3.723
`3.345
`3.282
`3.046
`3.017
`2.788
`
`14.7
`8.3
`28.0
`21.7
`34.4
`22.5
`21.6
`13.5
`34.7
`34.4
`10.5
`11.7
`29.9
`100.0
`23.1
`13.3
`52.9
`26.2
`11.3
`12.9
`
`Thermal analysis of Form VII is presented in Example 6
`Form VIII
`Form VIII is a novel crystalline form of 5-azacytidine.
`Form VIII was recovered by recrystallizing 5-azacytidine
`Form I from a N-methyl-2-pyrrolidone (NMP) single solvent
`system (see Example 4). The XRPD powder pattern of Form
`VIII is shown in FIG. 8 along with some of the 28 values.
`Table 8 provides the most prominent 28 angles, d-spacing and
`relative intensities for this material. The XRPD pattern for
`Form VIII is distinctly different from that of any other form.
`
`TABLES
`
`5-azacyticline, Form VIII - the most prominent 20 angles,
`cl-spacing and relative intensities (Cu Ka radiation)
`
`20Angle ( 0
`
`)
`
`cl-spacing (A)
`
`Relative Intensity
`
`6.599
`10.660
`12.600
`13.358
`15.849
`17.275
`20.243
`20.851
`21.770
`22.649
`25.554
`
`13.384
`8.292
`7.020
`6.623
`5.587
`5.129
`4.383
`4.257
`4.079
`3.923
`3.483
`
`2.9
`2.2
`23.4
`2.6
`2.0
`4.2
`5.8
`7.8
`74.4
`32.1
`100.0
`
`20
`
`25
`
`Amorphous 5-azacytidine
`Amorphous 5-azacytidine may be recovered from equilib(cid:173)
`rium saturated solutions of 5-azacytidine in propylene glycol,
`polyethylene glycol and DMSO. See Example 8.
`Pharmaceutical Formulations
`For the most effective administration of drug substance of
`the present invention, it is preferred to prepare a pharmaceu(cid:173)
`tical formulation (also known as the "drug product") prefer(cid:173)
`ably in unit dose form, comprising one or more of the 5-aza(cid:173)
`cytidine forms of the present invention and one or more
`pharmaceutically acceptable carrier, diluent, or excipient.
`Such pharmaceutical formulation may, without being lim-
`ited by the teachings set forth herein, include a solid form of
`the present invention which is blended with at least one phar(cid:173)
`maceutically acceptable excipient, diluted by an excipient or
`enclosed within such a carrier that can be in the form of a
`30 capsule, sachet, tablet, buccal, lozenge, paper, or other con(cid:173)
`tainer. 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,
`35 elixirs, suspensions, emulsions, solutions, syrups, capsules
`(such as, for example, soft and hard gelatin capsules), sup(cid:173)
`positories, sterile injectable solutions, and sterile packaged
`powders.
`Examples of suitable excipients include, but are not limited
`40 to, starches, gum arabic, calcium silicate, microcrystalline
`cellulose, polyvinylpyrrolidone, cellulose, water, syrup, and
`methyl cellulose. The formulations can additionally include
`lubricating agents such as, for example, talc, magnesium
`stearate and mineral oil; wetting agents; emulsifying and
`45 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, maltose, glucose, lactose, dextrose,
`50 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 composi-
`55 tions may include other components such as bulling agents
`and/or granulating agents, and the like. The compositions of
`the invention can be formulated so as to provide quick, sus(cid:173)
`tained, controlled, or delayed release of the drug substance
`after administration to the patient by employing procedures
`60 well known in the art.
`In certain embodiments of the invention, the 5-azacytidine
`forms(s) may be made into the form of dosage units for oral
`administration. The 5-azacytidine forms(s) may be mixed
`with a solid, pulverant carrier such as, for example, lactose,
`65 saccharose, sorbitol, mannitol, starch, amylopectin, cellulose
`derivatives or gelatin, as well as with an antifriction agent
`such as for example, magnesium stearate, calcium stearate,
`
`

`

`US 7,772,199 B2
`
`10
`quantity of the 5-azacytidine polymorph calculated to pro(cid:173)
`duce the desired therapeutic effect, in association with pref(cid:173)
`erably, at least one pharmaceutically acceptable carrier, dilu(cid:173)
`ent, or excipient.
`The following examples are provided for illustrative pur(cid:173)
`poses only, and are not to be construed as limiting the scope of
`the claims in any way.
`
`EXAMPLES
`
`Example 1
`
`Prior Art Procedure for Synthesis of 5-azacytidine
`Drug Substance
`
`10
`
`Using commercially available 5-azacytosine (1) and 1,2,3,
`5-Tetra-O-~-acetyl-ribofuranose (2) (RTA), 5-azacytidine (3)
`20 may be synthesized according to the pathway below.
`
`9
`and polyethylene glycol waxes. The mixture is then pressed
`into tablets or filled into capsules. If coated tablets, capsules,
`or pulvules are desired, such tablets, capsules, or pulvules
`may be coated with a concentrated solution of sugar, which
`may contain gum arabic, 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.
`Soft gelatin capsules may be prepared in which capsules
`contain a mixture of the 5-azacytidine form(s) and vegetable
`oil or non-aqueous, water miscible materials such as, for
`example, polyethylene glycol and the like. Hard gelatin cap(cid:173)
`sules may c