PCT
`INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`WO 98/32466
`
`WORLD INTELLECTUAL PROPERTY ORGANIZATION
`International Bureau
`
`(51) International Patent Classification 6 :
`A61K 47/48
`
`Al
`
`(11) International Publication Number:
`
`(43) International Publication Date:
`
`30 July 1998 (30.07.98)
`
`(21) International Application Number:
`
`PCT/GB98/00253
`
`(22) International Filing Date:
`
`28 January 1998 (28.01.98)
`
`(30) Priority Data:
`9701800.6
`9701804.8
`9704653.6
`9708055.0
`
`29 January 1997 (29.01.97)
`29 January 1997 (29.01.97)
`6 March 1997 (06.03.97)
`22 April 1997 (22.04.97)
`
`GB
`GB
`GB
`GB
`
`(81) Designated States: AL, AM, AT, AU, AZ, BA, BB, BG, BR,
`BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, GE,
`GH, GM, GW, HU, ID, IL, IS, JP, KE, KG, KP, KR, KZ,
`LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW,
`MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, SL,
`TJ, TM, TR, TT, UA, UG, US, UZ, VN, YU, ZW, ARIPO
`patent (GH, GM, KE, LS, MW, SD, SZ, UG, ZW), Eurasian
`patent (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), European
`patent (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT,
`LU, MC, NL, PT, SE), OAPI patent (BF, BJ, CF, CG, CI,
`CM, GA, GN, ML, MR, NE, SN, TD, TG).
`
`(71) Applicant (for all designated States except US): POL YMASC
`PHARMACEUTICALS PLC [GB/GB]; Fleet Road, London Published
`NW3 2EZ (GB).
`With international search report.
`Before the expiration of the time limit for amending the
`claims and to be republished in the event of the receipt of
`amendments.
`
`(72) Inventors; and
`(75) Inventors/Applicants (for US only): FRANCIS, Gillian, Eliz(cid:173)
`abeth [GB/GB]; Summer Cottage, Cane End, Reading,
`Berkshire RG4 9GH (GB). FISHER, Derek [GB/GB]; 34
`Corinium Gate, St. Albans, Hertfordshire AL3 4HY
`(GB). MALIK, Farooq [GB/GB]; 67 Gracefield Gardens,
`Streatham, London SW16 2TS (GB).
`
`(74) Agent: NACHSHEN, Neil, Jacob; D. Young & Co., 21 New
`Fetter Lane, London EC4A IDA (GB).
`
`(54) Title: PEGYLATION PROCESS
`
`(57) Abstract
`
`The present invention relates to the attachment of a polyethylene glycol (PEG) moiety to a target substrate. Processes for such
`attachment will be hereinafter referred to as ''PEGylation" of the substrate. In particular, the present invention relates to a process for direct
`covalent PEGylation of a substrate, comprising the reaction of a halogenated PEG with the substrate wherein the halogen of the halogenated
`PEG acts as a leaving group in the PEGylation reaction.
`
`Novo Nordisk Exhibit 2005
`Mylan Pharms. Inc.v. Novo Nordisk A/S
`IPR2023-00722
`Page 00001
`
`

`

`FOR THE PURPOSES OF INFORMATION ONLY
`
`Codes used to identify States party to the PCT on the front pages of pamphlets publishing international applications under the PCT.
`
`AL
`AM
`AT
`AU
`AZ
`BA
`BB
`BE
`BF
`BG
`BJ
`BR
`BY
`CA
`CF
`CG
`CH
`CI
`CM
`CN
`cu
`CZ
`DE
`DK
`EE
`
`Albania
`Am1enia
`Austria
`Australia
`Azerbaijan
`Bosnia and Herzegovina
`Barbados
`Belgium
`Burkina Faso
`Bulgaria
`Benin
`Brazil
`Belarus
`Canada
`Central African Republic
`Congo
`Switzerland
`Cbte d'Ivoire
`Cameroon
`China
`Cuba
`Czech Republic
`Germany
`Denmark
`Estonia
`
`ES
`FI
`FR
`GA
`GB
`GE
`GH
`GN
`GR
`HU
`IE
`IL
`IS
`IT
`JP
`KE
`KG
`KP
`
`KR
`KZ
`LC
`LI
`LK
`LR
`
`Spain
`Finland
`France
`Gabon
`United Kingdom
`Georgia
`Ghana
`Guinea
`Greece
`Hungary
`Ireland
`Israel
`Iceland
`Italy
`Japan
`Kenya
`Kyrgyzstan
`Democratic People's
`Republic of Korea
`Republic of Korea
`Kazakstan
`Saint Lucia
`Liechtenstein
`Sri Lanka
`Liberia
`
`LS
`LT
`LU
`LV
`MC
`MD
`MG
`MK
`
`ML
`MN
`MR
`MW
`MX
`NE
`NL
`NO
`NZ
`PL
`PT
`RO
`RU
`SD
`SE
`SG
`
`Lesotho
`Lithuania
`Luxembourg
`Latvia
`Monaco
`Republic of Moldova
`Madagascar
`The former Yugoslav
`Republic of Macedonia
`Mali
`Mongolia
`Mauritania
`Malawi
`Mexico
`Niger
`Netherlands
`Norway
`New Zealand
`Poland
`Portugal
`Romania
`Russian Federation
`Sudan
`Sweden
`Singapore
`
`SI
`SK
`SN
`sz
`TD
`TG
`TJ
`TM
`TR
`TT
`UA
`VG
`us
`uz
`VN
`YU
`zw
`
`Slovenia
`Slovakia
`Senegal
`Swaziland
`Chad
`Togo
`Tajikistan
`Turkmenistan
`Turkey
`Trinidad and Tobago
`Ukraine
`Uganda
`United States of America
`Uzbekistan
`Viet Nam
`Yugoslavia
`Zimbabwe
`
`Novo Nordisk Exhibit 2005
`Mylan Pharms. Inc.v. Novo Nordisk A/S
`IPR2023-00722
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`WO 98/32466
`
`PCT/GB98/00253
`
`PEGYLATION PROCESS
`
`5
`
`The present invention relates to the attachment of a polyethylene glycol
`
`(PEG) moiety to a target substrate. Processes for such attachment will be
`
`hereinafter referred to as "PEGylation" of the substrate.
`
`In particular, the
`
`present invention relates to a process for direct covalent PEGylation of a
`
`10
`
`substrate. comprising the reaction of a halogenated PEG with the substrate
`
`wherein the halogen of the halogenated PEG acts as a leaving group in the
`
`PEGylation reaction.
`
`Covalent attachment of PEG to molecules such as proteins or structures
`
`15
`
`such as liposomes is well known to improve their pharmacological and
`
`physiological properties.
`
`EP-A-354855 describes a liposome which comprises a PEG-bound
`
`phospholipid wherein the PEG moiety is bonded to a phospholipid present in
`
`20
`
`the liposome membrane. This is claimed to provide a reduction in the
`
`absorption of proteins to the liposome in vivo and hence an increase in its in
`
`vivo stability.
`
`EP-A-154316 describes
`
`a method
`
`for
`
`chemically modifying
`
`25
`
`lymphokines by attachment of a PEG moiety wherein the PEG is bonded to at
`
`least one primary amino group of the lymphokine. This is claimed to result in
`
`the delayed clearance of lymphokines when used as drugs and to decrease their
`
`antigenicity.
`
`Novo Nordisk Exhibit 2005
`Mylan Pharms. Inc.v. Novo Nordisk A/S
`IPR2023-00722
`Page 00003
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`WO98/32466
`
`PCT/GB98/00253
`
`2
`
`There are many methods for achieving covalent coupling of PEG to
`
`substrates. All such methods require the activation of the PEG by attachment
`
`of a group usually referred to as an "activating moiety" or by converting a
`
`terminal moiety of the PEG into an activating moiety. This is followed by a
`
`5
`
`second step where the PEG couples to the target molecule, usually via a
`
`residual portion of the activating moiety which may be referred to as the
`
`"coupling moiety".
`
`Examples of known techniques include:
`
`10
`
`Succinimidyl Active Ester Methods: see e.g. US Patent 4,412,989;
`WO 86/04145; WO 87/00056; EP-A-0 247 860, C. Monfardini, 0.
`
`Shiavon, P. Caliceti, M. Morpurgo, J. M. Harris, and F. M.
`
`Veronese, "A branched monomethoxypoly(ethylene glycol) for protein
`
`15
`
`modification," Bioconjugate Chem., 6,62-69 (1995), Zalipsky, S. et al.
`
`(1991) in "Polymeric Drugs and Drug Delivery Systems" (R. L. Dunn
`
`& R. M. Ottenbrite, eds.) ACS, Washington, DC, Chapter 10,
`
`Zalipsky, S. et al. (1992) Biotechnol. Appl. Biochem. 15:100, Chiu,
`
`H.-C. et al. (1993) Biocoujugate Chem. 4:290, Sirokman, G. &
`
`20
`
`Fasman, G. (1993) Protein Sci. 2:1161, Veronese, F. M. et al (1989)
`J. Controlled Release 10: 145, Abuchowski, A. et al (1984) Cancer
`
`(1979)
`Biochem. Biophys. 7:175, Joppich, M. & Luisi, P.L.
`Macromol. Chem. 180:1381, Klibanov, A. L. et al (1990) .EEB.s.
`
`Letters 268:235, Sartore, L. et al (1991) Appl. Biochem. Biotech.
`
`25
`
`31:213
`
`Carbonyldiimidazole Method: see e.g. EP-A-0 154 432.
`
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`WO98/32466
`
`PCT/GB98/00253
`
`3
`
`Phenylchloroformate Methods: see e.g. WO 89/06546 and WO
`
`90/15628.
`
`PEG-Succinate Mixed Anhydride Methods: see e.g. Ahlstedt et al
`(1983) Int. Arch. Allergy Appl. Immunol., 71,228-232; Richter and
`
`Akerblom (1983) Int. Arch. Allergy Appl. lmmunol, 70, 124-131;
`
`Organic Sulphonyl Halide Methods: see e.g. US Patent 4,415,665.
`
`PEG-Maleimide and Related Methods: see e.g. Goodson & Katre
`(1990) Biotechnology, 8, 343-346.
`
`Phenylglyoxal Method: see e.g. EP-A-0 340 741
`
`Succinimide Carbonate Method: see e.g. WO 90/13540; WO
`91/07190
`
`Cyanogen Bromide Method: see USP 4,301,144
`
`Poly-PEG Maleic Acid Anhydride Method: Yoshimoto et al (1987)
`Biochem. and Biophy. Res. Commun. ill, 876-882.
`
`5
`
`10
`
`15
`
`20
`
`Cyanuric chloride method: Abuchowski, A. van Es, T., Palczuk,
`N.C., & David, F.F. (1977). Alteration of immunological properties
`
`25
`
`of bovine serum albumin by covalent attachment of polyethylene
`
`glycol. J. Biol. Chem., 252, 3578-3581.
`
`PEG acetaldehyde methods: Royer, G.P. US 4,002,531 EP-A-
`0154316. Harris, J.M., Yoshinaga, K. Paley, M.S., & Herati, M. R.
`
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`WO 98/32466
`
`PCT/GB98/00253
`
`4
`
`(1989). New activated PEG derivatives for affinity partitioning. In D.
`
`Fisher & I.A. Sutherland (Eds) Separations Using Aqueous Phase
`
`Systems. Applications in Cell Biology and Biotechnology (pp. 203-
`
`210). London Plenum Press.
`
`Amine acylation methods {both PEG-COOH and PEG-NH~: see
`e.g. EP007211 l and EP 0401384.
`
`Vinylsulfone method: M. Morpurgo, F. M. Veronese, D. Kachensky
`and J.M. Harris, J. Biocortj. Chem., 1, 363-368 (1996).
`
`PEG epoxide methods: Elling, L. & Kula, M-R. (1991) Biotech.
`
`Appl. Biochem. 13,354.
`
`PEG isocyanate method: R. B. Greenwald. A. Pendri and D. Bolikal,
`J. Org. Chem., 60, 331-336 (1995).
`
`PEG orthopyridyl-disulphide: C. Woghiren, B. Sharma and S. Stein,
`Bioconj. Chem., 4,314 (1993).
`
`5
`
`10
`
`15
`
`20
`
`PEG-proprionaldehyde: Harris, J.M., Dust, J.M., McGill, Harris,
`
`P.A., Edgell, M.J., Sedaghat-Herati, R.M., Karr, L.J., & Donnelly,
`
`D.L. (1991). New polyethylene glycols for biomedical applications.
`
`Chapter 27 in S. W. Shalaby, C. L. McCormick, & G. B. Butler
`
`25
`
`(Eds.), Water-Soluble Polymers Washington D. C.: American Chemical
`
`Society.
`
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`WO98/32466
`
`PCT/GB98/00253
`
`5
`
`These methods suffer from one or more of the following defects:
`
`Substantial loss of biological activity (e.g. 20-95 % loss of bio-activity) is
`
`frequently seen with the cyanuric chloride method:
`
`5
`
`Savoca KV, Abuchowski A, van Es T, Davis FF, Palczuk NC (1979),
`
`Biochem Biopbys Acta 578: 47-53, Ashihara Y, Kono T, Yamazaki S, Inada
`
`Y (1978) Biochern Biophys Res Commun 83:385-391, Kamisaki Y, Wada H,
`Yagura T, Matsushima A, lnada Y (1981) J Pharmacol Exp Ther 216: 410-
`414, Wieder K. J. Palczuk NC, van Es T, Davis F F (1979) J Biol Chem
`
`10
`
`254:12579-12587, Nishimura H, Matsushima A, Inada Y (1981) Enzyme
`
`26:49-53 and Pyatak PS, Abuchowski A, Davis FF (1980) Res Commun
`
`Chem Pathol Pharmacol 29:113-127
`
`The coupling of PEG (or other polymers) to proteins (or other target
`
`15
`
`molecules) is, with few exceptions, in a manner which leaves part of the
`
`activating moiety, a coupling moiety, between the PEG and the target
`
`molecule. Of the above methods, only the organic sulphonyl halide methods
`
`and PEG-acetaldehyde methods disclosed in Royer US 4002531 (1977) and
`
`Harris (1989, ibid) couple PEG directly without coupling moieties i.e. to
`
`20
`
`produce a "linkerless" PEGylated product. With the exception of some other
`
`PEG acetaldehyde methods where the coupling moiety is ethylene oxide (and
`
`thus indistinguishable from PEG itself) and the direct coupling methods above,
`
`all other coupling methods incorporate a coupling moiety distinct from the
`
`polymer and the target and are thus regarded as "indirect" coupling methods.
`
`25
`
`The incorporation of a coupling moiety generates further problems
`
`depending on the nature of the coupling moiety, thus
`
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`WO98/32466
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`PCT/GB98/00253
`
`6
`
`(i)
`
`some coupling moieties provide targets for enzymatic
`
`cleavage or hydrolysis (see below);
`
`(ii)
`
`some
`
`coupling
`
`moieties
`
`provide
`
`an
`
`5
`
`immunogenic/antigenic group (e.g. the triazine ring of
`
`the cyanuric chloride method or the succinyl group of
`
`the succinimidyl succinate method and PEG succinate
`
`mixed anhydride method);
`
`10
`
`(iii)
`
`some coupling moieties are potentially toxic or are
`
`themselves of unknown toxicity but derived from a
`
`compound known to be toxic (e.g. the triazine ring of
`
`the cyanuric chloride method and reagents
`
`in the
`
`phenylchloroformate method); and
`
`15
`
`20
`
`(iv)
`
`some coupling moieties provide reactive groups capable
`
`of linking further molecules to the PEG-target construct
`
`via the coupling moiety (e.g. the triazine ring of the
`
`cyanuric chloride method, Leonard, M. ~ al.,
`
`Tetrahedron, :!Q: 1585 (1984)) and,
`
`(v)
`
`Some coupling groups alter surface charge at the site of
`
`attachment of the polymer.
`
`25
`
`Coupling in some instances is thus via an unstable bond liable to be
`
`cleaved by enzymes present in serum, plasma, cells or other biological
`
`materials or by procedures applied to the PEG-target product. This has two
`
`possible deleterious consequences,
`
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`WO 98/32466
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`PCT/GB98/00253
`
`7
`
`(i)
`
`the PEG-target construct is degraded enzymatically or by the
`
`conditions required for subsequent reaction steps; the former
`
`occurs particularly with methods generating ester bonds and
`
`probably also with amide bonds; and
`
`(ii)
`
`removal of the PEG moiety alters the target molecule; this
`
`occurs with some succinimidyl active ester and mixed
`
`anhydride methods,
`
`5
`
`10
`
`and either or both of these can occur.
`
`Many of the above methods recommend long coupling times and/or a
`
`non physiological pH for the PEGylation reaction, thus rendering some target
`
`molecules
`
`less
`
`active
`
`or
`
`inactive
`
`(cf.
`
`the
`
`cyanuric
`
`chloride,
`
`15
`
`phenylchloroformate, acetaldehyde and propionaldehyde methods).
`
`Many of these methods use activated PEG species and/or produce co(cid:173)
`
`products which are toxic m a wide range of bioassays and which are
`toxic in .YiYQ
`
`potentially
`
`if not separated from the product (e.g.
`
`the
`
`20
`
`phenylchloroformate, cyanuric chloride methods).
`
`Some methods are unsuitable for use in aqueous solution, thus limiting
`
`the target molecules to those which will tolerate non-aqueous conditions (cf.
`
`the organic sulphonyl halide method using
`
`trifluoromethanesulphonyl
`
`25
`
`chloride).
`
`Some of the activated PEG-target constructs are unstable, for instance
`
`being subject to hydrolysis during either the activation or coupling reactions
`
`(cf. the phenylchloroformate method). For example, PEG acetaldehyde is
`
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`WO 98/32466
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`PCT/GB98/00253
`
`8
`
`sensitive to decomposition under basic conditions and can give inconsistent
`
`results.
`
`Ouchi T., et al [(1987) J. Macromol. Sci. Chem. A24 1011-1032]
`
`5
`
`discusses
`
`the PEGylation of 5-fluorouracil with various methoxy-PEG
`
`derivatives to generate methoxy-PEG ether, ester or amide-linked constructs.
`
`The preparation of methoxy-PEG-ether-5-fluorouracil from a methoxy-PEG(cid:173)
`
`Br derivative in chlorobenzene using tetra-n-butylammonium bromide as a
`
`phase-transfer catalyst is described. None of the methoxy-PEG-ether-5-
`
`10
`
`fluorouracil derivatives thus produced showed bioactivity (i.e. anti-tumour
`
`activity).
`
`Zheng Hu et al (1987) Acta Pharmaceutical Sinica, 22 (8) 637 - 640
`
`discusses
`
`the synthesis of PEG-estrogen compounds
`
`from chlorinated
`
`15
`
`polyethylene in non-aqueous solvents using the Williamson reaction.
`
`Probably the most advantageous PEGylation method employed hitherto
`
`is the TMPEG method, mentioned in WOA-90/04606, which comprises
`
`activation
`
`of monomethoxy
`
`PEG
`
`("MPEG")
`
`with
`
`2,2,2-
`
`20
`
`trifluoroethanesulphonyl chloride (tresyl chloride) to produce tresyl MPEG
`
`("TMPEG") which is subsequently reacted with a target protein molecule to
`
`produce monomethoxy PEGylated products. At physiological pH the TMPEG
`
`method is a "direct" coupling method in that the PEG moiety is coupled
`
`directly to the target substrate without a coupling or linker moiety. A similar
`
`25
`
`technique is described in WO 90/04650 for coupling monomethoxy PEG
`
`moieties to DNA/protein complexes.
`
`It is also known that the use of TMPEG as the activated PEG for use
`
`in PEGylation can, particularly at very high pH, result in the elimination of
`
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`PCT/GB98/00253
`
`9
`
`HF by an alternate pathway. This alternative elimination pathway may occur
`
`for example when reacting TMPEG with a protein and
`
`involves
`
`the
`
`elimination of HF which converts TMPEG into an intermediate alkene
`
`followed by hydration and further elimination of HF to create the acyl fluoride
`
`5
`
`which is converted to the a-sulphonate acid via further hydrolysis. The alkene
`
`and acyl fluoride MPEG derivatives can react with target molecules to form a
`
`sulphonate amide derivative.
`
`It is clearly desirable to develop a functionalised PEG which is simple
`
`10
`
`and cheap to prepare, which can be used to PEGylate a wide range of potential
`
`substrates, which generates a linkerless or directly coupled PEGylated
`
`substrate, is capable of pegylating a substrate in both aqueous and non-aqueous
`
`solvents and which does not result in any of the undesirable side effects listed
`
`above.
`
`It is also desirable to have a PEGylation process which functions
`
`15
`
`rapidly under physiological conditions since this is critical for retention of
`
`biological activity in the PEGylation of many proteins.
`
`Hence there is provided according to the present invention a process
`
`for the PEGylation of a substrate comprising the reaction of a halogenated
`
`20
`
`PEG with the substrate wherein the halogen of the halogenated PEG acts as a
`
`leaving group and the PEG is bound directly to the substrate.
`
`In a preferred embodiment of the present invention the halogenated
`
`PEG for use in the process of the invention is of general formula I or general
`
`25
`
`formula IL
`
`wherein:
`
`X-PEG-Y
`
`(PEG)0-Yi
`
`(I)
`
`(II)
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`WO98/32466
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`
`10
`
`In formula I PEG is a bivalent group of formula -(-CH2CH20-)m(cid:173)
`CH2CHr, where m is equal or greater than 1, derived from a polyethylene
`
`glycol;
`
`5
`
`X is a halogen atom, a blocking group or an activating group capable
`
`of coupling the PEG moiety to another moiety; Y is a halogen; n represents
`
`the number of PEG termini and n is equal or greater than 2; i is equal or less
`
`than n; and i/n PEG termini are substituted by Yin compounds of formula II.
`
`10
`
`Halogenated PEGs of Formula
`
`I may be monofunctional,
`
`homobifunctional, or heterobifunctional activated PEGs i.e. a halogenated
`
`PEG of formula I may have two terminal halogens (X and Y are halogens
`
`which may be the same or different), or when only one terminal halogen is
`
`present the other terminal group X may be either a blocking group or an
`
`15
`
`activating group. Halogenated PEGs of Formula II may be of branched,
`
`cruciform or stellate structure.
`
`In preferred embodiments of the present
`
`invention, X is a blocking group selected from methyl, t-butyl and benzyl
`
`ethers.
`
`20
`
`In further preferred embodiments of the present invention, X is an
`
`activating group having an atom that is susceptible to nucleophilic attack or is
`
`capable of rendering the terminal carbon atom of the PEG susceptible to
`
`nucleophilic attack or equivalent alternative substitution and is preferably a
`
`sulphonate ester, a substituted triazine, a N-hydroxysucinimide active ester, an
`
`25
`
`anhydride,
`
`a
`
`substituted phenyl carbonate, oxycarbonylimidazole,
`
`a
`
`maleimide, an aldehyde, a glyoxal, carboxylate, a vinyl sulphone, an epoxide,
`
`an isocyanate, a disulphide, an acrylate, an allyl ether, a silane or a cyanate
`
`ester. More preferably X is an activating group selected from
`
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`IPR2023-00722
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`11
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`2,2,2-trifluoroethanesulphonate,
`
`pentafluorobenzenesulphonate,
`
`fluorosulphonate,
`
`2,4,5-trifluorobenzenesulphonate,
`
`5
`
`2, 4-difluorobenzenesulphonate,
`
`2-chloro-4-fluorobenzenesulphonate,
`
`3-chloro-4-fluorobenzenesulphonate,
`
`4-amino-3-chlorobenzenesulphonate,
`
`4-amino-3-fluorobenzenesulphonate,
`
`10
`
`o-trifluoromethy lbenzenesuplphonate,
`
`m-trifluoromethy lbenzenesulphonate,
`
`p-trifluoromethy lbenzenesulphonate,
`
`2-trifluoromethoxybenzenesulphonate,
`
`4-trifluoromethoxybenzenesulphonate,
`
`15
`
`5-fluoro-2-methylbenzenesulphonate,
`
`4,6-dichlorotriazine,
`
`6-chlorotriazine,
`
`N-hydroxysuccinimidyl succinate,
`
`N-hydroxysuccinimidy l glutarate,
`
`20
`
`N-hydroxysuccinimidyl succinamide,
`
`N-hydroxysuccinimidylalkanedioicamides,
`
`N-hydroxysuccinimidyl derivatives of carboxymethylated polymers,
`
`succinimidy lcarbonate,
`
`N-hydroxysuccinimidyl esters of amino acids,
`
`25
`
`succinate mixed anhydride,
`
`succinic anydride,
`
`trichlorophenyl carbonate,
`
`nitrophenyl carbonate,
`
`maleimide,
`
`Novo Nordisk Exhibit 2005
`Mylan Pharms. Inc.v. Novo Nordisk A/S
`IPR2023-00722
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`WO 98/32466
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`PCT/GB98/00253
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`12
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`N-substituted maleimide,
`
`acetaldehyde,
`
`propionaldehyde and chemically equivalent sulphur analogues,
`
`glyoxal,
`
`5
`
`phenylglyoxal,
`
`acrylate,
`
`methacrylate.
`
`Preferred halogens for the groups X and Y include chlorine, bromine
`
`10
`
`and iodine. Chlorine is most preferred.
`
`In the most preferred embodiments of the present invention, the
`
`halogenated PEG is one of
`
`15
`
`20
`
`monomethoxy PEG-Cl
`
`monomethoxy PEG-Br
`
`monomethoxy PEG-I
`
`Cl-PEG-Cl
`
`Br-PEG-Br
`
`I-PEG-I
`
`Although some halogenated PEGs are known compounds,
`
`it
`
`is
`
`particularly surprising that they have utility as PEG derivatives suitable for
`
`direct use in a PEGylation reaction. It was previously believed that halogens
`
`25
`
`would be of vastly inferior reactivity to known leaving groups such as tosylate
`
`or tresylate. For example McMurry J. in "Organic Chemistry" 4th Ed. (1996)
`
`cites chlorine as having 300 times less reactivity than tosylate as a leaving
`
`group. When considering that tresylate is described as having 100 fold greater
`
`reactivity than tosylate (March J. Advanced Organic Chemistry Reactions,
`
`Novo Nordisk Exhibit 2005
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`IPR2023-00722
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`WO98/32466
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`Mechanisms and Structure, 4th Ed. [1992]), it may be concluded that chlorine
`
`would be expected to have 30,000 fold less reactivity than tresylate.
`
`Halogenated PEGs may be synthesised by methods well known in the
`
`5
`
`art. PEG may be synthesised or purchased commercially and then derivatised
`
`with halogen, activating groups or blocking groups, as required, using
`
`methods disclosed in e.g. Bayer E. et al, Polymer Bulletin .8.,585 - 592 (1982);
`Zalipsky, S et al, Eur. Polym. J. Vol 12 No. 12 pp 1177-1183 (1983);
`Buckmann A. F., Morr. M and Johansson G., Makromol. Chem. l.81, 1379-
`1384 (1981); Harris, J.M. J. Macromol. Sci., Rev. Polym. Chem. Phys.
`
`10
`
`C25(3) 325-373 (1985); Harris, J.M., Struck, E. C., Case, M. G., et al. J.
`
`Poly. Sci, Poly. Chem. Ed. 22, 341-352 (1984); Zalipsky, S. & Lee, C. in
`
`Poly(Ethylene Glycol) Chemistry: Biotechnical and Biomedical applications
`
`(ed Harris, J. M.) 347-370 (Plenum Press, New York, 1992); and as reviewed
`in Zalipsky S. Bioconjugate Chem. (1995) .6 150-165 where MPEG-Cl was
`
`15
`
`used to prepare activated PEGs which were subsequently linked to substrates.
`
`Multi-halogenated PEGs can be constructed using either naturally
`
`branched PEGs, such as the cruciform PEG found in some preparations of
`
`20
`
`high molecular weight PEGs, or from proprietary multibranched PEGs known
`
`as "star" PEGs. Derivatisation of the free PEG termini with halogen is
`
`achieved as for halogenated PEGs above.
`
`Reaction conditions for the process of the present invention will clearly
`
`25
`
`depend upon the nature of X, Y and of the substrate.
`
`As indicated above, the PEG moieties of the halogenated PEG's used
`
`in accordance with the invention, may desirably be derived from commercially
`
`available PEGs. These materials are generally characterised by their number
`
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`Mylan Pharms. Inc.v. Novo Nordisk A/S
`IPR2023-00722
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`PCT/GB98/00253
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`14
`
`and weight average molecular weight.
`
`For example, PEG-5000 is a
`
`polyethylene glycol having a number average molecular weight of about 5000.
`
`The size of the PEG moiety to be attached to the target substrate will usually
`
`be chosen according to the nature of the substrate and how its properties are
`
`5
`
`desired to be modified by the attachment of the PEG moiety. For example, if
`
`the target substrate is a liposome for administration to an animal and it is
`
`desired to increase the circulation half life of the liposome after administration,
`
`a PEG of molecular weight 1000 to 5000 may be selected. It should be noted,
`
`however, that the process of the present invention is generally applicable to the
`
`10
`
`attachment of PEG moieties of any size to target substrates.
`
`PEGylated substrates generated according to the present invention
`
`particularly include those which do not lose their bioactivity relative to the
`
`unPEGylated substrate. Thus PEGylation according to the present invention
`
`15
`
`may maintain or increase the specific activity of a substrate or it may increase
`
`the in vivo half-life of a substrate which has had its specific activity decreased,
`
`maintained or increased by PEGylation. Additionally PEGylation according to
`
`the present invention may differentially modify the specific activity of
`
`pleiotropic substrates such as certain proteins.
`
`20
`
`The term
`
`II substrate II as used herein is
`
`intended to include any
`
`molecule, macromolecule or structure which is capable of being covalently
`
`attached to a PEG moiety and which thereby may have its chemical,
`
`biological, physiological or physical properties modified. It is not intended to
`
`25
`
`encompass molecules which when reacted with halogenated PEG merely
`
`produce a further activated PEG derivative which is to be used as an
`
`intermediate to couple the PEG moiety to another substrate. The substrate is
`
`not a steroid.
`
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`IPR2023-00722
`Page 00016
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`15
`
`Suitable substrates to which PEG can be attached in accordance with
`
`the present invention include materials having biological activity which are
`
`useful in, for instance diagnosis or therapy and which are all well known to
`
`those skilled in the art. They all contain at least one group capable of reacting
`
`5
`
`with the halogenated PEG. Examples of such reactive groups include primary,
`
`secondary and tertiary amino groups, thiol groups and aromatic hydroxy
`
`groups.
`
`More specifically, substrates for use according to the present invention
`
`10
`
`include proteins, peptides, amino acids and
`
`their derivatives such as:
`
`antibodies and fragments thereof; cytokines and derivatives or fragments
`
`thereof, for example, the interleukins (IL) and especially the IL-1, IL-2, IL-3,
`
`IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 and IL-12 subtypes thereof;
`
`colony stimulating factors,
`
`for example granulocyte-macrophage colony
`
`15
`
`stimulating factor, granulocyte-colony stimulating factor (alpha and beta
`
`forms), macrophage colony stimulating factor (also known as CSF-1);
`
`haemopoietins, for example erythropoietin, haemopoietin-alpha and kit-ligand
`
`(also known as stem cell factor or Steel factor); interferons (IFNS), for
`
`example IFNalpha, IFNbeta and IFNgamma; growth factors and bifunctional
`
`20
`
`growth modulators, for example epidermal growth factor, platelet derived
`
`growth
`
`factor,
`
`transforming growth
`
`factor
`
`(alpha and beta
`
`forms),
`
`amphiregulin, somatomedin-C, bone growth factor, fibroblast growth factors,
`
`insulin-like growth factors, heparin binding growth factors and tumour growth
`
`factors; differentiation factors and
`
`the
`
`like,
`
`for example macrophage
`
`25
`
`differentiating factor, differentiation inducing factor (DIF) and leukaemia
`
`inhibitory factor; activating factors, for example platelet activating factor and
`
`macrophage
`
`activation
`
`factor;
`
`coagulation
`
`factors
`
`such
`
`as
`
`fibrinolytic/anticoagulant agents including heparin and proteases and their pro(cid:173)
`
`factors, for example clotting factors VII, VIII, IX, X, XI and XII,
`
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`IPR2023-00722
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`16
`
`antithrombin ill, protein C, protein S, streptokinase, urokinase, prourokinase,
`
`tissue plasminogen activator, fibrinogen and hirudin; peptide hormones, for
`
`example
`
`insulin, growth hormone, gonadotrophins,
`
`follicle stimulating
`
`hormone,
`
`leutenising hormone, growth hormone releasing hormone and
`
`5
`
`calcitonin; enzymes such as superoxide dismutase, glucocerebrosidase,
`
`asparaginase and adenosine deaminase; vaccines, for example hepatitis-B
`
`vaccine, malaria vaccine, melanoma vaccine and HIV-1 vaccine; transcription
`
`factors and transcriptional modulators; carbohydrates, glycosoaminoglycans,
`
`glycoproteins and polysaccharides;
`
`lipids,
`
`for example phosphatidyl-
`
`10
`
`ethanolamine, phosphtidylserine and derivatives thereof; sphingosine; and
`
`derivatives thereof; nucleotides, nucleosides, heterocyclic bases, DNA, RNA,
`
`synthetic and non-synthetic oligonucleotides including those with nuclease
`
`resistant backbones; vitamins; antibiotics including lantibiotics; bacteristatic
`
`and bactericidal agents; antifungal, anthelminthic and other agents effective
`
`15
`
`against
`
`infective agents
`
`including unicellular pathogens; small effector
`
`molecules such as noradrenalin, alpha adrenergic receptor ligands, dopamine
`
`receptor ligands, histamine receptor ligands, GABA/benzodiazepine receptor
`
`ligands, serotonin receptor ligands, leukotrienes and triodothyronine; cytotoxic
`
`agents such as doxorubicin, methotrexate and derivatives thereof.
`
`20
`
`The substrate may also be part of a larger multi-molecular structure.
`
`These include cells or parts thereof, for instance erythrocytes, erythrocyte
`
`"ghosts" and leukocytes, viruses, unicellular organisms, liposomes such as
`
`multilamellar vesicles and unilamellar vesicles, micelles and micelle-like
`
`25
`
`structures, and aggregates, microemulsions, coacervates, emulsions and
`
`suspensions of the foregoing. The substrate may also be a surface on a device
`
`such as a catheter, stent, contact lens or artificial valve.
`
`Novo Nordisk Exhibit 2005
`Mylan Pharms. Inc.v. Novo Nordisk A/S
`IPR2023-00722
`Page 00018
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`PCT/GB98/00253
`
`17
`
`It will be appreciated that when the substrate is part of suc.h a structure
`
`there will generally be many reactive groups in each structure; treatment
`
`according to the invention may therefore produce a structure bearing many
`
`PEG moieties. When the PEG is bi- or multi-valent, reaction with a
`
`5
`
`multimolecular substrate may result in intermolecular cross-linking by the
`
`PEG between molecules of the same target structure and/or between molecules
`
`of different target structures as well as intramolecular bonding of the PEG to
`
`more than one position on the same molecule of a target structure.
`
`10
`
`Substrates lacking a reactive group may be modified so as to create one
`
`or more reactive groups; this is within the ability of those skilled in the art and
`
`can be achieved by well-known techniques.
`
`Some substrates (e.g. RNA and single stranded DNA) pose special
`
`15
`
`problems because they may provide too many reactive groups to which the
`
`PEG would attach in a standard reaction. Therefore, if desired, some groups
`
`may be temporarily protected by involvement in an appropriate conformation
`
`precluding nucleophilic attack on the halogenated PEG, as for example by the
`
`hydrogen bonding associated with base pairing of DNA (see below).
`
`20
`
`25
`
`The term "blocking group" as used herein is intended to imply a
`
`moiety which when covalently bound to a PEG terminus,
`
`is capable of
`
`preventing the attachment of an activating group to that terminus during the
`
`activation process.
`
`An embodiment of
`
`the present process
`
`involves
`
`site-specific
`
`modification of DNA, RNA and synthetic oligonucleotide targets (or of any
`
`molecule containing and amino or other reactive group which can participate
`
`in interactions such as hydrogen bonding with another molecule or compound)
`
`Novo Nordisk Exh