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USOO88651 49B2

(12) United States Patent (10) Patent No.: US 8,865,149 B2 McManus et al. (45) Date of Patent: Oct. 21, 2014

(54) POLYMERIC REAGENTS COMPRISINGA 5,932,462 A 8, 1999 Harris et al. KETONE OR A RELATED FUNCTIONAL 5,932,641 A 8/1999 Blanchard et al. GROUP 6,048,698 A 4, 2000 Eaton et al. 6,102.997 A 8/2000 Helling et al. 6,258,774 B1 7/2001 Stein et al. (75) Inventors: Samuel P. McManus, Huntsville, AL 6,348,558 B1 2, 2002 E"al. (US); Antoni Kozlowski, Huntsvill, AL 6,362.254 B2 3/2002 Harris et al. (US); Xiaoming Shen, Madison, AL &E R 3.39: E. l (US); Daniel C. Cook, Harvest, AL (US) 6,642,035I w B2 11/2003 Jandaenley et etal. al. (73) Assignee: Nektar Therapeutics, San Francisco, 20067,208,145 B2Rio 4/200758. McME". tal. 52.5/56 CA (US) 2003. O157582 A1 8, 2003 Roisin et al. (*) Notice: Subject to any disclaimer, the term of this FOREIGN PATENT DOCUMENTS patent is extended or adjusted under 35 U.S.C. 154(b) by 1902 days. CA 1148,166 6, 1983 DE 3334209 4f1985 DE 101.26158 12/2002 (21) Appl. No.: 11/714,322 EP O361704 4f1990 EP O4004.86 12/1990 (22) Filed: Mar. 6, 2007 EP O42O232 4f1991 EP 0457128 11, 1991 (65) Prior Publication Data EP O605963 T 1994 EP O634393 1, 1995 US 2007/O166277 A1 Jul. 19, 2007 EP 1174436 1, 2002 EP 1310533 5, 2003 JP 60212435 10, 1985 Related U.S. Application Data JP O125562O 10, 1989 JP O2283659 11, 1990 (63) Continuation of application No. 10/751,009, filed on JP 69737 1, 1994 Dec. 31, 2003, now Pat. No. 7,208,145. JP 649471 2, 1994 JP O6324421 11, 1994 (60) Provisional application No. 60/437,325, filed on Dec. JP 7622.79 3, 1995 31, 2002. JP T62594 3, 1995 s JP O7159917 6, 1995 (51) Int. Cl JP 2OOO129568 5, 2000 A6 IK3I/74 (2006.01) (Continued) C08G 65/33 (2006.01) OTHER PUBLICATIONS A6 IK 47/48 (2006.01) C08G 65/329 (2006.01) European Search Report and Examination Report, (Aug. 27, 2007). C08G 65/334 (2006.01) Australian Examination Report corresponding to Australian Patent 52) U.S. C Application No. 2003303519 dated Jun. 5, 2008. (52) AV e. we Indian Examination Report corresponding to Indian Patent Applica CPC ...... A61K 47/48215 (2013.01); C08G 65/331 tion No. 2863/DELNP/2005 dated Oct. 5, 2007. (2013.01); C08G 65/329 (2013.01); C08G Japanese Office Action corresponding to Japanese Patent Application 65/3344 (2013.01); C08L 2203/02 (2013.01) No. 2004-564925 mailing date Aug. 11, 2009. USPC ...... 424/78.27 424/78.26: 424/78.37: Mexican Office Action corresponding to Mexican Patent Application s 424/78.08 No. PA/a/2005/007165 Dated Apr. 16, 2009. PCT International Search Report corresponding to PCT International (58) Field of Classification Search Application No. PCT/U52003/041743 date of mailing Nov. 18, 2004. USPC ...... 424/78.27 PCT Written Opinion corresponding to PCT International Applica See application file for complete search history. tion No. PCT/US2003/041743 date of mailing Dec. 9, 2004. (56) References Cited (Continued) U.S. PATENT DOCUMENTS Primary Examiner —James Rogers (74) Attorney, Agent, or Firm — Mark A. Wilson; Timothy 3,551384 A 12, 1970 Zeh A. Marquart 3,668,183 A 6/1972 Hoy et al. 3,779,986 A 12/1973 Smith et al. 4,980,514 A 12/1990 Sanderson et al. (57) ABSTRACT 4,992,204 A 2, 1991 Kluger et al. Polymeric reagents comprising a polymer attached, either 5,002,998 A 3/1991 Carey et al. 5,045,396 A 9, 1991 Lin et al. directly or through one or more atoms, to a ketone or a related 5,138,095 A 8/1992 Speranza et al. functional group Such as ketone hydrate, thione, monothio 5,149,806 A 9, 1992 Moren et al. hydrate, dithiohydrate, hemiketal, monothiohemiketal, 5,151,470 A 9, 1992 Sanders et al. dithiohemiketal, ketal, or dithioketal are provided. The poly 5,382,657 A * 1/1995 Karasiewicz et al...... 530,351 meric reagents are useful for, among other things, forming 5,580,923 A 12/1996 Yeung et al. 5,674,531 A 10, 1997 Ahlers et al. polymer-active agent conjugates. Related methods, compo 5,723,289 A 3, 1998 Eaton et al. sitions, preparations, and so forth are also provided. 5,733,968 A 3, 1998 MOSZner et al. 5,858,660 A 1/1999 Eaton et al. 7 Claims, 3 Drawing Sheets US 8,865,149 B2 Page 2

(56) References Cited NOF Corporation, “PEG Derivatives, Phospholipid and Drug Deliv ery Materials for Pharmaceuticals', pp. 1-46, (Catalogue 2003-1). NOF Corporation, “PEG Derivatives, Phospholipid and Drug Deliv FOREIGN PATENT DOCUMENTS ery Materials for Pharmaceuticals”, pp. 1-50, (Catalogue 2003-2"). JP 2001.25562O 9, 2001 Pande, et al., “Camphorquinone-10-Sulfonic acid and derivatives: JP 2002234940 8, 2002 Convenient reagents for reversible modification of arginine resi WO 90,00049 1, 1990 dues”. Proc. Natl. Acad. Sci. USA, 77(2);895-899, Feb. 1980, Bio WO 95/21631 8, 1995 chemistry. WO 96.33,156 10, 1996 Parr, et al., “Factors influencing the retention and chemical stability WO 96.331.57 10, 1996 of poly()-lipid conjugates incorporated into large WO 97,49664 12/1997 WO 98/53857 12/1998 unilamellar vesicles'. Biophysica Acta 1195 (1994), pp. 21-30. WO OOf 17398 3, 2000 Seliger, et al., “Two-carrier liquid-phase synthesis of main-chain WO 0044808 8, 2000 liquid crystalline oligomers and characterization of the products'. WO 01.04179 1, 2001 Fresenius J. Anal. Chem. (1995), 351:260-270. WO 01.041.83 1, 2001 Shearwater Polymers, Inc., p. 2-49, (Catalog Mar. 1995). WO O1/92584 12/2001 Shearwater Polymers, Inc., and Derivatives, pp. WO WOO192584 A1 * 12/2001 1-53, (Catalog Jul. 1997). WO O3,004471 1, 2003 Shearwater Polymers, Inc., Polyethylene Glycol and Derivatives: WO O3,OO8646 1, 2003 Functionalized Biocompatible Polymers for Research and Pharma WO O3,O34995 5, 2003 ceuticals, pp. 1-50, (Catalog 2000). WO 2004/060406 T 2004 Shearwater Corporation, Polyethylene Glycol and Derivatives for OTHER PUBLICATIONS Biomedical Applications, pp. 1-17. (Catalog 2001). PCT International Preliminary Examination Report corresponding to Chinese First Office Action corresponding to Chinese Patent Appli PCT International Application No. PCT/US2003/041743 date of cation No. 20038.0109450.4 dated Mar. 2, 2007. mailing Apr. 8, 2005. Chinese Second Office Action corresponding to Chinese Patent Cordova, et al., “A Highly Chemo- and Stereoselective Synthesis of Application No. 20038.0109450.4 dated Oct. 24, 2008. B-Keto Esters via a Polymer-Supported Lipase Catalyzed Chinese Third Office Action corresponding to Chinese Patent Appli Transesterfication”. J. Org. Chem. (2001), 66:1906-1909. cation No. 20038.0109450.4 dated Oct. 10, 2009. Haag, et al., “New Polyethylene Glycol Polymers as Ketal Protecting European Communication corresponding to European Patent Appli Groups—A Polymer Supported Approach to Symmetrically Substi cation No. 038150 12.4 dated Nov. 21, 2006. tuted Spiroketals'. Synthetic Communications (2001), 31(19):2965 PCT Invitation to Pay Additional Fees/Partial International Search 2977. corresponding to PCT Application No. PCT/US2003/041743 dated Hai, et al., “Polymerization to Diaspirin Cross-Linked Hemoglobin Aug. 10, 2003. (DCLHb) with Water-Soluble, Nonimmunogenic Polyamide Cross Office Action corresponding to Canadian Patent Application No. Linking Agents'. Bioconjugate Chem. (1999), 10:1013-1020. 2,509,248 dated Aug. 2, 2010. Herman, et al., “Poly(Ethylene Glycol) with Reactive Endgroups: I. Office Action corresponding to Canadian Patent Application No. Modification of Proteins”. J. of Bioactive and Compatible Polymers 2,509,248 dated Apr. 6, 2011. (Apr. 1995), 10:145-187. Notification of the Fourth Office Action corresponding to Chinese Huang, et al., “The Synthesis of Polyethylene Glycol (PEG) with Patent Application No. 20038.0109450.4 dated Aug. 5, 2011. Different Functional Groups at Each End. II. Synthesis of a Series of Communication corresponding to European Patent Application No. Monosubstituted PEG via Polyvinyl (PVA) as Support”. J. of 03 815 012.4-2404 dated Nov. 26, 2012. Applied Polymer Sci. (1993), 47: 1503-1511. Communication corresponding to European Patent Application No. Mohseni, et al., “Condution and electrooptical properties of vana 03 815 0124-1408 dated Apr. 26, 2013. dium-containing polymer electrolyte networks from Sol-gel methods Notice of Reasons for Rejection mailed Jun. 15, 2010 corresponding with modified poly(ethylene glycol)S and poly(tetramethylene to Japanese Patent Application No. 2004-564925. oxide), J. Mater. Chem. (1999) 9:1363-1367. Notice of Reasons for Rejection mailed Nov. 1, 2011 corresponding Moszner, et al., “Reaction behaviour of monomeric B-ketoesters'. to Japanese Patent Application No. 2004-564925. Polymer Bulletin (1994), 32:411-417. Notice of Grounds for Rejection issued Nov. 5, 2010 corresponding Naka, et al., “Synthesis of a star-shaped polymer having tris to Korean Patent Application No. 2005-70 12459. (B-diketonato)chromium(III) at the center core”. Polymer Bulletin Summons to Attend Oral Proceedings pursuant to Rule 115(1) EPC (1998), 41:263-266. dated Dec. 3, 2013 corresponding to European Patent Application NEKTARTM Transforming Therapeutics, Nektar Molecule Engi No. 03815O124-1408 1581260. neering: Polyethylene Glycol and Derivatives for Advanced PEGyla tion, pp. 1-20, (Catalog 2003). * cited by examiner U.S. Patent Oct. 21, 2014 Sheet 1 of 3 US 8,865,149 B2

FIGURE 1

O H2O OH Polymeric Reagent - Polymeric Reagent - 12 OH

- HNH Active Agent HN- Active Agent

OH Polymeric Reagent s-s-H2O Polymeric Reagent H2O HN-Active Agent N- Active Agent U.S. Patent Oct. 21, 2014 Sheet 2 of 3 US 8,865,149 B2

O

Polymeric Reagent H', olo CHOH,-H

CH ob O1 CHOH, -H O-CH Polymeric Reagent? H, -CHOH Polymeric Reagent

-H-NH Active Agent HNH Active Agent

ob H, -CH-OH,-H Rymric Rev. NH Active Agent H, CH-OH,-H Active Agent U.S. Patent Oct. 21, 2014 Sheet 3 of 3 US 8,865,149 B2

FIGURE 3

O R-C-R" HN- Activee Agent polymeric reagent

nucleophilic addition/ condensation

N Active Agent R-C-R" (imine intermediate)

reduction

secondary amine

polymer-active agent conjugate US 8,865,149 B2 1. 2 POLYMERC REAGENTS COMPRISINGA cially available PEGylated polypeptides include PEGA KETONE OR A RELATED FUNCTIONAL SYSR PEGylated interferon alpha-2a (Hoffmann-La Roche, GROUP Nutley, N.J.), PEG-INTRONR PEGylated interferon alpha 2b (Schering Corp., Kennilworth, N.J.), SOMAVERTR CROSS REFERENCE TO RELATED PEGylated human growth hormone receptor antagonist, and APPLICATIONS NEULASTAR PEG-filgrastim (Amgen Inc., Thousand Oaks, Calif.). PEGylated small molecules such as dis This application is a continuation of U.S. patent applica tearoylphosphatidylethanolamine (Zalipsky (1993) Biocon tion Ser. No. 10/751,009, filed Dec. 31, 2003, now U.S. Pat. jug. Chem. 4(4):296-299) and fluorouracil (Ouchi et al. No. 7,208,145, which claims the benefit of priority to U.S. 10 (1992) Drug Des. Discov. 9(1):93-105) have also been pre Provisional Patent Application Ser. No. 60/437,325, filed pared. Dec. 31, 2002, each of which is incorporated herein by ref Despite these successes, conjugation of a polymer to an CCC. active agent remains challenging. In particular, the conjuga tion reaction is relatively imprecise in that a relatively dis FIELD OF THE INVENTION 15 perse mixture of products results. For example, the conjuga tion reaction often results in a mixture of singly substituted, The present invention relates generally to novel polymeric disubstituted, and poly Substituted conjugate forms. More reagents comprising a ketone or a related functional group over, the possible number of different conjugate forms Such as, for example, a ketone hydrate, thione, monothiohy increases inasmuch as different attachment sites of an active drate, dithiohydrate, hemiketal, monothiohemiketal, dithio agent can result in different arrangements of attachments. For hemiketal, ketal, and dithioketal. In addition, the invention example, a singly substituted active agent having three dif relates to conjugates formed from the attachment of a poly ferent sites for attachment to a polymer can have three differ meric reagent described herein to another Substance. Such as ent forms: each form having the single polymer attached to an active agent. Furthermore, the invention relates to methods one of the three different attachment sites. Recognizing that for synthesizing polymeric reagents, methods, for conjugat 25 each conjugate form can have a unique pharmacodynamic ing a polymeric reagent to another Substance, compositions and pharmacokinetic profile only serves to underscore the comprising the polymeric reagents, and the like. complexity associated with providing polymer-active agent conjugates that are Suited for use as therapeutic agents. Thus, BACKGROUND OF THE INVENTION there is a desire to provide polymeric reagents that are “site 30 directed so that the desired conjugates can be more predict Scientists and clinicians face a number of challenges in ably formed. their attempts to develop active agents into forms suited for Some degree of selectivity in the conjugation reaction is delivery to a patient. Active agents that are polypeptides, for achieved based on the presence of the particular functional example, are often delivered by injection rather than orally. In group present on the polymeric reagent. For example, poly this way, the polypeptide is introduced into the systemic 35 (ethylene glycol) derivatives bearing an aldehyde derivative circulation without exposure to the proteolytic environment (shown below) undergo reductive amination reactions with of the stomach. Injection of polypeptides, however, has sev primary amines of for example, a polypeptide. eral drawbacks. For example, many polypeptides have a rela tively short half-life, thereby necessitating the need for repeated injections, which are often inconvenient and painful. 40 O Moreover, Some polypeptides may elicit one or more immune responses with the consequence that the patient’s immune HCO-(CH2CH2O)-CHCHCHCH system may be activated to degrade or inactivate the polypep tide. Thus, the delivery of polypeptides and other active a poly(ethylene glycol) derivative bearing an aldehyde agents is often problematic even when these agents are 45 derivative wherein (n) represents the number of repeat administered by injection. ing ethylene oxide units Some Success in addressing the problems of delivering As a consequence, a degree of selectivity is achieved in that active agents via injection has been achieved. For example, conjugation typically occurs only between the relatively reac conjugating the active agent to a water-soluble polymer has tive and readily accessible primary amine groups of the resulted in polymer-active agent conjugates having reduced 50 polypeptide and the aldehyde of the polymeric reagent. For immunogenicity and antigenicity. In addition, these conju polypeptides having many relatively reactive primary amine gates often have greatly increased half-lives compared to their groups (e.g., a polypeptide having many primary amine-con unconjugated counterparts as a result of decreased clearance taining lysine residues), however, the conjugation reaction through the kidney and/or decreased enzymatic degradation nonetheless results in a relatively polydisperse mixture of in vivo. As a result of having a greater half-life, the conjugate 55 conjugate forms. requires less frequent dosing, which in turn reduces the over In order to provide an alternative to polymeric reagents all number of painful injections and inconvenient visits to a bearing aldehyde derivatives, the present invention provides healthcare professional. Moreover, active agents that are only polymeric reagents comprising a ketone or a related func marginally soluble often demonstrate a significant increase in tional group (e.g., a ketone hydrate, thione, monothiohydrate, water Solubility when conjugated to a water-soluble polymer. 60 dithiohydrate, hemiketal, monothiohemiketal, dithiohe Due to its documented safety as well as its approval by the miketal, ketal, or dithioketal). FDA for both topical and internal use, poly(ethylene glycol) Others have described polymers comprising a ketone or a has been conjugated to active agents. When an active agent is related functional group for use in variety of contexts. Typi conjugated to a polymer of poly(ethylene glycol) or “PEG'. cally, however, the described polymers are unsuited for con the conjugated active agent is conventionally referred to as 65 jugation. having been "PEGylated.” The commercial success of PEGy For example, WO 96/33 156 describes poly(alkylene gly lated active agents attests to their value. Exemplary commer col) derivatives of benzophenone and related aromatic moi US 8,865,149 B2 3 4 eties. The derivatives are described as being useful as photo It is another object of the invention to provide such a initiators. Aromatic rings, however, are preferably absent in conjugate wherein the Substance is an active agent. polymeric reagents useful for conjugation to an active agent It is yet another object of the invention to provide a phar because the hydrophobic nature of aromatic moieties unde maceutical composition comprising a polymer-active agent sirably decreases aqueous solubility. In addition, aromatic conjugate as described herein. rings contained in a conjugate may be metabolized in vivo It is an additional object of the invention to provide a into arene oxide intermediates, which can covalently bind method for administering the polymer conjugates described with nucleophilic groups present on proteins, DNA and RNA, herein. thereby leading to cellular toxicity. See Daly et al. (1979) Additional objects, advantages and novel features of the Experientia 28(10): 1129-1149. 10 invention will be set forth in the description that follows, and U.S. Pat. No. 5,149,806 describes a carbon acid useful as a in part, will become apparent to those skilled in the art upon Michael donor. One of the described structures is provided the following, or may be learned by practice of the invention. In one embodiment of the invention, then, a polymeric below: reagent is provided comprising a functional group and a 15 water-soluble polymer segment. The functional group and the CH3 CH3 water-soluble polymer segment are attached through either a direct covalent bond or through one or more atoms. The CHO(CH2CHO)17-CHCH-NHCCHCCH functional group is selected from the group consisting of ketone, ketone hydrate, thione, monothiohydrate, dithiohy CH CH drate, hemiketal, monothiohemiketal, dithiohemiketal, ketal, and dithioketal. Among other things, the polymeric reagent CHO(CHCHO)-CHCH-NHCCHCCH can form polymer-containing conjugates with another Sub CH3 CH3 stance (e.g., an active agent). In another embodiment, a polymeric reagent is provided CHO(CHCHO)-CHCH-NHCCHCCH. 25 comprising a functional group and a water-soluble polymer segment having a weight average molecular weight of greater Even if structures like these could be used for conjugation than 2,200, wherein the functional group is attached to the reactions, the propylene oxide polymers are not suitably water-soluble polymer segment through either a direct cova water-soluble to provide a conjugate acceptable for in vivo lent bond or through one or more atoms, and further wherein 30 (a) the functional group is selected from the group consisting administration. of ketone, ketone hydrate, thione, monothiohydrate, dithio Other previously described polymeric reagents suffer from hydrate, hemiketal, monothiohemiketal, dithiohemiketal, similar deficiencies. ketal, and dithioketal, and (b) the polymeric reagent lacks Thus, there remains a need in the art to provide polymeric each of the following: an aromatic moiety; a residue of pro reagents that are useful for providing, among other things, 35 line; a cyclic dienone, a Saturated hydrocarbon chain of 8 conjugates with other Substances. The present invention carbons or greater; and a —ONH group, with the proviso that addresses this and other needs in the art by providing, inter the polymeric reagent is none of alia, novel polymeric reagents comprising a ketone or a related functional group (e.g., a ketone hydrate, thione, monothiohydrate, dithiohydrate, hemiketal, monothiohe 40 O O | miketal, dithiohemiketal, ketal, or dithioketal). HCO--CHCHO-H-C-CH-C-CH3, SUMMARY OF THE INVENTION mWS000 O Accordingly, it is a primary object of this invention to 45 provide a polymeric reagent comprising a functional group HCO--CHCHO-H-CH-C-CH3, and a water-soluble polymer segment, wherein the functional mWS000 group is attached to the water-soluble polymer segment O through one or more atoms, wherein the functional group is HCO-CHCHO-H-CH-C-CH. selected from the group consisting of ketone, ketone hydrate, 50 thione, monothiohydrate, dithiohydrate, hemiketal, mono mWS000 thiohemiketal, dithiohemiketal, ketal, and dithioketal. It is another object of the invention to provide such a and a ketal derivative of polymeric reagent wherein the water-soluble polymer seg In another embodiment of the invention, a polymeric ment has a molecular weight of greater than 2.200. 55 reagent is provided comprised of a functional group and a It is an additional object of the invention to provide such a water-soluble polymer segment, wherein the functional polymeric reagent wherein the functional group is selected group is attached to the water-soluble polymer segment from the group consisting of ketone, ketone hydrate, through either a direct covalent bond or through one or more hemiketal, and ketal. atoms, and further wherein (a) the functional group is selected It is still another object of the invention to provide such a 60 from the group consisting of thione, monothiohydrate, dithio polymeric reagent wherein the water-soluble polymer seg hydrate, monothiohemiketal, dithiohemiketal, and ment is a poly(ethylene glycol). dithioketal, and (b) the polymeric reagent lacks a Saturated It is a further object of the invention to provide methods for hydrocarbon chain of 8 carbons or greater. preparing the polymeric reagent described herein. In yet another embodiment of the invention, a polymeric It is still a further object of the invention to provide a 65 reagent is provided comprising a water-soluble polymer seg conjugate of a Substance and a polymeric reagent as described ment and a functional group, wherein the functional group is herein. part of a cyclic structure and further wherein the cyclic struc US 8,865,149 B2 5 6 ture is attached to the water-soluble polymer segment through taining two functional groups (either the same or different). either a direct covalent bond or through one or more atoms, Exemplary bifunctional polymeric reagents encompassed by wherein the functional group is selected from the group con the invention comprise the following structure: sisting of ketone, ketone hydrate, thione, monothiohydrate, dithiohydrate, hemiketal, monothiohemiketal, dithiohe miketal, ketal, and dithioketal, and further wherein the poly (Formula II) meric reagent lacks a cyclic dienone. As will be explained in greater detail below, the polymeric reagents of the invention encompass many different forms. For example, the polymeric reagent can be monofunctional. 10 Exemplary monofunctional polymeric reagents encom passed by the invention comprise the following structure: wherein: (Formula I) 15 POLY is a water-soluble polymer segment; R2 (a) is either Zero or one; (b) is either Zero or one; (c) is either Zero or one; (d) is either Zero or one; Z X', when present, is a spacer moiety; X, when present, is a spacer moiety; wherein: EW', when present, is an electron-withdrawing group; POLY is a water-soluble polymer segment having a ter EW’, when present, is an electron-withdrawing group; minus defined as —O R: (Z) is 0 or a positive integer, e.g., 1, 2, 3, 4, 5, 6, or 7. (a) is either Zero or one; 25 (y) is 0 or a positive integer, e.g., 1, 2, 3, 4, 5, 6, or 7. (b) is either Zero or one: each R, when present, is independently H or a organic X', when present, is a spacer moiety; radical, for example, H or a nonaromatic-containing organic EW', when present, is an electron-withdrawing group; radical; (Z) is 0 or a positive integer, e.g., 1, 2, 3, 4, 5, 6, or 7: each R, when present, is independently H or a organic each R, when present, is independently H or a organic 30 radical, for example, H or a nonaromatic-containing organic radical, for example, H or a nonaromatic-containing organic radical; radical; each R', when present, is independently H or a organic each R, when present, is independently H or a organic radical, for example, H or a nonaromatic-containing organic radical, for example, H or a nonaromatic-containing organic radical; radical; 35 each R, when present, is independently H or a organic R’ is Hora organic radical, for example, H or a nonaro radical, for example, H or a nonaromatic-containing organic matic-containing organic radical; radical; FG is a functional group selected from ketone, ketone —FG-R' is either (i) a functional group selected from the hydrate, thione, monothiohydrate, dithiohydrate, hemiketal, group consisting of ketone, ketone hydrate, thione, monothio monothiohemiketal, dithiohemiketal, ketal, and dithioketal; 40 hydrate, dithiohydrate, hemiketal, monothiohemiketal, and dithiohemiketal, ketal, and dithioketal having a terminus R" is an organic radical, for example, a nonaromatic-con defined as R', wherein R' is an organic radical, or (ii) a taining organic radical; nondienone cyclic structure wherein a functional group and further wherein the selected from the group consisting of ketone, ketone hydrate, 45 thione, monothiohydrate, dithiohydrate, hemiketal, mono thiohemiketal, dithiohemiketal, ketal, and dithioketal is part R2 of the cyclic structure; and -FG-R is either (i) a functional group selected from the C group consisting of ketone, ketone hydrate, thione, monothio R3 50 hydrate, dithiohydrate, hemiketal, monothiohemiketal, Z dithiohemiketal, ketal, and dithioketal having a terminus defined as R, wherein R is an organic radical, or (ii) a moiety optionally contains one or more double bonds, with nondienone cyclic structure wherein a functional group the proviso that the polymeric reagent is neither selected from the group consisting of ketone, ketone hydrate, 55 thione, monothiohydrate, dithiohydrate, hemiketal, mono thiohemiketal, dithiohemiketal, ketal, and dithioketal is part O O of the cyclic structure. HO-(CH2CHO) su-s-s-s-sso Optionally, each of the H 60 O R2 R4 HCO-(CH2CH2O)-CH-CH-CH-C-CH. -- a - mw = 2000

65 Z Z The polymeric reagents of the invention can also be pro vided in the form of a bifunctional polymeric reagent con moieties in Formula II contains one or more double bonds. US 8,865,149 B2 7 In still another embodiment, the invention provides poly and a ketal derivative of meric reagents comprising two or more water-soluble poly mer segments (either the same or different) and a functional group selected from the group consisting of ketone, ketone O hydrate, thione, monothiohydrate, dithiohydrate, hemiketal, monothiohemiketal, dithiohemiketal, ketal, and dithioketal. HCO--CHCHO-H-CH-CH-CH-C-CH. For example, the polymeric reagent can comprise a first mW 2000 water-soluble polymer segment, a second water-soluble poly mer segment, and a functional group selected from the group The current invention also provides methods for making consisting of ketone, ketone hydrate, thione, monothiohy 10 polymeric reagents. Thus, in another embodiment of the drate, dithiohydrate, hemiketal, monothiohemiketal, dithio invention, a method is provided comprised of the steps of (i) hemiketal, ketal, and dithioketal, wherein each of the first and providing a precursor molecule comprised of at least one second water-soluble polymer segments are attached, either active anionic site Suitable for initiating polymerization and a through a direct covalent bond or through one or more atoms, functional group or a protected form thereof, wherein the to the functional group and further wherein when the func 15 functional group is selected from the group consisting of tional group is a ketone, ketone hydrate, hemiketal or ketal: ketone, ketone hydrate, thione, monothiohydrate, dithiohy (a) the polymeric reagent lacks an aromatic moiety; or (b) drate, hemiketal, monothiohemiketal, dithiohemiketal, ketal, each water-soluble polymer segment in the polymeric reagent and dithioketal; (ii) contacting the anionic site of the precur Sor molecule with a reactive monomer capable of polymeriZ has a weight average molecule weight of 1000 Daltons or ing, to thereby initiate polymerization of the reactive mono greater. As will be explained in greater detail below, a poly mer onto the precursor molecule; (iii) adding additional meric reagent as described herein comprising two water reactive monomers to the precursor molecule to form one or soluble polymer segments can be linear and as taking the form more polymer chains; (iv) allowing said adding to continue of the letter “V” such that each line segment of the letter until a desired length of the one or more polymer chains is comprises one single chain of atoms comprising a water reached; (v) terminating the reaction, thereby resulting in a soluble polymer segment and each chain is attached a carbon 25 polymeric reagent comprised of the functional group or pro atom of the functional group. Nonlinear forms of polymeric tected form thereof; and (vi) optionally, when the functional reagents comprising two or more water-soluble polymer seg group is in the protected form, deprotecting the functional ments are provided as well. group. In another embodiment of the invention, compositions Another method for forming polymeric reagents is pro comprising a polymeric reagent are provided. Preferably, the 30 vided comprising the steps of (i) providing a precursor mol polymeric reagent compositions are free or substantially free ecule comprised of a protected secondary alcohol or thiol and of oxidation side products. An exemplary polymeric reagent at least one anionic site suitable for initiating polymerization; composition comprises a polymeric reagent comprised of the (ii) contacting the anionic site of the precursor molecule with following structure: a reactive monomer capable of polymerizing, to thereby ini 35 tiate polymerization of the reactive monomer onto the pre cursor molecule; (iii) adding additional reactive monomers to the precursor molecule to form one or more polymer chains; i (iv) allowing said contacting to continue until a desired length HCO-CHCH-OCHCH, OCHCH-IX) -(Ew)--C-CH of the one or more polymer chains is reached; (v) terminating R3 40 the reaction, thereby resulting in an intermediate comprised of the protected secondary alcohol or thiol: (vi) deprotecting the protected secondary alcohol or thiol of the intermediate to wherein: form an unprotected secondary alcohol or thiol; (vii) oxidiz (a) is either Zero or one; ing the unprotected secondary alcohol or thiol to provide a (b) is either Zero or one: 45 polymeric reagent comprised of a ketone when the precursor molecule comprised a secondary alcohol or a thione when the X', when present, is a spacer moiety; precursor molecule comprised a secondary thiol, and (viii) EW', when present, is an electron-withdrawing group; optionally further modifying the ketone or thione to result in (Z) is from o or a positive integer, e.g., 1, 2, 3, 4, 5, 6, or 7: a functional group selected from the group consisting of each R, when present, is independently H or an organic 50 ketone, ketone hydrate, thione, monothiohydrate, dithiohy radical, for example, H or a nonaromatic-containing organic drate, hemiketal, monothiohemiketal, dithiohemiketal, ketal, radical; and dithioketal. each R, when present, is independently H or an organic In another embodiment of the invention, a method com radical, for example, H or a nonaromatic-containing organic prising the steps of (i) providing a precursor molecule com 55 prised of at least one isocyanate moiety and a functional radical; and group or a protected form thereof, wherein the functional (m) is a positive integer, e.g., from 11 to about 3000, group is selected from the group consisting of ketone, ketone wherein the composition is substantially free of oxidation hydrate, thione, monothiohydrate, dithiohydrate, hemiketal, side products. Preferably the composition also lacks f-keto monothiohemiketal, dithiohemiketal, ketal, and dithioketal; esters, 60 and (ii) contacting, under Suitable reaction conditions, the precursor molecule and the water-soluble polymer segment bearing at least one hydroxyl group to thereby form a poly O meric reagent comprising the functional group or protected form thereof, and (iii) optionally, when the functional group is HCO--CHCHO-H-CH-CH-CH-C-CH, 65 in the protected form, deprotecting the functional group. mW 2000 In another embodiment of the invention for providing poly meric reagent, a method is described comprising the steps of US 8,865,149 B2 10 (i) providing a water-soluble polymer segment having at least functional group and its hydrate in a conjugation reaction. A one available nucleophilic group; (ii) providing a precursor second equilibria representing condensation between a molecule comprised of at least one activating group and a carbinolamine and the corresponding imine is also depicted. functional group or a protected form thereof, wherein the FIG. 2 is a schematic representation illustrating the forma functional group is selected from the group consisting of 5 tion of a ketal from a polymeric reagent bearing a ketone ketone, ketone hydrate, thione, monothiohydrate, dithiohy functional group, and of a conjugate from a polymeric reagent drate, hemiketal, monothiohemiketal, dithiohemiketal, ketal, and dithioketal; (iii) contacting, under Suitable reaction con bearing ketal functional group. ditions, the precursor molecule with the water-soluble poly FIG. 3 is a schematic representation of a conjugation mer segment, thereby forming a polymeric reagent compris method according to the present invention. ing the functional group or protected form thereof, and (iv) 10 optionally, when the functional group is in the protected form, DETAILED DESCRIPTION OF THE INVENTION deprotecting the functional group. In yet another embodiment for making polymeric reagents, I. Definitions and Overview a method is provided comprising the steps of: (i) providing a water-soluble polymer segment having at least one available 15 Before describing the present invention in detail, it is to be leaving group; (ii) providing a precursor molecule comprised understood that this invention is not limited to the particular of at least one nucleophilic group and a functional group or a polymers, synthetic techniques, active agents, and the like, as protected form thereof, wherein the functional group is Such may vary. selected from the group consisting of ketone, ketone hydrate, It must be noted that, as used in this specification and the thione, monothiohydrate, dithiohydrate, hemiketal, mono claims, the singular forms “a,” “an,” and “the include plural thiohemiketal, dithiohemiketal, ketal, and dithioketal; (iii) referents unless the context clearly dictates otherwise. Thus, contacting, under Suitable reaction conditions, the precursor for example, reference to “polymeric reagent' includes a molecule with the water-soluble polymer segment, thereby single polymeric reagent as well as two or more of the same forming a polymeric reagent comprising the functional group or different polymeric reagents, reference to a "conjugate' or protected form thereof; and (iv) optionally, when the func 25 includes a single conjugate as well as two or more of the same tional group is in the protected form, deprotecting the func of different conjugates, reference to an "' includes a tional group. single excipient as well as two or more of the same or different In still another embodiment of the invention, a method for making a polymeric reagent is provided wherein the method , and the like. comprises the steps of reacting a water-soluble polymer seg In describing and claiming the present invention, the fol ment having at least one alkoxide ion or thiolate ion with a 30 lowing terminology will be used in accordance with the defi precursor molecule comprised of at least one leaving group nitions described below. and a functional group or a protected form thereof, wherein “PEG.,” “polyethylene glycol” and “poly(ethylene glycol)” the functional group is selected from the group consisting of as used herein, are meant to encompass any water-soluble ketone, ketone hydrate, thione, monothiohydrate, dithiohy poly(ethylene oxide). Typically, PEGs for use in accordance drate, hemiketal, monothiohemiketal, dithiohemiketal, ketal, 35 with the invention comprise the following structure “ O and dithioketal, thereby providing a polymeric reagent. (CH2CH2O), where (m) is 2 to 4000. As used herein, In still another embodiment of the invention, a method of PEG also includes “ CHCH O(CH2CH2O), preparing a conjugate is provided comprising the step of CH2CH2 and '-(CH2CH2O), depending upon contacting a polymeric reagent as provided herein with an whether or not the terminal oxygens have been displaced. active agent under conditions Suitable to provide a conjugate. 40 When the PEG further comprises a spacer moiety (to be In still another embodiment of the invention, pharmaceu described in greater detail below), the atoms comprising the tical preparations are provided comprising a conjugate of the spacer moiety, when covalently attached to a water-soluble invention in combination with a pharmaceutical excipient. polymer segment, do not result in the formation of an oxygen The pharmaceutical preparations encompass all types of for oxygen bond (i.e., an '-O-O or peroxide linkage). mulations and in particular those that are Suited for injection, 45 Throughout the specification and claims, it should be remem e.g., powders that can be reconstituted as well as Suspensions bered that the term “PEG' includes structures having various and solutions. terminal or “end capping groups and so forth. The term In yet another embodiment, polymer-active agent conju “PEG” also means a polymer that contains a majority, that is gates are provided. Thus, for example, the invention provides to say, greater than 50%, of —CH2CHO— repeating Sub a conjugate comprising a covalent bond between a nitrogen 50 units. With respect to specific forms, the PEG can take any atom of an active agent to a secondary carbon atom, wherein number of a variety of molecular weights, as well as struc the secondary carbon atom is attached through one or more tures or geometries such as “branched.” “linear,” “forked.” atoms to a water-soluble polymer segment and the one or “multifunctional.” and the like, to be described in greater more atoms lacks a carbonyl moiety beta to the secondary detail below. carbon. 55 The terms "end-capped' and “terminally capped' are inter In an additional embodiment of the invention, a method for changeably used herein to refer to a terminal or endpoint of a administering a conjugate is provided comprising the step of polymer having an end-capping moiety. Typically, although administering to a patient a conjugate provided herein. The not necessarily, the end-capping moiety comprises a hydroxy conjugate is often provided as part of a pharmaceutical prepa or C-2 alkoxy group, more preferably a Coalkoxy group. ration. Any approach to administer the conjugate can be used 60 and more preferably a Cls alkoxy group. Thus, examples of and the invention is not limited in this regard. It is preferred, end-capping moieties include alkoxy (e.g., methoxy, ethoxy however, that the conjugate is administered via injection. and benzyloxy), as well as aryl, heteroaryl, cyclo, heterocy clo, and the like. In addition, Saturated, unsaturated, Substi BRIEF DESCRIPTION OF THE DRAWINGS tuted and unsubstituted forms of each of the foregoing are 65 envisioned. Moreover, the end-capping group can also be a FIG. 1 is a schematic representation illustrating of the silane. The end-capping group can also advantageously com equilibria between a polymeric reagent bearing a ketone prise a detectable label. When the polymer has an end-cap US 8,865,149 B2 11 12 ping group comprising a detectable label, the amount or loca The terms “protected.” “protecting group' and “protective tion of the polymer and/or the moiety (e.g., active agent) to group' refer to the presence of a moiety (i.e., the protecting which the polymer is coupled to can be determined by using group) that prevents or blocks reaction of a particular chemi a suitable detector. Such labels include, without limitation, cally reactive functional group in a molecule under certain fluorescers, chemiluminescers, moieties used in enzyme reaction conditions. The protecting group will vary depend labeling, colorimetric (e.g., dyes), metal ions, radioactive ing upon the type of chemically reactive group being pro moieties, and the like. Suitable detectors include photom tected as well as the reaction conditions to be employed and eters, films, spectrometers, and the like. The end-capping the presence of additional reactive or protecting groups in the group can also advantageously comprise a phospholipid. molecule, if any. Protecting groups known in the art can be When the polymer has an end-capping group comprising a 10 phospholipid, unique properties (such as the ability to form found in Greene, T. W., et al., PROTECTIVE GROUPS IN ORGANIC organized structures with similarly end-capped polymers) are SYNTHESIS, 3rd ed., John Wiley & Sons, Inc., New York, N.Y. imparted to the polymer. Exemplary phospholipids include, (1999). without limitation, those selected from the class of phospho As used herein, the term “functional group' or any syn lipids called phosphatidylcholines. Specific phospholipids 15 onym thereof is meant to encompass protected forms thereof. include, without limitation, those selected from the group An “organic radical in the context describing a structure, consisting of dilauroylphosphatidylcholine, dioleylphos a structural formula, a molecule, and so forth, refers to a phatidylcholine, dipalmitoylphosphatidylcholine, dis carbon-containing moiety wherein a carbon atom provides a teroylphosphatidylcholine, behenoylphosphatidylcholine, point of attachment. Exemplary organic radicals include, arachidoylphosphatidylcholine, and lecithin. alkyl (e.g., lower alkyl). Substituted alkyl (including het "Non-naturally occurring” means a polymer that in its eroalkyl, and chain-substituted heteroalkyl), alkenyl, Substi entirety is not found in nature. A non-naturally occurring tuted alkenyl, alkynyl, Substituted alkynyl, aryl, Substituted polymer may, however, contain one or more Subunits or seg aryl, heteroaryl, substituted heteroaryl, heterocyclicyl, sub ments of Subunits that are naturally occurring, so long as the stituted heterocyclicyl, and so forth. overall polymer structure is not found in nature. 25 A“nonaromatic-containing organic radical in the context The term “water soluble' as in a “water-soluble polymer of describing a structure, a structural formula, a molecule, and segment and “water-soluble polymer is any segment or so forth, refers to any “organic radical.” as described above polymer that is soluble in water at room temperature. Typi but excluding aryl, substituted aryl, heteroaryl, substituted cally, a water-soluble polymer or segment will transmit at, heteroaryl, and alkyl or other groups that contain any aro least about 75%, more preferably at least about 95% of light, 30 matic group, e.g., aryl, Substituted aryl, heteroaryl, Substi transmitted by the same solution after filtering. On a weight tuted heteroaryl, and the like. basis, a water-soluble polymer or segment thereof will pref “Alkyl refers to a hydrocarbon chain, typically ranging erably be at least about 35% (by weight) soluble in water, from about 1 to 15 atoms in length. Such hydrocarbon chains more preferably at least about 50% (by weight) soluble in are preferably but not necessarily Saturated and may be water, still more preferably about 70% (by weight) soluble in 35 branched or straight chain, althoughtypically straight chain is water, and still more preferably about 85% (by weight) preferred. Exemplary alkyl groups include methyl, ethyl, pro soluble in water. It is most preferred, however, that the water pyl, butyl, pentyl, 1-methylbutyl, 1-ethylpropyl, 3-methyl soluble polymer or segment is about 95% (by weight) soluble pentyl, and the like. As used herein, “alkyl includes-cy in water or completely soluble in water. cloalkyl as well as cycloalkylene-containing alkyl. “Nominal average molecular weight' in the context of a 40 “Lower alkyl refers to an alkyl group containing from 1 to water-soluble, non-naturally occurring polymer of the inven 6 carbon atoms, and may be straight chain or branched, as tion such as PEG, refers to the mass average molecular exemplified by methyl, ethyl, n-butyl, i-butyl, and t-butyl. weight, of the polymer, typically determined by size exclu “Cycloalkyl refers to a saturated or unsaturated cyclic sion chromatography, light scattering techniques, or intrinsic hydrocarbon chain, including bridged, fused, or spiro cyclic velocity determination in 1,2,4-trichlorobenzene. The poly 45 compounds, preferably made up of 3 to about 12 carbon mers of the invention are typically polydisperse, possessing atoms, more preferably 3 to about 8 carbon atoms. low polydispersity values of preferably less than about 1.2, “Cycloalkylene' refers to a cycloalkyl group that is inserted more preferably less than about 1.15, still more preferably into an alkyl chain by bonding of the chain at any two carbons less than about 1.10, yet still more preferably less than about in the cyclic ring system. 1.05, and most preferably less than about 1.03. 50 “Alkoxy' refers to an —O—R group, wherein R is alkyl or "Thiol derivative in the context of a water-soluble, non Substituted alkyl, preferably Calkyl (e.g., methoxy, ethoxy, naturally occurring water Soluble polymer, means such a propyloxy, etc.). polymer having at least one terminus that is a thiol group As used herein, “alkenyl refers to a branched or (—SH), a thiolate (-S ) or a protected thiol, that is to say, unbranched hydrocarbon group of 2 to 15 atoms in length, a thiol group in its protected form. Typical thiol protecting 55 containing at least one double bond, such as ethenyl, n-pro groups include thioether, thioester, or disulfide. Exemplary penyl, isopropenyl. n-butenyl, isobutenyl, octenyl, decenyl, protecting groups for thiols can be found in Greene, T., and tetradecenyl, and the like. Wuts, Peter G.M., “PROTECTIVE GROUPS IN ORGANIC SYNTHESIS, The term “alkynyl as used herein refers to branched or Chapter 6, 3' Edition, John Wiley and Sons, Inc., New York, unbranched hydrocarbon group of 2 to 15 atoms in length, 1999. 60 containing at least one triple bond, and includes ethynyl, The terms “active' and “activated when used in conjunc propynyl, butynyl, octynyl, decynyl, and so forth. tion with a particular functional group, refers to a reactive "Aryl means one or more aromatic rings, each of 5 or 6 functional group that reacts readily with an electrophile or a core carbonatoms. Aryl includes multiple aryl rings that may nucleophile on another molecule. This is in contrast to those be fused, as in naphthyl, or unfused, as in biphenyl. Aryl rings groups that require strong catalysts or highly impractical 65 may also be fused or unfused with one or more cyclic hydro reaction conditions in order to react (i.e., a “nonreactive' or carbon, heteroaryl, or heterocyclic rings. As used herein, “inert' group). “aryl' includes heteroaryl. US 8,865,149 B2 13 14 “Heteroaryl' is an aryl group containing from one to four or pharmacologically active Substance that produces a local heteroatoms, wherein each heteroatom in the heteroaryl ring ized or systemic effect in a patient. is preferably a Sulfur, oxygen, or nitrogen atom. Heteroaryl "Pharmaceutically acceptable excipient or carrier' refers rings may also be fused with one or more cyclic hydrocarbon, to an excipient that may optionally be included in the com heterocyclic, aryl, or heteroaryl rings. positions of the invention and that causes no significant “Heterocyclicyl” or "heterocyclic' means a group of one or adverse toxicological effects to thepatient. “Pharmacologi more rings of 5-12 atoms, preferably 5-7 atoms, with or cally effective amount,” “physiologically effective amount.” without unsaturation or aromatic character and having at least and “therapeutically effective amount are used interchange one ring atom which is not a carbon. Preferred heteroatoms ably herein to mean the amount of a polymer-active agent include Sulfur, oxygen, and nitrogen. 10 conjugate that is needed to provide a desired level of active The term “substituted as in, for example, “substituted agent in the bloodstream or in the target tissue. The precise alkyl refers to a moiety (e.g., an alkyl group) Substituted amount will depend upon numerous factors, e.g., the particu with one or more non-interfering Substituents, such as, but not lar drug or therapeutic agent, the components and physical limited to: Css cycloalkyl, e.g., cyclopropyl, cyclobutyl, and characteristics of the therapeutic composition, intended the like; halo, e.g., fluoro, chloro, bromo, and iodo; cyano; 15 patient population, individual patient considerations, and the alkoxy; lower phenyl; substituted phenyl; and the like. “Sub like, and can readily be determined by one skilled in the art, stituted aryl' is aryl having one or more honinterfering groups based upon the information provided herein. as a Substituent. For Substitutions on a phenyl ring, the Sub “Multi-functional means a polymer having 3 or more stituents may be in any orientation (i.e., ortho, meta, or para). functional groups contained therein, where the functional “Substituted heteroaryl is heteroaryl having one or more groups may be the same or different. Multi-functional poly noninterfering groups as Substituents. meric reagents of the invention will typically contain from “Substituted heterocyclicyl is a heterocyclicyl group hav about 3-100 functional groups, or from 3-50 functional ing one or more side chains formed from noninterfering Sub groups, or from 3-25 functional groups, or from 3-15 func stituents. tional groups, or from 3 to 10 functional groups, or will “Noninterfering substituents' are those groups that, when 25 contain 3, 4, 5, 6, 7, 8, 9 or 10 functional groups within the present in a molecule, are typically nonreactive with other polymer backbone. functional groups contained within the molecule. A basic or acidic reactant described herein includes neu “Electrophile” refers to an ion or atom or collection of tral, charged, and any corresponding salt forms thereof. atoms, that may be ionic, having an electrophilic center, i.e., The term "patient, refers to a living organism Suffering a center that is electron seeking, capable of reacting with a 30 from or prone to a condition that can be prevented or treated nucleophile. by administration of an active agent, and includes both "Nucleophile” refers to an ion or atom or collection of humans and animals. atoms that may be ionic having a nucleophilic center, i.e., a “Optional' or “optionally’ means that the subsequently center that is seeking an electrophilic center or with an elec described circumstance may or may not occur, so that the trophile. 35 description includes instances where the circumstance occurs A “physiologically cleavable' or “hydrolyzable' or and instances where it does not. “degradable bond is a relatively weak bond that reacts with The polymeric reagents described hereincomprise a water water (i.e., is hydrolyzed) under physiological conditions. soluble polymer segment and a functional group. The func Preferred are bonds that have a half-life at pH 8 tional group is selected from the group consisting of ketone, and 25°C. of less than about 30 minutes. The tendency of a 40 ketone hydrate, thione, monothiohydrate, dithiohydrate, bond to hydrolyze in water will depend not only on the gen hemiketal, monothiohemiketal, dithiohemiketal, ketal, and eral type of linkage connecting two central atoms but also on dithioketal. The functional group can be attached to the water the Substituents attached to these central atoms. Appropriate soluble polymer segment through a through a direct covalent hydrolytically unstable or weak linkages include, but are not bond. More typically, however, the functional group is limited to, carboxylate ester, phosphate ester, anhydrides, 45 attached to the water-soluble polymer segment through one or acyloxyalkyl ether, imines, and oligonucleotide bonds. more atoms. Thus, in its most elemental form, the polymeric An "enzymatically degradable linkage” means a linkage reagents described herein all share a core structure compris that is Subject to degradation by one or more enzymes. 1ng: A“hydrolytically stable' linkage or bond refers to a chemi POLY------FG cal bond, typically a covalent bond, that is substantially stable 50 in water, that is to say, does not undergo hydrolysis under wherein POLY is a water-soluble polymer segment, FG is a physiological conditions to any appreciable extent over an functional group selected from the group consisting of extended period of time. Examples of hydrolytically stable ketone, ketone hydrate, thione, monothiohydrate, dithiohy linkages include, but are not limited to, the following: carbon drate, hemiketal, monothiohemiketal, dithiohemiketal, ketal, carbon bonds (e.g., in aliphatic chains), ethers, amides, ure 55 and dithioketal, and the dashed line represents a direct cova thanes, and the like. Generally, a hydrolytically stable linkage lent bond or one or more atoms serving to attach the func is one that exhibits a rate of hydrolysis of less than about 1-2% tional group to the water-soluble polymer segment. As will be per day under physiological conditions. Hydrolysis rates of seen in more detail below, the polymeric reagents described representative chemical bonds can be found in most standard herein can have more complex structures. chemistry textbooks. 60 Although not wishing to be bound by theory, it is believed 'Active agent, as described herein, includes any agent, that polymeric reagents bearing a ketone, ketone hydrate, drug, compound, composition of matter, or mixture that pro thione, monothiohydrate, dithiohydrate, hemiketal, mono vides some pharmacologic, often beneficial, effect that can be thiohemiketal, dithiohemiketal, ketal, or dithioketal are more demonstrated in-vivo or in vitro. This includes , selective for use in conjugating to another Substance (e.g., Supplements, peptides, nutrients, nutriceuticals, drugs, vac 65 active agent) than a polymer bearing, for example, an alde cines, antibodies, vitamins, and other beneficial agents. As hyde. One factor contributing to the increased selectivity of used herein, these terms further include any physiologically the polymeric reagents described herein is the relative stabil US 8,865,149 B2 15 16 ity associated with the functional group(s). As a result of hydrate, dithiohydrate, hemiketal, monothiohemiketal, being more stable, the functional group(s) of the polymeric dithiohemiketal, ketal, and dithioketal. Structurally, each reagent tend to only react with relatively more reactive moi functional group (“FG') is shown below. In the structures, R eties, thereby providing a greater degree of selectivity. stands for an organic radical, e.g., alkyl, and the “functional The increased stability of ketones, ketone hydrates, group carbon' is designated with an asterisk. thiones, monothiohydrates, dithiohydrates, hemiketals, monothiohemiketals, dithiohemiketals, ketals, and dithioket als compared to other functional groups such as aldehydes is O OH SH believed to be a function of two effects: electronic effects and | l | l C- -C- C- -C- steric effects. With respect to electronic effects, crowding at 10 : : the functional group effectively provides the ability of the ketone OH ione OH functional group to “share the burden any ionization or ketone monothiohydrate “excess charge' that may occur during a reaction. Thus, for hydrate example, a ketone—having two carbon atoms attached on SH OH SR either side of a carbonyl carbon can better absorb the elec 15 tron-rich environment associated with a carbonyl moiety than o f o o – o – the corresponding aldehyde, which has only one carbon. SH OR OH With respect to steric effects, nucleophilc addition to the dithiohydrate hemiketal monothiohemiketal of both aldehydes and ketones changes the carbonyl carbon from sphybridization to sphybridization. SR OR SR The presence of a second—and relatively large—carbon atom associated with a ketone, however, slows the approach - f f of the relatively bulky active agent to the sp’ hybridized SH OR SR carbonyl carbon and sterically crowds the forming sphybrid dithiohemiketal ketal dithioketal ized carbon much more than the relatively small hydrogen 25 associated with an aldehyde. As a consequence, the combi Although not shown, a carbon atom must be attached on each nation of both electronic and steric effects increases the acti side of the functional group carbon in order to provide the Vation energy of ketones and related functional groups by named functional group. decreasing their starting energy state (i.e., by providing a Advantageously, the described functional groups are often stabilized carbonyl carbon, an electronic effect) and raising 30 easily transformed into each other. Thus, for example, some the energy of the transition state (i.e., the change from sp° to or all of a ketone can be transformed into a ketone hydrate by sphybridization, a steric effect). Thus, ketones and related the addition of water. Moreover, in the presence of water, a functional groups are relatively more stable and hence less ketone generally exists in an equilibrium with the correspond reactive. ing hydrate. This is especially true with ketones having a Surprisingly, however, it has been found that the increased 35 proximate electron-withdrawing group. Because of equilib stability of ketone-, ketone hydrate-, thione-, monothiohy rium, as the amount of water in a ketone-containing system is drate-, dithiohydrate-, hemiketal-, monothiohemiketal-, decreased, the amount of the ketone hydrate species dithiohemiketal-, ketal-, or dithioketal-bearing polymeric decreases. Thus, by controlling the amount of water in the reagents is advantageous. For example, an active agent may system, it is possible to influence the amount (or ratio) of the have several amine groups, each having its own reactivity 40 two forms (e.g., ketone and ketone hydrate). Other factors, toward a carbonyl moiety present on a polymeric reagent. By Such as the presence or absence of electron-withdrawing and providing a relatively less reactive ketone-bearing polymeric electron-donating groups and proximal steric hindrance, also reagent, conjugation will occur more preferentially between influence the amount of and ratio between the two forms. the least sterically hindered and most reactive amine(s) on the Thione and monothiohydrate forms can also be provided in a active agent and the carbonyl moiety of the ketone, thereby 45 similar fashion. reducing the number of different conjugate forms. Usefully, conjugation reactions employing the presently Another advantage of the polymeric reagents described described polymeric reagents can often proceed without herein is that the functional group can be between two poly regard to controlling the specific Solvation status of the func mer chains (which may be linear), thereby providing the tional group. For example, it makes no difference whether the ability to attach two polymer chains through a single atom 50 predominating species of the polymeric reagent is a ketone or that is directly attached to an active agent, Surface, or other a ketone hydrate; using one, the other, or both under conju moiety. Previously described branched polymeric reagents gation conditions will result in the formation of polymer comprise a spacer moiety having a reactive group (e.g., alde active agent conjugates. While not wishing to be bound by hyde) for conjugation and separate and distinct branching theory, ketone hydrates—in equilibrium with the correspond atom that serves to link two polymer chains. In this way, the 55 ing ketone—can be used because as the ketone species is water-soluble polymer segments can be brought relatively being depleted during the conjugation reaction with the active closer to the active agent, thereby decreasing or eliminating agent, the equilibrium causes additional ketone hydrate spe entirely small “pendent groups left behind on an active agent cies to be transformed into the corresponding ketone. The when a water-soluble polymer segment is cleaved from the series of reactions is depicted in FIG.1. As shown therein, two conjugate. 60 equilibria reactions are involved: the equilibria between the ketone and ketone hydrate in a polymeric reagent and the II. Features of the Polymeric Reagents condensation processes involving an amine group on an active agent to form the carbinolamine and corresponding A. The Functional Group (“FG') imine. Thus, while conjugation may proceed via the ketone, As previously stated, the polymeric reagents of the inven 65 use of the corresponding ketone hydrate form will also result tion are associated with a functional group selected from the in formation of the desired conjugate. Again, while not wish group consisting of ketone, ketone hydrate, thione, monothio ing to be bound by theory, it is believed that increased stability US 8,865,149 B2 17 18 of the hydrate form (as compared to the corresponding ketone ene glycol) (“PEG') or a derivative thereof. It should be form) corresponds to increased reactivity of the ketone form understood, however, that related polymers are also suited for to conjugation to an active agent. use in the practice of this invention and that the use of the term In addition, ketones can be converted into hemiketals “PEG” or “poly(ethylene glycol) is intended to be inclusive through the addition of an alcohol. Again, as the amount of and not exclusive in this respect. Consequently, the term alcohol to a ketone-containing system is decreased, the “PEG' includes poly(ethylene glycol) in any of its linear, amount of hemiketal species decreases. In this way, in equi branched or multi-arm forms, including alkoxy PEG, bifunc libria reactions similar to those discussed above, it is possible tional PEG, forked PEG, branched PEG, pendant PEG, or to provide a hemiketal by adding alcohol to a ketone-contain PEG with degradable linkages therein, to be more fully ing system and providing a ketone by removing alcohol from 10 described below. a hemiketal-containing system. Moreover, in the presence of In one form useful in the present invention, free or non an acid catalyst, additional quantities of alcohol will trans bound PEG is a linear polymer terminated at each end with form a hemiketal into a ketal. In addition, ketones can be hydroxyl groups: directly transformed into ketals by the addition of a diol, e.g., HO CHCH-OH, in the presence of an acid catalyst. 15 HO CHCH-O-(CH2CH2O). CHCH-OH Advantageously, these and other ketals often serve as ketone (m') typically ranges from Zero to about 4,000. protecting groups. The above polymer, alpha-, omega-dihydroxylpoly(ethyl FIG. 2 schematically depicts the formation of a ketal from ene glycol), can be represented in brief form as HO-PEG-OH a polymeric reagent bearing a ketone functional group, and of where it is understood that the -PEG- symbol can represent a conjugate from a polymeric reagent bearing a ketal func the following structural unit: tional group. In FIG. 2, a polymeric reagent bearing a ketone functional group can be initially converted into a reactive hemiketal intermediate, and then into the ketal (in this Figure, where (m') is as defined as above. a dimethyl ketal). FIG. 2 also illustrates, through a series of Another type of PEG useful in the present invention is equilibria, how ketals can be utilized directly in conjugation 25 methoxy-PEG-OH, or mPEG in brief, in which one terminus reactions with an amine-containing active agent. is the relatively inert methoxy group, while the other terminus In a similar way, thiones can be transformed into dithiohy is a hydroxyl group. The structure of mPEG is given below. drates (sometimes referred to as dithiosolvates) by Solvating the thione in a Sulfur-containing (e.g., hydrogen Sulfide) envi ronment. Thiones can also be transformed into dithiohe 30 where (m') is as described above. miketals in the presence of thiols, and into dithioketals in the Multi-armed or branched PEG molecules, such as those presence of excess thiol. Inaway analgous to ketones, thiones described in U.S. Pat. No. 5,932,462, can also be used as the can be directly transformed into dithioketals by the addition PEG polymer. For example, PEG can have the structure: of a dithiol, e.g., HS CHCH SH. Dithioketals often serve as thione protecting groups as well. 35 Although exemplary methods for transforming a given polya-P functional group into another functional group have been provided, the polymeric reagents are not limited in the man ner in which the functional group is formed. Those of ordi R--poly-Q nary skill in the art know of other approaches for providing 40 the functional groups described herein. Moreover, reference to any given functional group encompasses both the func wherein: tional group per se, as well as to protected forms of the poly, and poly, are PEG backbones (either the same or functional group. different). Such as methoxy poly(ethylene glycol); B. The Water-Soluble Polymer Segment (“POLY” e.g., 45 R" is a nonreactive moiety, such as H. methyl or a PEG “POLY', “POLY2 etc.) backbone; and The polymeric reagents of the invention also comprise at P and Q are nonreactive linkages. In a preferred embodi least one water-soluble polymer segment. Water-soluble ment, the branched PEG polymer is methoxy poly(ethylene polymer segments that are nonpeptidic and water-soluble, glycol) disubstituted lysine. with from 2 to about 300 termini, are particularly useful in the 50 These polymers may be linear, or may be in any of the invention. Examples of Suitable water-soluble polymer seg above-described forms (e.g., branched, forked, and the like). ments include, but are not limited to, poly(alkylene glycols), In addition, the PEG can comprise a forked PEG. An such as poly(ethylene glycol) (“PEG'), copolymers of ethyl example of a forked PEG is represented by the following ene glycol and propylene glycol having water-solubility, poly Structure: (olefinic alcohol), poly(vinylpyrrolidone), poly(hydroxy 55 alkylmethacrylamide), poly(hydroxyalkylmethacrylate), poly(saccharides), poly(C.-hydroxy acid), poly(vinyl alco hol), polyphosphaZene, polyoxazoline, poly(N-acryloylmor PEG-X-C-H pholine), such as described in U.S. Pat. No. 5,629,384, and copolymers, terpolymers, and mixtures thereof. For purposes 60 of the present invention, poly(propylene glycol) has insuffi cient water-solubility to act as a water-soluble polymer seg wherein: X is a spacer moiety and each Z is an activated ment. Consequently, it is preferred that water-soluble poly terminal group linked to CH by a chain of atoms of defined mer segments described herein do not contain propylene length. International Application No. PCT/US99/05333, dis OOCS. 65 closes various forked PEG structures capable of use in the It is preferred, however, that the water-soluble polymer present invention. The chain of atoms linking the Z functional segment is preferably, although not necessarily, a poly(ethyl groups to the branching carbon atom serve as a tethering US 8,865,149 B2 19 20 group and may comprise, for example, alkyl chains, ether from about 2,000 Daltons to about 25,000 Daltons; and from chains, ester chains, amide chains and combinations thereof. about 5,000 Daltons to about 20,000 Daltons. The PEG polymer may comprise a pendant PEG molecule Exemplary nominal average molecular weights for the having reactive groups, such as carboxyl, covalently attached water-soluble polymer segment include about 100 Daltons, along the length of the PEG rather than at the end of the PEG 5 about 200 Daltons, about 300 Daltons, about 400 Daltons, chain. The pendant reactive groups can be attached to the about 500 Daltons, about 600 Daltons, about 700 Daltons, PEG directly or through a spacer moiety, Such as an alkylene about 750 Daltons, about 800 Daltons, about 900 Daltons, group. about 1,000 Daltons, about 2,000 Daltons, about 2,200 Dal In addition to the above-described forms of PEG, the poly tons, about 2,500 Daltons, about 3,000 Daltons, about 4,000 mer can also be prepared with one or more weak or degrad 10 able linkages in the polymer, including any of the above Daltons, about 4,400 Daltons, about 5,000 Daltons, about described polymers. For example, PEG can be prepared with 6,000 Daltons, about 7,000 Daltons, about 7,500 Daltons, ester linkages in the polymer that are subject to hydrolysis. As about 8,000 Daltons, about 9,000 Daltons, about 10,000 Dal shown below, this hydrolysis results in cleavage of the poly tons, about 11,000 Daltons, about 12,000 Daltons, about mer into fragments of lower molecular weight: 15 13,000 Daltons, about 14,000 Daltons, about 15,000 Daltons, about 20,000 Daltons, about 22,500 Daltons, about 25,000 Daltons, about 30,000 Daltons, about 40,000 Daltons, about Other hydrolytically degradable linkages, useful as a 50,000 Daltons, about 60,000 Daltons, and about 75,000 degradable linkage within a polymer backbone, include: car Daltons. bonate linkages; imine linkages resulting, for example, from With respect to PEG, wherein a structure corresponding to reaction of an amine and an aldehyde (see, e.g., Ouchi et al. * CHCH-O-(CHCHO), CHCH can be pro (1997) Polymer Preprints 38(1):582-3): phosphate ester link vided, preferred values for (m) include: from about 3 to about ages formed, for example, by reacting an alcohol with a 3,000; from about 10 to about 3,000; from about 15 to about phosphate group; hydrazone linkages which are typically 3,000; from about 20 to about 3,000; from about 25 to about formed by reaction of a hydrazide and an aldehyde; acetal 25 3,000; from about 30 to about 3,000; from about 40 to about linkages that are typically formed by reaction between an 3,000; from about 50 to about 3,000; from about 55 to about aldehyde and an alcohol; orthoester linkages that are, for 3,000; from about 75 to about 3,000; from about 100 to about example, formed by reaction between a formate and an alco 3,000; and from about 225 to about 3,000. hol; amide linkages formed by an amine group, e.g., at an end As used herein, the term “water-soluble polymer segment' of a polymer Such as PEG, and a carboxyl group of another 30 includes those water-soluble polymer segments that are bio PEG chain; urethane linkages formed from reaction of, e.g., a PEG with a terminal isocyanate group and a PEG alcohol: compatible and nonimmunogenic and specifically excludes peptide linkages formed by an amine group, e.g., at an end of any water-soluble polymer segments that are not biocompat a polymer Such as PEG, and a carboxyl group of a peptide; ible and nonimmunogenic. With respect to biocompatibility, and oligonucleotide linkages formed by, for example, a phos 35 a substance is considered biocompatible if the beneficial phoramidite group, e.g., at the end of a polymer, and a 5' effects associated with use of the substance alone or with hydroxyl group of an oligonucleotide. another Substance (e.g., active agent) in connection with liv It is understood by those of ordinary skill in the art that the ing tissues (e.g., administration to a patient) outweighs any term poly(ethylene glycol) or PEG represents or includes all deleterious effects as evaluated by a clinician, e.g., a physi the above forms of PEG. 40 cian. With respect to non-immunogenicity, a Substance is Although the nominal average molecular weight of the considered nonimmunogenic if the intended use of the Sub water-soluble polymer segment can vary, the nominal aver stance in Vivo does not produce an undesired immune age molecular weight will satisfy one or more of the follow response (e.g., the formation of antibodies) or, if an immune ing values: greater than 100 Daltons; greater than 400 Dal response is produced, that such a response is not deemed tons; greater than 500 Daltons, greater than 750 Daltons; 45 clinically significant or important as evaluated by a clinician. greater than 900 Daltons; greater than 1,000 Daltons, greater It is particularly preferred that the water-soluble polymer than 1,400 Daltons; greater than 1,500 Daltons, greater than segments described herein as well as conjugates are biocom 1,900 Daltons; greater than 2,000 Daltons; greater than 2,200 patible and nonimmunogenic. As used herein, the term Daltons; greater than 2,500 Daltons; greater than 3,000 Dal “water-soluble polymer segment also excludes those water tons; greater than 4,000 Daltons; greater than 4,900 Daltons; 50 soluble polymer segments made of monomers comprising greater than 5,000 Daltons; greater than 6,000 Daltons; one or more ketone moieties. greater than 7,000 Daltons; greater than 7,500 Daltons, Those of ordinary skill in the art will recognize that the greater than 9,000 Daltons; greater than 10,000 Daltons; foregoing discussion concerning Substantially water-soluble greater than 11,000 Daltons; greater than greater than 14,000 polymer segments is by no means exhaustive and is merely Daltons, greater than 15,000 Daltons; greater than 16,000 55 illustrative, and that all polymeric materials having the quali Daltons; 19,000 Daltons; greater than 20,000 Daltons; greater ties described above are contemplated. As used herein, the than 21,000 Daltons; greater than 22,000 Daltons, greater term “polymeric reagent generally refers to an entire mol than 25,000 Daltons; and greater than 30,000 Daltons. It is ecule, which can comprise a water-soluble polymer segment understood that the maximum limit of molecular weight for and a functional group. The term “water-soluble polymer any given water-soluble polymer segment useful herein is less 60 segment is generally reserved for use in discussing one than about 300,000 Daltons. portion of a larger molecular structure such as a polymeric The nominal average molecular weight of the water reagent, precursor molecule, conjugate, and so forth. soluble polymer segment can also be expressed as being a C. Attachment Through One or More Atoms value within a range of nominal average molecular weights. Each portion (e.g., functional group, active agent, water Exemplary ranges include: from about 100 Daltons to about 65 soluble polymer segment, and so forth) of the polymeric 100,000 Daltons; from about 500 Daltons to about 80,000 reagent and other structures described herein can be directly Daltons; from about 1,000 Daltons to about 50,000 Daltons; attached to each other via a direct covalent bond. More typi US 8,865,149 B2 21 22 cally, however, each portion is attached through one or more Another exemplary form includes defining the carbon atoms serving to tether each portion together into a unified chain as having an organic radical (e.g., a lower alkyl) whole. attached to the carbon in the chain C. to the functional group Preferred atoms through which the various portions of the carbon. Advantageously, the presence of Such an organic polymeric reagents and other structures described herein group in this position increases the selectivity of the poly include a chain of atoms made of carbon, nitrogen, oxygen, meric reagent. While not wishing to be bound by theory, it is and/or Sulfur atoms. Attached to this chain of atoms, can be thought that the steric effects of the organic radical further one or more other atoms such as carbon, nitrogen, oxygen, increases the activation energy necessary for the functional Sulfur, and hydrogen. The chain can be short and comprise as group to react. An exemplary carbon chain (attached to a few as a chain of two to five atoms. Longer chains, for 10 ketonehaving a terminal methyl group) showing the C. carbon example, a chain of atoms often, fifteen, or more in length are and a B carbon is provided below, wherein a methyl group is also contemplated. Thus, a great number of different combi attached to the C. carbon and all other substituents in the nations of atoms are possible for attaching portions of the carbon chain are hydrogen. molecules described herein. Typically, however, three differ 15 ent groups of atoms are associated with linking the various portions of the polymeric reagents and other molecules H th described herein: a carbon chain; an electron withdrawing -C-C-C-CH, group; and a spacer moiety. Each will be discussed in turn. (i) The Carbon Chain, (e.g., H. H. Preferably, when an organic radical is attached to a carbon R2 R4 R8 C. to the functional group carbon, the atom B to the functional group carbon is or is part of an electron-withdrawing group. C s C , and C ) 25 Thus, exemplary arrangements are provided below wherein k an oxygen atom is used as the electron-withdrawing group Z y X.

Optionally, one or more carbon chains is included in the H O CH. H. CHO polymeric reagent (as well as the corresponding conjugates, 30 precursor molecules to form the polymeric reagents, and so -o-,-,-,-,-, -o---ch. forth). Typically, but not necessarily, the carbon chain is located immediately adjacent to the functional group of the H O CH polymeric reagent. Notwithstanding the absence of a carbon chain, however, a carbon atom (from, for example, a spacer 35 -o-,---o-, a moiety, organic radical, electron-withdraing group, and so forth) must be attached on each side of the functional group CH3 O CH3 carbon. When present, the carbon chain can have any number of -o-,---0- carbon atoms So long as the overall polymeric reagent 40 remains water soluble. Typically, however, there will be 1, 2, 3, 4, 5, 6, or 7 carbons in the carbon chain. Preferably, the Although the above structures use oxygen as an electron polymeric reagent (as well as the corresponding precursor withdrawing group, electron-withdrawing groups other than molecule, conjugate, and so forth) will lack a hydrocarbon oxygen (e.g., carbonyl) can be used. chain of 8 carbons or greater. Thus, with respect to the various 45 When the carbon C. to the functional group carbon bears an carbon chains structurally defined herein, each of (Z), (y), and organic radical (e.g., methyl), the resulting polymeric reagent (X) can independently be Zero, a positive integer, such as 1, 2, may comprise a chiral center. Specific chirality, however, is 3, 4, 5, 6, and 7, a positive integer from 1 to 7, a positive not explicitly illustrated herein with respect to any compound integer from 2 to 7, and so forth. or structure comprising one or more chiral centers. Thus, the Due to its tetravalent character, each carbon in the carbon 50 present discussion encompasses both the isomerically pure chain has two groups attached to it. As structurally defined forms of any optically active compound described herein, as herein, these groups have been designated as R. R. R. R. well as enantiomeric mixtures, including a racemic mixture, Rand R. Each of these groups, when present, is indepen thereof. dently either hydrogen oran organic radical. A preferred form Optionally, when two or more carbons are in the carbon includes each occurrence of R. R. R. R. RandR, when 55 chain, the carbon chain can include a carbon-carbon double present, as hydrogen. bond. Such unsaturated groups can offer additional possibili Exemplary classes of moieties from which any of R. R. ties for reactions, such as the ability of the polymeric reagents R. R. RandR can independently be chosen from include to provide conjugate addition of a nucleophile to the B carbon H, alkyl, substituted alkyl, alkenyl, substituted alkenyl, alky of an O-3 unsaturated ketone, as shown below. nyl, Substituted alkynyl, aryl, and Substituted aryl. Although, 60 as further explained below, aromatic moieties are preferably absent. Specific moieties from which any of R,R,R,R,R and R can independently be chosen from include H. methyl, ethyl, n-propyl, isopropyl. n-butyl, sec-butyl, isobutyl, tert M HN Active Agent Po yme ic Reage C=C butyl, and vinyl. In addition, and two of R. R. R. R. Rand 65 (C agen V R can be joined to form a cyclic ring system, which may bridge certain carbon chain atoms. US 8,865,149 B2 23 24 -continued of -O-, -NH , —C(O)NH-, -NHC(O) , OC O (O) , OC(O) , NH OC(O) , OC(O) NH-, H. H. H. C(O)— —C(S)— and —C(OR)H , wherein OR is an / alkoxy or hydroxy substituent. A preferred electron-with S-CV 5 drawing group is —C(O)—, either alone or as part of amide HN or carbamate. A particularly preferred electron-withdrawing group is —O—. An O-B unsaturated ketone is often called an enone. Thus, (iii) The Spacer Moiety (“X” e.g., “X'.” “X” “X” “X”, while one or more carbon-carbon double bonds can be “X” etc.) 10 Optionally, the polymeric reagents provided herein (as included at any position of the carbon chain, it is preferred well as the corresponding precursor molecules, conjugates, that when a double bond is present, the double bond is located and so forth) include a spacer moiety. Exemplary moieties between the terminal C. and B carbons relative to the func suited for any spacer moiety designated as X and X’ are tional group carbon (e.g., the carbonyl carbon). As will be those independently selected from the group consisting of discussed below, however, enones may preferably be absent 15 from the polymeric reagents described herein. (ii) The Electron Withdrawing Groups (“EW, e.g., “EW'.” “EW.2. “EW’, etc.) An electron-withdrawing group is optionally located within the polymeric reagent (or corresponding precursor molecule, conjugate, and so forth) and can comprise the atoms that serve to link various portions of a larger molecule together. An electron-withdrawing group is any atom or group of atoms including electronegative atom(s) and in some cases, having resonance interactions among some of the 25 atoms in the electron-withdrawing group that create an enhanced electron-withdrawing effect. Electrons are attracted to the electronegative atom(s) of the electron-with drawing group, thereby influencing the chemistry of a nearby functional group. The presence of an electron-withdrawing 30 group adjacent (i.e., “C.”) to or within two to six atoms distant CH-CH CH-CH , —CH2—C(O)—NH CH from the functional group carbon effectively destabilizes this CH-CH , —CH2—CH2—C(O)—NH-CH CH . carbon by withdrawing electrons, thereby increasing the CH, CH, CH, C(O) NH-CH , CH, overall reactivity of the functional group. Thus, for example, CH-CH C(O) NH-CH CH , —CH2—CH the presence of an electron-withdrawing group near a ketone 35 group will increase its reactivity toward amines in a reductive amination reaction and other reactions wherein nucleophilic CH2—, C(O)—O—CH2—CH2—, NH C(O) addition takes place. CH , —CH NH CO)—CH2—, —CH2—CH It is preferred, however, that the presence and/or location of NH C(O)-CH , NH C(O) CH-CH , the electron-withdrawing group does not render the func 40 CH NH C(O)—CH, CH, , CH, CH, tional group overly reactive, thereby frustrating the ability to NH C(O) CH-CH , C(O) NH-CH , provide relative selectivity during, for example, conjugation C(O) NH CH, CH, , —O C(O) NH-CH , reactions. One of ordinary skill in the art can determine O C(O) NH-CH CH , O C(O) NH whether any given electron-withdrawing group as well as CH, CH, CH , —NH-CH —NH CH whether the location of the electron-withdrawing group ren 45 ders the functional group overly reactive. For example, con jugation techniques discussed herein can be performed using different polymeric reagents, each having a different elec tron-withdrawing group and/or an electron-withdrawing group at different distances from the functional group carbon. 50 Those polymeric reagents providing desired reactivities as evidence through, for example, single conjugation to a pro tein, are not overly reactive. Any electron-withdrawing group can be included and the invention is not limited in this regard. Generically, electron 55 withdrawing groups include —O— —NH —NHC(O)—, C(O)NH-, - OC(O)— —OC(O) , OC(O) NH-, NH OC(O)— —C(O)— —C(S)—and —C(OR)H , moieties suited for any spacer moiety designated as X, X. wherein OR is an alkoxy or hydroxyl substituent. With and X include those independently selected from the group respect to the structures then, electron-withdrawing groups 60 consisting of —O— —S—, —C(O)— —O—C(O)—, designated as “EW' are preferably selected from the group C(O) NH , NH C(O) NH , NH C(O) consisting of O— —NH —NHC(O)— —C(O)NH . O—, C(S)—, —CH2—, CH2—CH2—, —CH2 OC(O) , OC(O) , —OC(O) NH-, -NH OC CH-CH . —CH2—CH2—CH2—CH2—, (O)— —C(O)— —C(S)—, and —C(OR)H , wherein OR O—CH2—, —CH2—O— —O—CH2—CH2, —CH2 is an alkoxy or hydroxyl substituent, while electron-with- 65 O—CH2—, CH2—CH2—O , O—CH2—CH drawing groups designated as “EW' and “EW are each CH2—, —CH2—O—CH2—CH2—, —CH2—CH2—O preferably independently selected from the group consisting CH2—, —CH2—CH2—CH2—O— —O—CH2—CH2 US 8,865,149 B2 26

CH-CH NH C(O) , —CH C(O) NH-CH structure. The presence of an aromatic structure is undesired because Such structures decrease water solubility and can result in harmful effects in vivo. For the same reasons, the 10 polymeric reagents also preferably lack halogen atoms (e.g., F, Cl, Br and I) in their structures. The presence of either halogen atoms or aromatic moieties can be warranted in cer tain circumstances, however, such as in a method for making a polymeric reagent wherein protection groups include aro 15 matic or halogen atoms. In addition, ionized forms of halo gens can also be useful to form salts, which are not typically harmful in moderate amounts. As used herein, a polymeric reagent lacking a halogen atom does not include instances wherein the polymeric reagent (or conjugate formed there from) is in a salt form having a halogen counterion. In addition, the polymeric reagents preferably lack a cyclic dienone. As used herein, a cyclic dienone is a ketone wherein the carbonyl carbon is part of a cyclic structure and the C.B carbons on either side of the carbonyl carbon are have a 25 double bond. Schematically, the core structure of cyclic dienones can be generically represented as:

30

35 Exemplary cyclic dienones lacking in the polymeric reagents In any of the above, a simple cycloalkylenegroup, e.g., 1.3- include the following: or 1.4-cyclohexylene, may replace any two, three, or four carbon alkylene group. 40 For purposes of the present disclosure, however, a series of atoms is not a spacer moiety when the series of atoms is and immediately adjacent to a water-soluble polymer segment and the series of atoms is but another monomer, such that the 45 proposed spacer moiety would represent a mere extension of the polymer chain. For example, given the partial structure “POLY-X-” and POLY is defined as “CHO(CHCH, O), wherein (m) is 2 to 4000 and X is defined as a spacer 50 HO OH. moiety, the spacer moiety Cannot be defined as “—CH2CH2O since such a definition would merely rep resent an extension of the polymer. In Sucha case, however, an acceptable spacer moiety could be defined as “ CH 55 It is believed that the presence of such doubly C.B-unsatur CH-.” ated structures in a polymeric reagent may result in conjugate D. Other Features addition of a nucleophile. This approach to conjugation may In addition to those features already described, the poly not, however, have the same selectivity as, for example, meric reagents (and corresponding precursor molecules and reductive amination through a ketone. Consequently, the conjugates) can have other optional features. 60 polymeric reagents preferably lack cyclic dienones. More over, it can also be the case that the polymeric reagents also For example, the entire polymeric reagent (as well as the lack an enone (i.e., an O.B-unsaturated ketone). When, how organic radicals present in the polymeric reagent) preferably ever, an alternative conjugation approach is desired, an enone lacks an aromatic moiety. As used herein, an entity lacks an can be present. aromatic moiety when the entity is completely free of a con 65 In addition, the polymeric reagents also preferably lack jugated system of TL electrons that closes upon itself. Typical other reactive groups that could impede the selectivity. For of aromatic moieties are those that comprise a benzene, example, the polymeric reagents preferably lack a terminal US 8,865,149 B2 27 28 oxylamine group (i.e., —ONH2). In addition, the polymeric and further wherein the reagents also preferably lack a terminal hydrazine group (i.e., NHNH). In some cases, the polymeric reagents of the invention are R2 not in certain forms. Thus, for example, the polymeric 5 reagents are not in the following forms: C R3 Z

10 moiety optionally contains one or more double bonds, with HCO-CHCHO-H-C-CH-C-CH, the proviso that the polymeric reagent is neither mw 5000

O O HCO-CHCHO-H-CH-C-CH3, 15 mW 5000 HO-(CH2CH2O) son1n N --- O O O H HO-(CH2CH2O) so --- O N-1\ s H HCO-(CH2CH2O)-CH-CH-CH-C-CH. O mw = 2000 HCO-(CH2CH2O)-CH-CH-CH-C-CH, mw = 2000 An exemplary polymeric reagent having a linear arrange ment and a ketone has the following structure: 25 and ketal derivatives of any of the foregoing. It will be under stood that “mw' refers to the weight average molecular (Formula Ia) weight of the ethylene oxide moiety, and that a range such as R2 O “1-50 provided adjacent to a monomer indicates that the moiety encompasses the monomer repeating from 1 to 50 30 R7-O-POLY1-(X)-(EW) C-R1 times, inclusive. E. Structurally Defined Polymeric Reagents Z As will be described in further detail below, the polymeric reagents of the invention can be provided in a number of wherein R. POLY', X', EW', R. R. R. (a), (b), and (z) are forms. 35 as previously defined with respect to Formula I. In some instances, the polymeric reagent is linear in form The corresponding ketal has the following structure: and comprises a single water-soluble polymer segment and a single functional group. An exemplary polymeric reagent of (Formula Ib) this form comprises the following structure: 40 R2 OR 10 (Formula I) R7-O-POLY-CXI)-(EW)7 C-R1 R2 R3 b. Z FG-RI 45 wherein R7, POLY', X', EW, R. R. (a), (b), (z) and R' are Z as previously defined with respect to Formula I, and further wherein R' is a organic radical (preferably nonaromatic), wherein: R'' is an organic radical (preferably nonaromatic), or R'' and POLY is a water-soluble polymer segment having a ter 50 R' combine to form a cyclic ketal (preferably nonaromatic, minus defined as —O R': e.g., an alkylene Such as an ethylene or Substituted ethylene). (a) is either Zero or one; An exemplary polymeric reagent having a linear arrange (b) is either Zero or one: ment and a ketone hydrate has the following structure: X', when present, is a spacer moiety; EW', when present, is an electron-withdrawing group; 55 (Z) is an integer from 2 to 7: (Formula Ic) each occurrence of R is independently H or a nonaro | pi matic-containing organic radical; R7-O-POLY-CX)-(EW)--C-HC-R1 | | | each occurrence of R is independently H or a nonaro 60 R3 OH matic-containing organic radical; Z R’ is H or a nonaromatic-containing organic radical; FG is a functional group selected from ketone, ketone wherein R. POLY', X', EW, R. R. R', (a), (b), and (z) are hydrate, thione, monothiohydrate, dithiohydrate, hemiketal, as previously defined with respect to Formula I. monothiohemiketal, dithiohemiketal, ketal, and dithioketal; 65 More specific (although nonlimiting) examples polymeric and reagents of this form include those selected from the group R" is a nonaromatic-containing organic radical; consisting of:

US 8,865,149 B2 31 32 Another example of a linear polymeric reagent of the invention is a polymeric reagent comprised of a carbon-con taining functional group and a water-soluble polymer seg ment, wherein the carbon of the carbon-containing functional group is part of a cyclic structure and further wherein the cyclic structure is attached to the water-soluble polymer seg ment through either a direct covalent bond or through a spacer moiety, wherein the functional group is selected from the and cyclooctyl, e.g., group consisting of ketone, ketone hydrate, thione, monothio hydrate, dithiohydrate, hemiketal, monothiohemiketal, 10 dithiohemiketal, ketal, and dithioketal. With respect to the cyclic structure, any nonaromatic ring can be used and the invention is not limited in this regard. Exemplary cyclic structures include: piperidinyl, e.g., 15

oxa and azaforms thereof and dioxa and diaza forms of any of the foregoing. o N C; Preferably, when one or more heteroatoms are present in the cyclic structure, at least one heteroatom is situated in the alpha or beta position of the functional group carbon. Exem cyclohexyl, e.g., plary heteroatom-containing cyclic structures wherein a het eroatom is situated at a beta position include: Oxacyclohexyl, 25 C.9. -OC; 30 cyclopentyl, e.g., azacyclopentyl, e.g., 35 bicyclo2.2.1]heptanyl, e.g., U/ 40 oXabicyclo[2.2.1]heptanyl, e.g., zf 45

4): 50 and dioxacyclooctyl, e.g., and

C 55 bicyclo[2.2.1]hexanyl, bicyclo[3.2.1]octanyl, bicyclo[3.3.1 nonanyl, cyclobutyl, e.g., O. 60 Although any carbon in the cyclic structure can be the functional group carbon, exemplary placements of the func l tional group carbon have been designated with an asterisk. 65 Diaza forms of cyclic structures, useful in reacting with polymeric active esters to form branched products, include cylcoheptyl, e.g., the following: US 8,865,149 B2 33 34 each occurrence of R, when present, is independently Hor NH an organic radical; N-7) each occurrence of R, when present, is independently Hor NH2 an organic radical; and NH2 NH2 5 R’ is Horan organic radical. T as l The corresponding ketal derivative has the following struc ture: H C -NH2 NH ANZ)1 10 (Formula IIa) NH2 R2 HC-NH OR 10 R7-O-POLY-(X)-(EW)--C-N OR 11 15 3 NH2 Z HN HC-NH wherein R7, POLY', X', EW', R. R. (a), (b), and (z) are as NH2 previously defined with respect to Formula II, and further HC-NH2 20 wherein R" is a organic radical (preferably nonaromatic), 2 R'' is an organic radical (preferably nonaromatic), or R'' and R' combine to form a cyclic ketal (preferably nonaromatic, HC-NH e.g., an alkylene Such as an ethylene or Substituted ethylene). HC-NH OH 2 2 An exemplary polymeric reagent having a linear arrange 25 ment and a ketone hydrate has the following structure CH HC 3 NH NH2 - ? (Formula IIb) H NH2 and R2 HC 30 OH V NH R7-O-POLY -(X)-(X)-(EW)--C OX CH-NH. HC 2 2 R3 Z OH

35 wherein R7, POLY', X', EW', R. R. (a), (b), and (z) are as previously defined with respect to Formula II. When a cyclic structure containing a functional group is NH2 present in the polymeric reagent, it is particularly preferred 40 that the cyclic structure is attached directly to an electron Again, any carbon in the cyclic structure can be the functional withdrawing or a spacer moiety. Thus, for example, with group carbon, wherein the functional group is selected from respect to each of Formulas II, IIa, and IIb, it is preferred that the group consisting of ketone, ketone hydrate, thione, mono- (z) is zero. Furthermore, it is particularly preferred that the thiohydrate, dithiohydrate, hemiketal, monothiohemiketal, electron withdrawing group (EW) is carbonyl. Thus, pre dithiohemiketal, ketal, and dithioketal. ferred polymeric reagents taking this form are encompassed Examples of polymeric reagents having a heteroatom in a by the following structures cyclic structure comprising a ketone are shown in Formula II. The cyclic structure in Formula II comprises a piperidonyl cyclic moiety. 50 r–orov-x--O- (Formula II) OR 10

55 R7-O-POLY1-(XI), (EW)--C-Ni O r-orso-vi-'-OXOR 11 O OH R3 Z R-O-POLY-CX)-(X)-C-N OH wherein: 60 POLY is a water-soluble polymer segment having a ter minus defined as —O R: wherein (a), R7, POLY', and X for each of Formulas IIc, IId, (a) is either Zero or one; and IIe are as previously defined with respect to Formula II, (b) is either Zero or one: and R'' and R' for Formula IId are as previously defined X', when present, is a spacer moiety; 65 with respect to Formula IIa. EW', when present, is an electron-withdrawing group; Exemplary polymeric reagents of this form include, but are (Z) is Zero or a positive an integer, not limited to, the following: US 8,865,149 B2 36 hydrate, dithiohydrate, hemiketal, monothiohemiketal, dithiohemiketal, ketal, and dithioketal having a terminus defined as R, wherein R is an organic radical, or (ii) a neo-clich-oches-orict--O-0. nondienone cyclic structure wherein a functional group 5 selected from the group consisting of ketone, ketone hydrate, R O thione, monothiohydrate, dithiohydrate, hemiketal, mono HCO-CHCH-(OCH2CH)-OCHCH-C-N ; and thiohemiketal, dithiohemiketal, ketal, and dithioketal is part O of the cyclic structure. OH: A particularly preferred polymeric reagent of this form HCO-CHCH-(OCH2CH)- ocic--OX 10 comprises the following structure: OH (Formula IIIa) wherein (m) is from about 3 to about 3000. In some instances, the polymeric reagent is linear in form 15 and comprises a single water-soluble polymer segment and more than one functional group. An exemplary polymeric reagent of this form comprises the following structure: Z wherein POLY', (a), (b), (c), (d), X', X, EW', EW’, (z), (y), (Formula III) R", R. R. R. R. and R are as previously defined with respect to Formula III. A corresponding ketal-containing version of Formula (IIIa) comprises the following structure: 25

(Formula IIIb) wherein: POLY is a water-soluble polymer segment having a OR 12 R4 R2 OR 10 molecular weight of greater than 2,200 Daltons; 30 R- ( (EW2)-(X2)-POLY-(X)-(EW) $ (-R (a) is either Zero or one; OR'13 RD.5 y R3 OR (b) is either Zero or one; (c) is either Zero or one; (d) is either Zero or one: wherein POLY', (a), (b), (c), (d), X', X, EW', EW’, (Z), (y), X', when present, is a spacer moiety; 35 R", R. R. R. R. and R area as previously defined with X, when present is a spacer moiety; respect to Formula (III), and further wherein R' is an organic EW', when present, is an electron-withdrawing group; radical (preferably nonaromatic), R'' is an organic radical EW’, when present, is an electron-withdrawing group; (preferably nonaromatic), R' is an organic radical (prefer (z) is zero or a positive integer from 1 to 7: ably nonaromatic), R' is an organic radical (preferably non (y) is Zero or a positive integer from 1 to 7: 40 aromatic), or R'' and R' combine to form a cyclic ketal each occurrence of R, when present, is independently Hor (preferably nonaromatic, e.g., an alkylene Such as ethylene or an organic radical; substituted ethylene), and R'' and R' combine to form a each occurrence of R, when present, is independently Hor cyclicketal (preferably nonaromatic, e.g., an alkylene Such as an organic radical; ethylene or substituted ethylene). each occurrence of R", when present, is independently Hor 45 The corresponding ketone hydrate of Formula III com an organic radical; prises the following structure: each occurrence of R, when present, is independently Hor an organic radical; FG-R' is either (i) a functional group selected from the (Formula IIIc) group consisting of ketone, ketone hydrate, thione, monothio 50 hydrate, dithiohydrate, hemiketal, monothiohemiketal, OHR R2 OH dithiohemiketal, ketal, and dithioketal having a terminus R- ( (EW2)-(X2)-POLY-(X)-(EW) $ g-R defined as R', wherein R' is an organic radical, or (ii) a OH R5 y R3 OH nondienone cyclic structure wherein a functional group Z selected from the group consisting of ketone, ketone hydrate, thione, monothiohydrate, dithiohydrate, hemiketal, mono 55 wherein POLY', (a), (b), (c), (d), X', X, EW', EW’, (z), (y), thiohemiketal, dithiohemiketal, ketal, and dithioketal is part R", R. R. R. R. and R are as previously defined with of the cyclic structure; and respect to Formula III. FG-R is either (i) a functional group selected from the Exemplary forms of the polymeric reagents in accordance group consisting of ketone, ketone hydrate, thione, monothio with Formula III include:

O O o-O--inch-octet-out-- O;

US 8,865,149 B2 39 -continued OH pi HC-C-CH-O-CHCH-(OCH2CH)-OCHCH-O-CH-C-CH3, OH OH wherein (m) is from about 55 to about 3000. Another class of reagents that may advantageously contain a ketone group or one of its forms are heterobifunctional 10 (Formula IV) polymeric reagents. Of most use are those that react with R2 R8 different types of biomolecules. For example, one may have a ketone that would react with an amine group in a protein or POLY-CXI)-(EW) FG $ (EW3)-(X)-POLY2 polypeptide and a maleimide functional group that would 3 9 react with a thiol group in a different protein or polypeptide. 15 RJ, RJ, An exemplary form of the aforementioned heterobifunctional polymeric reagent is shown below,

O O N-C-C –––clien-one-occu--O-

wherein (m) is from about 3 to about 3000. wherein: These and other heterobifunctional forms wherein one 30 POLY is the first water-soluble polymer segment; functional group of the polymeric reagent is selected from the POLY is the second water-soluble polymer segment; group consisting of ketone, ketone hydrate, thione, monothio (a) is either Zero or one; hydrate, dithiohydrate, hemiketal, monothiohemiketal, (b) is either Zero or one; dithiohemiketal, ketal, and dithioketal, are encompassed (e) is either Zero or one; 35 (f) is either Zero or one; within the present invention. X', when present, is a spacer moiety; Advantageously, the polymeric reagents of the invention X, when present is a spacer moiety; can comprise more than a single water-soluble polymer seg EW', when present, is an electron-withdrawing group; ment. Often, such polymeric reagents are conventionally EW, when present, is an electron-withdrawing group; referred to as “branched.” As will be seen in further detail 40 (z) is zero or a positive integer from 1 to 7: below, branching can be effected in the currently described (x) is zero or a positive integer from 1 to 7: polymers at the functional group carbon or at a point other each occurrence of R, when present, is independently Hor than the functional group carbon. Branching effected at the an organic radical; functional group carbon provides a linear and branched poly each occurrence of R, when present, is independently Hor meric reagent, thereby providing a 'V' arrangement. Branch 45 an organic radical; ing effected by a moiety other than the functional group each occurrence of R, when present, is independently Hor carbon is typically nonlinear and the resulting polymeric an organic radical; reagent is often in the form of a letter “Y” each occurrence of R, when present, is independently Hor 50 an organic radical; and An exemplary branched polymeric reagent (linear or oth FG is the functional group selected from ketone, ketone erwise) is comprised of a first water-soluble polymer seg hydrate, thione, monothiohydrate, dithiohydrate, hemiketal, ment, a second water-soluble polymer segment, and a func monothiohemiketal, dithiohemiketal, ketal, and dithioketal. tional group selected from the group consisting of ketone, A ketone-containing polymeric reagent of Formula IV ketone hydrate, thione, monothiohydrate, dithiohydrate, 55 comprises the following structure: hemiketal, monothiohemiketal, dithiohemiketal, ketal, and dithioketal, wherein each of the first and second water (Formula IVa) soluble polymer segments are attached, either through a direct covalent bond or through one or more atoms, to the POLY1-(X)-(EW) | C | $ (EW)-(X)-POLY2 functional group and further wherein when the functional 60 group is a ketone, ketone hydrate, hemiketal or ketal: (a) the Z X. polymeric reagent lacks an aromatic moiety; or (b) each wherein POLY', POLY, (a), (b), (e), (f), (Z), (x), X', EW', water-soluble polymer segment in the polymeric reagent has EW, and X are as previously defined with respect to For a weight average molecule weight of 1000 Daltons or greater. mula IV. Thus, for example, the branched polymeric reagents of the The ketal-containing version of Formula IV comprises the invention include those comprising the following structure: following structure: US 8,865,149 B2 41 42 i y (Formula IVb) POLY-CX)-(EW)--C C (EW)-(X)-POLY2 b I R9 Z X.

wherein POLY', POLY', (a), (b), (e), (f), (z), (x), X', EW', 10 A preferred branching moiety to provide branched poly EW, and X’ are as previously defined with respect to For meric reagents of the invention comprises the following struc mula IV, and further wherein R' is a organic radical (prefer ture ably nonaromatic), R'' is an organic radical (preferably non aromatic), or R'' and R' combine to form a cyclic ketal POLY 1-X (preferably nonaromatic, e.g., an alkylene Such as an ethylene 15 R"-C- or substituted ethylene). POLY2-X5 Exemplary polymeric reagents of Formula IV wherein the functional group is a ketone hydrate comprise the following wherein each of POLY' and POLY is a water-soluble poly Structure: mersegment (either the same or different), R" is a nonreactive

(Formula IVc) i pi |f POLY-CXI)-(EW) (EW)-(X)-POLY2 R3 OH R9 Z X. wherein POLY', POLY', (a), (b), (e), (f), (z), (x), X', EW', moiety, Such as H. methyl, or a water-soluble polymer seg EW, and X are as previously defined. 30 ment; and each of X and X is a spacer moiety (either the As is evident from the polymeric reagents of Formulas IV. same or different). IVa., IVb, and IVc, the functional group carbon serves as the branching point through which each of two water-soluble Preferred branched moieties are selected from the group polymer segments is ultimately attached. In this way, the consisting of polymeric reagent has a form resembling the letter “V” or a 35 flattened form thereof. Each water-soluble polymer segment POLY ICH.) POLY-O--CH can also be further branched, thereby providing a polymeric reagent with four or more water-soluble polymer segments. The functional group carbon of a branched polymeric 2 reagent can also be a part of a more complex moiety, as shown 40 POLY ICHle, POLY4-O--CH, below:

7 N, N Y(X)-CH-CH,-(OCH-CH)n-OCHCH-OCHs. wherein (m) is from about 3 to about 3000, (a) is either Zero -continued or one, (e) is either Zero or one, X is a spacer moiety when present, and X is a spacer moiety when present. Diaza cyclic 55 O structures other than the 3,7-diaza-bicyclo[3.3.1 nonan-9- POLY-O-C-CH) one shown above can also provide a moiety Suitable for branching. a t In some instances of the branched polymeric reagent forms 60 POLY'-O-C-CH), and of the present invention, a moiety other than the functional O group carbon serves as the branching moiety. In Such a case, a however, the branching moiety must provide at least three POLY o-c-N-ty, attachment sites, one attachment site providing a linkage O HC (either directly or through one or more atoms) to each of the 65 | H functional group, the first water-soluble polymer segment, POLY2-O-C-N-CH), and the second water-soluble polymer segment. US 8,865,149 B2 43 44 wherein, in each instance, POLY' and POLY are as previ In other instances, FG-R'' represents a cyclic structure ously defined, (d) is Zero, 1, 2, or 3, and (e) is Zero, 1, 2, or 3. wherein a functional group selected from the group consist An example of a branched structure of this type is com ing of ketone, ketone hydrate, thione, monothiohydrate, prised of the following structure: dithiohydrate, hemiketal, monothiohemiketal, dithiohe miketal, ketal, and dithioketal is part of the cyclic structure. (Formula V) Exemplary polymeric reagents wherein the cyclic structure is a piperidonyl structure comprise the following: POLY1-X R2 polyk--ee-r Z 10 PO LY 1-X4 R2 wherein: i--X-EW N O; PO LY 1-X5 R3 POLY is the first water-soluble polymer segment; Z POLY is the second water-soluble polymer segment; 15 PO ty (a) is either Zero or one; (b) is either Zero or one: i-i-o-citchcic- O; PO LY2-X5 X', when present, is a spacer moiety; POLY 1-X O X is a spacer moiety; OH X is a spacer-moiety; H-C-O-CHCHCHC-N EW', when present, is an electron-withdrawing group; OH: (z) is zero or a positive integer from 1 to 7: PO LY2-X5 each occurrence of R, when present, is independently Hor PO ty an organic radical; 25 it--o-clich- O; and each occurrence of R, when present, is independently Hor PO LY2-X5 an organic radical; POLY 1-X O R" is H or a noninterfering organic radical; and OH FG-R'' is either (i) a functional group selected from the H-C-O-CHCHC-N group consisting of ketone, ketone hydrate, thione, monothio OH, hydrate, dithiohydrate, hemiketal, monothiohemiketal, 30 PO LY2-X5 dithiohemiketal, ketal, and dithioketal having a terminus defined as R'', wherein R'' is an organic radical, or (ii) a wherein in each instance, POLY', POLY, R, R, X, X, X, cyclic structure wherein a functional group selected from the (a), EW, and (b) are as previously defined with respect to group consisting of ketone, ketone hydrate, thione, monothio Formula V. hydrate, dithiohydrate, hemiketal, monothiohemiketal, 35 Exemplary branched polymers suitable for use as a water dithiohemiketal, ketal, and dithioketal is part of the cyclic soluble polymer segment herein are described in U.S. Pat. No. Structure. 5,932,462 and are based on a disubstituted lysine residue. In some instances, FG-R'' represents a functional group Two specific examples of lysine-based branched polymers selected from the group consisting of ketone, ketone hydrate, include an ester-containing version: thione, monothiohydrate, dithiohydrate, hemiketal, mono 40 thiohemiketal, dithiohemiketal, ketal, and dithioketal having a terminus defined as R'', wherein R'' is an organic radical. O O Thus, exemplary forms of FG-R'' (as well as FG-R' and a FG-R) include POLY -o-c-s-p-c-o- 45 fl. fl. -C-CH, -C-CHCH, -C-CH=CH, O CH 50 POLY2-O---H, --ch. ––clich. --ch-ch. OH OH OH an amide-containing version: 13 chich ch 55 --ch. --ch, s ––clei. O O OCH OCH2CH OCH in H2CH3 it. POLY -o-c-s-p-c-n- ––clich. --ch-ch. 60 l OCH2CH OCH fl. H2CH3 CH O CH --ch-ch. and --ch POLY2-O---H, OCH2CH3 O CH 65 Thus, for example, polymeric reagents encompassed by the following structure result by substituting PEG for each of US 8,865,149 B2 45 46 POLY' and POLY and using the amide-containing, lysine polymer. Thus, for example, a polymeric reagent comprising based branched structure as the branching moiety with two water-soluble polymer segments wherein each segment respect to Formula V: has a nominal average molecular weight of about 5,000 Dal

O O R2 HCO-CHCH-(OCH2CH)-OCHCH-O-C-N-CH-C- NH-(X)-(EW) FG-R14, R3 CH Z CH O CH HCO-CHCH-(OCH2CH)-OCHCH-O-C-N-CH wherein each of (a), (b), (Z), X', EW', R. R. R', FG, is as tons will have a total molecular weight of about 10,000 Dal previously defined with respect to Formula V. and (m) is from tOnS. 3 to about 3,000. In the context of delivering a conjugate formed from a In addition, polymeric reagents encompassed by the fol polymeric derivative comprised of two or more water-soluble lowing structure result by substituting PEG for each of segments, individual water-soluble polymer segments can be POLY' and POLY and using the amide-containing, lysine cleaved in vivo. As one or more water-soluble polymer seg based branched structure as the branching moiety with ments are cleaved from the conjugate, the Smaller size of the respect to Formula IV: resulting conjugate often results in improved clearance of the

R2 R8 (X)-(EW) FG (EW)-(X)3 3 R3 R9

O O HCO-CHCH-(OCH2CH)-OCHCH-O-C-N-CH-C-NH f". f". Oif it CH HCO-CHCH-(OCH2CH)-OCHCH-O-C-N-CH O O -C-CH-N-C-O-CHCHO-(CH2CH2O)-CHCH-OCH

CH

CH CH2| H O CH-N-C-O-CHCH-O-(CH2CH2O)-CHCH-OCH wherein each of R. R. R. R. (a), (b), (e), (f), (Z), (x), X', conjugate from the body. By selecting the molecular weight EW, FG, EW, and X are as previously defined, and (m) is (i.e., size) of the water-soluble polymer segment and/or cleav from 3 to about 3000. able linkage in the polymeric reagent, it is possible to modu Polymeric reagents comprising two or more water-soluble 65 late the clearance profile of a conjugate. One of ordinary skill polymer segments advantageously provide higher molecular in the art can determine the proper molecular weight of the weight species without having to use a relatively long single water-soluble polymer segment as well as the cleavable link US 8,865,149 B2 47 48 age based on routine experimentation by, for example, pre Among other problems, oxidation side products can render paring a variety of polymeric reagents with different water a polymeric-reagent composition unsuited for use as a soluble polymer segments weights and cleavable linkages, reagent in a conjugation reaction. For example, an oxidation conjugating each of the polymeric reagents with an active side product can be a cleaved portion of a water-soluble agent, administering each of the conjugates to a patient, and 5 polymer segment such as PEG, which can occur when a then obtaining the clearance profile (e.g., through periodic water-soluble polymer segment has been exposed to oxidiz blood or urine sampling). Thereafter, it is possible to deter ing conditions. In addition, an oxidation side product can be mine which polymeric reagent (based on the corresponding an oxidizing agent itself, which was not completely removed conjugate) provides the desired clearance profile. following a synthetic step. The agent can continue to oxidize The polymeric reagents can also take other forms. For 10 example, the polymeric reagent can be comprised of a func the products of the composition, thereby initiating undesired tional group attached, either through a direct covalent bond or chain cleavage of the water-soluble polymer segment. Thus, through one or more atoms, to a water-soluble polymer seg oxidation side products are preferably absent from the com ment, wherein: (a) the functional group is selected from the positions described herein. group consisting ketone, ketone hydrate, thione, monothio 15 Thus, the invention also encompasses compositions com hydrate, dithiohydrate, hemiketal, monothiohemiketal, prising a polymeric reagent comprised of a functional group dithiohemiketal, ketal, and dithioketal of thione, monothio attached, either through a direct covalent bond or through one hydrate, dithiohydrate, monothiohemiketal, dithiohemiketal, and dithioketal, and (b) the polymeric reagent lacks Saturated or more atoms, to a water-soluble polymer segment, wherein hydrocarbon segments having 8 carbon atoms or more. It is the composition is substantially free of oxidation side prod preferred, that the functional group is selected from the group uctS. consisting of thione, monothiohydrate, dithiohydrate, mono An example of a polymeric reagent that can (although not thiohemiketal, dithiohemiketal, and dithioketal. necessarily) be a component of a composition that is Substan The polymeric reagents of the invention can be provided in tially free of oxidation side products is comprised of the the form of a composition. Typically a composition com following structure:

(Formula VI) R2 O | | || HCO-CHCH-(OCH2CH)-OCHCH-CX)-(EW)--C C-CH

Z prises two or more of the same or different polymeric reagents 35 wherein: in addition to one or more optional components. While it may (a) is either Zero or one; be impossible in some contexts to remove all undesirable components of a composition, it is preferred that the compo (b) is either Zero or one; sitions of the polymeric reagents described herein are free or X', when present, is a spacer moiety; substantially free of undesirable components. Examples of 40 EW', when present, is an electron-withdrawing group; undesirable components will be known to those of ordinary (z) is from 1 to 7: skill and are often dependent upon the intended uses of the each occurrence of R is independently H or a nonaro composition and/or polymeric reagent. Specific examples, matic-containing organic radical; however, typically include impurities, components that each occurrence of R is independently H or a nonaro would cause harm (i.e., be toxic) if administered to a patient, 45 matic-containing organic radical; and unreacted materials remaining from previous synthetic steps, and so forth. (m) is from 3 to about 3000, more preferably from 11 to Preferably, the composition will be substantially free or, about 3000, more preferably, entirely free of oxidation side products. wherein the composition is substantially free of oxidation Compositions can often be rendered substantially free or side products, 3-keto esters, entirely free of oxidation side products by purifying a com 50 position through, for example, chromatography, to remove any undesired oxidation side products. Another approach is to O provide the polymeric reagent and any other components comprising the composition without using an oxidation step. HCO--CHCHO-H-CH-CH-CH-C-CH In addition, care must be taken not to allow atmospheric 55 mW 2000 oxygen to react with the composition or any of its compo nents, thereby allowing for the creation of oxidation side products in the composition. In the present context, a com and a ketal derivative of position that is Substantially free of oxidation side products is at least 75% (by weight) free of oxidation side products, more 60 preferably at least 85% (by weight) free of oxidation side O products, still more preferably at least 90% (by weight) free HCO--CHCHO-H-CH-CH-CH-C-CH. of oxidation side products, yet more preferably at least 95% free of oxidation side products, even more preferably at least mW 2000 98% (by weight) of oxidation side products, with at least 65 99.9% (by weight) free of oxidation side products being most The functional groups other than ketone discussed herein, preferred. (i.e., ketone hydrate, thione, monothiohydrate, dithiohydrate, US 8,865,149 B2 49 50 hemiketal, monothiohemiketal, dithiohemiketal, ketal, and The anionic site serves as an initiator site for polymeriza dithioketal) can be substituted for the ketone functional group tion and is typically, although not necessarily, an alkoxide in Formula VI. moiety. Alkoxide moieties are advantageously obtained by Subjecting the corresponding alcohol to a deprotonating base, III. Methods for Making Polymeric Reagents thereby removing the hydrogen atom from the alcohol to yield the desired alkoxide moiety. Before discussing the various methods for making poly In order to form a polymeric reagent corresponding to meric reagents, it must be pointed out that the methods, at Formula IV, a precursor molecule comprising the following times, rely on the use of a protected form of a functional structure can be used: group. Some functional groups, such as ketals, may not 10 require protecting. Moreover, some functional groups serve as a protected form for another functional group associated with the presently described polymeric reagent (e.g., ketals (Formula VIIa) often serve as a protected form for ketones). Those of ordi nary skill in the art know suitable protecting forms for each of 15 the functional groups associated with the presently described polymeric reagents. For example, protected forms of a ketone include ketals (either cyclic or of a diether type). A ketal can beformed by reacting a ketone with an alcohol, diol, thiol, or dithiol in the presence of an acid catalyst. The ketal so formed wherein (a), (b), (e), (f), (x), (z), EW', EW. R. R. R. and (as well as other ketals) can be transformed into a ketone by Rare as previously defined with respect to Formula IV, and acid hydrolysis. Other protected forms, as well as protecting further wherein pFG is a functional group or a protected form and deprotecting the various functional groups discussed thereof, wherein the functional group is selected from the herein, are provided in Greene et al. Supra. group consisting of ketone, ketone hydrate, thione, monothio The various steps for making a polymeric reagent are car 25 hydrate, dithiohydrate, hemiketal, monothiohemiketal, ried out in a suitable solvent. One of ordinary skill in the art dithiohemiketal, ketal, and dithioketal. can determine whether any specific solvent is appropriate for Polymeric reagents corresponding to Formula I can be any given reaction step. Often, however, the solvent is pref formed through a method comprising this approach wherein erably a nonpolar solvent or a polar aprotic solvent. Nonlim the precursor molecule comprises the following structure: iting examples of nonpolar solvents include benzene, Xylene, 30 dioxane, tetrahydrofuran (THF), t-butyl alcohol and toluene. Particularly preferred nonpolar solvents include toluene, (Formula VIIb) Xylene, dioxane, tetrahydrofuran, and t-butyl alcohol. Exem R2 plary polar aprotic solvents include, but are not limited to, DMSO (dimethylsulfoxide), HMPA (hexamethylphosphora 35 O-(X)-(EW) mide), DMF (dimethylformamide), DMA (dimethylaceta mide), and NMP (N-methylpyrrolidinone). Z The polymeric reagents of the invention can be synthesized using a variety of routes. It should be noted that common wherein (a), (b), (Z), EW, R', R, and R, are as previously techniques discussed with respect to one method often apply 40 defined with respect to Formula I (in the Summary of the to similar techniques discussed in other methods. Conse Invention Section), and further wherein pFG is a functional quently, discussion of any specific technique for a given group or a protected form thereof, wherein the functional method is not limited to that method and therefore applies to group is selected from the group consisting of ketone, ketone each method utilizing that technique. hydrate, thione, monothiohydrate, dithiohydrate, hemiketal, In some routes, the polymeric reagent is formed via a 45 polymerization approach. Thus, for example, a polymeric monothiohemiketal, dithiohemiketal, ketal, and dithioketal. reagent can be formed by a method comprising the steps of If the diol corresponding to Formula VIIa is used or the (i) providing a precursor molecule comprised of at least alcohol corresponding to Formula VIIb is used (wherein a hydrogenatom is attached to eachalkoxide moiety), an added one active anionic site Suitable for initiating polymerization step of removing the hydrogen (by, for example, treating the and a functional group or a protected form thereof, wherein 50 the functional group is selected from the group consisting of alcohol with a deprotonating base) is required in order to yield ketone, ketone hydrate, thione, monothiohydrate, dithiohy the alkoxide initiator sites. In addition, thiolates (i.e., —S) drate, hemiketal, monothiohemiketal, dithiohemiketal, ketal, can be used in the place of alkoxide moieties. In Such a case and dithioketal; then, the corresponding thiol or dithiol can be placed in a (ii) contacting the anionic site of the precursor molecule 55 based to provide the necessary ionic site Suited for polymer with a reactive monomer capable of polymerizing, to thereby ization. initiate polymerization of the reactive monomer onto the pre Specific molecules Suited for use as precursor molecules in cursor molecule; this approach include the following: (iii) adding additional reactive monomers to the precursor molecule to form one or more polymer chains; 60 (iv) allowing said adding to continue until a desired length O O of the one or more polymer chains is reached; 1 (v) terminating the reaction, thereby resulting in a poly -O-CH Sc4 -CHs. meric reagent comprised of the functional group or protected form thereof, and 65 O O (vi) optionally, when the functional group is in the pro -O-CH-CH-O-CH Sc4 -CH, tected form, deprotecting the functional group. US 8,865,149 B2 51 52 -continued chain extension while, at the same time, adding a hydrolyti cally cleavable unit. The latter point is important with drug it i delivery of active agents, such as pharmacologically active proteins, which may have undesirable clearance profiles from Oit O is the body. A generalized example of this chain extending -O-CH-CH-O-CH, SC4 -CH, capping comprises reacting a monosubstituted PEG deriva tive that gives a carbonate, urethane or similar functional O O group linkage with the living polymer. Attachment of the -O-CH-SC4 -CH-O polymer, however, must not result in a polymer conjugate that 10 is neither water-soluble nor immunogenic. Having formed the water-soluble polymer segments in the O O polymeric reagent, an end-capping group optionally can be -O-CH-SC4 -CH-CH-O, and added using conventional techniques. For example, an alkyl halide (e.g., methyl halide or methyl p-toluenesulfonate) can O O 15 be reacted with the exposed terminal (the terminal or termini distal to the functional group or protected form thereof) of the polymer chain. In addition, the one or more polymer chains can be capped with an additional polymer. - O O. Optionally, when the polymeric reagent bears a protected form a functional group, the functional group can be removed These and other molecules Suited for use as precursor mol using art known methods. For example, a ketal moiety (either ecules can be obtained commercially (either as shown above cyclic or a diether type) can be removed by undergoing or in their corresponding alcohol forms) and/or may be Syn hydrolysis. Again, reference is made to Greene et al. Supra for thesized using conventional techniques. methods of removing other protecting groups. Having provided a precursor molecule with an initiator site 25 An advantage of this ethoxylation-based approach for pro (e.g., an anionic site) Suited for polymerization, the next step viding polymeric reagents having a central functional group a in this approach comprises the step of contacting the initiator water-soluble polymer segment on each side of the functional site of the precursor molecule with a reactive monomer group is the flexibility of molecular weight control and in the capable of polymerizing, to thereby initiate polymerization of capping step. For example, one may polymerize to give a 30 living polymer with an average molecular weight of 5,000 the reactive monomer onto the precursor molecule. Any reac Daltons and cap the hydroxy end groups by adding a methy tive monomer can be used to 'grow” the polymer chain(s) so lating reagent. This gives a polymer with a molecular weight long as the resulting polymer chain is water soluble. It is of about 5,000 Daltons that has stable (relatively unreactive) particularly preferred, however, that the reactive monomer is end groups but with a functional moiety roughly in the center ethylene oxide, thereby providing poly(ethylene oxide) 35 of the polymer derivative. In addition, one may cap the living chain(s). These and other polymerization techniques are polymer with chain extenders to give a higher molecular known to those of ordinary skill in the art and are referenced weight or with functionally reactive ether groups, such as in, for example, Odian, Chap. 7, Principles of Polymeriza benzyl to give a removable group that could lead to the poten tion, 3" Ed., McGraw-Hill, 1991. tial of chemical modification of the end groups. Once polymerization is initiated, additional reactive mono 40 Another polymerization-based approach for making poly mers are added to the precursor molecule to form one or more meric reagents involves the use of a protected secondary polymer chains. The addition of the reactive monomers effec alcohol. In particular, this approach the method comprises the tively allows the one or more polymer chains to “grow.” steps of Growth of the polymer chain(s) continues until a desired (i) providing a precursor molecule comprised of a pro molecular weight is achieved, at which time the reaction is 45 tected secondary alcohol or thiol and at least one anionic site terminated. Termination can occur through any of a number Suitable for initiating polymerization; of art known methods. For example, neutralizing the reaction (ii) contacting the anionic site of the precursor molecule medium will halt the growth of the polymer chain(s). In with a reactive monomer capable of polymerizing, to thereby addition, adding a specific weight or amount of the reactive initiate polymerization of the reactive monomer onto the pre monomerand allowing the polymerization to proceed until all 50 cursor molecule: reactive monomer is exhausted results in a polymer chain (iii) adding additional reactive monomers to the precursor having a certain and determinable molecular weight. The molecule to form one or more polymer chains; result is a polymeric reagent comprising a functional group or (iv) allowing said contacting to continue until a desired protected form thereof. length of the one or more polymer chains is reached; Optionally and prior to carrying out an end-capping step, 55 (v) terminating the reaction, thereby resulting in an inter an electrophilically reactive polymer (either hydrophobic or mediate comprised of the protected secondary alcohol or hydrophilic) can be added to the polymer chain(s). thiol: An added benefit of the ethoxylation route is that it allows (vi) deprotecting the protected secondary alcohol or thiol for capping the living polymer end (i.e., the terminal of the of the intermediate to form an unprotected secondary alcohol polymer where additional monomers can be added) with vari 60 or thiol: ous moieties including other polymers. This allows for the (vii) oxidizing the unprotected secondary alcohol or thiol generation of polymers with different properties. Thus, it is to provide a polymeric reagent comprised of a ketone when possible to cap the already formed water-soluble polymer the precursor molecule comprised a secondary alcohol or a segment with a hydrophobic polymer and generate a final thione when the precursor molecule comprised a secondary polymer having hydrophilic and hydrophobic regions. More 65 thiol, and importantly for many applications, it is possible to add an (viii) optionally further modifying the ketone or thione to electrophilically reactive PEG derivative, thereby providing result in a functional group selected from the group consisting US 8,865,149 B2 53 54 of ketone, ketone hydrate, thione, monothiohydrate, dithio hydrate, hemiketal, monothiohemiketal, dithiohemiketal, ketal, and dithioketal. st-oil Thus, with respect to steps (ii), (iii), (iv), (v), involving the ti-ye use of polymerization techniques, the same polymerization 5 CH-OH.: techniques discussed above apply equally as well here. As above, the anionic site serves as an initiator site for polymer wherein Y is oxygen or Sulfur, "pg is a protecting group ization and is typically, although not necessarily, an alkoxide Suitable for protecting a secondary alcohol when Y is oxygen moiety. Alkoxide moieties are advantageously obtained by or thiol when Y is sulfur. Subjecting the corresponding alcohol to a deprotonating base, 10 A particularly preferred monoprotected is 2-ben thereby removing the hydrogen atom from the alcohol to Zyloxy-1,3-propanediol shown below: yield the desired alkoxide moiety. In order to form a branched polymeric reagent of Formula HO IV using this approach, the precursor molecule will comprise 15 the following structure: - O (Formula VIIIa) 2-benzyloxy-1,3-propanediol

"O-(X)-(EW) it is (EW3)-(X)-O- These and other monoprotected are available from commercial Suppliers such as Sigma (St. Louis, Mo.) and/or R3 H R9 can be synthesized using conventional techniques. 25 Having formed the water-soluble polymer segment, along with optionally additionally attaching an electrophilic poly wherein (a), (b), (e), (f), (x), (z), X, X, EW', EW. R. R. mer and optionally alkylating the terminal or termini of the R, and Rare as previously defined with respect to Formula water-soluble polymer segment or segments, the method IV, and further wherein Y is oxygen or sulfur and “pg is a comprises the step of deprotecting the protected secondary protecting group for a secondary alcohol when Y is oxygen alcohol or thiol to form an unprotected secondary alcohol or and a protecting group for a secondary thiol when Y is Sulfur. 30 thiol. The protecting group can be removed using art known Polymeric reagents corresponding to Formula I can be methods. For example, the deprotecting step can be effected formed through a method comprising this approach wherein by exposing the intermediate to H and a metal catalyst such the precursor molecule comprises the following structure: as palladium, palladium oxide, nickel, platinum, or lead. After the deprotecting step, the method comprises carrying 35 out an oxidizing step, which can be performed in any conven tional manner and the invention is not limited in this regard. (Formula VIIIb) Examples of suitable oxidizing techniques comprise contact fre ing the intermediate to be oxidized with an oxidizing reagent "O-(X)-(EW) i-R selected from the group consisting of KMnO, MnO, 40 KCrOz, CrO., pyridinium chlorochromate, pyridinium R3 H Z fluorochromate, pyridinium dichromate, RuO, RuCls, tetra n-propylammonium perruthenate, dimethyl Sulfoxide, N-chlorosuccinimide, AgCOs, AGO, and Dess-Martin wherein (a), (b), (Z), X', EW', R', R, and R are as previ periodinane reagents. ously defined with respect to Formula I (in the Summary of 45 Other approaches for forming the polymeric reagents of the Invention Section), and further wherein Y is oxygen or the invention are not based on a polymerization approach. In Sulfur and “pg is a protecting group for a secondary alcohol one nonpolymerization approach for providing a polymeric when Y is oxygen and a protecting group for a secondary thiol reagent of the invention, the method comprises the steps of (i) when Y is sulfur. providing a precursor molecule comprised of at least one As previously indicated, the precursor molecule comprises 50 isocyanate moiety and a functional group or a protected form a protected secondary alcohol or thiol. Any suitable, art thereof, wherein the functional group is selected from the known protecting group Suitable for protecting a secondary group consisting of ketone, ketone hydrate, thione, monothio alcohol or thiol can be used, and include, for example, benzyl hydrate, dithiohydrate, hemiketal, monothiohemiketal, ether, methyoxymethyl ether (MOM), methylthiomethyl dithiohemiketal, ketal, and dithioketal; and (ii) contacting, ether (MTM), tetrahydropyranyl ether (THP), 4-methoxytet 55 under Suitable reaction conditions, the precursor molecule rahydropyranyl ether, tetrahydrofuranyl ether, 1-ethoxyethyl and a water-soluble polymer segment bearing at least one ether, 1-methyl-1-methoxyethyl ether, 2-(phenylselenyl) hydroxyl or amine group to thereby form a polymeric reagent ethyl ether, allyl ether, o-nitrobenzyl ether, triphenylmethyl comprising the functional group or protected form thereof. ether, C.-naphthyldiphenylmethyl ether, p-methyoxyphenyl and (iii) optionally, when the functional group is in the pro diphenylmethyl ether, 9-(9-phenyl-10-oxo)anthryl ether (tri 60 tected form, deprotecting the functional group. tylone), isopropyldimethylsilyl ether, t-butyldimethylsilyl In this third approach, a number of precursor molecules ether (TBDMS), tribenzylsilyl ether, and triisopropylsilyl comprising at least one isocyanate moiety can a functional ether. A particularly preferred protecting group for use with group or protected form thereof can be used. the method is benzyl ether. In order to form a branched polymeric reagent of Formula Specific molecules (in the corresponding alcohol forms) 65 IV using this approach, the reaction will comprise contacting: Suited for use as precursor molecule in this approach include (a) a precursor molecule comprised of the following struc a monoprotected glycerol having the following formula: ture: US 8,865,149 B2 56

(Formula DXa)

wherein (a), (b), (e), (f), (x), (z), X, X, EW', EW. R. R. 10 or protected form thereof; and (iv) optionally, when the func R, and Rare as previously defined with respect to Formula tional group is in the protected form, deprotecting the func IV and further wherein pFG is a functional group or a pro tional group. tected form thereof, wherein the functional group is selected The water-soluble polymer segment having at least one from the group consisting of ketone, ketone hydrate, thione, available nucleophilic group can be any polymer terminating monothiohydrate, dithiohydrate, hemiketal, monothiohe 15 in a nucleophilic group. Such polymers include, without limi miketal, dithiohemiketal, ketal, and dithioketal; with (b) a tation: polymers bearing a terminal amine (e.g., amine-termi water-soluble polymer segment bearing one terminal nated PEG, available from, for example, Nektar Therapeu hydroxyl group. tics, Huntsville, Ala.). The activating group of the precursor molecule can be any Suitable activating group as that term is The water-soluble polymer segment will be added in at understood by those of ordinary skill in the art, but an acti least a 2:1 molar to the isocyanate precursor molecule. It is Vating group selected from the group consisting of halide preferred, however that an excess of the water-soluble poly (e.g., fluoride, chloride, iodide), Sulfonate esters (e.g., meth mer segment is present. anSulfonate, p-toluenesulfonate, and so forth), and N-Succin In order to form a polymeric reagent of Formula I using this imidyl is preferred. approach, the reaction comprises contacting: (a) a precursor 25 Branched polymeric reagents of Formula IV can beformed molecule comprised of the following structure: using this approach by contacting a water-soluble polymer segment having at least one available nucleophilic group; with a precursor molecule comprised of the following struc (Formula DXb) ture: 30 (Formula Xa) R R AC-CX)-(EW)--C-HpFGC-H(EW) (X) AC 35 wherein (a), (b), (Z), X', EW', R', R, and R are as previ is LR), ously defined with respect to Formula I (in the Summary of the Invention Section), and further wherein pFG is a func wherein (a), (b), (e), (f), (x), (Z), X', X, EW', EW. R. R. tional group or a protected form thereof, wherein the func R, and Rare as previously defined with respect to Formula tional group is selected from the group consisting of ketone, 40 IV, and further wherein each AC is an activating group and ketone hydrate, thione, monothiohydrate, dithiohydrate, pFG is a functional group or a protected form thereof, hemiketal, monothiohemiketal, dithiohemiketal, ketal, and wherein the functional group is selected from the group con dithioketal; with (b) a water-soluble polymer segment bear sisting of ketone, ketone hydrate, thione, monothiohydrate, ing one terminal hydroxyl or amine group. dithiohydrate, hemiketal, monothiohemiketal, dithiohe 45 miketal, ketal, and dithioketal. The water-soluble polymer segment will be added in at The water-soluble polymer segment is added in at least a least a 1:1 molar ratio of water-soluble polymeric reagent to 2:1 molar ratio to the precursor molecule. Preferably, how isocyanate precursor molecule. It is preferred, however that ever, an excess of the water-soluble polymer segment is an excess of the isocyanate precursor molecule is present. added. In order to form a homobifunctional polymeric reagent, the 50 Polymeric reagents of Formula I can also be prepared using reaction will comprise contacting: (a) a precursor molecule of this approach. Thus, a water-soluble polymer segment having Formula IXb; with (b) a water-soluble polymer segment bear at least one available nucleophilic group is contacted with a ing two terminal hydroxyl groups. The water-soluble poly precursor molecule comprised of the following structure: mer segment is typically in no more than a 1:2 molar ratio to isocyanate precursor molecule. 55 In a fourth approach for preparing a polymeric reagent, a (Formula Xb) method is provided comprising the steps of (i) providing a water-soluble polymer segment having at least one available nucleophilic group; (ii) providing a precursor molecule com prised of at least one activating group and a functional group 60 or a protected form thereof, wherein the functional group is selected from the group consisting of ketone, ketone hydrate, thione, monothiohydrate, dithiohydrate, hemiketal, mono wherein (a), (b), (Z), X', EW', R', R, and Rare as previ thiohemiketal, dithiohemiketal, ketal, and dithioketal; (iii) ously defined with respect to Formula I (in the Summary of contacting, under Suitable reaction conditions, the precursor 65 the Invention Section), and further wherein AC is an activat molecule with the water-soluble polymer segment, thereby ing group and pFG is a functional group or a protected form forming a polymeric reagent comprising the functional group thereof, wherein the functional group is selected from the US 8,865,149 B2 57 58 group consisting of ketone, ketone hydrate, thione, monothio -continued hydrate, dithiohydrate, hemiketal, monothiohemiketal, dithiohemiketal, ketal, and dithioketal. The water-soluble it it polymer segment having at least one available nucleophilic it is group is added in amount equaling at least a 1:1 molar ratio of 5 O O water-soluble polymer segment to the precursor molecule. It -O-CH-CH-O-CH, SC4 -CH, is preferred, however, that an excess of the water-soluble segment is present. O O A homobifunctional polymeric reagent can also be pre -O-CH-SC4 -CH-O- pared by reacting a water-soluble polymer segment having at 10 least two available nucleophilic groups with a precursor mol O O ecule having a structure corresponding to Formula Xb. In this -O-CH-SC4 -CH-CH-O; and instance, the molar ratio of water-soluble polymer segment to O O precursor molecule is typically no more than 1:2, and typi 15 cally an excess of the precursor molecule is present. A fifth approach for making a polymeric reagent comprises the steps of: (i) providing a water-soluble polymer segment having at - O O. least one available leaving group; (ii) providing a precursor molecule comprised of at least In a sixth approach for providing a polymeric reagent of the one nucleophilic group and a functional group or a protected invention, a method is provided comprising the step of react form thereof, wherein the functional group is selected from ing a water-soluble polymer segment having at least one the group consisting of ketone, ketone hydrate, thione, mono alkoxide ion or thiolate ion with a precursor molecule com thiohydrate, dithiohydrate, hemiketal, monothiohemiketal, 25 prised of at least one leaving group and a functional group or dithiohemiketal, ketal, and dithioketal; a protected form thereof, wherein the functional group is (iii) contacting, under Suitable reaction conditions, the pre selected from the group consisting of ketone, ketone hydrate, cursor molecule with the water-soluble polymer segment, thione, monothiohydrate, dithiohydrate, hemiketal, mono thereby forming a polymeric reagent comprising the func thiohemiketal, dithiohemiketal, ketal, and dithioketal, tional group or protected form thereof, and 30 thereby providing a polymeric reagent. (iv) optionally, when the functional group is in the pro Conveniently, the water-soluble polymer segment having tected form, deprotecting the functional group. at least one alkoxide or thiolate ion is prepared by combining Preferred leaving groups on the water-soluble polymer a water-soluble polymer segment having at least one segment include those that are primary (e.g., a primary halo), 35 hydroxyl or thiol group in the presence of a Suitable base. although leaving groups that are secondary may also be used. Thus, the base transforms POLY-OH into POLY-O and Examples of Suitable leaving groups include halogens and POLY-SH into POLY-ST. It is further believed that the water Sulfonate esters. Among the halogens, bromo, chloro, and soluble polymer, now bearing an alkoxide or thiolate moiety, iodo are preferred, with bromo and chloro being particularly in turn reacts via a S2 reaction mechanism with the precur preferred. With respect to sulfonate esters, methanesulfonate 40 Sormolecule comprised of a Suitable leaving group. As will be (abbreviated “Ms), trifluoromethanesulfonate, trichlo recognized by those of ordinary skill in the art, this approach romethanesulfonate, 2.2.2-trifluoroethanesulfonate, 2.2.2- corresponds to Williamson ether synthesis, and the principles trichloroethanesulfonate, nonafluorobutanesulfonate, p-bro mobenzenesulfonate, p-nitrobenzenesulfonate, and and techniques generally used in a Williamson ether synthesis p-toluenesulfonate are particularly preferred, although other are applicable here as well. 45 Nonlimiting examples of bases suitable to form alkoxides Sulfonate esters and similarly constituted leaving groups and thiolates include Sodium, Sodium hydroxide, potassium, known to those of ordinary skill in the art can be used as well. potassium hydroxide, sodium hydride, potassium hydride, With respect to the precursor molecule bearing, among Sodium methoxide, potassium methoxide, sodium tert-butox other things, a nucleophilic group, the nucleophilic group can ide, potassium tert-butoxide, Sodium carbonate, and potas be any suitable nucleophilic group that can react with the 50 sium carbonate. Preferred bases for use in accordance with leaving group(s) associated with water-soluble polymer seg this step, however, include those selected from the group ment. Typically, the nucleophilic group is alkoxide (R—O) consisting of sodium, potassium, Sodium hydride, potassium or thiolate (R—S), derived from removing the hydrogen hydride, Sodium methoxide, potassium methoxide, Sodium from the corresponding alcohol or thiol forms, respectively. tert-butoxide, and potassium tert-butoxide. Thus, precursor molecules comprising structures correspond 55 In addition, the water-soluble polymer segment having at ing to Formulas VIIa and VIIb can be used. As previously least one alkoxide or thiolate ion can conveniently be pro indicated, specific examples of alkoxide moieties include the vided via a polymerization reaction, as previously described. following: In this approach for providing the water-soluble polymer segment, it is preferred that the water-soluble polymer seg 60 ment has at least one alkoxide or thiolate ion. Generally, although not necessarily, an excess of the pre O O / cursor molecule is allowed to react with the water-soluble -O-CH Sc4 -CHs. polymer bearing at least one alkoxide or thiolate ion. Typi cally, the amount of the precursor molecule represents at least O O 65 a molar equivalent to the number available hydroxyl and/or -O-CH-CH-O-CH Sc4 -CH: thiolate groups of the water-soluble polymer molecule. Het erofunctional polymer species (i.e., species bearing two or US 8,865,149 B2 59 60 more different terminal functional groups) can be prepared by Exemplary conjugation conditions include carrying out the using nonstoichiometric amounts of the precursor molecule. conjugation reaction at a pH of from about 4 to about 10, and That is, heterofunctional species are formed when the total at, for example, a pH of about 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, number of moles of available hydroxyl or thiolate groups on 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0. The reaction is allowed to the water-soluble polymer segment exceeds the total number proceed from about 5 minutes to about 72 hours, preferably of moles of the precursor molecule. from about 30 minutes to about 48 hours, and more preferably A water-soluble polymer segment bearing an alkoxide from about 4 hours to about 24 hours. The temperature under moiety can be prepared synthetically via a polymerization which conjugation can take place is typically, although not process. For example, an alkoxide Salt such as sodium necessarily, in the range of from about 0°C. to about 40°C., 10 and is often at room temperature or less. The conjugation 2-methoxy ethanolate (Na:OCHCHOCH) can initiate reactions are often carried out using a phosphate buffer solu polymerization of ethylene oxide. Assuming that the final tion, Sodium acetate, or similar system. monomer added to the polymer chain leaves a reactive group With respect to reagent concentration, an excess of the Such as an alkoxide (as in the case of ethylene oxide), the polymeric reagent is typically combined with the active polymer chain can then be reacted with the precursor mol 15 agent. In some cases, however, it is preferred to have stoichi ecule as discussed above. ometic amounts of reactive groups on the polymeric reagent Any water-soluble polymer having at least one hydroxyl or to the reactive groups of the active agent. Thus, for example, thiol group (to provide, for example, a water-soluble polymer two moles of a polymeric reagent having two ketone groups having at least one alkoxide ion or thiolate ion, respectively) are combined for every mole of active agent. Exemplary can be used. Although water-soluble polymers bearing only a ratios of polymeric reagent to active agent include molar single hydroxyl or thiol can be used, polymers bearing two, ratios of about 1:1 (polymeric reagent:active agent), 1.5:1, three, four, five, six, seven, eight, nine, ten, eleven, twelve or 2:1, 3:1, 4:1, 5:1, 6:1.8:1, or 10:1. The conjugation reaction is more hydroxyl and/or thiol moieties can be used. Advanta allowed to proceed until Substantially no further conjugation geously, as the number of hydroxyl or thiol moieties on the occurs, which can generally be determined by monitoring the water-polymer segment increases, the number of available 25 progress of the reaction over time. sites for providing ketone, ketone hydrate, thione, monothio Progress of the reaction can be monitored by withdrawing hydrate, dithiohydrate, hemiketal, monothiohemiketal, aliquots from the reaction mixture at various time points and dithiohemiketal, ketal, and dithioketal moieties increases. analyzing the reaction mixture by SDS-PAGE or MALDI Nonlimiting examples of the upper limit of the number of TOF mass spectrometry or any other suitable analytical hydroxyl and/or thiol moieties associated with the water 30 method. Once a plateau is reached with respect to the amount soluble polymer segment include 500, 100, 80 and 40. of conjugate formed or the amount of unconjugated polymer remaining, the reaction is assumed to be complete. Typically, IV. Conjugates the conjugation reaction takes anywhere from minutes to several hours (e.g., from 5 minutes to 24 hours or more). The The polymeric reagents described herein are useful for 35 resulting product mixture is preferably, but not necessarily conjugation to biologically active agents or Surfaces. Exem purified, to separate out excess polymeric reagent, unconju plary groups Suited for reaction with the polymeric reagents gated reactants (e.g., active agent), and undesired multi-con described herein are primary amine groups (e.g., primary jugated species. The resulting conjugates can then be further amines from the side chain of alysine residue), (e.g., characterized using analytical methods such as MALDI, cap a from the side chain of a serine or threonine 40 illary electrophoresis, gel electrophoresis, and/or chromatog residue), hydrazines, and hydrazides. Additional functional raphy. groups are reactive with ketones (and related functional In another embodiment of the invention, a method is pro groups) and can be employed in conjugation reactions when vided for preparing a polymer-active agent conjugate. Any present on another molecule, e.g., active agent. Such groups conjugating method that employs a functional group selected suited to react with the polymeric reagents described herein 45 from the group consisting of ketone, ketone hydrate, thione, are known to those of ordinary skill in the art. Thus, the monothiohydrate, dithiohydrate, hemiketal, monothiohe invention provides a method for making a conjugate compris miketal, dithiohemiketal, ketal, and dithioketal can be used. A ing the step of contacting, under conjugation conditions, an preferred conjugation approach for preparing polymer-active active agent with a polymeric reagent comprising a functional agent conjugates is reductive animation. While not wishing to group selected from the group consisting of ketone, ketone 50 be bound by theory, the reductive animation approach is hydrate, thione, monothiohydrate, dithiohydrate, hemiketal, believed to comprise nucleophilic addition of an amine-con monothiohemiketal, dithiohemiketal, ketal, and dithioketal. taining active agent to the functional group of a polymeric Preferred polymeric reagents used in the conjugation reaction reagent, wherein the functional group is selected from the include those described herein (e.g., the polymeric reagents group consisting of ketone, ketone hydrate, thione, monothio of Formulas I, II, III, IV, V, and VI). 55 hydrate, dithiohydrate, hemiketal, monothiohemiketal, Suitable conjugation conditions are those conditions of dithiohemiketal, ketal, and dithioketal. With respect to reduc time, temperature, pH, reagent concentration, reagent func tive animation, preferred functional groups of the polymeric tional group(s), available functional groups on the active reagent include ketone, ketone hydrate, hemiketal, and ketal. agent, Solvent, and the like Sufficient to effect conjugation Those of ordinary skill in the art are familiar with reductive between a polymeric reagent and an active agent. As is known 60 animation techniques. in the art, the specific conditions depend upon, among other Briefly, and without wishing to be bound by theory, an things, the active agent, the type of conjugation desired, the exemplary reductive animation (wherein the active agent presence of other materials in the reaction mixture, and so bears an amine and the polymeric reagent comprises a ketone) forth. Sufficient conditions for effecting conjugation in any first proceeds via a carbinolamine intermediate. The carbino particular case can be determined by one of ordinary skill in 65 lamine then dehydrates (condenses) to result in an imine. The the art upon a reading of the disclosure herein, reference to the imine, in turn, can then be reduced using a Suitable reducing relevant literature, and/or through routine experimentation. agent to form the polymer-active agent conjugate. An exem US 8,865,149 B2 61 62 plary reducing agent is sodium cyano borohydride. In some nucleophile becomes attached to the f carbon, and the C. instances, the reducing agents such as Zinc in the presence of carbon accepts a proton from the solvent or from the conju HCl, sodium borohydride, iron pentacarbonyl in the presence gate acid of the nucleophile. The structure of the reagent will of alcoholic KOH, hydrogen (H) in the presence of a metal largely dictate how the reaction will occur. With an O.B- catalyst, and formic acid can be used. The reaction is sche unsaturated ketone on the vinyl carbons and sterically hin matically depicted in FIG. 3. dered on the other alpha carbon, conjugate addition (1,4- Of particular note is the linkage between the active agent addition) is expected to prevail. and polymeric reagent: the amine of the active agent is Regardless of the approach used in their formation, the attached to a secondary carbonatom of the polymeric reagent. polymer-active agent conjugates can be purified to obtain/ Thus, in another embodiment of the invention, a conjugate is 10 isolate different conjugated species. Alternatively, and more provided comprising a covalent bond between a nitrogen preferably for lower molecular weight (e.g., less than about atom of an active agent and a secondary carbon atom derived 20,000 Dalton, more preferably less than about 10,000 Dal from the polymeric reagent. A secondary carbon atom is a ton) polymeric reagents used to form conjugates, the product carbon atom that is directly bonded to two other carbons mixture can be purified to obtain the distribution of water (represented as R' and R" in FIG. 3). With respect to the 15 soluble polymer segments per active agent. For example, the remaining part of the conjugate, the secondary carbonatomis product mixture can be purified to obtain an average of any attached, either directly or through one or more atoms, to a where from one to five PEGs per active agent (e.g., protein), water-soluble polymer segment. Thus, the secondary carbon typically an average of about 3 PEGs per active agent (e.g., atom is attached to a first carbonatom (as in R' in FIG. 3) and protein). The strategy for purification of the final conjugate a second carbon atom (as in R" in FIG. 3) wherein at least one reaction mixture will depend upon a number of factors, of the first carbon atom or second carbon atom is attached, including, for example, the molecular weight of the poly either directly or through one or more atoms, to a water meric reagent employed, the particular active agent, the soluble polymer segment. desired dosing regimen, and the residual activity and in vivo Depending on the polymeric reagent used to form the con properties of the individual conjugate(s). jugate, the atom or atoms attached to the secondary carbon 25 If desired, conjugates having different molecular weights (other than the nitrogen from the active agent), will vary. In can be isolated using gel filtration chromatography. That is to Some instances, a methyl group is attached to the secondary say, gel filtration chromatography is used to fractionate dif carbon (i.e., one of the first carbon atom or second carbon ferently numbered polymer-to-active agent ratios (e.g., atom attached to the secondary carbon atom is methyl). In 1-mer, 2-mer, 3-mer, and so forth, wherein" 1-mer indicates other instances, a water-soluble polymer segment is attached 30 1 polymeric reagent to active agent, “2-mer indicates two to each one of the two carbonatoms (i.e., the first carbonatom polymeric reagents to active agent, and so on) on the basis of and second carbon atom attached to the secondary carbon their differing molecular weights (where the difference cor atom) attached to the secondary carbonatom, thereby provid responds essentially to the average molecular weight of the ing two polymer-containing 'arms’ connected through the water-soluble polymer segments). For example, in an exem secondary carbon atom. In these instances, the conjugate 35 plary reaction where a 100,000 Dalton protein is randomly comprises a second water-soluble polymer segment attached, conjugated to a ketone-containing PEG having a molecular either directly or though one or more atoms, to the secondary weight of about 20,000 Daltons, the resulting reaction mix carbon atom. In still other instances, the water-soluble poly ture may contain unmodified protein (having a molecular meric segment itself may be branched, such when a poly weight of about 100,000 Daltons), monoPEGylated protein meric reagent shown in Formula V is conjugated to an active 40 (having a molecular weight of about 120,000 Daltons), diP agent. EGylated protein (having a molecular weight of about 140, With respect to reductive amination, the conjugates pref 000 Daltons), and so forth. erably lack a hydroxyl moiety attached to the secondary car While this approach can be used to separate PEG and other bon atom, which can result from direct addition (or “1.2 polymer-active agent conjugates having different molecular addition') of a nucleophile to, for example, a carbonyl of an 45 weights, this approach is generally ineffective for separating C.f3-unsaturated carbonyl. In addition, the conjugates prefer positional isomers having different polymer attachment sites ably lack a carbonyl attached to a carbon adjacent to the within the protein. For example, gel filtration chromatogra secondary carbon atom (i.e., lacking a carbonyl to the phy can be used to separate from each other mixtures of PEG secondary carbon atom), which can result from conjugate 1-mers, 2-mers, 3-mers, and so forth, although each of the addition (or "1.4 addition'). 50 recovered PEG-mer compositions may contain PEGs As shown in FIG. 3, a nitrogen from a primary amine attached to different reactive amino groups (e.g., lysine resi containing active agent (e.g., the primary amine from alysine dues) within the active agent. side chain of a protein, the N-terminal amine of a protein, and Gel filtration columns suitable for carrying out this type of So forth) was used to form the conjugate. Conjugation via separation include SuperdexTM and SephadexTM columns reductive animation can also occur when the active agent 55 available from Amersham Biosciences (Piscataway, N.J.). comprises a secondary amine. In this case, the resulting con Selection of a particular column will depend upon the desired jugate comprises a tertiary amine (derived from the active fractionation range desired. Elution is generally carried out agent) linked to a secondary carbon (derived from the poly using a Suitable buffer, such as phosphate, acetate, or the like. meric reagent). The collected fractions may be analyzed by a number of Other approaches for conjugating the polymeric reagents 60 different methods, for example, (i) optical density (OD) at to active agents can also be used. For example, polymeric 280 nm for protein content, (ii) bovine serum albumin (BSA) reagents having an O.B-unsaturated functional group (e.g., an protein analysis, (iii) iodine testing for PEG content (Sims et C.f3-unsaturated ketone) can participate in direct addition, al. (1980) Anal. Biochem, 107:60-63), and (iv) sodium dode wherein a nucleophile (e.g., amine) can react by nucleophilic cyl sulfate polyacrylamide gel electrophoresis (SDS PAGE), addition via direct addition (or "1.2 addition') to the func 65 followed by staining with barium iodide. tional group. In addition, these polymeric reagents can par Separation of positional isomers is carried out by reverse ticipate in conjugate addition (or "1.4 addition'), wherein the phase chromatography using a reverse phase-high perfor US 8,865,149 B2 63 64 mance liquid chromatography (RP-HPLC) C18 column (Am bone morphogenic proteins such as bone morphogenic pro ersham BioSciences or Vydac) or by ion exchange chroma tein-2, bone morphogenic protein-6, OP-1; acidic fibroblast tography using an ion exchange column, e.g., a Sepharose"M growth factor, basic fibroblast growth factor, CD-40 ligand, ion exchange columnavailable from Amersham BioSciences. heparin, human serum albumin, low molecular weight hep Either approach can be used to separate polymer-active agent arin (LMWH), interferons such as interferon alpha, interferon isomers having the same molecular weight (positional iso beta, interferon gamma, interferon omega, interferon tau, mers). consensus interferon; interleukins and interleukin receptors The polymeric reagents described herein can be attached, Such as interleukin-1 receptor, interleukin-2, interluekin-2 either covalently or noncovalently, to a number of entities fusion proteins, interleukin-1 receptor antagonist, interleu including films, chemical separation and purification Sur 10 kin-3, interleukin-4, interleukin-4 receptor, interleukin-6. faces, Solid Supports, metal Surfaces such as gold, titanium, interleukin-8, interleukin-12, interleukin-13 receptor, inter tantalum, niobium, aluminum, Steel, and their oxides, silicon leukin-17 receptor; lactoferrin and lactoferrin fragments, oxide, macromolecules (e.g., proteins, polypeptides, and so luteinizing hormone releasing hormone (LHRH), insulin, forth), and Small molecules. Additionally, the polymeric pro-insulin, insulin analogues (e.g., mono-acylated insulinas reagents can also be used in biochemical sensors, bioelec 15 described in U.S. Pat. No. 5,922,675), amylin, C-peptide, tronic Switches, and gates. The polymeric reagents can also be Somatostatin, Somatostatin analogs including octreotide, employed as carriers for peptide synthesis, for the preparation vasopressin, follicle stimulating hormone (FSH), influenza of polymer-coated Surfaces and polymer grafts, to prepare vaccine, insulin-like growth factor (IGF), insulintropin, mac polymer-ligand conjugates for affinity partitioning, to pre rophage colony stimulating factor (M-CSF), plasminogen pare cross-linked or non-cross-linked hydrogels, and to pre activators such as alteplase, urokinase, reteplase, streptoki pare polymer-cofactor adducts for bioreactors. nase, pamiteplase, lanoteplase, and teneteplase; nerve growth A biologically active agent for use in coupling to a poly factor (NGF), osteoprotegerin, platelet-derived growth fac meric reagent as presented herein may be any one or more of tor, tissue growth factors, transforming growth factor-1, Vas the following. Suitable agents can be selected from, for cular endothelial growth factor, leukemia inhibiting factor, example, hypnotics and sedatives, psychic energizers, tran 25 keratinocyte growth factor (KGF), glial growth factor (GGF), quilizers, respiratory drugs, anticonvulsants, muscle relax T Cell receptors, CD molecules/antigens, tumor necrosis fac ants, antiparkinson agents (dopamine antagnonists), analge tor (TNF), monocyte chemoattractant protein-1, endothelial sics, anti-inflammatories, antianxiety drugs (anxiolytics), growth factors, parathyroid hormone (PTH), glucagon-like appetite Suppressants, antimigraine agents, muscle contrac peptide. Somatotropin, thymosin alpha 1, rasburicase, thy tants, anti-infectives (, antivirals, antifungals, vac 30 mosin alpha 1 IIb/IIIa inhibitor, thymosin beta 10, thymosin cines) antiarthritics, antimalarials, antiemetics, anepileptics, beta 9, thymosin beta 4, alpha-1 antitrypsin, phosphodi bronchodilators, cytokines, growth factors, anti-cancer esterase (PDE) compounds, VLA-4 (very late antigen-4), agents, antithrombotic agents, antihypertensives, cardiovas VLA-4 inhibitors, bisphosponates, respiratory syncytial cular drugs, antiarrhythmics, antioxicants, anti-asthma virus antibody, cystic fibrosis transmembrane regulator agents, hormonal agents including contraceptives, sympatho 35 (CFTR) gene, deoxyreibonuclease (Dnase), bactericidal/per mimetics, diuretics, lipid regulating agents, antiandrogenic meability increasing protein (BPI), and anti-CMV antibody. agents, antiparasitics, anticoagulants, neoplastics, antine Exemplary monoclonal antibodies include etanercept (a oplastics, hypoglycemics, nutritional agents and Supple dimeric fusion protein consisting of the extracellular ligand ments, growth Supplements, antienteritis agents, vaccines, binding portion of the human 75 kD TNF receptor linked to antibodies, diagnostic agents, and contrasting agents. 40 the Fc portion of IgG1), abciximab, adalimumab, afelimo More particularly, the active agent may fall into one of a mab, alemtuzumab, antibody to B-lymphocyte, atlizumab, number of structural classes, including but not limited to basiliximab, bevacizumab, biciromab, bertilimumab, CDP Small molecules (preferably insoluble Small molecules), pep 571, CDP-860, CDP-870, cetuximab, clenoliximab, dacli tides, polypeptides, proteins, antibodies, antibody fragments, Zumab, eculizumab, edrecolomab, efalizumab, epratuZumab, polysaccharides, steroids, nucleotides, oligonucleotides, 45 fontolizumab, gavilimomab, gemtuzumab ozogamicin, ibri polynucleotides, fats, electrolytes, and the like. Preferably, an tumomab tiuXetan, infliximab, inolimomab, keliximab, active agent for coupling to a polymer as described herein labetuzumab, lerdelimumab, olizumab, radiolabeled lym-1, possesses a native amino group, or alternatively, is modified metelimumab, mepolizumab, mitumomab, muromonad to contain at least one reactive amino group Suitable for con CD3, nebacumab, natalizumab, odulimomab, omalizumab, jugating to a polymer described herein. 50 oregovomab, palivizumab, pemtumomab, pexelizumab, Specific examples of active agents Suitable for covalent rhuMAb-VEGF, rituximab, satumomab pendetide, attachment include but are not limited to aspariginase, Sevirumab, siplizumab, to situmomab, I'' to situmomab, tras amdoxovir (DAPD), antide, becaplermin, calcitonins, tuZumab, tuvirumab, visilizumab, tacrine, memantine, cyanovirin, denileukin diftitox, erythropoietin (EPO), EPO rivastigmine, galantamine, donepezil, levetiracetam, repa agonists (e.g., peptides from about 10-40 amino acids in 55 glinide, atorvastatin, alefacept, Vardenafil. Sildenafil, and length and comprising a particular core sequence as described Valacyclovir. in WO 96/40749), dornase alpha, erythropoiesis stimulating Additional agents suitable for covalent attachment include, protein (NESP), coagulation factors such as Factor V. Factor but are not limited to, adefovir, alosetron, amifostine, amio VII, Factor VIIa, Factor VIII, Factor IX, Factor X, Factor XII, darone, aminocaproic acid, aminohippurate sodium, amino Factor XIII, von Willebrand factor; ceredase, cerezyme, 60 glutethimide, aminolevulinic acid, aminosalicylic acid, alpha-glucosidase, collagen, cyclosporin, alpha defensins, amsacrine, anagrelide, anastroZole, aripiprazole, asparagi beta defensins, exedin-4, granulocyte colony stimulating fac nase, anthracyclines, bexarotene, bicalutamide, bleomycin, tor (GCSF), thrombopoietin (TPO), alpha-1 proteinase buserelin, buSulfan, cabergoline, capecitabine, carboplatin, inhibitor, elcatonin, granulocyte macrophage colony stimu carmustine, chlorambucin, cilastatin Sodium, cisplatin, lating factor (GMCSF), fibrinogen, filgrastim, growth hor 65 cladribine, clodronate, cyclophosphamide, cyproterone, cyt mones human growth hormone (hGH), growth hormone arabine, camptothecins, 13-cis retinoic acid, all trans retinoic releasing hormone (GHRH), GRO-beta, GRO-beta antibody, acid; dacarbazine, dactinomycin, daunorubicin, deferoxam US 8,865,149 B2 65 66 ine, dexamethasone, diclofenac, diethylstilbestrol, docetaxel, Preferred peptides or proteins for coupling to a polymeras doxorubicin, dutasteride, epirubicin, estramustine, etopo described herein include EPO, IFN-C, IFN-B, consensus IFN, side, exemestane, eZetimibe, feXofenadine, fludarabine, Factor VIII, Factor IX, GCSF, GMCSF, hCH, insulin, FSH, fludrocortisone, fluorouracil, fluoxymesterone, flutamide, and PTH. fondaparinux, fulvestrant, gamma-hydroxybutyrate, gemcit The above exemplary biologically active agents are meant abine, epinephrine, L-Dopa, hydroxyurea, idarubicin, ifosfa to encompass, where applicable, analogues, agonists, antago mide, imatinib, irinotecan, itraconazole, goserelin, letrozole, nists, inhibitors, isomers, and pharmaceutically acceptable leucovorin, levamisole, lisinopril, lovothyroxine Sodium, salt forms thereof. In reference to peptides and proteins, the lomustine, mechlorethamine, medroxyprogesterone, mege invention is intended to encompass synthetic, recombinant, strol, melphalan, mercaptopurine, metaraminol bitartrate, 10 methotrexate, metoclopramide, mexiletine, mitomycin, native, glycosylated, and non-glycosylated forms, as well as mitotane, mitoxantrone, , nicotine, nilutamide, biologically active fragments thereof. In addition, the term nitisinone, octreotide, oxaliplatin, pamidronate, pentostatin, “active agent' is intended to encompass the active agent prior pilcamycin, porfimer, prednisone, procarbazine, prochlor to conjugation as well as the active agent “residue' following perazine, ondansetron, palonosetron, oxaliplatin, raltitrexed, 15 conjugation. Sirolimus, Streptozocin, tacrolimus, pimecrolimus, tamox ifen, , temozolomide, teniposide, testosterone, tet V. Pharmaceutical Preparations rahydrocannabinol, thalidomide, thioguanine, thiotepa, topo tecan, treprostinil, tretinoin, Valdecoxib, celecoxib, The present invention also includes pharmaceutical prepa rofecoxib, valrubicin, vinblastine, Vincristine, vindesine, rations comprising a conjugate as provided herein in combi Vinorelbine, Voriconazole, dolasetron, granisetron; formot nation with a pharmaceutical excipient. Generally, the con erol, fluticaSone, leuprolide, midazolam, alprazolam, ampho jugate itself will be in a solid form (e.g., a precipitate), which tericin B, podophylotoxins, nucleoside antivirals, aroyl can be combined with a suitable pharmaceutical excipient hydrazones, Sumatriptan, eletriptan, macrollides such as that can be in either solid or liquid form. erythromycin, oleandomycin, troleandomycin, roXithromy 25 Exemplary excipients include, without limitation, those cin, clarithromycin, davercin, azithromycin, flurithromycin, selected from the group consisting of , inor dirithromycin, josamycin, spiromycin, midecamycin, lorata ganic salts, antimicrobial agents, , Surfactants, dine, desloratadine, leucomycin, miocamycin, rokitamycin, buffers, acids, bases, and combinations thereof. andazithromycin, and Swinolide A, fluoroquinolones such as A Such as a Sugar, a derivatized Sugar Such as ciprofloxacin, ofloxacin, levofloxacin, trovafloxacin, alatrof 30 an alditol, aldonic acid, an esterified Sugar, and/or a Sugar loxacin, moxifloxicin, norfloxacin, enoxacin, grepafloxacin, polymer may be present as an excipient. Specific carbohy gatifloxacin, lomefloxacin, sparfloxacin, temafloxacin, drate excipients include, for example: monosaccharides, such pefloxacin, amifloxacin, fleroxacin, to Sufloxacin, prulifloxa as fructose, , galactose, glucose, D-mannose, Sorbose, cin, irloxacin, paZufloxacin, clinafloxacin, and sitafloxacin; and the like; , such as , , treha aminoglycosides Such as gentamicin, netilmicin, paramecin, 35 lose, cellobiose, and the like; polysaccharides, such as raffi tobramycin, amikacin, kanamycin, neomycin, and strepto nose, melezitose, maltodextrins, dextrans, , and the mycin, Vancomycin, teicoplanin, rampolanin, mideplanin, like; and alditols, such as . , , , colistin, daptomycin, gramicidin, colistimethate; polymixins Xylitol, (glucitol), pyranosyl Sorbitol, myoinositol, Such as polymixin B, capreomycin, bacitracin, penems; peni and the like. cillins including peniclinase-sensitive agents like penicillin 40 The excipient can also include an inorganic salt or buffer G. penicillin V: peniclinase-resistant agents like methicillin, Such as citric acid, Sodium chloride, potassium chloride, oxacillin, cloxacillin, dicloxacillin, floxacillin, nafcillin; , potassium nitrate, sodium phosphate gram negative microorganism active agents like amplicillin, monobasic, sodium phosphate dibasic, and combinations amoxicillin, and hetacillin, cillin, and galampicillin; antip thereof. seudomonal penicillins like carbenicillin, ticarcillin, azlocil 45 The preparation can also include an antimicrobial agent for lin, mezlocillin, and piperacillin; cephalosporins like ce?po preventing or deterring microbial growth. Nonlimiting doXime, cefprozil, ceftbuten, ceftizoxime, ceftriaxone, examples of antimicrobial agents suitable for the present cephalothin, cephapirin, cephalexin, cephradrine, cefoxitin, invention include benzalkonium chloride, benzethonium cefamandole, cefazolin, cephaloridine, cefaclor, cefadroxil, chloride, , cetylpyridinium chloride, chlorobu cephaloglycin, cefuroxime, ceforanide, cefotaxime, cefatriz 50 tanol, phenol, phenylethyl alcohol, phenylmercuric nitrate, ine, cephacetrile, cefepime, cefixime, cefonicid, cefopera thimersol, and combinations thereof. Zone, cefotetan, cefimetazole, ceftazidime, loracarbef, and An can be present in the preparation as well. moxalactam, monobactams like aztreonam; and carbapen Antioxidants are used to prevent oxidation, thereby prevent ems such as imipenem, meropenem, and ertapenem, penta ing the deterioration of the conjugate or other components of midine isetionate, albuterol Sulfate, lidocaine, metaproter 55 the preparation. Suitable antioxidants for use in the present enol Sulfate, beclomethasone diprepionate, triamcinolone invention include, for example, ascorbyl palmitate, butylated acetamide, budesonide acetonide, fluticaSone, ipratropium hydroxyanisole, butylated hydroxytoluene, hypophospho bromide, flunisolide, cromolyn Sodium, and ergotamine tar rous acid, monothioglycerol, propyl gallate, sodium bisulfite, trate; taxanes Such as paclitaxel, SN-38, and tyrphostines. sodium formaldehyde sulfoxylate, sodium metabisulfite, and Preferred Small molecules for coupling to a polymer as 60 combinations thereof. described herein are those having at least one naturally occur A Surfactant can be present as an excipient. Exemplary ring amino group. Preferred molecules such as these include surfactants include: polysorbates, such as “Tween 20' and aminohippurate Sodium, amphotericin B, doxorubicin, ami “Tween 80 and pluronics such as F68 and F88 (both of nocaproic acid, aminolevulinic acid, aminosalicylic acid, which are available from BASF, Mount Olive, N.J.); sorbitan metaraminol bitartrate, pamidronate disodium, daunorubi 65 esters; lipids, such as phospholipids such as lecithin and other cin, levothyroxine Sodium, lisinopril, cilastatin Sodium, phosphatidylcholines, phosphatidylethanolamines (although mexiletine, cephalexin, deferoxamine, and amifostine. preferably not in liposomal form), fatty acids and fatty esters; US 8,865,149 B2 67 68 steroids, Such as cholesterol; and chelating agents, such as vehicle prior to use, and emulsions and liquid concentrates for EDTA, Zinc and other such suitable cations. dilution prior to administration, among others. Acids or bases can be present as an excipient in the prepa ration. Nonlimiting examples of acids that can be used VI. Methods of Administering include those acids selected from the group consisting of 5 hydrochloric acid, acetic acid, phosphoric acid, citric acid, The invention also provides a method for administering a malic acid, lactic acid, formic acid, trichloroacetic acid, nitric conjugate as provided herein to a patient Suffering from a acid, perchloric acid, phosphoric acid, Sulfuric acid, fumaric condition that is responsive to treatment with conjugate. The acid, and combinations thereof. Examples of Suitable bases method comprises administering, generally via injection, a include, without limitation, bases selected from the group 10 therapeutically effective amount of the conjugate (preferably consisting of sodium hydroxide, Sodium acetate, ammonium provided as part of a pharmaceutical preparation). The hydroxide, potassium hydroxide, ammonium acetate, potas method of administering may be used to treat any condition sium acetate, sodium phosphate, potassium phosphate, that can be remedied or prevented by administration of the Sodium citrate, sodium formate, sodium Sulfate, potassium 15 particular conjugate. Those of ordinary skill in the art appre Sulfate, potassium fumerate, and combinations thereof. ciate which conditions a specific conjugate can effectively The pharmaceutical preparations encompass all types of treat. The actual dose to be administered will vary depend formulations and in particular those that are Suited for injec upon the age, weight, and general condition of the Subject as tion, e.g., powders that can be reconstituted as well as Sus well as the severity of the condition being treated, the judg pensions and solutions. The amount of the conjugate (i.e., the ment of the health care professional, and conjugate being conjugate formed between the active agent and the polymer administered. Therapeutically effective amounts are known described herein) in the composition will vary depending on to those skilled in the art and/or are described in the pertinent a number of factors, but will optimally be a therapeutically reference texts and literature. Generally, a therapeutically effective dose when the composition is stored in a unit dose effective amount will range from about 0.001 mg to 100 mg. container (e.g., a vial). In addition, the pharmaceutical prepa 25 preferably in doses from 0.01 mg/day to 75 mg/day, and more ration can be housed in a syringe. A therapeutically effective preferably in doses from 0.10 mg/day to 50 mg/day. dose can be determined experimentally by repeated adminis The unit dosage of any given conjugate (again, preferably tration of increasing amounts of the conjugate in order to provided as part of a pharmaceutical preparation) can be determine which amount produces a clinically desired end administered in a variety of dosing schedules depending on point. 30 the judgment of the clinician, needs of the patient, and so The amount of any individual excipient in the composition forth. The specific dosing schedule will be known by those of will vary depending on the activity of the excipient and par ordinary skill in the art or can be determined experimentally ticular needs of the composition. Typically, the optimal using routine methods. Exemplary dosing schedules include, amount of any individual excipient is determined through without limitation, administration five times a day, four times routine experimentation, i.e., by preparing compositions con 35 a day, three times a day, twice daily, once daily, three times taining varying amounts of the excipient (ranging from low to weekly, twice weekly, once weekly, twice monthly, once high), examining the stability and other parameters, and then monthly, and any combination thereof. Once the clinical end determining the range at which optimal performance is point has been achieved, dosing of the composition is halted. attained with no significant adverse effects. One advantage of administering the conjugates of the Generally, however, the excipient will be present in the 40 present invention is that individual water-soluble polymer composition in an amount of about 1% to about 99% by portions can be cleaved off. Such a result is advantageous weight, preferably from about 5%-98% by weight, more pref when clearance from the body is potentially a problem erably from about 15-95% by weight of the excipient, with because of the polymer size. Optimally, cleavage of each concentrations less than 30% by weight most preferred. water-soluble polymer portion is facilitated through the use of These foregoing pharmaceutical excipients along with 45 physiologically cleavable and/or enzymatically degradable other excipients are described in “Remington: The Science & linkages such as urethane, amide, carbonate or ester-contain Practice of Pharmacy”, 19' ed., Williams & Williams, ing linkages. In this way, clearance of the conjugate (via (1995), the “Physician's Desk Reference”, 52" ed., Medical cleavage of individual water-soluble polymer portions) can Economics, Montvale, N.J. (1998), and Kibbe, A. H., Hand be modulated by selecting the polymer molecular size and the book of Pharmaceutical Excipients, 3" Edition, American 50 type functional group that would provide the desired clear Pharmaceutical Association, Washington, D.C., 2000. ance properties. One of ordinary skill in the art can determine The pharmaceutical preparations of the present invention the proper molecular size of the polymeras well as the cleav are typically, although not necessarily, administered via able functional group. For example, one of ordinary skill in injection and are therefore generally liquid Solutions or Sus the art, using routine experimentation, can determine a proper pensions immediately prior to administration. The pharma 55 molecular size and cleavable functional group by first prepar ceutical preparation can also take other forms such as syrups, ing a variety of polymer derivatives with different polymer creams, ointments, tablets, powders, and the like. Other weights and cleavable functional groups, and then obtaining modes of administration are also included, such as pulmo the clearance profile (e.g., through periodic blood or urine nary, rectal, transdermal, transmucosal, oral, intrathecal, Sub sampling) by administering the polymer derivative to a cutaneous, intra-arterial, and so forth. 60 patient and taking periodic blood and/or urine sampling. As previously described, the conjugates can be adminis Once a series of clearance profiles have been obtained for tered injected parenterally by intravenous injection, or less each tested conjugate, a Suitable conjugate can be identified. preferably by intramuscular or by Subcutaneous injection. It is to be understood that while the invention has been Suitable formulation types for parenteral administration described in conjunction with the preferred specific embodi include ready-for-injection solutions, dry powders for com 65 ments thereof, that the foregoing description as well as the bination with a solvent prior to use, Suspensions ready for examples that follow are intended to illustrate and not limit injection, dry insoluble compositions for combination with a the scope of the invention. Other aspects, advantages and US 8,865,149 B2 69 70 modifications within the scope of the invention will be appar Formation of mPEG (2K Da)-methyl Ketone ent to those skilled in the art to which the invention pertains. To a stirred Suspension of molecular sieves 4A (0.5 g), All articles, books, patents and other publications refer potassium carbonate (0.34g, 2.46 mmol), and N-chlorosuc enced herein are hereby incorporated by reference in their cinimide (0.037g, 0.277 mmol) in 5 mL of anhydrous dichlo 5 romethane were successively added mPEG (2K Da)-2-Hy entireties. droxypropane (0.5 g., 0.25 mmol) and N-tert VII. Experimental butylbenzenesulfenamide (0.009 g, 0.050 mmol) at 0°C. The reaction mixture was stirred at the same temperature for 2 The practice of the invention will employ, unless otherwise hours. Next, the solvent was evaporated to dryness under 10 reduced pressure. The residue was dissolved in 20 mL water, indicated, conventional techniques of organic synthesis and the pH was adjusted to 4.5 with 10% of HPO, and the the like, which are within the skill of the art. Such techniques desired product was extracted with dichloromethane (20 are fully explained in the literature. See, for example, J. mLx2). The extract was dried with anhydrous sodium sulfate, March, Advanced Organic Chemistry: Reactions Mecha concentrated under vacuum, and precipitated with 20 ml of a nisms and Structure, 4th Ed. (New York: Wiley-Interscience, 15 mixture of and diethyl ether (1:1). The 1992), supra. precipitated product was collected through vacuum filtration In the following examples, efforts have been made to and dried under vacuum overnight. Yield: 0.35 g NMR (de ensure accuracy with respect to numbers used (e.g., amounts, DMSO): 2.04 ppm (s, —CH, 3H), 3.24 ppm (s, —OCH, temperatures, etc.) but some experimental error and deviation 3H), 3.51 ppm (s, polymer backbone), 4.11 ppm (s. —OCH should be accounted for. Unless indicated otherwise, tem (C=O) , 2H). perature is in degrees C and pressure is at or near atmospheric Depending on the conditions, the ketone exists in variable pressure at sea level. All reagents were obtained commer amounts of ketone and ketone hydrate. The ketone and ketone cially unless otherwise indicated. All molecular weights are hydrate are in equilibrium in the presence of water, both of in terms of weight average molecular weight and are which can be used (either alone or in combination) to form expressed in units of Daltons (“Da') unless otherwise noted. 25 polymer-active agent conjugates. The following abbreviations are used herein and elsewhere in PEGylation of Lysozyme the specification: Lysozyme (3.0 mg) was dissolved in 1 mL of 20 mM DCC dicyclohexyl carbodiimide sodium phosphate buffer (pH 7.5) and MPEG (2K Da)-me HOBT 1-hydroxybenzotriazole thyl ketone (8.4 mg. 20 fold molar excess) was added. After 30 15 minutes, 0.159 M solution of NaCNBH (66 uL, 50 fold MALDI-TOF matrix-assisted laser desorption/ionization molar excess) was then added and the solution was stirred for time of flight 17 hours at room temperature. Analysis of the reaction mix SDS-PAGE -polyacrylamide gel ture by SDS-PAGE showed that PEGylated lysozyme was electrophoresis formed. K—when used in the context of describing molecular weight, 35 This example is repeated several times using different sizes e.g., (2K), indicates thousand of polymer. First, mPEG mesylate of different sizes are used NMR-nuclear magnetic resonance in place of mPEG mesylate to provide a variety of mPEG Example 1 methyl ketones having different sizes. Specifically, the fol lowing different sizes of mPEG mesylate are used: mPEGs 40 mesylate; mPEGomesylate; and mPEGomesylate. Once the mPEG methyl ketones having a variety of different sizes are formed, each is conjugated to lysozyme using the proce Method A: mPEG(2K Da)-Methyl Ketone with C. Ether Bond dure described above. Results similar those obtained for the NAH mPEG methyl ketone are obtained. mPEGK-OMs + CH-CH-CH-OH - - - Toluene 45 OH Example 2 mPEG2k-O o CHCH CH3 mPEG (5K Da)-Methyl Ketone OH mPEG2k-O-CH2C-CH 50 Method B: mPEG (5K Da)-Methyl Ketone with C. O Ether Bond

Formation of mPEG (2K Da)-2-Hydroxypropane Formation of Ethyl 2-benzyloxylactate To an azeotropically dried solution of mPEG, Mesylate (mPEGOMS. Nektar Therapeutics, 2 g, 1 mmole) in 50 mL 55 of anhydrous toluene, 1,2-propanediol (1.522 g, 20 mmole) OH O and sodium hydride (60% in , 0.800g, 20 mmole) HC-CH-C-O-CH-CH He were added. The reaction mixture was stirred for 20 hours at 80° C. under nitrogen atmosphere. The insoluble precipitate ethyl lactate was filtered off and the filtrate was concentrated under 60 CH vacuum to dryness. The crude product was dissolved in 3 mL of dichloromethane and precipitated with 100 mL of isopro ( )—ci-o-o-c-ol pyl alcohol-diethyl ether (1:1) mixture. The final product was ethyl 2-benzyloxylactate collected using vacuum filtration and dried under vacuum overnight. Yield: 1.4 g. NMR (d-DMSO): 1.01 ppm (d. 65 —CH, 3H), 3.24 ppm (s, —OCH 3H), 3.51 ppm (s, poly Trifluoromethanesulfonic acid (1.0 mL) was added gradu merbackbone), 4.54 ppm (d. —OH, 1H). ally to a stirred mixture of ethyl lactate (8.90 g, 75 mmole), US 8,865,149 B2 71 72 benzyl 2.2.2-trichloroacetimidate (22.7 g, 90 mmole), anhy hydride (60% dispersion in mineral oil, 0.36 g, 9 mmole) drous cyclohexane (100 mL), and anhydrous dichlo were added and the reaction mixture was stirred at 85°C. romethane (50 mL). The mixture was stirred overnight at overnight under a nitrogen atmosphere. After cooling to room room temperature under argon atmosphere. The mixture was temperature, the mixture was filtered and the solvent was filtered, and the filtrate was washed with saturated solution of 5 distilled off under reduced pressure. The residue was dis sodium hydrogen carbonate (250 mL), and with distilled solved in dichloromethane (20 mL) and precipitated with 350 water (250 mL). Then, the solution was dried with anhydrous mL isopropyl alcohol at 0-5°C. The precipitated product was sodium sulfate and the solvents were distilled under reduced filtered off and dried under reduced pressure. Yield: 12.7 g. pressure using a rotary evaporator. The residue was subjected NMR (d-DMSO): 1.10 ppm (d. —CH, 3H), 3.24 ppm (s. to fractional vacuum distillation giving 9.6 g of colorless 10 —OCh. 3H), 3.51 ppm (s, polymer backbone), 4.53 ppm (s. product. NMR (CDC1): 1.21 ppm (t, —CHCH 3H), 1.32 —CH2—, 2H), 7.33 ppm (m, 5H). ppm (d. —CH2—, 3H), 4.13 ppm (m, —CH- and —CH Formation of mPEG (5K Da)-2-hydroxypropane CH, 3H), 4.50 ppm (m, —CH2—, 2H), 7.32 ppm (m, 5H). Formation of 2-benzyloxy-1-propanol A solution of ethyl 2-benzyloxylactate (9.5g, 45.6 mmole) 15 in anhydrous tetrahydrofuran (30 mL) was added dropwise during 30 minutes to a stirred solution of lithium aluminum 1-c-() -e- hydride (1.06 g. 28 mmole) in anhydrous tetrahydrofuran (56 mPEGsk-OCH2CHCH mL) at room temperature under argon atmosphere. Next, the mPEG (5K Da)-2-benzyloxypropane mixture was stirred for 3 hours at 60°C. After cooling to 30° OH C., freshly prepared saturated solution of sodium sulfate (10 mPEGsk-OCH2CHCH mL) was slowly added. The mixture was filtered and the mPEG (5K Da)-2-hydroxypropane solvent was distilled under reduced pressure. The residue was dissolved in dichloromethane (100 mL) and the solution was 25 dried with anhydrous . The solvent then A mixture of mPEG (5K Da)-2-benzyloxypropane (8.8 g), was distilled under reduced pressure. The residue was sub ethyl alcohol (120 mL), and palladium (10% on active car jected to fractional vacuum distillation giving 6.2 g of color bon, 0.8 g) was hydrogenated overnight under 45 psi of less liquid product. NMR (d-DMSO): 1.90 ppm (d. —CH, hydrogen. The mixture was filtered and the solvent was dis 3H), 3.20-3.50 ppm (bm, —CH- and —CH-, 3H), 4.53 30 tilled off under reduced pressure. The crude product was ppm (s. —CH2—, 2H), 4.63 ppm (t, —OH, 1H), 7.33 ppm dissolved in dichloromethane (15 mL) and precipitated with (m, 5H). 300 mL isopropyl alcohol at 0-5°C. The precipitated product Formation of mPEG (5 K Da)-mesylate was filtered off and dried under reduced pressure. Yield: 6.1 g. A solution of mPEG (5K Da)-OH (25.0 g, 5 mmole) in NMR (d-DMSO): 1.01 ppm (d. —CH, 3H), 3.24 ppm (s. toluene (150 mL) was azeotropically dried by distilling off 35 —OCH,3H), 3.51 ppm (s, polymer backbone), 4.54 ppm (d. toluene under reduced pressure. The dried mPEG (5K Da)- OH, 1H). OH was dissolved in a mixture of anhydrous toluene (150 Formation of mPEG (5K Da)-Methyl Ketone mL) and anhydrous dichloromethane (30 mL). Next, triethy lamine (0.9 mL, 6.5 mmole) and methanesulfonyl chloride (0.45 mL, 5.8 mmole) were added and the mixture was stirred 40 OH overnight at room temperature under nitrogen atmosphere. Dess-Martin periodinane The solvents were then distilled off under reduced pressure. mPEGSK-OCH2CHCH -e- Thereafter, the residue was dissolved in dichloromethane (40 mPEG (5K Da)-2-hydroxypropane mL) and 650 mL isopropyl alcohol was added. The mixture was stirred for 30 minutes at 0°C. and a precipitated product 45 mPEGSK-OCH2CHCH was filtered off and dried under vacuum to yield 22 g of the mesylate derivative shown by NMR to be 100% substituted. mPEG (5K Da)-methyl ketone Formation of mPEG (5K Da)-2-benzyloxypropane mPEG (5K Da)-2-Hydroxypropane (4.0 g) was added to a 50 solution of Dess-Martin periodinane (0.44 g) in dichlo romethane (20 mL) and the mixture was stirred for 6 hours at room temperature. Next, the solvent was distilled off under NaH reduced pressure. The residue was then dissolved in warm + CHCHCH-OH--() Her mPEGSK-OMs toluene isopropyl alcohol (100 mL), and ethyl ether (50 mL) was mPEGSK-mesylate 2-benzyloxy-1-propanol 55 added. The mixture was stirred for 15 minutes. The precipi tated product was filtered off and dried under reduced pres sure. Yield: 3.2g. NMR (d-DMSO): 2.04 ppm (s. —CH, 3H), 3.24 ppm (s. —OCH3H), 3.51 ppm (s, polymer back mPEG2k-OCH2CHCH--( ) bone), 4.11 ppm (s. —OCH (C=O)—, 2H). 60 Depending on the conditions, the ketone exists in variable mPEG (5K Da)-2-benzyloxypropane amounts of ketone and ketone hydrate. The ketone and ketone hydrate are in equilibrium in the presence of water, both of A solution of mPEG (5K Da)-mesylate (15g, 3 mmole) in which can be used (either alone or in combination) to form toluene (150 mL) was azeotropically dried by distilling off polymer-active agent conjugates. toluene under reduced pressure. The dried mPEG (5K Da)- 65 PEGylation of Lysozyme mesylate was dissolved in anhydrous toluene (150 mL), and Lysozyme (3.0 mg) was dissolved in 1 mL of 20 mM 2-benzyloxy-1-propanol (1.5 g, 9 mmole), and sodium sodium phosphate buffer (pH 7.5) and mPEG (5K Da)-me US 8,865,149 B2 73 74 thyl ketone (7.5 mg) was added. After 15 minutes, 0.1 M molar excess to the lysozyme) was added into the lysozyme solution of NaCNBH (42 uL) was added and the solution was solution. The vial was placed on a Roto MixTM orbital shaker stirred for 18 hours at room temperature. Analysis of the (Thermolyne Corp., Dubuque, Iowa) set at slow speed to reaction mixture by SDS-PAGE showed that PEGylated allow reaction to proceed at room temperature. After 15 min lysozyme was formed. utes, 0.066 mL of 159 mM aqueous NaCNBH (available This example is repeated several times using different sizes from, for example, Aldrich, St. Louis, Mo., product number of polymer. First, mPEG mesylate of different sizes are used 15,615-9) was added. This amount of NaCNBH represented in place of mPEGs mesylate to provide a variety of mPEG a 50-fold molar excess to the lysozyme. Aliquots (20 uL) methyl ketones having different sizes. Specifically, the fol were withdrawn at timed intervals (17 hours and 41 hours) lowing different sizes of mPEG mesylate are used: mPEG 10 from the reaction mixture and were analyzed by SDS-PAGE mesylate; mPEGomesylate; and mPEGomesylate. Once using a precast gel electrophoresis system based on a 10% the mPEG methyl ketones having a variety of different sizes Tris-HCl Ready GelTM precast gel (both available from Bio are formed, each is conjugated to lysozyme using the proce Rad Laboratories, Hercules, Calif.) and MALDI-TOF Mass dure described above. Results similar those obtained for the Spectroscopy using an OmniFLEX(R) MALDI-TOF Mass 15 Spectrometer (Bruker Daltonics, Inc., Bellerica, Mass.). mPEGs methyl ketone are obtained SDS-PAGE analysis indicated the presence of PEG deriva tives of lysozyme having 1, 2, and 3 PEG moieties attached. Example 3 MALDI-TOF mass spectrometry displayed peaks for three PEGylated derivatives of lysozyme at 19,660 Da, 25,069 Da, mPEG (5K Da)-Piperidone and its Corresponding and 30,568 Da, differing in mass by approximately 5000 Da. Ketone Hydrate The mass of unmodified lysozyme by MALDI-TOF was 14,198 Da. This example is repeated several times using different sizes HCI of polymer. First, mPEG-SPA of different sizes are used in 25 place of mPEG-SPA to provide a variety of MPEG piperi OH dones having different sizes. Specifically, the following dif mPEGs-SPA + HN He ferent sizes of mPEG-SPA are used: mPEG-SPA; OH mPEGo-SPA; and mPEG-SPA. Once the mPEG-piperi 4-Piperidone monohydrate hydrochloride dones having a variety of different sizes are formed, each is O 30 conjugated to lysozyme using the procedure described above. OH Results similar those obtained for the mPEGs-piperidone mPEGs-OCHCHC-N are obtained. OH Example 4 Formation of mPEG-piperidone 35 To a solution of the succinimidyl ester of mPEG (5K Da)- mPEG Methyl Ketone propionic acid (Nektar Therapeutics, mPEGs-SPA, 1.0g, 2 mmole.) in dichloromethane (20 mL), triethylamine (0.084 Method C: mPEG (5K Da) Methyl Ketone with B mL, 6 mmole) and 4-piperidone monohydrate hydrochloride Carbamido Group (0.077 g. 5 mmole) were added. The reaction mixture was 40 stirred at room temperature under nitrogen atmosphere over night. Insoluble precipitate was filtered and filtrate was evaporated to dryness. The crude product was dissolved in O | HOBT, DCC dichloromethane and precipitated with isopropyl alcohol. mPEGSK-NH2 + CHCCH2CH2COOH -->TEA The final product was collected through vacuum filtration and 45 Levulinic Acid dried under vacuum overnight. Yield: 0.88 g. NMR (de O O DMSO): 2.32 ppm (t, —CH C(OH), CH ), 2.42 ppm | (t, —CH2—C(OH)2CH ), 2.66 ppm (t, —OCH2 mPEGSK-N CCHCHCCH3 CHCON ), 3.24 ppm (S. —OCH), 3.51 ppm (S. polymer H backbone). 50 Depending on the conditions, the ketone hydrate exists in Levulinic acid (0.07 g. 6 mmole) was dissolved in anhy variable amounts of ketone hydrate and ketone. The ketone drous dichloromethane (20 mL). Next, mPEG (5K Da)-amine hydrate and ketone are in equilibrium in the presence of (Nektar Therapeutics, 2.0 g, 4 mmole), triethylamine (0.028 water, both of which can be used (either alone or in combi mL, 2 mmoles), 1-hydroxybenzotriazole (0.027 g, 2 mmole), nation) to form polymer-active agent conjugates. 55 and 1,3-dicyclohexylcarbodiimide (0.12 g. 6 mmole) were PEGylation of Lysozyme added. The reaction mixture was stirred at room temperature The protein lysozyme is used as a model to demonstrate the under nitrogen atmosphere overnight. Insoluble precipitate ability of the a given polymeric reagent to form conjugations was filtered off and the filtrate was evaporated to dryness. The with a protein. Lysozyme is a 129 amino acid secretory crude product was dissolved in dichloromethane and precipi enzyme containing six lysine residues, each having a side 60 tated with isopropyl alcohol (50 mL). The final product was chain terminating in an amine residue. Amine residues rep collected through vacuum filtration and dried under vacuum resent potential attachment sites for the polymeric reagents of overnight. Yield: 1.72 g. NMR (d-DMSO): 2.28 ppm (t, the invention. NH COCHCOCH, 2H), 2.32 ppm (s, -CH3H), 2.62 Lysozyme from chicken egg white (3 mg, Sigma, St. Louis, ppm (t, NH COCH-CHCOCH, 2H), 3.16 ppm (q. Mo., product number L-6876) was dissolved in 1 ml of 20 65 —CHCH-NH CO. , 2H), 3.24 ppm (s. —OCH, 3H), mM sodium phosphate buffer (pH 7.5, J. T. Baker, Phillips 3.51 ppm (s, polymer backbone), 7.87 ppm (t, —N burg, N.J.). m-PEGs-Ketone of Example 3 (21 mg, a 20-fold H CO CH 1H). US 8,865,149 B2 75 76 Depending on the conditions, the ketone exists in variable Methylation of 2-benzyloxy-1,3-bis(a)-hydroxypoly(ethyl amounts of ketone and ketone hydrate. The ketone and ketone ene glycol))propane, MW=6KDa hydrate are in equilibrium in the presence of water, both of CH-O-PEG-OH which can be used (either alone or in combination) to form CH-O 2 Methyl p-toluenesulfonate polymer-active agent conjugates. 5 CH-O-PEG-OH potassium tert-butoxide PEGylation of Lysozyme 2-benzyloxy-1,3-bis(omega Lysozyme (3.0 mg) was dissolved in 1 mL of 20 mM hydroxypoly(ethylene glycol)propane, sodium phosphate buffer (pH 7.5) and mPEG (5K Da)-Ke- MW = 6KDa CH-O-PEG-OCH tone (21 mg, 20 fold excess) prepared from this example was 10 2 u-r J-uur 13 added. After stirring for 15 minutes, 0.159 M solution of CH-O-( NaCNBH (66 uL, 50 fold molar excess) was added and the CH-O-PEG-O-CH solution was stirred 20hat room temperature. Analysis of the 2-benzyloxy-1,3-bis(omega reaction mixture by SDS-PAGE showed that PEGylated methoxypoly(ethylene glycol))propane, lysozyme was formed. 15 MW = 6KDa This example is repeated several times using different sizes 2-benzyloxy-1,3-bis(a)-hydroxypoly(ethylene glycol)) of polymer. First, mPEG-amine of different sizes are used in propane, MW=6K Da (10g, 1.67 mmol) in 400 mL of toluene place of mPEGs--amine to provide a variety of differently was azeotropically dried by distilling off toluene under sized mPEG methyl ketones with a? carbamido group. Spe- 20 reduced pressure. The residue was redissolved in 700 mL of cifically, the following different sizes of mPEG-amine are anhydrous toluene. To the Solution were added potassium used: mPEG-amine,- - - MPEGo-amine;- - - and mPEGo- tert-butoxidemmol) and methyl (1.0 Mp-toluenesulfonate solution in tert-, (3.5 mL, 1623.3 mL, mmol). 16.7 amine. Once the differently sized mPEG methyl ketones with The reaction mixture was stirred at 45° C. under nitrogen a B carbamido group are formed, each is conjugated to atmosphere overnight. The mixture was filtered and the fil lysozyme using the procedure described above. Results simi- 25 trate was evaporated to dryness under reduced pressure. The lar those obtained for the mPEGs methyl ketone with a B residue was dissolved in 500 mL of H.O. NaCl (50 g) was carbamido group are obtained. added and the pH of the solution was adjusted to 7.5. The product was extracted with dichloromethane (250 mLx2). The extract was dried with magnesium sulfate, concentrated Example 5 under vacuum and the product was precipitated with a mix "ture of isopropyl alcohol and diethyl ether (7:3, 500 mL). Next, the product was dried under vacuum overnight. Yield: PEG2 Ketone (Linkerless PEG2 Ketone) via 7.8 g. NMR (d6-DMSO): 3.24 ppm (s, OCH 6H), 3.51 Ethoxylation: PEG2(6K Da) Ketone ppm (S. polymer backbone), 4.61 ppm (S. —OCHCHs 2H), 7.33 ppm (m, —OCH2CHs 5H). 35 Formation of 2-hydroxy-1,3-bis(a)-methoxypoly(ethylene Formation of 2-benzyloxy-1,3-bis(a)-hydroxypoly(ethylene glycol))propane glycol))propane

CH-O-PEG-OCH H2C-OH 40 ( y CH-O ( O - NEM - CH-O-PEG-O-CH - ci? r , 2 H-C-OH O 2-benzyloxy-1,3-bis(omega-methoxypoly 2 (ethylene glycol) 2-benzyloxyglycerol then, neutralization 45 propane, MW = 6kDa CH-O-PEG-OCH p 'ye (OQ-NOH H-O ()-ci? N 50 CH-O-PEG-O-CH H-C-O2 ( N-1)son 2-hydroxy-1,3-bis(omega methoxypoly(ethylene glycol)) 2-benzyloxy-1,3-bis(omega propane, hydroxypoly(ethylene glycol))propane, MW = 6KDa 2-Benzyloxy-1,3-bis(c)-methoxypoly(ethylene glycol)) propane, MW=6K Da (5.0 g, 0.83 mmol) was dissolved in 2-Benzyloxyglycerol, available from Sigma-Aldrich (St. 100 mL of ethyl alcohol, and Pd(OH) (5% on active carbon, Louis, Mo.) was ethoxylated to form 2-benzyloxy-1,3-bis(a)- 0.25 g), and cyclohexene (0.84 mL, s: mmol) were added. hydroxypoly(ethylene glycol)propane, MW=6K Da. The 60 The suspension was refluxed for 2 hours. The catalyst was variable (m) in the above structures is a number to provide a filtered and filtrate was evaporated to dryness. The residue polymer weight of 3,000 Daltons, thereby providing a total was dissolved in dichloromethane (5 mL) and precipitated Weight of essentially 6,000 Daltons. NMR analysis of the with 300 mL of diethylether. The final product was collected product confirmed its structure and HPLC and GPC analysis through vacuum filtration and dried under vacuum overnight. confirmed its purity and molecular weight. This polymer was 65 Yield: 4.6 g. NMR (d-DMSO): 3.24 ppm (s, OCH 6H), used at the raw material for the ketone formed by the reaction 3.51 ppm (S. polymer backbone), 4.76 ppm (d. —CH-OH, scheme shown below. 1H). US 8,865,149 B2 77 78 Formation of mPEG2-(6K Da)-Ketone 0.159 M solution of NaCNBH (105 uL, 50 fold molar excess) was added and the solution was stirred for 20 hours at room temperature. Analysis of the reaction mixture by SDS CH-O-PEG-OCH PAGE showed that PEGylated lysozyme was formed. Dess-Martin periodinane 5 H-O -e- This example is repeated several times using different sizes of mPEG2-(6K Da)-ketone. The following different sizes of CH-O-PEG-O-CH mPEG2-(6KDa)-ketone are used: mPEG2-(1OKDa)-ketone; 2-benzyloxy-1,3-bis(omega mPEG2-(20K Da)-ketone; and mPEG2-(40K Da)-ketone. In methoxypoly(ethylene glycol))propane, each case, similar results to the mPEG2-(6K Da)-ketone are MW = 6KDa 10 obtained.

CH-O-PEG-OCH Example 6 CH-O-PEG-O-CH 15 mPEG-(6K-Da)-ketone MW = 6KDa mPEG2(40K Da)-Piperidone and its Hydrate Formation of mPEG2(40K)-Piperidone and its Hydrate

O mPEGok-O-C-NH HCI (CH2)4 O OH CH -- HN mPEGok-O--NH1 -o-, OH O O Succinimidyl ester of PEG-(40K)acid 4-Piperidone monohydrate hydrochloride

O O mPEGok-O-C-NH mPEGok-O-C-NH -HO (CH2)4 2 (CH2)4 O H2O O mPEGok-O--NH1 CH Sc-O-N OH mPEGok-O--NH1 CH YC1 N()- O OH O O

45 2-Hydroxy-1,3-bis(c)-methoxypoly(ethylene glycol))pro To a solution of succinimidyl ester of PEG2-(40K Da) acid pane, MW=6K Da (4.0 g, 0.67 mmol) was added to a stirred (Nektar Therapeutics, 10 g, 0.25 mmole) in dichloromethane (200 mL), triethylamine (0.100 ml, 0.72 mmole) and 4-pip solution of Dess-Martin periodinane (0.37g, 0.867 mmol) in eridone monohydrate hydrochloride (0.100 g, 0.65 mmole) dichloromethane (20 mL). The reaction mixture was stirred were added. The reaction mixture was stirred at room tem for 6 hours at the room temperature. Thereafter, it was con 50 perature under nitrogen atmosphere overnight. Next the Sol centrated and the product was precipitated with a mixture of vent was distilled offunder reduced pressure. The residue was isopropyl alcohol and diethyl ether (2:1, 300 mL). The pre dissolved in dichloromethane and precipitated with a mixture of isopropyl alcohol and ethyl ether. The product was col cipitate was collected through vacuum filtration and dried lected through vacuum filtration and dried under vacuum under vacuum overnight. Yield: 3.8 g. NMR (d-DMSO): ss overnight. Yield: 9.5 g. NMR (d-DMSO): 2.33 ppm and 2.43 3.24 ppm (s, —OCH, 6H), 3.51 ppm (s, polymer backbone), ppm (t, —CH C(OH)—CH2—, 4H), 3.24 ppm (s. 4.22 ppm (S. —O CH-C-O, 4H). —OCH, 6H), 3.51 ppm (s, polymer backbone), 4.36 ppm Depending on the conditions, the ketone exists in variable (m, —CHOCONH-, 4H), 7.18 ppm (t, —NH lysine, amounts of ketone and ketone hydrate. The ketone and ketone 1H), 8.04 ppm (d. —NH lysine, 1H). hydrate are in equilibrium in the presence of water, both of 60 As shown schematically above, the ketone exists in vari which can be used (either alone or in combination) to form able amounts of ketone and ketone hydrate. The ketone and polymer-active agent conjugates. ketone hydrate are in equilibrium in the presence of water, both of which can be used (either alone or in combination) to PEGylation of Lysozyme form polymer-active agent conjugates. Lysozyme (3.0 mg) was dissolved in 1 mL of 20 mM 65 PEGylation of Lysozyme sodium phosphate buffer (pH 7.5) and mPEG2-(6K Da)- Lysozyme (3.0 mg) was dissolved in 1 ml of 20 mM ketone (25 mg, 20 fold excess) was added. After 15 minutes, sodium phosphate buffer (pH 7.5) and PEG2(40K Da)-pip US 8,865,149 B2 79 80 eridone (168 mg, 20 fold excess) was added. After 15 min -continued utes, 0.159 M solution of NaCNBH (66 uL, 50 fold molar O excess) was added and the solution was stirred for 24 hours at room temperature. Analysis of the reaction mixture by SDS HCl·H2N No lsch, - PAGE showed that PEGylated lysozyme was formed. H 1-amino-2-popanol Example 7 O O mPEGs-OCH2C-NH-CH2-C-CH PEG2 Ketone: mPEG2(40K Da)-Ketone with two C. 10 mPEG(5 K)-methyl ketone Amido Linkers To a solution of succinimidyl ester of mPEG (5K Da) carboxylic acid (10g, 2 mmole) in acetonitrile (100 mL), 15 1-amino-2-propanol (0.9 g, 12 mmole), and 4-(dimethy lamino)pyridine (1.5 g) were added. The reaction mixture mPEGo-OCH2 C-O-N was stirred overnight at room temperature under nitrogen atmosphere. Next, the solvent was distilled offunder reduced O pressure. The residue was dissolved in 200 ml distilled water. O NaCl (30 g) was added and the product was extracted with Succinimidyl ester of mPEG20kcarboxylic acid dichloromethane (80 and 40 mL). The extract was dried with anhydrous sodium sulfate and it was concentrated under -- -- reduced pressure. Thereafter, the product was precipitated O with a mixture of isopropyl alcohol and ethyl ether and was 25 collected through vacuum filtration and dried under vacuum overnight. Yield: 9.5 g. Icissulu-Nielic The above product (7.0 g, 1.4 mmole) was dissolved in 1,3-diaminoacetone dihydrochloride O O O dichloromethane (70 mL) and Dess-Martin periodinane (1.17 30 g) was added. The reaction mixture was stirred overnight at mPEGo-OCH2C-NH-CH-C-CH-NH-C-CH2O-PEGom room temperature under nitrogen atmosphere. Next, the reac tion mixture was washed with 15%-NaCl solution (200 mL), mPEG2(40 KDa)-ketone dried with anhydrous sodium Sulfate, and concentrated under reduced pressure. Thereafter, the product was precipitated To a solution of succinimidyl ester of mPEG (20K Da) 35 with a mixture ofisopropyl alcohol and ethyl ether giving 6.2 carboxylic acid (12 g, 0.6 mmole) in acetonitrile (50 ml), g of white solid after drying under reduced pressure. NMR 1,3-diaminoacetone dihydrochloride monohydrate (0.054g, (d-DMSO): 2.08 ppm (s, —CH3H), 3.24 ppm (s. —OCH, 0.3 mmole), and 4-(dimethylamino)pyridine (0.909 g) were 3H), 3.51 ppm (s, polymer backbone), 3.94 ppm (s. —O added. The reaction mixture was stirred for 48 hours at room CHCONH , 2H), 3.97 ppm (d. NHCH (C=O) , 2H), temperature under nitrogen atmosphere. Next, the Solvent 40 7.98 ppm (t, —NH CH(C=O) 1H). was distilled off under reduced pressure. The residue was Depending on the conditions, the ketone exists in variable dissolved in dichloromethane and precipitated with a mixture amounts of ketone and ketone hydrate. The ketone and ketone of isopropyl alcohol and ethyl ether. The final product was hydrate are in equilibrium in the presence of water, both of collected through vacuum filtration and dried under vacuum which can be used (either alone or in combination) to form overnight. Yield: 9.5 g. NMR (d-DMSO): 3.24 ppm (s. 45 polymer-active agent conjugates. —OCH, 6H), 3.51 ppm (s, polymer backbone), 4.00 ppm (s. —OCHCONH , 4H), 4.29 ppm (d. NH-CH (C=O) Example 9 CH NH 2H), 6.68 ppm (t, NH CH (C=O)CH2— NH 2H). PEG (5K Da)-C.-Hydroxy-()-2-Propanone Diethyl 50 Ketal (with Alpha Ether Bond) Example 8 Formation of Ethyl Pyruvate Diethyl Ketal A mixture of ethylpyruvate (13.92g, 0.120 moles), triethyl mPEG (5K Da) Methyl Ketone, Method A: mPEG orthoformate (19.44 g., 0.131 moles), ethyl alcohol (9.0 g), (5K Da)-Methyl Ketone with an O. Amido Linker and p-toluenesulfonic acid monohydrate (0.0432 g, 0.00227 moles) was stirred at 45° C. overnight under nitrogen atmo sphere. Next, after cooling to room temperature, NaCO O (1.20 g) was added and the mixture was stirred for 15 minutes. 60 The reaction mixture was then filtered and ethyl alcohol and residual triethyl orthoformate were distilled off under reduced pressure. The residue was subjected to fractional negoch-i-o-n -- vacuum distillation giving 18.2 g of pure ethyl pyruvate O diethyl ketal. NMR (d-DMSO): 1.27 ppm (t, —CH, ethyl O 65 (ketal), 6H) 1.34 ppm (t, —CH, ethyl (ester), 3H), 1.40 ppm Succinimidyl ester of mPEG5kcarboxylic acid (S. —CH C-COO-, 3H), 3.45 ppm (q. —OCHCH (ketal), 4H), 4.29 ppm (q, —OCHCH (ester), 2H). US 8,865,149 B2 81 82 Formation of 2,2-Diethoxypropan-1-ol 2-propanone diethyl ketal; PEG (10K Da)-O-hydroxy-co-2- A solution of ethyl pyruvate diethyl ketal (16.0 g, 0.084 propanone diethyl ketal; PEG (20K Da)-O-hydroxy-co-2- moles) in ethyl ether (60 mL) was added dropwise during 30 propanone diethyl ketal; and PEG (40K Da)-O-hydroxy-co-2- minto a stirred solution of lithium aluminum hydride (1.90 g, propanone diethyl ketal. In each case, similar results to the 0.050 moles) in ethyl ether (150 mL). Next the mixture was PEG (5K)-O-hydroxy-()-2-propanone diethyl ketal are stirred 1 h at room temperature under nitrogen atmosphere. obtained. Saturated solution of sodium sulfate (20 mL) was added and the mixture was stirred 15 min. Next the mixture was filtered, Example 10 dried with anhydrous magnesium sulfate, and the solvent was distilled off. The residue was subjected to fractional vacuum 10 Preparation of Polymer-EPO Conjugate-Random distillation giving 9.5 g of pure 2,2-diethoxypropan-1-ol. PEGylation of EPO NMR (d-DMSO): 1.17 ppm (t, —CH, ethyl (ketal), 6H), 1.31 ppm (s, —CH C CH-OH, 3H), 3.37 ppm (q - O Recombinant erythropoietin, “EPO" (produced in E. coli, CHCH (ketal), 4H), 3.86 ppm (d. —CHOH, 2H), 4.45 mammalian cells Such as Chinese hamster ovary cells, or ppm (t, —OH, 1H). 15 another source) is coupled to mPEG (5K)-methyl ketone PEG (5K Da)-C.-Hydroxy-()-2-Propanone Diethyl Ketal (prepared as described in Example 2). EPO (-2 mg) is dissolved in 1 ml of 50 mM phosphate Ethyl buffer (pH 7.6) and mPEG (5K) methyl ketone is added at 5x / Ethyl the molar EPO concentration. A reducing agent, NaCNBH, O KNp, Ethylene Oxide is added and the solution stirred for 24 hours at room tem O He then neutralization perature to couple the mPEG (5K)-methyl ketone reagent to HO the protein via an amine linkage. CH3 The reaction mixture is analyzed by SDS-PAGE to deter 2,2-Diethoxypropan-1-ol 25 mine the degree of PEGylation. Confirmation of the degree of PEGylation, 1-mer, 2-mers, etc. is done by Matrix Assisted ly Ethyl O / Laser Desorption Ionization Time-of-Flight (MALDI-TOF) O mass spectrometry. The displayed peaks for native and monoPEGylated species differ by approximately 5,000 Dal HO -(N- 30 tons. The resulting reaction mixture contains a mixture of CH3 native and monoPEGylated protein. Increasing the ratio of PEG(5K)-2-propanone diethyl ketal PEG reagent to protein increases the degree of polyPEGyla tion, that is to say, the formation of 2-mers, 3-mers, and so on. 2,2-Diethoxypropan-1-ol (0.366 g., 0.00247 moles), THF The above demonstrates random PEGylation of an illus (200 mL), and potassium naphthalene 0.3 mol/L-tetrahydro 35 trative protein of the invention to yield a distribution of PEGy furan (THF) solution (20 mL, 0.006 moles) were added to a lated EPO products. If desired, the reaction mixture can be glass reactor and stirred for 3 minutes in an nitrogen atmo further separated to isolate the individual isomers as sphere. Ethylene oxide (12.6 g 0.286 moles) was added to this described below. solution and the reaction mixture was stirred for 48 hours at PEG conjugates having different molecular weights are room temperature. Next, the mixture was purged with nitro 40 separated by gel filtration chromatography. The different gen and 0.1M phosphate buffer (pH=8, 150 ml) was added. PEG conjugates (1-mer, 2-mer, 3-mer, and so forth) are frac The THF layer was separated and discarded. Naphthalene tionated on the basis of their different molecular weights (in was removed from the solution by ethyl ether extraction. The this case, varying by approximately 5,000 Daltons). Specifi product was extracted from the residue with dichloromethane cally, the separation is performed by using a serial column (3x100 mL). The extract was dried with anhydrous sodium 45 system suitable for effective separation of products in the sulfate and concentrated to about 50 mL. Next, ethyl ether molecular weight range observed, e.g., a SuperdexTM200 col (300 mL) was added and the mixture was stirred for 15 umn (Amersham Biosciences). The products are eluted with minutes at 0°C. The precipitated product was filtered off and 10 ml acetate bufferata flow rate of 1.5 ml/min. The collected dried under reduced pressure. The variable (m) in the above fractions (1 ml) are analyzed by OD at 280 nm for protein structure is a number to provide a polymer weight of 5,000 50 content and also using an iodine test for PEG content (Sims et Daltons. Yield 11.7 g. NMR (d-DMSO): 1.17 ppm (t, al. (1980) Anal Biochem. 107:60-63). In addition, the results —CH, ethyl (ketal), 6H), 1.31 ppm (S. —CH can be visualized by running an SDS PAGE gel, followed by —C CH-O-, 3H), 3.51 ppm (s, polymer backbone), 4.57 staining with barium iodide. Fractions corresponding to the ppm (t—OH, 1H). eluted peaks are collected, concentrated by ultrafiltration PEGylation of Lysozyme 55 using a membrane, and lyophilized. This method results in Lysozyme (3.0 mg) was dissolved in 1 mL of 20 mM separation/purification of conjugates having the same sodium phosphate buffer (pH 5.0) and PEG (5K Da)-O-hy molecular weights but does not provide separation of conju droxy-()-2-propanone diethyl ketal (21 mg, 20 fold excess) gates having the same molecular weight but different PEGy was added. After 15 minutes, 0.159 M solution of NaCNBH lation sites (i.e., positional isomers). (66 uL, 50 fold molar excess) was added and the solution was 60 Separation of positional isomers is carried out by reverse stirred for 20 hours at room temperature. Analysis of the phase chromatography using an RP-HPLCC18 column (Am reaction mixture by SDS-PAGE showed that PEGylated ersham Biosciences or Vydac). This procedure is effective for lysozyme was formed. separating PEG-biomolecule isomers having the same This example is repeated several times using different sizes molecular weight (positional isomers). The reverse-phase of PEG (5K Da)-O-hydroxy-()-2-propanone diethyl ketal. 65 chromatography is carried out using a RP-HPLC C18 pre The following different sizes of PEG-O-hydroxy-co-pro parative column and eluted with a gradient of water/0.05% panone diethyl ketal are used: PEG (2K Da)-O-hydroxy-co TFA (Eluent A) and acetonitrile/0.05% TFA (Eluent B). US 8,865,149 B2 83 84 Fractions corresponding to the eluted peaks are collected, conducted by peptide mapping. Increasing the ratio of PEG to evaporated to eliminate acetonitrile and TFA, followed by protein increases the degree of PEGylation yielding polyP removal of solvent to isolate the individual positional PEG EGylated protein. isomers. Example 13 Example 11 N-terminal PEGylation of Interferon-C. Preparation of Polymer-EPO Conjugate N-terminal Recombinant interferon-alfa, “IFN-C” (produced in E. PEGylation of EPO coli, mammaliancells such as Chinese hamster ovary cells, or 10 another source) is coupled to mPEG (5K Da)-methyl ketone Recombinant erythropoietin, “EPO" (produced in E. coli, (prepared as described in Example 8). mammalian cells Such as Chinese hamster ovary cells, or IFN-O (-2. mg) is dissolved in 1 ml of 0.1 mM sodium another source) is coupled to mPEG (5K)-methyl ketone acetate (pH 5) and mPEG (5K Da)-methyl ketone is added at (prepared as described in Example 2). 5x the molar IFN-C concentration. A reducing agent, NaC 15 NBH, is added and the solution stirred for 24 hours at 4°C. EPO (-2 mg) is dissolved in 1 ml of 0.1 mM sodium acetate to couple the mPEG (5K Da)-methyl ketone reagent to the (pH 5) and mPEG (5K)-methyl ketone is added at 5x the protein via an amine linkage. molar EPO concentration. A reducing agent, NaCNBH, is The reaction mixture is analyzed by SDS-PAGE to deter added and the solution is stirred for 24 hours at 4°C. to couple mine the degree of PEGylation. Confirmation of the degree of the mPEG (5K)-methyl ketone reagent to the protein via an PEGylation, 1-mer, 2-mers etc. is carried out by Matrix amine linkage. Assisted Laser Desorption Ionization Time-of-Flight The reaction mixture is analyzed by SDS-PAGE to deter (MALDI-TOF) mass spectrometry. The displayed peaks for mine the degree of PEGylation. Confirmation of the degree of native and monoPEGylated species differ by approximately PEGylation, 1-mer, 2-mers, and so one, is carried out by 5,000 Daltons. The resulting reaction mixture primarily con tains a mixture of native and monoPEGylated protein. The Matrix Assisted Laser Desorption Ionization Time-of-Flight 25 monoPEGylated species are purified by column chromatog (MALDI-TOF) mass spectrometry. The displayed peaks for raphy to remove free interferon-C. and higher molecular native and mono-PEGylated species differ by approximately weight species. Confirmation of N-terminal PEGylation is 5,000 Daltons. The resulting reaction mixture primarily con conducted by peptide mapping. Increasing the ratio of PEG to tains a mixture of native and monoPEGylated protein. The protein increases the degree of PEGylation yielding polyP monoPEGylated species are purified by column chromatog 30 EGylated IFN-C. raphy to remove free EPO and higher molecular weight spe C1GS. Example 14 Confirmation of N-terminal PEGylation is carried out by peptide mapping. Increasing the ratio of PEG to protein N-terminal PEGylation of Human Growth Hormone increases the degree of PEGylation, yielding polyPEGylated 35 protein. Recombinant human growth hormone, “hoH (produced The above demonstrates PEGylation of an illustrative pro in E. coli, mammalian cells Such as Chinese hamster ovary tein of the invention to yield a predominantly N-terminal cells, or another source) is coupled to mPEG2(40K Da)- single PEGylated protein. ketone (prepared as described in Example 7). 40 hGH (-2 mg) is dissolved in 1 ml of 0.1 mM sodium acetate (pH 5) and mPEG(40K Da)-ketone is added at 5x the molar Example 12 hGH concentration. A5 to 20-fold molar excess of the reduc ing agent, NaCNBH, is added and the solution is stirred for N-terminal PEGylation of GCSF 24 hours at 4°C. to couple the mPEG (40K)-ketone reagent 45 to the protein via an amine linkage. Recombinant granulocyte colony stimulating factor, Progress of the reaction is analyzed by SDS-PAGE or “GCSF (produced in E. coli, mammalian cells such as Chi MALDI-TOF mass spectrometry to determine the degree of nese hamster ovary cells, or another source) is coupled to PEGylation. Confirmation of the degree of PEGylation, mPEG (5K)-methyl ketone (prepared as described in 1-mer, 2-mers, and so forth, is carried out by Matrix Assisted Example 8). 50 Laser Desorption Ionization Time-of-Flight (MALDI-TOF) GCSF (-2. mg) is dissolved in 1 ml of 0.1 mM sodium mass spectrometry. The displayed peaks for native and acetate (pH 5) and mPEG (5K)-methylketone (from Example monoPEGylated and other species differ by approximately 2) is added at 5x the molar GCSF concentration. The reducing 40,000 Daltons. The resulting reaction mixture primarily agent, NaCNBH, is added and the solution stirred for 24 contains a mixture of native and monoPEGylated protein. The hours at 4° C. to couple the mPEG (5K)-methyl ketone 55 monoPEGylated species are purified by column chromatog reagent to the protein via an amine linkage. raphy to remove free hCH and higher molecular weight spe The resulting reaction mixture is analyzed by SDS-PAGE cies. Confirmation of N-terminal PEGylation is conducted by to determine the degree of PEGylation. Confirmation of the peptide mapping. Increasing the ratio of mPEG (40K)-ke degree of PEGylation, 1-mer, 2-mers, and so forth, is carried tone to protein increases the degree of PEGylation yielding a out by Matrix Assisted Laser Desorption Ionization Time-of 60 population of polyPEGylated hCH. Flight (MALDI-TOF) mass spectrometry. The displayed peaks for native and mono-PEGylated species differ by Example 15 approximately 5,000 Daltons. The resulting reaction mixture primarily contains a mixture of native and monoPEGylated N-terminal PEGylation of Interferon-f GCSF. The monoPEGylated species are purified by column 65 chromatography to remove free GCSF and higher molecular Recombinant interferon-B, “IFN-B” (produced in E. coli, weight species. Confirmation of N-terminal PEGylation is mammalian cells Such as Chinese hamster ovary cells, or US 8,865,149 B2 85 86 another source) is coupled to mPEG2(40K Da)-ketone (pre stirred for 24 hours at 4°C. to couple the PEG (5K Da)-diethyl pared as described in Example 7). ketal reagent to the protein via an amine linkage. IFN-B (-2 mg) is dissolved in 1 ml of 0.1 mM sodium Progress of the reaction is analyzed by SDS-PAGE or acetate (pH 5) and mPEG2(40KDa)-ketone is added at 5x the MALDI-TOF mass spectrometry to determine the extent of molar IFN-B concentration. A5 to 20-fold molar excess of the 5 PEGylation. Confirmation of the degree of PEGylation, reducing agent, NaCNBH, is added and the solution is 1-mer, 2-mers, and so forth, is carried out by Matrix Assisted stirred for 24 hours at 4°C. to couple the mPEG-(40K Da)- Laser Desorption Ionization Time-of-Flight (MALDI-TOF) ketone reagent to the protein via an amine linkage. mass spectrometry. The displayed peaks for native and Progress of the reaction is analyzed by SDS-PAGE or monoPEGylated and other species differ by approximately 10 5,000 Daltons. The resulting reaction mixture, primarily con MALDI-TOF mass spectrometry to determine the degree of tains a mixture of native and monoPEGylated protein, in PEGylation. Confirmation of the degree of PEGylation, particular due to the PEG (5K)-diethyl ketal reagent. The 1-mer, 2-mers, and so forth, is carried out by Matrix Assisted monoPEGylated species are purified by column chromatog Laser Desorption Ionization Time-of-Flight (MALDI-TOF) raphy to remove free hCH and higher molecular weight spe mass spectrometry. The displayed peaks for native and mono cies. Confirmation of N-terminal PEGylation is conducted by PEGylated and other species differ by approximately 40,000 peptide mapping. Daltons. The resulting reaction mixture primarily contains a mixture of native and monoPEGylated protein. The monoP Example 18 EGylated species are purified by column chromatography to remove free IFN-B and higher molecular weight species. PEGylation of Amphotericin B Confirmation of N-terminal PEGylation is conducted by pep tide mapping. Increasing the ratio of mPEG2(40K Da)-ke The amino group of a small molecule, amphotericin B, is tone to protein increases the degree of PEGylation yielding a modified by attachment of a mPEG (2K Da)-methyl ketone. population of polyPEGylated IFN-B. To a solution of amphotericin B HCl in deionized water is 25 added a 2-fold molar excess of mPEG (2KDa)-methyl ketone Example 16 (Example 1) dissolved in 0.1 M phosphate buffer at pH 6.5. To this mixture is added a solution of NaCNBH (at a 1.5 to N-terminal PEGylation of FSH 10-fold molar excess) in phosphate buffer at pH 6.5, and the resulting solution is stirred at room temperature overnight Recombinant follicle stimulating hormone, “FSH (pro 30 under an argon atmosphere. Aliquots of the reaction mixture duced in E. coli, mammalian cells Such as Chinese hamster are withdrawn at various time intervals to monitor the ovary cells, or another source) is coupled to mPEG2(40K progress of the reaction by H NMR. Upon completion, the Da)-ketone (prepared as described in Example 7). reaction mixture is further diluted by addition of water, and FSH (-2 mg) is dissolved in 1 mL of 0.1 mM sodium NaCl is added to achieve saturation. The product is then acetate (pH 5) and mPEG2(40KDa)-ketone is added at 5x the 35 extracted with dichloromethane, and the combined organic molar FSH concentration. A 5 to 20-fold molar excess of the extracts are dried over anhydrous sodium sulfate, filtered to reducing agent, NaCNBH, is added and the solution is remove drying agent, and the solvent evaporated by rotary stirred for 24 hour at 4°C. to couple the mPEG (40K Da)- evaporation. The product is then precipitated by addition of ketone reagent to the protein via an amine linkage. diethyl ether, and dried under vacuum overnight. The recov Progress of the reaction is analyzed by SDS-PAGE or 40 ered product is analyzed by gel permeation chromatography MALDI-TOF mass spectrometry to determine the degree of to determine the extent of conjugation. PEGylation. Confirmation of the degree of PEGylation, The crude product is purified by cation exchange chroma 1-mer, 2-mers, and so forth, is carried out by Matrix Assisted tography using Poros 50 HS cation exchange resin (PerSep Laser Desorption Ionization Time-of-Flight (MALDI-TOF) tive BioSystems, Framingham, Mass.). Following washing of mass spectrometry. The displayed peaks for native and mono 45 the column with deionized water, the product is eluted with PEGylated and other species differ by approximately 40,000 1N NaCl Solution. The conjugate containing extracts are Daltons. The resulting reaction mixture primarily contains a combined, and the product is extracted with dichlo mixture of native and monoPEGylated protein. The monoP romethane. The organic Solution is dried over anhydrous EGylated species are purified by column chromatography to sodium sulfate, filtered, and the solvent evaporated by rotary remove free FSH and higher molecular weight species. Con 50 evaporation. The purified conjugate is purified by the addition firmation of N-terminal PEGylation is conducted by peptide of diethyl ether. mapping. Increasing the ratio of mPEG2(40K Da)-ketone to If necessary, the product is further purified by reverse phase protein increases the degree of PEGylation yielding a popu HPLC chromatography using a Betasil C18 column (Key lation of polyPEGylated FSH. stone Scientific). 55 Example 17 What is claimed is: 1. A conjugate, wherein the conjugate has the structure N-terminal PEGylation of hCH

Recombinant human growth hCH (produced in E. coli, 60 mammalian cells Such as Chinese hamster ovary cells, or O NH another source) is coupled to PEG (5K Da)-diethyl ketal (prepared as described in Example 9). mPEG-OCHCNH-CH-CH-CH hGH (-2 mg) is dissolved in 1 mL of 0.1 mM sodium acetate (pH 5) and PEG (5K Da)-diethyl ketal is added at 5x 65 wherein mPEG is a methoxy capped poly(ethylene glycol) the molar hCH concentration. A5 to 20-fold molar excess of having a nominal average molecular weight of from about the reducing agent, NaCNBH, is added and the solution is 2,500 Daltons to about 100,000 Daltons and US 8,865,149 B2 87 88 HN-Active Agent is an amine-containing active agent. 5 2. The conjugate of claim 1, wherein the mPEG has a nominal average molecular weight of from about 4,900 Dal tons to about 40,000 Daltons. 3. The conjugate of claim 2, wherein the mPEG has a nominal average molecular weight of from about 9,900 Dal 10 tons to about 25,000 Daltons. 4. The conjugate of claim 1, wherein the mPEG has a nominal average molecular weight of about 5,000 Daltons. 5. The conjugate of claim 1, wherein the active agent is selected from erythropoietin, granulocyte macrophage 15 colony stimulating factor (GMCSF), granulocyte colony stimulating factor (GCSF), interferon alpha, interferon beta, human growth hormone, amphotericin B, and follicle stimu lating hormone. 6. The conjugate of claim 1, wherein the active agent is a polypeptide. 7. A pharmaceutical preparation comprising the conjugate of claim 1 and a pharmaceutically acceptable excipient.

k k k k k