USOO8765432B2
(12) United States Patent (10) Patent No.: US 8,765,432 B2 Charles et al. (45) Date of Patent: Jul. 1, 2014
(54) TARGETED DRUG PHOSPHORYLCHOLINE 6,627.314 B2 9/2003 Matyjaszewski et al. POLYMER CONUGATES 6,759,491 B2 7/2004 Matyjaszewski et al. 6,790,919 B2 9/2004 Matyjaszewski et al. 6,852,816 B2 2/2005 Lewis et al. (75) Inventors: Stephen A. Charles, San Jose, CA (US); 6,887.962 B2 5/2005 Matyjaszewski et al. D. Victor Perlroth, Palo Alto, CA (US) 7,019,082 B2 3/2006 Matyjaszewski et al. 7,049,373 B2 5/2006 Matyjaszewski et al. (73) Assignee: Oligasis, LLC, Palo Alto, CA (US) 7,056,455 B2 6/2006 Matyjaszewski et al. 7,064,166 B2 6/2006 Matyjaszewski et al. (*) Notice: Subject to any disclaimer, the term of this 7,157,5307,125,938 B2 10/20061/2007 Matyjaszewski et al. patent is extended or adjusted under 35 7,300,990 B2 11/2007 Lewis et al. U.S.C. 154(b) by 0 days. 7,348,424 B2 3/2008 Miyazawa et al. 7,569,655 B2 8/2009 Pacetti et al. (21) Appl. No.: 13/515,913 2003/0143596 A1 7/2003 Bentley et al. 2004/0063881 A1 4/2004 Lewis et al. 1-1. 2004/0253596 A1 12, 2004 Pawlak et al. (22) PCT Filed: May 10, 2010 2005, 0112088 A1 5.2005 Zhao et al. 2005, 01235O1 A1 6, 2005 Lewis (86). PCT No.: PCT/US2O1 O/O34252 2005. O159556 A1 7, 2005 Lewis et al. 371 (c)(1 2005, 0180945 A1 8, 2005 Chaikofetal. S371 (c)(1), 2006/0135714 A1 6/2006 Lewis et al. (2), (4) Date: Aug. 27, 2012 2006/0165804 A1 7/2006 Lewis et al. 2006/0217285 A1 9, 2006 DeStarac (87) PCT Pub. No.: WO2011/075185 2007, 0141 104 A1 6/2007 Hauenstein 2008/O124450 A1 5.2008 Pacetti PCT Pub. Date: Jun. 23, 2011 2008. O147178 A1 6, 2008 Pacetti et al. 2010, O15885.0 A1 6/2010 Baker, Jr. et al. (65) Prior Publication Data US 2013/OO34517 A1 Feb. 7, 2013 FOREIGN PATENT DOCUMENTS EP 1465933 B1 8, 2007 O O EP 1592.719 B1 3, 2008 Related U.S. Application Data EP 1732621 B1 12, 2009 (60) Provisional application No. 61/288,127, filed on Dec. EP 2260873 A1 12/2010 18, 2009 JP 2010-117.189 A 5, 2010 s WO WOOO. 59968 A1 10, 2000 WO WOO2,28929 A1 4/2002 (51) Int. Cl. WO WOO3,062290 A1 T 2003 A6IP35/00 (2006.01) WO WOO3,O74O26 A1 9, 2003 CO7D 49/22 (2006.01) (Continued) CO7D 49/47 (2006.01) CI2N 9/96 (2006.01) OTHER PUBLICATIONS A6 IK 47/48 (2006.01) A61 K31/41 (2006.01) Venditto, et al. (Cancer Therapies Utilizing the Camptothecins: A (52) U.S. Cl. Review of the in Vivo Literature, 7 Molecular Pharm. 307-49 CPC, C12N 9/96 (2013.01); A61K 31/41 (2013.01); (2010)." A61K47/48169 (2013.01) Chen, et al., Polymeric phosphorylcholine-camptothecin conjugates USPC 435/188: 514/283.546/48: 546/51 prepared by controlled free radical polymerization and click chem istry, 20 Bioconjugate Chem. 2331-41 (2009).* (58) Field of Classification Search Lewis, et al., Poly(2-methacryloyloxyethyl phosphorylcholine) for None protein conjugation, 19 Bioconjugate Chem. 2144-55 (2008).* See application file for complete search history. Bontempo, et al., “Cysteine-Reactive Polymers Synthesized by Atom Transfer Radical Polymerization for Conjugation to Proteins.”.J. Am. (56) References Cited Chem. Soc., (2004), 126, pp. 15372-15373. Chen, et al., “Lubrication at Physiological Pressures by PolyZwit U.S. PATENT DOCUMENTS terionic Brushes.” Science, (2009), 323, pp. 1698-1701. 5,741,923 A 4, 1998 Driver et al. (Continued) 5,763,548 A 6/1998 Matyjaszewski et al. 5,789.487 A 8/1998 Matyjaszewski et al. 5,807,937 A 9/1998 Matyjaszewski et al. Primary Examiner — H. Sarah Park 6,111,0225,945,491 A 8/20008/1999 Matyjaszewski et al. (74) Attorney, Agent, or Firmriva — Alston & Bird LLP 6,121,371 A 9/2000 Matyjaszewski et al. 6,124,411 A 9/2000 Matyjaszewski et al. 6,162,882 A 12/2000 Matyjaszewski et al. (57) ABSTRACT 6,407,1876,512,060 B1 6/20021/2003 Matyjaszewski et al. The present invention provides random copolymers COnta1nin 6,538,091 B1 3/2003 Matyjaszewski et al. 1ng phosphorylcholine and one or more functional agents, and 6,541,580 B1 4/2003 Matyjaszewski et al. methods of preparing Such random copolymers. 6,555,593 B1 4/2003 Hoyle et al. 6,583,272 B1 6, 2003 Bailon 6,624,262 B2 9/2003 Matyjaszewski et al. 2 Claims, 1 Drawing Sheet US 8,765.432 B2 Page 2
(56) References Cited Lena, et al., “Investigation of metal ligand affinities of atom transfer radical polymerization catalysts with a quadrupole ion trap. Dalton Transactions, (2009), 41, pp. 8884-8889. FOREIGN PATENT DOCUMENTS Lewis, et al., “Crosslinkable coatings from phosphorylcholine-based WO WOO3,O74090 A1 9, 2003 polymers.” Biomaterials, (2001), 22, pp. 99-111. WO WO 2004/063237 A1 T 2004 Lewis, et al., “Poly(2-methacryloyloxyethyl phosphorylcholine) for WO WO 2004/113394 A2 12/2004 Protein Conjugation.” Bioconjugate Chem... (2008), 19:11, pp. 2144 WO WO 2005/028539 A2 3, 2005 2155. WO WO 2005/058367 A2 6, 2005 Lutz, et al., “Preparation of Ideal PEG Analogues with a Tunable WO WO 2007/005253 A1 1, 2007 WO WO 2007/075534 A2 7/2007 Thermosensitivity by Controlled Radical Copolymerization of 2-(2- WO 2007/100.902 A2 9, 2007 Methoxyethoxy)ethyl Methacrylate and Oligo(ethylene glycol) WO WO 2008/098.930 A1 8, 2008 Methacrylate.” Macromolecules, (2006), 39:2, pp. 893-896. WO WO 2008, 112257 A1 9, 2008 Ma, et al., “Synthesis of Biocompatible Polymers. 1. WO WO 2008, 112289 A2 9, 2008 Homopolymerization of 2-Methacryloyloxyethyl WO WO 2010/068862 A2 6, 2010 Phosphorylcholine via ATRP in Protic Solvents: An Optimization WO WO 2010/068864 A2 6, 2010 WO WO 2010.068864 A2 * 6, 2010 Study.” Macromolecules, (2002), 35:25, pp. 9306-93.14. WO WO 2011 O75185 A1 6, 2011 Ma, et al., “Well-Defined Biocompatible Block Copolymers via WO WO 2011 O75736 A2 6, 2011 Atom Transfer Radical Polymerization of 2-Methacryloyloxyethyl WO WO 2011, 130694 A2 10/2011 Phosphorylcholine in Protic Media.” Macromolecules, (2003), WO WO 2013,059.137 A1 4/2013 36:10, pp. 3475-3484. Mantovani, et al., “Design and Synthesis of N-Maleimido OTHER PUBLICATIONS Functionalized Hydrophilic Polymers via Copper-Mediated Living Radical Polymerization: A Suitable Alternative to PEGylation.” J. Chen, et al., “Polymeric Phosphorylcholine-Camptothecin Conju Am. Chem. Soc., (2005), 127, pp. 2966-2973. gates Prepared by Controlled Free Radical Polymerization and Click Matyjaszewski, et al., “Diminishing catalyst concentration in atom Chemistry.” Bioconjugate Chem... (2009), 20:12, pp. 2331-2341. transfer radical polymerization with reducing agents.” PNAS, (Oct. Crowe, et al., “Recombinant human respiratory syncytial virus 17, 2006), 103:42, pp. 15309-15314. (RSV) monoclonal antibody Fab is effective therapeutically when Min, et al., “Use of Ascorbic Acid as Reducing Agent for Synthesis of introduced directly into the lungs of RSV-infected mice.” Proc. Natl. Well-Defined Polymers by ARGET ATRP” Macromolecules, (2007), 40:6. pp. 1789-1791. Acad. Sci. USA, (1994) 91 pp. 1386-1390. Miyamoto, et al., “Effect of water-soluble phospholipid polymers Da Pieve, et al., “Conjugation of PolyPEG(R), Linear PEG and conjugated with papain on the enzymatic stability.” Biomaterials, Branched PEG to a Thiol-Modified Aptamer.” Poster, Warwick (2004), 25, pp. 71-76. Effect Polymers Ltd, retrieved from (56) References Cited U.S. Appl. No. 13/516, 173, Restriction Requirement mailed Sep. 3, 2013. OTHER PUBLICATIONS U.S. Appl. No. 13/959,563, Office Action mailed Oct. 10, 2013. Wang, et al., “Controlled 'Living Radical Polymerization. Atom Sayers, et al., “The Reduced Viscosity of PolyPEG(R) Compared with Transfer Radical Polymerization in the Presence of Transition-Metal Linear PEG.” Poster, Warwick Effect Polymers Ltd, retrieved from 10 In some embodiments when there are multiple comono LGI-I lyr mers M, one of the comonomers M can be linked to a group (CH2), I S other than a linking group LG, a functional agent A or an initiator I. In other embodiments, at least one R group is H. 15 Co alkyl, Calkenyl, C- alkynyl, Chaloalkyl, C. LG-L-I rty-fi heteroalkyl, C.s cycloalkyl, Cls heterocycloalkyl, aryl, or (CH2), I S heteroaryl. For example, such structures include the follow 1ng: Al-L-I yartyr (CH2). LG - T - 2 2. i ZW A-L-I y}{y} (y } - 1- T - 2 i (CH2) R2a A-L I rty 1, I S. 25 ZW A2b (CH2), I S LG-L-I MI } M2a M2b I 'yla 'ylb LG-L-I yet M-II (CH2) R2a L2b s and ZW A2b (CH2). LG S 30 ZW Al-L-I MI M2a M2b I yla 'ylb LG-L-I rty-fi (CH), R2 LG2b (CH2), I S. s ZW 35 wherein R* can be H. Coalkyl, Calkenyl, Calkynyl, The linking groups LG facilitate the "clicking on of func Chaloalkyl, Cheteroalkyl, Css cycloalkyl, C.s hetero tional agents and initiator groups following polymerization. cycloalkyl, aryl, or heteroaryl. In other embodiments, R” can 40 be a species having one or more positive or negative charges, When a plurality of comonomers M is present, the Such as aspartic acid, glutamic acid, lysine, histidine, argin comonomers can be linked to either a functional agent or a ine, choline or hyaluronic acid. linking group, for example as shown in the following for When R of some comonomers M is the initiator I, more mula: complex architectures can be prepared of the random copoly 45 mers. For example, comb polymers, hyperbranched polymers and dendrimers can be prepared. When initiator I is present A-L-I y }. y I on a comonomer M. polymerization using initiator I typi (CH). LG cally occurs following polymerization using initiator I-I". In S. some embodiments, polymerization via I-I" and I can be w A2b 50 simultaneous. Moreover, the initiator I can be linked to the comonomer M. via a cleavable or non-cleavable linker L. In Some embodiments, the random copolymers of the In addition, the linking group can be present on the initiator present invention can be modified via a Subsequent polymer fragment I while functional agents A are linked to the ization with one or more additional monomers. For example, comonomers M. Alternatively, when the linking group LG is 55 in formula III above, monomers M' and M" can be copoly linked to the initiator fragment I, a second linking group LG merized in a first polymerization, and monomer M can be can be linked to one of the comonomers M: polymerized in a second polymerization. A block copolymer would be formed having two blocks, the first block being a random copolymer of M" and M', and the second block a - T - 2 2. i 60 homopolymer of M. Alternatively, following polymeriza LG-L-I y}{y} (y } - tion of monomers M and M', monomer M can be copo (CH2). LG lymerized with monomer M*, thus forming a block copoly S. mer where the first block is a random copolymer of M" and M", and the second block is a random copolymer of Mand 65 M. Additional polymer structures can be prepared by copo Moreover, a functional agent A' can be linked to the initiator lymerizing monomers M', M and M' in a first polymer fragment I while linking groups LG are linked to the comono ization, followed by copolymerization of monomers M. US 8,765,432 B2 21 22 M", and others, in a second copolymerization. Additional Other groups are useful for the initiator fragment I' of the blocks can be prepared by yet a third polymerization using present invention. In some embodiments, the initiator frag additional monomers. Such polymers provide blocks of ment I' is bromine. copolymers that can have different properties, drugs and Initiators employed for ATRP reactions can be hydroxy functional agents. 5 lated. In some embodiments, the initiators employed for A. Initiators ATRP reactions to prepare random copolymers of the inven The random copolymers of the present invention are poly tion are selected from alkanes, cycloalkanes, alkyl carboxylic merized using any Suitable initiator. Initiators useful in the acids or esters thereof, cycloalkylcarboxylic acids or esters present invention can be described by the formula: I-(I), thereof, ethers, cyclic alkyl ethers, alkyl aryl groups, alkyl where subscript m is an integer from 1 to 20. The initiator 10 fragment I can be any group that initiates the polymerization. amides, alkyl-aryl carboxylic acids and esters thereof, bear The initiator fragment I' can be any group that will reversibly ing a hydroxyl group, and also bearing one initiator fragment terminate the growing polymer chain. The initiator fragment I' where unbranched random copolymers are to be prepared, I' can be a halogen Such as bromine, allowing the end of the or alternatively, more than one initiator fragment I' where polymer to be functionalized after polymerization. In addi 15 branched molecules are to be prepared. tion, the initiator fragment I can optionally be functionalized Initiators employed for ATRP reactions can bear one or with an R' group that can include a variety of functional more amine groups. In some embodiments, the initiators groups to tune the functionality of the random copolymer. employed for ATRP reactions to prepare random copolymers Initiators useful in the present invention can have a single of the invention are alkanes, cycloalkanes, alkyl carboxylic initiator fragment I", or any Suitable number of branches Such acids or esters thereof, cycloalkylcarboxylic acids or esters that there are multiple initiator fragments I" each capable of thereof, ethers, cyclic alkyl ethers alkyl aryl groups, alkyl reversibly terminating a growing polymer chain. When the amides, alkyl-aryl carboxylic acids and esters thereof, bear initiator fragment I is branched and is capable of initiating ing an amine group and also bearing one initiator fragment I' multiple polymer chains, Subscript misgreater than one Such where unbranched random copolymers are to be prepared, or that there are as many initiator fragments I" as there are grow 25 alternatively, more than one initiator fragment I' where ing polymer chains. branched molecules are to be prepared. The bond between initiator fragments I and I' is labile, such Alkylcarboxylic acids, including alkyl dicarboxylic acids, that during the polymerization process monomers M' and having at least one initiator fragment I", and Substituted with comonomers M are inserted between initiator fragments I amino or hydroxy groups can also be employed as initiators. and I'. For example, during a free radical polymerization, 30 In some embodiments of the invention where ATRP is Such as ATRP, initiator fragments I and I'dissociate, as shown employed to prepare random copolymers of the present in FIG. 1, to form radicals of I and I'. The radical of initiator invention, the initiators can be alkylcarboxylic acids bearing fragment I then reacts with the monomers in Solution to grow one or more halogens selected from chlorine and bromine. the polymer and forms a propagating polymer radical (spe Alkanes substituted with two or more groups selected from cies A and species C of FIG. 1). During the polymerization 35 —COOH, -OH and NH, and at least one initiator frag process, the radical of initiator fragment I' will reversibly ment I', can also be employed as initiators for the preparation react with the propagating polymer radical to temporarily of random copolymers where ATRP is employed to prepare stop polymer growth. The bond between the monomer and the random copolymers of the present invention. initiator fragment I' is also labile, such that the bond can Initiators can also contain one or more groups including, cleave and allow the propagating polymer radical to react 40 but not limited to, —OH, amino, monoalkylamino, dialky with additional monomer to grow the polymer. The end result lamino, —O-alkyl, -COOH, -COO-alkyl, or phosphate of the polymerization process is that initiator fragment I is at groups (or protected forms thereof). one end of the polymer chain and initiator fragment I' is at the A broad variety of initiators are commercially available, opposite end of the polymer chain. for example bromoacetic acid N-hydroxysuccinimide ester The radical of initiator fragment I is typically on a second 45 available from Sigma-Aldrich (St. Louis, Mo.). Suitably pro ary or tertiary carbon, and can be stabilized by an adjacent tected forms of those initiators can be prepared using standard carbonyl carbon. The initiator fragment I' is typically a halo methods in the art as necessary. gen, Such as bromine, chlorine or iodine. Together, initiator Other initiators include thermal, redox or photo initiator, fragments I and I' form the initiators I and I useful in the including, for example, alkyl peroxide, Substituted alkyl per preparation of the random copolymers of the present inven 50 oxides, aryl peroxides, Substituted aryl peroxides, acyl per tion. oxides, alkyl hydroperoxides, Substituted aryl hydroperoX A broad variety of initiators can be used to prepare the ides, aryl hydroperoxides, Substituted aryl hydroperoxides, random copolymers of the invention, including a number of heteroalkyl peroxides, substituted heteroalkyl peroxides, het initiators set forth in U.S. Pat. No. 6,852,816 (incorporated eroalkyl hydroperoxides, substituted heteroalkyl hydroper herein by reference). In some embodiments, the initiators 55 oxides, heteroaryl peroxides, substituted heteroaryl perox employed for ATRP reactions to prepare random copolymers ides, heteroaryl hydroperoxides, substituted heteroaryl of the invention are selected from alkanes, cycloalkanes, alkyl hydroperoxides, alkyl peresters, Substituted alkyl peresters, carboxylic acids or esters thereof, cycloalkylcarboxylic acids aryl peresters, Substituted aryl peresters, azo compounds and or esters thereof, ethers and cyclic alkyl ethers, alkyl aryl halide compounds. Specific initiators include cumene hydro groups, alkyl amides, alkyl-aryl carboxylic acids and esters 60 peroxide (CHP), tert-butyl hydroperoxide (TBHP), tert-butyl thereof, and also bearing one initiator fragment I' where perbenzoate, (TBPB), sodium carbonateperoxide, benzoyl unbranched random copolymers are prepared, and more than peroxide (BPO), lauroyl peroxide (LPO), methylethyl ketone one initiator fragment I' where branched molecules are pre 45%, potassium persulfate, ammonium persulfate, 2,2-azobis pared. (2,4-dimethyl-Valeronitrile), 1,1-azobis(cyclo-hexanecarbo Initiator fragments I useful in the present invention 65 nitrile), 2,2-azobis(N,N-dimethyleneisobutyramidine)dihy include, but are not limited to, halogens, such as Br, Cl and I. drochloride, and 2,2-azobis(2-amido-propane) thiocyanate ( SCN) and isothiocyanate ( N=C=S). dihydrochloride. Redox pairs such as persulfate/sulfite and Fe US 8,765,432 B2 23 24 (2+) peroxide or ammonium persulfate and N.N.N'N'-tetram- -continued ethylethylenediamine (TEMED). A O O Still other initiators useful for preparing the random YK Or Yx copolymers of the present invention, are branched. Suitable 5 NN NN initiators having a single branch point include the following: E. H B A. H r I O O In some embodiments, the initiator can be: W O R 10 N-- € O Br R O O O O where radical R can be any of the following:- - - Nn-1\o O O , O 2O O X-(- ? O Br Y& O Br, Y& O Cl, p F which is a protected maleimide that can be deprotected after polymerization to form the maleimide for reaction with addi O O ° tional functional groups. Y& I, Y& s Additional branched initiators include, but are not limited O O to, the following, where radical R is as defined above: C A. O A. O 30 Y& O Y& O O / R Br I R F O F O 35 OHC Yx O --- Cl, Y&? O-- Br, OHC P O O ' 40 Y& I, Y{ Cl, R 'O ... O H ^-O \, 45 O O In some embodiments, the branched initiators include, but are not limited to, the following: Y& O Br, Y& O I, 50 O Br O O O 55 O / X X f H H OHC C Br 60 O O \ H I ? H C 65 )-(- s US 8,765,432 B2 26 -continued -continued O Br Br O O O O OHC H 1- 1 (C O 10 { O O Br Br, and In other embodiments, the initiator can have several branch O Br points to afford a plurality of polymer arms, such as: 15 O O R H O -(- O O ^- O O O O s O Br N-1 no- { O R O Other branched initiators useful for preparing the random 25 copolymers of the present invention include the following: O where radical R is as defined above. In some other embodi R 30 ments, the initiator can have the following structure: O Br 35 )-(- R where radical R is as defined above, and radical X can be 40 CHO, SOC1, SOCH=CH, NHCOCHI, N=C=O and O N=C=S, among others. Additional X groups can include Br the O O 45 O O O Br 50 As described above, the initiator can be added to the poly merization mixture separately, or can be incorporated into Still other initiators include, but are not limited to, the follow another molecule. Such as a monomer (hyperbranched struc ing: 55 ture) or a polymer fragment (Such as graft copolymers). Ini tiation of the polymerization can be accomplished by heat, UV light, or other methods known to one of skill in the art. O Br B. Monomers 60 Monomers useful for preparing the random copolymers of O O O)-(- the present invention include any monomer capable of radical polymerization. Typically, Such monomers have a vinyl N-O O N-1 no J-C group. Suitable monomers include, but are not limited to, O acrylate, methacrylate, acrylamide, methacrylamide, styrene, O O -(- O 65 vinyl-pyridine and vinyl-pyrrolidone monomers. Monomers, O Br M', containing the Zwitterionic moiety, ZW, include, but are not limited to, the following: US 8,765,432 B2 27 28 In still other embodiments, the random copolymer has the R4 formula: R4 R4 R4 21 I O I O R-I , (CH2) (CH2) SX (CH2) O O O ZW ZW ZW 10 L2 N." PC O y wherein A is camptothecin. 15 In other embodiments, the random copolymer has the for mula: YS,ZW Monomers, M, containing the linking group or functional agent include, but are not limited to, the following structures: S 25 PC wherein R* and Rare each independently selected from H, Co alkyl, Calkenyl, C- alkynyl, Chaloalkyl, C. heteroalkyl, C.s cycloalkyl, C-sheterocycloalkyl, aryl, het 30 eroaryl, A. L-A: LG, or L-LG; each of R, R and R' are independently selected from H or Ce alkyl; Subscripts y' and y are each independently an integer of from 1 to 1000; and PC is phosphatidylcholine. In still yet other embodiments, the random copolymer can have the following 35 formula: Other monomers are well-knownto one of skill in the art, and include vinyl acetate and derivatives thereof. In some embodiments, the monomers are acrylate or meth acrylate monomers. In other embodiments, the random A-L-I copolymer has the formula: 40 S 45 wherein each of L and L' is a linker, and each of A* and A is a functional agent. In other embodiments, the random 50 copolymer can have the following formula: PC wherein the groups R and R are independently selected from Hor C. alkyl, and PC is phosphatidylcholine. In some other embodiments, the random copolymer has the formula: 55 60 S wherein each of L and L' is a linker, and each of A* and A is a functional agent. 65 C. Zwitterions The Zwitterions of the present invention include any com pound having both a negative charge and a positive charge. US 8,765,432 B2 29 Groups having a negative charge and Suitable for use in the Zwitterions of the present invention include, but are not lim ited to, phosphate, Sulfate, other oxoanions, etc. Groups hav ing a positive charge and Suitable for use in the Zwitterions of the present invention include, but are not limited to, ammo 5 nium ions. In some embodiments, the Zwitterion can be phos phorylcholine. D. Linkers The random copolymers of the present invention can also incorporate any suitable linker L. The linkers provide for 10 attachment of the functional agents to the initiator fragment I and the comonomers M. The linkers can be cleavable or non-cleavable, homobifunctional or heterobifunctional. Other linkers can be both heterobifunctional and cleavable, or homobifunctional and cleavable. 15 Cleavable linkers include those that are hydrolyzable link ers, enzymatically cleavable linkers, pH sensitive linkers, disulfide linkers and photolabile linkers, among others. Hydrolyzable linkers include those that have an ester, carbon ate or carbamate functional group in the linker Such that reaction with water cleaves the linker. Enzymatically cleav able linkers include those that are cleaved by enzymes and can include an ester, amide, or carbamate functional group in the linker. pH sensitive linkers include those that are stable at one pH but are labile at another pH. For pH sensitive linkers, the 25 change in pH can be from acidic to basic conditions, from basic to acidic conditions, from mildly acidic to strongly acidic conditions, or from mildly basic to strongly basic con ditions. Suitable pH sensitive linkers are known to one of skill in the art and include, but are not limited to, ketals, acetals, 30 imines or imminiums, siloxanes, silaZanes, silanes, male amates-amide bonds, ortho esters, hydrazones, activated car —(CH-)o-phenyl-(CH2). , —N=C(R)— —(C. boxylic acid derivatives and vinyl ethers. Disulfide linkers are alkyl)-C(R)—N—(C. alkyl)-. —(C. alkyl)-(aryl)-C(R) characterized by having a disulfide bond in the linker and are =N-(C. alkyl)-, —(C. alkyl)-C(R)=N-(aryl)-(C. cleaved under reducing conditions. Photolabile linkers 35 alkyl)-, and —(Calkyl)-O-P(O)(OH)—O—(Coalkyl)- include those that are cleaved upon exposure to light, Such as wherein R is H. C. alkyl, C. cycloalkyl, or an aryl group visible, infrared, ultraviolet, or electromagnetic radiation at having 5-8 endocyclic atoms. other wavelengths. In some otherembodiments, linkers L'and L’ can be any of Other linkers useful in the present invention include those the following: —C-C alkyl-, -C-C cycloalkyl-, described in U.S. Patent Application Nos. 2008/0241102 (as 40 (—(CH). O—(CH) ) -, (-(CH2). NH signed to Ascendis/Complex Biosystems) and 2008/0152661 (CH2)4)1-12 , -(CH2)2O , (—(CH2). O— (assigned to Mirus) (incorporated in their entirety herein). (CH2)4)-12-O-, —(CH)—(CO)—O—, Mirus linkers useful in the present invention include, but are —(CH2)2 -(CO)—NH-, -(CH2). O—(CO)—, not limited to, the following: —(CH2). NH-(CO)—, (—(CH2). O— O O O O polymer R -OH NN R. polymer-NH3 + O - H H O O O 55 Other linkers include those described in Bioconjugate Tech (CH) -4) 1-1 O—(CH2) 1-23 —(CH2) 1-1 —(CO)—O— niques, Greg T. Hermanson, Academic Press, 2d ed., 2008 (CH2)-12 , —(CH) (CO)—NH-(CH2) , (incorporated in its entirety herein), and those described in —(CH2). O—(CO)—(CH2) , -(CH2)2– Angew. Chem. Int. Ed. 2009, 48,6974-6998 (Bertozzi, C. R. NH-(CO)—(CH) , —(C-C cycloalkyl)-, —(C- and Sletten, E. M) (incorporated in its entirety herein). 60 Calkyl)-(C-C cycloalkyl)-. —(C-C cycloalkyl)-(Cs In some embodiments, linkers L and L’ can have a length alkyl)-, —(Csalkyl)-(C-C cycloalkyl)-(Csalkyl)-, and of up to 30 atoms, each atom independently C, N, O, S, and P. In other embodiments, the linkers L and L’ can be any of the -(CH2)o-s-aryl-(CH2)o-s—. following: —C alkyl-, -C cycloalkyl-, -(Cs In still other embodiments, each of linkers L' and L is a alkyl)-(C. cycloalkyl)-(Cols alkyl)-, -(CH2)2O , 65 cleavable linker independently selected from hydrolyzable (—(CH2). O—(CH2): ) , (-(CH2). NH linkers, enzymatically cleavable linkers, pH sensitive linkers, (CH2)-4)1-12 . (—(CH2). O—(CH2)). O—, disulfide linkers and photolabile linkers. US 8,765,432 B2 31 32 E. Linking Groups LG triazole ring via the Huisgencycloaddition process (see U.S. The linkers and functional agents of the present invention Pat. No. 7,375,234, incorporated herein in its entirety). The can react with a linking group on the initiator fragment I or the maleimide chemistry involves reaction of the maleimide ole comonomers M to formabond. The linking groups LG of the fin with a nucleophile, such as —OH, -SH or NH, to present invention can be any Suitable functional group form a stable bond. Other linking groups include those capable of forming a bond to another functional group, described in Bioconjugate Techniques, Greg T. Hermanson, thereby linking the two groups together. For example, linking Academic Press, 2d ed., 2008 (incorporated in its entirety groups LG useful in the present invention include those used herein). in click chemistry, maleimide chemistry, and NHS-esters, Some non-limiting examples of the reaction of the linking among others. Linking groups involved in click chemistry 10 groups and some groups typically found or introduced into include, but are not limited to, azides and alkynes that form a functional agents are set forth in Table I. TABLE I Illustrative Groups Exemplary Reactive that may react with Linking Groups a linking group (LG) (shown as appended to -X) Producty-X Y-COOH HO-X Y-C(=O)C-X (hydroxyl or activated forms hereof (e.g., tresylate, meSylate etc.)) Y-COOH Y-C(=O)S-X HS-X (thiol) Y-SH Y S S-X Y-SH R-S S-X Y S S-X (disulfide) Y-SH (pyridyl)-S-S-X Y S S-X (dithiopyridyl) H(O-)C-X Y-N-CH-X aldehyde O Y-NH-CH2-X following reduction (HO), HC-X Y-N-CH-X aldehyde hydrate O Y-NH-CH2-X following reduction (RO)2CH-X or Y-N-CH-X O H O Y-NH-CH2-X following X- reduction O acetal ROCH(OH)-X or Y-N-CH-X hemiacetal O Y-NH CH-X following reduction R'(O-)C-X Y-N-CR-X ketone O Y-NH C(R)H-X following reduction (R'O),C(R)-X or Y-N=C(R)-X O R O Y-NH C(R)H-X following X- reduction O ketal ROC(R)(OH)-X Y-N=C(R)-X hemiketal O Y-NH C(R)H-X following reduction R'(S-)C-X ketone O thione (thioketone) Y-NH C(R)H-X following redcuction O Y-NH C(R)H-X following reduction monothioketal US 8,765,432 B2 34 TABLE I-continued Illustrative Groups Exemplary Reactive that may react with Linking Groups Y-(R")CH NH (C=O) NH-X following reduction US 8,765,432 B2 37 38 R" is C alkyl, C. cycloalkyl, or an aryl group having 5-8 agents to preformed random copolymers can be used where endocyclic atoms; the functional agent is not compatible with conditions Suit R" is H. C. alkyl, C. cycloalkyl, or an aryl group having able for polymerization. In addition, where cost makes the 5-8 endocyclic atoms: loss of an agent to imperfect synthetic yields, oftentimes R" is a carbonyl derivative *-(C=O) , *-(C=O)— 5 encountered particularly in multistep synthetic reactions, coupling of functional agent to preformed random copoly mers of the present invention can be employed. Where a functional agent is not compatible with the con NH-(C=O)—, or alternatively, R" is carbonyl derivative of ditions employed for polymerization reactions, it can be 10 desirable to introduce the functional agent Subsequent to the polymerization reaction. Bioactive agents, A, can be broadly selected. In some embodiments the bioactive agents can be selected from one or —NH (C=O)—, where “*” indicates the point of attach more drugs, vaccines, aptamers, avimer scaffolds based on ment to Succinimidyl or benzotriazolyl groups; 15 human A domain scaffolds, diabodies, camelids, shark X and Y are each the active agent, linker, monomer or initiator IgNAR antibodies, fibronectin type III scaffolds with modi fragment I. fied specificities, antibodies, antibody fragments, vitamins C(O)NR'R'', NR'R'', C alkyl-NR'R'', and cofactors, polysaccharides, carbohydrates, Steroids, lip N(R')C(O)R', N(R')C(O)OR, N(R')C(O) ids, fats, proteins, peptides, polypeptides, nucleotides, oligo NR'R'', OP(O)(OR), S(O)OR', S(O), nucleotides, polynucleotides, and nucleic acids (e.g., mRNA, NR'R'', CN, NO, cycloalkyl, heterocycloalkyl, aryl tRNA, snRNA, RNAi, microRNA, DNA, cDNA, antisense and heteroaryl constructs, ribozymes, etc., and combinations thereof). In one F. Functional Agents embodiment, the bioactive agents can be selected from pro Functional agents useful in the random copolymers of the teins, peptides, polypeptides, soluble or cell-bound, extracel present invention include any biological agent or synthetic 25 lular or intracellular, kinesins, molecular motors, enzymes, compound capable of targeting a particular ligand, receptor, extracellular matrix materials and combinations thereof. In complex, organelle, cell, tissue, epithelial sheet, or organ, or another embodiment, bioactive agents can be selected from of treating aparticular condition or disease state. Of particular nucleotides, oligonucleotides, polynucleotides, and nucleic interest, is a combination of bioactive agents that together acids (e.g., mRNA, tRNA, snRNA, RNAi, DNA, cDNA, anti target mechanisms common to a particular disease. For 30 sense constructs, ribozymes etc and combinations thereof). In example, a first bioactive agent (stably attached) that is a another embodiment, bioactive agents can be selected from biopharmaceutical agent that binds to a proteinupregulated in steroids, lipids, fats and combinations thereof. For example, a disease; a second bioactive agent (stably attached) that is a the bioactive agent can bind to the extracellular matrix, Such peptide that binds to an extracellular matrix tissue constituent as when the extracellular matrix is hyaluronic acid or heparin Such as heparin Sulfate; a third bioactive agent (unstably 35 Sulfate proteoglycan and the bioactive agent is a positively attached) that is a small molecule drug that releases over time charged moiety Such as choline for non-specific, electrostatic, and exerts a local, intracellular effect, for example, an anti Velcro type binding interactions. In another embodiment, the proliferative effect. In some embodiments, the bioactive bioactive agent can be a peptide sequence that binds non agent is a drug, a therapeutic protein, a small molecule, a specifically or specifically. peptide, a peptoid, an oligonucleotide (aptamer, siRNA, 40 Bioactive agents can be designed and/or selected to have a microRNA), a nanoparticle, a carbohydrate, a lipid, a gly full activity (Such as a high level of agonism or antagonism). colipid, a phospholipid, or a targeting agent. The ratio of Alternatively, a multifunctional bioactive agent can be comonomers is chosen based on predefined Stoichiometry selected to modulate one target protein's activity while (for example, to match a biological avidity; to match a bio impacting fully another. logical Stoichiometry; to impart a gearing effect). Other 45 Just as mosaic proteins contain extracellular binding functional agents useful in the random copolymers of the domains or sub-domains (example, VEGF and Heparin Bind present invention include, but are not limited to, radiolabels, ing Epidermal Growth Factor), sequences from these binding fluorophores and dyes. sites can be replicated as a bioactive agent for polymerattach The functional agents can be linked to the initiator frag ment. More broadly, mosaic proteins represent strings of ment I or the comonomers M, or both, of the random copoly 50 domains of many functions (target binding, extracellular mers. The functional agents can be linked to the initiator matrix binding, spacers, avidity increases, enzymatic). The fragment I or the comonomers Meither before or after poly set of bioactives chosen for a particular application can be merization via cleavable or non-cleavable linkers described assembled in similar fashion to replicate a set of desired above. The functional agent can also be physisorbed or ioni functional activities. cally absorbed to the random copolymer instead of covalently 55 Other functional agents, A, include charged species such as attached. choline, lysine and hyaluronic acid, among others. The The preparation of the random copolymers of the present charged species are useful for facilitating ionic attachment, to invention linked to a functional agent can be conducted by vitreous for example. first linking the functional agent to a linking group attached to Therapeutic Proteins and Antibodies a monomer and Subjecting the coupled functional agent to 60 In one particularly useful embodiment, the functional conditions suitable for synthesis of the inventive random agent is a therapeutic protein. Numerous therapeutic proteins copolymers. In those cases, a Suitable linking group can be an are disclosed throughout the application Such as, and without initiator (e.g., iodinated, brominated or chlorinated com limitation, erythropoietin, granulocyte colony stimulating pound/group) for use in ATRP reactions. Such a reaction factor (G-CSF), GM-CSF, interferon alpha, interferon beta, scheme is possible where the functional agent is compatible 65 human growth hormone, and imiglucerase. with the polymer polymerization reactions and any Subse In one embodiment, the functional agents can be selected quent workup required. However, coupling of functional from specifically identified polysaccharide, protein or pep US 8,765,432 B2 39 40 tide bioactive agents, including, but not limited to: AB, agal imab, labetuzumab, lerdelimumab, olizumab, radiolabeled sidase, alefacept, alkaline phosphatase, aspariginase, amdox lym-1, metelimumab, mepolizumab, mitumomab, ovir (DAPD), antide, becaplermin, botulinum toxin including muromonad-CD3, nebacumab, natalizumab, odulimomab, types A and B and lower molecular weight compounds with omalizumab, oregovomab, palivizumab, pemtumomab, pex botulinum toxin activity, calcitonins, cyanovirin, denileukin 5 elizumab, rhuMAb-VEGF, rituximab, satumomab pendetide, diftitox, erythropoietin (EPO), EPO agonists, dornase alpha, sevirumab, siplizumab, to situmomab, I'"to situmomab, tras erythropoiesis stimulating protein (NESP), coagulation fac tuZumab, tuvirumab, visilizumab, and fragments and mimet tors such as Factor V. Factor VII, Factor VIIa, Factor VIII, ics thereof. Factor IX, Factor X, Factor XII, Factor XIII, von Willebrand In one embodiment, the bioactive agent is a fusion protein. factor, ceredase, cerezyme, alpha-glucosidase, N-Acetylga 10 For example, and without limitation, the bioactive component lactosamine-6-sulfate Sulfatase, collagen, cyclosporin, alpha can be an immunoglobulin or portion of an immunoglobulin defensins, beta defensins, desmopressin, exendin-4, cytok fused to one or more certain useful peptide sequences. For ines, cytokine receptors, granulocyte colony stimulating fac example, the bioactive agent may contain an antibody Fc tor (G-CSF), thrombopoietin (TPO), alpha-1 proteinase fragment. In one embodiment, the bioactive agent is a CTLA4 inhibitor, elcatonin, granulocyte macrophage colony stimu 15 fusion protein. For example, the bioactive agent can be an lating factor (GM-CSF), fibrinogen, filgrastim, growth hor Fc-CTLA4 fusion protein. mones human growth hormone (hGH). Somatropin, growth In one particularly useful embodiment, the bioactive agent hormone releasing hormone (GHRH), GRO-beta, GRO-beta is a human protein or human polypeptide, for example, a antibody, bone morphogenic proteins such as bone morpho heterologously produced human protein or human polypep genic protein-2, bone morphogenic protein-6, parathyroid tide. Numerous proteins and polypeptides are disclosed hormone, parathyroid hormone related peptide, OP-1; acidic herein for which there is a corresponding human form (i.e., fibroblast growth factor, basic fibroblast growth factor, Fibro the protein or peptide is normally produced in human cells in blast Growth Factor 21, CD-40 ligand, heparin, human serum the human body). Therefore, in one embodiment, the bioac albumin, low molecular weight heparin (LMWH), interferon tive agent is the human form of each of the proteins and alpha, interferon beta, interferon gamma, interferon omega, 25 polypeptides disclosed herein for which there is a human interferon tau, consensus interferon; interleukins and inter form. Examples of Such human proteins include, without leukin receptors such as interleukin-1 receptor, interleukin-2, limitation, human antibodies, human enzymes, human hor interleukin-2 fusion proteins, interleukin-1 receptor antago mones and human cytokines such as granulocyte colony nist, interleukin-3, interleukin-4, interleukin-4 receptor, stimulation factor, granulocyte macrophage colony stimula interleukin-6, interleukin-8, interleukin-12, interleukin-17, 30 tion factor, interferons (e.g., alpha interferons and beta inter interleukin-21, interleukin-13 receptor, interleukin-17 recep ferons), human growth hormone and erythropoietin. tor; lactoferrin and lactoferrin fragments, luteinizing hor Other examples of therapeutic proteins which may serve as mone releasing hormone (LHRH), insulin, pro-insulin, insu bioactive agents include, without limitation, factor VIII, lin analogues, amylin, C-peptide, Somatostatin, Somatostatin b-domain deleted factor VIII, factor VIIa, factor IX, antico analogs including octreotide, Vasopressin, follicle stimulat 35 agulants; hirudin, alteplase, tpa, reteplase, tpa, tpa-3 of 5 ing hormone (FSH), imiglucerase, influenza vaccine, insulin domains deleted, insulin, insulin lispro, insulinaspart, insulin like growth factor (IGF), insulintropin, macrophage colony glargine, long-acting insulin analogs, hgh, glucagons, tsh, stimulating factor (M-CSF), plasminogen activators such as follitropin-beta, fish, gm-csf.pdgh, ifinalpha2, ifn alpha2a, ifn alteplase, urokinase, reteplase, streptokinase, pamiteplase, alpha2b, inf-aphal, consensus ifn, ifn-beta, ifn-beta 1b, ifn lanoteplase, and teneteplase; nerve growth factor (NGF), 40 beta1a, ifn-gamma (e.g., 1 and 2), ifn-lambda, ifn-delta, il-2. osteoprotegerin, platelet-derived growth factor, tissue growth ill-11, hbsag, ospa, murine mab directed against t-lymphocyte factors, transforming growth factor-1, vascular endothelial antigen, murine mab directed against tag-72, tumor-associ growth factor, leukemia inhibiting factor, keratinocyte ated glycoprotein, fab fragments derived from chimeric mab growth factor (KGF), glial growth factor (GGF), T Cell recep directed against platelet Surface receptor gpII(b)/III(a), tors, CD molecules/antigens, tumor necrosis factor (TNF) 45 murine mab fragment directed against tumor-associated anti (e.g., TNF-C. and TNF-B), TNF receptors (e.g., TNF-C. recep gen cal25, murine mab fragment directed against human tor and TNF-B receptor), CTLA4, CTLA4 receptor, mono carcinoembryonic antigen, cea, murine mab fragment cyte chemoattractant protein-1, endothelial growth factors, directed against human cardiac myosin, murine mab frag parathyroid hormone (PTH), glucagon-like peptide. Soma ment directed against tumor Surface antigen psma, murine totropin, thymosin alpha 1, rasburicase, thymosin alpha 1 50 mab fragments (fab/fab2 mix) directed against hmw-maa, IIb/IIIa inhibitor, thymosin beta 10, thymosin beta 9, thy murine mab fragment (fab) directed against carcinoma-asso mosin beta 4, alpha-1 antitrypsin, phosphodiesterase (PDE) ciated antigen, mab fragments (fab) directed against nca 90, a compounds, VLA-4 (very late antigen-4), VLA-4 inhibitors, Surface granulocyte nonspecific cross reacting antigen, chi bisphosphonates, respiratory syncytial virus antibody, cystic meric mab directed against cd20 antigen found on Surface of fibrosis transmembrane regulator (CFTR) gene, deoxyribo 55 b lymphocytes, humanized mab directed against the alpha nuclease (Dnase), bactericidal/permeability increasing pro chain of the il2 receptor, chimeric mab directed against the tein (BPI), and anti-CMV antibody. Exemplary monoclonal alpha chain of the il2 receptor, chimeric mab directed against antibodies include etanercept (a dimeric fusion protein con tnf-alpha, humanized mab directed against an epitope on the sisting of the extracellular ligand-binding portion of the Surface of respiratory synctial virus, humanized mab directed human 75 kDTNF receptor linked to the Fc portion of IgG1), 60 against her 2, human epidermal growth factor receptor 2, abciximab, adalimumab, afelimomab, alemtuzumab, anti human mab directed against cytokeratin tumor-associated body to B-lymphocyte, atlizumab, basiliximab, bevaci antigen anti-ctla4, chimeric mab directed against cd 20 Sur Zumab, biciromab, bertilimumab, CDP-484, CDP-571, CDP face antigen ofb lymphocytes dornase-alpha dinase, beta glu 791, CDP-860, CDP-870, cetuximab, clenoliximab, cocerebrosidase, tinf-alpha, il-2-diptheria toxin fusion pro daclizumab, eculizumab, edrecolomab, efalizumab, epratu 65 tein, thfr-lgg fragment fusion protein laronidase, dnaases, Zumab, fontolizumab, gavilimomab, gemtuzumab ozogami alefacept, darbepoetin alpha (colony stimulating factor), tosi cin, ibritumomab tiuxetan, infliximab, inolimomab, kelix tumomab, murine mab, alemtuzumab, rasburicase, agalsi US 8,765,432 B2 41 42 dase beta, teriparatide, parathyroid hormone derivatives, Zumab (Genentech) is a chimeric (mouse and human) mono adalimumab (1ggl), anakinra, biological modifier, nesiritide, clonal antibody used to treat macular degeneration. human b-type natriuretic peptide (hbnp), colony stimulating Proteins, Peptides and Amino Acids factors, pegvisomant, human growth hormone receptor Proteins and peptides for use as bioactive agents as dis antagonist, recombinant activated protein c, omalizumab, 5 closed herein can be produced by any useful method includ immunoglobulin e (lge) blocker, Ibritumomab tiuxetan, ing production by in vitro synthesis and by production in ACTH, glucagon, Somatostatin, Somatotropin, thymosin, par biological systems. Typical examples of in vitro synthesis athyroid hormone, pigmentary hormones, somatomedin, methods which are well known in the art include solid-phase erythropoietin, luteinizing hormone, chorionic gonadotropin, synthesis (“SPPS) and solid-phase fragment condensation hypothalmic releasing factors, etanercept, antidiuretic hor 10 (“SPFC). Biological systems used for the production of mones, prolactin and thyroid stimulating hormone. And any proteins are also well known in the art. Bacteria (e.g., E. coli of these can be modified to have a site-specific conjugation and Bacillus sp.) and yeast (e.g., Saccharomyces cerevisiae point (a N-terminus, or C-terminus, or other location) using and Pichia pastoris) are widely used for the production of natural (for example, a serine to cysteine Substitution) (for 15 heterologous proteins. In addition, heterologous gene expres example, formylaldehyde per method of Redwood Bio sion for the production of bioactive agents for use as disclosed Sciences) or non-natural amino acid. herein can be accomplished using animal cell lines Such as Examples of therapeutic antibodies that may serve as bio mammalian cell lines (e.g., CHO cells). In one particularly active agents include, but are not limited, to HERCEPTINTM useful embodiment, the bioactive agents are produced in (Trastuzumab) (Genentech, CA) which is a humanized anti transgenic or cloned animals such as cows, sheep, goats and HER2 monoclonal antibody for the treatment of patients with birds (e.g., chicken, quail, ducks and turkey), each as is under metastatic breast cancer; REOPROTM (abciximab) (Cento stood in the art. See, for example, U.S. Pat. No. 6,781,030, cor) which is an anti-glycoprotein IIb/IIIa receptor on the issued Aug. 24, 2004, the disclosure of which is incorporated platelets for the prevention of clot formation: ZENAPAXTM in its entirety herein by reference. (daclizumab) (Roche Pharmaceuticals, Switzerland) which is 25 Bioactive agents such as proteins produced in domesti an immunosuppressive, humanized anti-CD25 monoclonal cated birds such as chickens can be referred to as “avian antibody for the prevention of acute renal allograft rejection; derived bioactive agents (e.g., avian derived therapeutic pro PANOREXTM which is a murine anti-17-IA cell Surface anti teins). Production of avian derived therapeutic proteins is gen IgG2a antibody (Glaxo Wellcome/Centocor); BEC2 known in the art and is described in, for example, U.S. Pat. which is a murine anti-idiotype (GD3 epitope) IgG antibody 30 No. 6,730,822, issued May 4, 2004, the disclosure of which is (ImClone System); IMC-C225 which is a chimeric anti incorporated in its entirety herein by reference. EGFR IgG antibody (ImClone System): VITAXINTM which In embodiments where the bioactive agent is a protein or is a humanized anti-CVB3 integrin antibody (Applied polypeptide, functional groups present in the amino acids of Molecular Evolution/MedImmune); Campath; Campath the protein polypeptide sequence can be used to link the agent 1H/LDP-03 which is a humanized anti CD52 IgG1 antibody 35 to the random copolymer. Linkages to protein or polypeptide (Leukosite); Smart M195 which is a humanized anti-CD33 bioactive agents can be made to naturally occurring amino IgG antibody (Protein Design Lab/Kanebo); RITUXANTM acids in their sequence or to naturally occurring amino acids which is a chimericanti-CD2O IgG1 antibody (IDEC Pharm/ that have either been added to the sequence or inserted in Genentech, Roche/Zettyaku); LYMPHOCIDETM which is a place of another amino acid, for example the replacement of humanized anti-CD22 IgG antibody (Immunomedics); ICM3 40 a serine by a cysteine. is a humanized anti-ICAM3 antibody (ICOS Pharm); IDEC Protein or polypeptide bioactive agents may also comprise 114 is a primate anti-CD80 antibody (IDEC Pharm/Mitsub non-naturally occurring amino acids in addition to the com ishi); ZEVALINTM is a radiolabelled murine anti-CD20 anti mon naturally occurring amino acids found in proteins and body (IDEC/Schering AG); IDEC-131 is a humanized anti polypeptides. In addition to being present for the purpose of CD40L antibody (IDEC/Eisai); IDEC-151 is a primatized 45 altering the properties of a polypeptide or protein, non-natu anti-CD4 antibody (IDEC); IDEC-152 is a primatized anti rally occurring amino acids can be introduced to provide a CD23 antibody (IDEC/Seikagaku): SMART anti-CD3 is a functional group that can be used to link the protein or humanized anti-CD3 IgG (Protein Design Lab); 5G1.1 is a polypeptide directly to random copolymer. Furthermore, humanized anti-complement factor 5 (CS) antibody (Alexion naturally occurring amino acids, e.g., cysteine, tyrosine, tryp Pharm); D2E7 is a humanized anti-TNF-C. antibody (CATI 50 tophan can be used in this way. BASF); CDP870 is a humanized anti-TNF-C. Fab fragment Non-naturally occurring amino acids can be introduced (Celltech); IDEC-151 is a primatized anti-CD4 IgG1 anti into proteins and peptides by a variety of means. Some of the body (IDEC Pharm/SmithKline Beecham); MDX-CD4 is a techniques for the introduction of non-natural amino acids are human anti-CD4 IgG antibody (Medarex/Eisai/Genmab); discussed in U.S. Pat. No. 5,162,218, the disclosure of which CDP571 is a humanized anti-TNF-C. IgG4 antibody 55 is incorporated in its entirety herein by reference. First, non (Celltech); LDP-02 is a humanized anti-C437 antibody (Leu naturally occurring amino acids can be introduced by chemi koSite/Genentech); OrthoClone OKT4A is a humanized anti cal modification of a polypeptide or protein on the amino acid CD4 IgG antibody (Ortho Biotech): ANTOVATM is a human side chain or at either the amino terminus or the carboxyl ized anti-CD40L IgG antibody (Biogen): ANTEGRENTM is a terminus. Non-limiting examples of chemical modification of humanized anti-VLA-4 IgG antibody (Elan); CAT-152, a 60 a protein or peptide might be methylation by agents such as human anti-TGF-B antibody (Cambridge Ab Tech); Cetux diazomethane, or the introduction of acetylation at an amino imab (BMS) is a monoclonal anti-EGF receptor (EGFr) anti group present in lysines side chain or at the amino terminus body; Bevacizuma (Genentech) is an anti-VEGF human of a peptide or protein. Another example of the protein/ monoclonal antibody; Infliximab (Centocore, JJ) is a chi polypeptide amino group modification to prepare a non-natu meric (mouse and human) monoclonal antibody used to treat 65 ralamino acid is the use of methyl 3-mercaptopropionimidate autoimmune disorders; Gemtuzumab ozogamicin (Wyeth) is ester or 2-iminothiolane to introduce a thiol (sulfhydryl, a monoclonal antibody used for chemotherapy; and Ranibi —SH) bearing functionality linked to positions in a protein or US 8,765,432 B2 43 44 polypeptide bearing a primary amine. Once introduced. Such functionality is unique or where other functionalities that groups can be employed to form a covalent linkage to the might react under the conditions of interest are hindered protein or polypeptide. either stereochemically or otherwise. Second, non-naturally occurring amino acids can be intro Drugs duced into proteins and polypeptides during chemical Syn In another embodiment, the bioactive agents can also be thesis. Synthetic methods are typically utilized for preparing selected from specifically identified drug or therapeutic polypeptides having fewer than about 200 amino acids, usu agents, including but not limited to: tacrine, memantine, ally having fewer than about 150 amino acids, and more rivastigmine, galantamine, donepezil, levetiracetam, repa usually having 100 or fewer amino acids. Shorter proteins or glinide, atorvastatin, alefacept, tadalafil. Vardenafil. Sildena polypeptides having less than about 75 or less than about 50 10 fil, foSamprenavir, oseltamivir, Valacyclovir and Valganciclo amino acids can be prepared by chemical synthesis. Vir, abarelix, adefovir, alfuZosin, alosetron, amifostine, The synthetic preparation methods that are particularly amiodarone, aminocaproic acid, aminohippurate Sodium, convenient for allowing the insertion of non-natural amino aminoglutethimide, aminolevulinic acid, aminosalicylic acids at a desired location are known in the art. Suitable acid, amlodipine, amsacrine, anagrelide, anastroZole, aprepi synthetic polypeptide preparation methods can be based on 15 tant, aripiprazole, asparaginase, atazanavir, atomoxetine, Merrifield solid-phase synthesis methods where amino acids anthracyclines, beXarotene, bicalutamide, bleomycin, bort are sequentially added to a growing chain (Merrifield (1963) eZomib, buserelin, buSulfan, cabergoline, capecitabine, car J. Am. Chem. Soc. 85:2149-2156). Automated systems for boplatin, carmustine, chlorambucin, cilastatin Sodium, cispl synthesizing polypeptides by Such techniques are now com atin, cladribine, clodronate, cyclophosphamide, cyproterone, mercially available from Suppliers such as Applied Biosys cytarabine, camptothecins, 13-cis retinoic acid, all trans ret tems, Inc., Foster City, Calif. 94.404; New Brunswick Scien inoic acid; dacarbazine, dactinomycin, daptomycin, dauno tific, Edison, N.J. 08818; and Pharmacia, Inc., Biotechnology rubicin, deferoxamine, dexamethasone, diclofenac, diethyl Group, Piscataway, N.J. 08854. stilbestrol, docetaxel, doxorubicin, dutasteride, eletriptan, Examples of non-naturally occurring amino acids that can emitricitabine, enfuvirtide, eplerenone, epirubicin, estramus be introduced during chemical synthesis of polypeptides 25 tine, ethinyl estradiol, etoposide, exemestane, eZetimibe, fen include, but are not limited to: D-amino acids and mixtures of tanyl, fexofenadine, fludarabine, fludrocortisone, fluorou D and L-forms of the 20 naturally occurring amino acids, racil, fluoxymesterone, flutamide, fluticaZone, fondaparinux, N-formyl glycine, ornithine, norleucine, hydroxyproline, fulvestrant, gamma-hydroxybutyrate, gefitinib, gemcitabine, beta-alanine, hydroxyvaline, norvaline, phenylglycine, epinephrine, L-Dopa, hydroxyurea, icodextrin, idarubicin, cyclohexylalanine, t-butylglycine (t-leucine, 2-amino-3,3- 30 ifosfamide, imatinib, irinotecan, itraconazole, goserelin, dimethylbutanoic acid), hydroxy-t-butylglycine, amino laronidase, lanSoprazole, letrozole, leucovorin, levamisole, butyric acid, cycloleucine, 4-hydroxyproline, pyroglutamic lisinopril, lovothyroxine sodium, lomustine, mechlore acid (5-oxoproline), aZetidine carboxylic acid, pipecolinic thamine, medroxyprogesterone, megestrol, melphalan, acid, indoline-2-carboxylic acid, tetrahydro-3-isoquinoline memantine, mercaptopurine, meduinol, metaraminol bitar carboxylic acid, 2,4-diaminobutyricacid, 2,6-diami 35 trate, methotrexate, metoclopramide, mexiletine, miglustat, nopimelic acid, 2,4-diaminobutyricacid, 2,6-diaminopimeli mitomycin, mitotane, mitoxantrone, modafinil, naloxone, cacid, 2,3-diaminopropionicacid, 5-hydroxylysine, naproxen, nevirapine, nicotine, nilutamide, nitazoxanide, neuraminic acid, and 3.5-diiodotyrosine. nitisinone, norethindrone, octreotide, Oxaliplatin, pal Third, non-naturally occurring amino acids can be intro onosetron, pamidronate, pemetrexed, pergolide, pentostatin, duced through biological synthesis in vivo or in vitro by 40 pilcamycin, porfimer, prednisone, procarbazine, prochlor insertion of a non-sense codon (e.g., an amber or ocher perazine, ondansetron, palonosetron, oxaliplatin, raltitrexed, codon) in a DNA sequence (e.g., the gene) encoding the rosuvastatin, sirolimus, Streptozocin, pimecrolimus, Serta polypeptide at the codon corresponding to the position where conazole, tacrolimus, tamoxifen, tegaserod, temozolomide, the non-natural amino acid is to be inserted. Such techniques teniposide, testosterone, tetrahydrocannabinol, thalidomide, are discussed for example in U.S. Pat. Nos. 5,162,218 and 45 thioguanine, thiotepa, tiotropium, topiramate, topotecan, tre 6,964,859, the disclosures of which are incorporated in their prostinil, tretinoin, Valdecoxib, celecoxib, rofecoxib, valru entirety herein by reference. A variety of methods can be used bicin, vinblastine, Vincristine, vindesine, vinorelbine, Vori to insert the mutant codon including oligonucleotide-directed conazole, dolasetron, granisetron, formoterol, fluticaSone, mutagenesis. The altered sequence is Subsequently tran leuprolide, midazolam, alprazolam, amphotericin B, podo scribed and translated, in vivo or in vitro in a system which 50 phylotoxins, nucleoside antivirals, aroyl hydrazones, provides a Suppressor tRNA, directed against the nonsense Sumatriptan, eletriptan; macrollides such as erythromycin, codon that has been chemically or enzymatically acylated oleandomycin, troleandomycin, roXithromycin, clarithromy with the desired non-naturally occurring amino acid. The cin, davercin, azithromycin, flurithromycin, dirithromycin, synthetic amino acid will be inserted at the location corre josamycin, spiromycin, midecamycin, loratadine, deslorata sponding to the nonsense codon. For the preparation of larger 55 dine, leucomycin, miocamycin, rokitamycin, andazithromy and/or glycosylated polypeptides, recombinant preparation cin, and Swinolide A, fluoroquinolones Such as ciprofloxacin, techniques of this type are usually preferred. Among the ofloxacin, levofloxacin, trovafloxacin, alatrofloxacin, moxi amino acids that can be introduced in this fashion are: formyl floxicin, norfloxacin, enoxacin, gatifloxacin, gemifloxacin, glycine, fluoroalanine, 2-Amino-3-mercapto-3-methylbu grepafloxacin, lomefloxacin, sparfloxacin, temafloxacin, tanoic acid, homocysteine, homoarginine and the like. Other 60 pefloxacin, amifloxacin, fleroxacin, to Sufloxacin, prulifloxa similar approaches to obtain non-natural amino acids in a cin, irloxacin, paZufloxacin, clinafloxacin, and sitafloxacin; protein include methionine Substitution methods. aminoglycosides such as gentamicin, netilmicin, paramecin, Where non-naturally occurring amino acids have a func tobramycin, amikacin, kanamycin, neomycin, and strepto tionality that is susceptible to selective modification, they are mycin, Vancomycin, teicoplanin, rampolanin, mideplanin, particularly useful for forming a covalent linkage to the pro 65 colistin, daptomycin, gramicidin, colistimethate; polymixins tein or polypeptide. Circumstances where a functionality is Such as polymixin B, capreomycin, bacitracin, penems; peni susceptible to selective modification include those where the cillins including peniclinase-sensitive agents like penicillin US 8,765,432 B2 45 46 G. penicillin V: penicillinase-resistant agents like methicillin, gallium 66 (Ga), copper 67 (Cu), gallium 67 ('Ga), gal oxacillin, cloxacillin, dicloxacillin, floxacillin, nafcillin; lium 68 (Ga), rubidium 82 (Rb), yttrium 86 (Y), yttrium gram negative microorganism active agents like amplicillin, 87 (7Y), strontium 89 (Sr), yttrium 90 (Y), rhodium 105 amoxicillin, and hetacillin, cillin, and galampicillin; antip ('Rh), silver 111 ('Ag), indium 111 ('''In), iodine 124 seudomonal penicillins like carbenicillin, ticarcillin, azlocil (I), iodine 125 (I), iodine 131 (I), tin 117m ('7"Sn), lin, mezlocillin, and piperacillin; cephalosporins like ce?po technetium 99m ("Tc), promethium 149 ('''Pm), doXime, cefprozil, ceftbuten, ceftizoxime, ceftriaxone, samarium 153 ('Sm), holmium 166 (166Ho), lutetium 177 cephalothin, cephapirin, cephalexin, cephradrine, cefoxitin, (77Lu), rhenium 186 (Re), rhenium 188 (Re), thallium cefamandole, cefazolin, cephaloridine, cefaclor, cefadroxil, 201 ('Tl), astatine 211 (At), and bismuth 212 ('Bi). As cephaloglycin, cefuroxime, ceforanide, cefotaxime, cefatriz 10 used herein, the “m” in '7"Sn and "Tc stands for meta ine, cephacetrile, cefepime, cefixime, cefonicid, cefopera state. Additionally, naturally occurring radioactive elements Zone, cefotetan, cefimetazole, ceftazidime, loracarbef, and Such as uranium, radium, and thorium, which typically rep moxalactam, monobactams like aztreonam; and carbapen resent mixtures of radioisotopes, are suitable examples of ems such as imipenem, meropenem, and ertapenem, penta radionuclides. 'Cu, ''I, 77Lu, and Re are beta- and midine isetionate, albuterol Sulfate, lidocaine, metaproter 15 gamma-emitting radionuclides. 'Bi is an alpha- and beta enol Sulfate, beclomethasone diprepionate, triamcinolone emitting radionuclide. 'At is an alpha-emitting radionu acetamide, budesonide acetonide, salmeterol, ipratropium clide. 32P *7Sc, 89Sr. 90Y, 105Rh, '''Ag, 117mSn, 149Pm, bromide, flunisolide, cromolyn Sodium, and ergotamine tar 'Sim, ''Ho, and Reare examples of beta-emitting radio trate; taxanes such as paclitaxel; SN-38, and tyrphostines. nuclides. Ga, '''In, "Tc, and 'Tl are examples of Bioactive agents may also be selected from the group con gamma-emitting radionuclides. Co, 'Cu, 'Cu, Cu, sisting of aminohippurate sodium, amphotericin B, doxoru Ga, Ga, Rb, and Y are examples of positron-emitting bicin, aminocaproic acid, aminolevulinic acid, aminosali radionuclides. Cu is a beta- and positron-emitting radionu cylic acid, metaraminol bitartrate, pamidronate disodium, clide. daunorubicin, levothyroxine sodium, lisinopril, cilastatin Nanoparticles Sodium, mexiletine, cephalexin, deferoxamine, and amifos 25 The functional agents can also include nanoparticles. tine in another embodiment. Nanoparticles useful in the present invention include particles Other bioactive agents useful in the present invention having a size ranging form 1 to 1000 nm. Nanoparticles can include extracellular matrix targeting agents, functional be beads, metallic particles or can in Some cases be micelles transport moieties and labeling agents. Extracellular matrix and in some other be liposomes. Other nanoparticles include targeting agents include, but are not limited to, heparin bind 30 carbon nanotubes, quantum dots and colloidal gold. Nano ing moieties, matrix metalloproteinase binding moieties, particles can be packed with diagnostic and/or therapeutic lysyl oxidase binding domains, negatively charged moieties agents. or positively charged moieties and hyaluronic acid. Func Those skilled in the art will also recognize that the inven tional transport moieties include, but are not limited to, blood tion can be used to enable coincident detection of more than brain barrier transport moieties, intracellular transport moi 35 one agent of the same or different type. Also, the use of eties, organelle transport moieties, epithelial transport flexible linker chemistries can also be used to witness the loss domains and tumor targeting moieties (folate, other). In some of one fluorescent label, for example as the molecule is taken embodiments, the targeting agents useful in the present inven up into the cell and into a low pH environment. tion target anti-TrkA, anti A-beta (peptide 1-40, peptide 1-42, monomeric form, oligomeric form), anti-IGF1-4, agonist 40 IV. Preparation of Phosphoryl-Containing Random RANK-L, anti-ApoE4 or anti-ApoA1, among others. Copolymers Diagnostic Agents Diagnostic agents useful in the random copolymers of the The random copolymers of the present invention can be present invention include imaging agents and detection prepared by any means known in the art. In some embodi agents such as radiolabels, fluorophores, dyes and contrast 45 ments, the present invention provides a process for preparing agents. a random copolymer of the present invention, the process Imaging agent refers to a label that is attached to the ran including the step of contacting a mixture of a first monomer dom copolymer of the present invention for imaging a tumor, and a second monomer with an initiator, I', under conditions organ, or tissue in a Subject. The imaging moiety can be Sufficient to prepare a random copolymer via free radical covalently or non-covalently attached to the random copoly 50 polymerization, wherein the first monomer comprises a phos mer. Examples of imaging moieties Suitable for use in the phorylcholine, and each of the second monomer and initiator present invention include, without limitation, radionuclides, independently comprise at least one of a functional agent or a fluorophores Such as fluorescein, rhodamine, Texas Red, Cy2, linking group for linking to the functional agent. Cy3, Cy5, and the AlexaFluor (Invitrogen, Carlsbad, Calif.) The mixture for preparing the random copolymers of the range offluorophores, antibodies, gadolinium, gold, nanoma 55 present invention can include a variety of other components. terials, horseradish peroxidase, alkaline phosphatase, deriva For example, the mixture can also include catalyst, ligand, tives thereof, and mixtures thereof. Solvent, and other additives. In some embodiments, the mix Radiolabel refers to a nuclide that exhibits radioactivity. A ture also includes a catalyst and a ligand. Suitable catalysts “nuclide” refers to a type of atom specified by its atomic and ligands are described in more detail below. number, atomic mass, and energy state, such as carbon 14 60 Any suitable monomer can be used in the process of the (''C). “Radioactivity” refers to the radiation, including alpha present invention, such as those described above. particles, beta particles, nucleons, electrons, positrons, neu The random copolymers of the present invention can be trinos, and gamma rays, emitted by a radioactive Substance. prepared by any Suitable polymerization method, such as by Radionuclides suitable for use in the present invention living radical polymerization. Living radical polymerization, include, but are not limited to, fluorine 18 ("F) phosphorus 65 discussed by Odian, G. in Principles of Polymerization, 4", 32 (PP), scandium 47 ('Sc), cobalt 55 (Co), copper 60 Wiley-Interscience John Wiley & Sons: New York, 2004, and ('Cu), copper 61 (Cu), copper 62 (Cu), copper 64 (Cu), applied to Zwitterionic polymers for example in U.S. Pat. No. US 8,765,432 B2 47 48 6,852,816. Several different living radical polymerization although immobilized catalysts can be employed. Suitable methodologies can be employed, including Stable Free Radi ligands include those pyridyl groups (including alkyl cal Polymerization (SFRP) and Radical Addition-Fragmen pyridines e.g., 4.4. dialkyl-2.2" bipyridines) and pyridyl tation Transfer (RAFT). In addition, Atom Transfer Radical groups bearing an alkyl Substituted imino group, where Polymerization (ATRP), provides a convenient method for 5 present, longer alkyl groups provide solubility in less polar the preparation of the random copolymers of the invention. monomer mixtures and solvent media. Triphenyl phosphines The preparation of polymers via ATRP involves the radical and other phosphorus ligands, in addition to indanyl, orcyclo polymerization of monomers beginning with an initiator pentadienyl ligands, can also be employed with transition bearing one or more halogens. The initiator is activated by a metal catalysts (e.g., Ru-halide or Fe"-halide complexes catalyst Such as a transition metal salt (CuBr) that can be 10 with triphenylphosphine, indanyl or cyclopentadienyl solubilized by a ligand (e.g., bipyridine). RAFT polymeriza ligands). tion uses thiocarbonylthio compounds, Such as dithioesters, An approximately stoichiometric amount of metal com dithiocarbamates, trithiocarbonates, and Xanthates, to medi pound and ligand in the catalyst, based on the molar ratios of ate the polymerization process via a reversible chain-transfer the components when the metal ion is fully complexed, is process. Other living radical processes useful in the prepara 15 employed in some embodiments. In other embodiments the tion of the inventive random copolymers include nitroxide ratio between metal compound and ligand is in the range mediated polymerization. 1:(0.5 to 2) or in the range 1:(0.8 to 1.25). Initiators Generally, where the catalyst is copper, bidentate or mul Initiators useful for the preparation of the random copoly tidentate nitrogen ligands produce more active catalysts. In mers of the present invention include any initiator suitable for addition, bridged or cyclic ligands and branched aliphatic polymerization via atom transfer radical polymerization polyamines provide more active catalysts than simple linear (ATRP), such as those described above. Other useful initia ligands. Where bromine is the counter ion, bidentate or one tors include those for nitroxide mediated radical polymeriza half tetradentate ligands are needed per Cut'. Where more tion (NMP), or reversible addition-fragmentation-termina complex counter ions are employed. Such as triflate or tion (RAFT or MADIX) polymerization. Still other 25 hexafluorophosphate, two bidentate or one tetradentate techniques to control a free-radical polymerization process ligand can be employed. The addition of metallic copper can can be used. Such as the use of iniferters, degenerative transfer be advantageous in some embodiments particularly where or telomerization process. Moreover, the initiators useful in faster polymerization is desired as metallic copper and Cu" the present invention include those having at least one branch may undergo redox reaction to form Cu'. The addition of point, Such as those described above. 30 some Cu" at the beginning of some ATRP reactions can be Random copolymers of the present invention having com employed to decrease the amount of normal termination. plex architectures including branched compounds having In some embodiments, the amount of catalyst employed in multiple polymer arms including, but not limited to, comb the polymerization reactions is the molar equivalent of the and star structures. Comb architectures can be achieved initiator that is present. Since catalyst is not consumed in the employing linear initiators bearing three or more halogen 35 reaction, however, it is not essential to include a quantity of atoms, preferably the halogens are chlorine, bromine, or catalyst as high as of initiator. The ratio of catalyst to initiator, iodine atoms, more preferably the halogens are chlorine or based on transition metal compound in some embodiments is bromine atoms. Star architectures can also be prepared from about 1:(1 to 50), and in other embodiments from about employing compounds bearing multiple halogens on a single 1:(1 to 10). carbon atom or cyclic molecules bearing multiple halogens. 40 Polymerization Conditions In some embodiments compounds having star architecture In some embodiments, the living radical polymerization have 3 polymer arms and in other embodiments they have 4 process of the invention is preferably carried out to achieve a polymer arms. See initiators described above. degree of polymerization in the range of 3 to about 2000, and Catalyst and Ligands in other embodiments from about 5 to about 500. The degree Catalyst for use in ATRP or group radical transfer poly 45 of polymerization in other embodiments is in the range 10 to merizations may include suitable salts of Cu'", Cui", Fe", 100, or alternatively in the range of about 10 to about 50. The Fe", Ru?", Ru.", Cr2", Cr", Mo?", Mo...", W2*. Wit, Mn2", degree of polymerization in group or atom transfer radical Mn?", Mn", Rh", Rh", Re?", Re", Co'", Co.", Co*, v2+, polymerization technique, is directly related to the initial ratio V", Zn.'", Zn, Ni", Ni, Au'', Au?", Ag'" and Ag". of initiator to monomer. Therefore, in some embodiments the Suitable salts include, but are not limited to: halogen, C-C- 50 initial ratios of initiator to monomer are in the range of 1:(3 to alkoxy, Sulfates, phosphate, triflate, hexafluorophosphate, about 2,000) or about 1:(5 to 500), or about 1:(10 to 100), or methaneSulphonate, arylsulphonate salts. In some embodi about 1:(10 to 50). ments the catalyst is a chloride, bromide salts of the above Polymerization reactions are typically carried out in the recited metalions. In other embodiments the catalyst is CuBr, liquid phase, employing a single homogeneous solution. The CuClor RuCl. 55 reaction may, however, be heterogeneous comprising a solid In some embodiments, the use of one or more ligands to and a liquid phase (e.g., a Suspension or aqueous emulsion). solubilize transition metal catalysts is desirable. Suitable In those embodiments where a non-polymerizable solvent is ligands are usefully used in combination with a variety of employed, the solvent employed is selected taking into con transition metal catalysts including where copper chloride or sideration the nature of the Zwitterionic monomer, the initia bromide, or ruthenium chloride transition metal salts are part 60 tor, the catalyst and its ligand; and in addition, any comono of the catalyst. The choice of a ligand affects the function of mer that can be employed. catalyst as ligands not only aid in solubilizing transition metal The solvent may comprise a single compound or a mixture catalysts in organic reaction media, but also adjust their redox of compounds. In some embodiments the solventis water, and potential. Selection of a ligand is also based upon the solu in other embodiments water is present in an amount from bility and separability of the catalyst from the product mix 65 about 10% to about 100% by weight, based on the weight of ture. Where polymerization is to be carried out in a liquid the monomers present in the reaction. In those embodiments phase soluble ligands/catalyst are generally desirable where a water insoluble comonomer is to be polymerized US 8,765,432 B2 49 50 with a Zwitterionic monomer, it can be desirable to employ a conjugation techniques are described in BertoZZi et al. Solvent or co-solvent (in conjunction with water) that permits Angewandte Chemie 2009, 48, 6974, and Gauthier et al. solubilization of all the monomers present. Suitable organic Chem. Commun. 2008, 2591, each incorporated by reference Solvents include, without limitation, formamides (e.g., dim in its entirety herein. ethylformamide), ethers (e.g., tetrahydrofuran), esters (ethyl Where, for example, the coupling requires the formation of acetate) and, most preferably, alcohols. In some embodi an ester or an amide, dehydration reactions between a car ments where a mixture of water and organic Solvent is to be boxylic acid and an alcohol oramine may employ a dehydrat employed, C-C water miscible alkyl alcohols (methanol, ing agent (e.g., a carbodiimide Such as dicyclohexylcar ethanol, propanol, isopropanol, butanol, isobutanol, and ter bodimide, DCC, or the water soluble agent 1-ethyl-3-(3- thutanol) are useful organic solvents. In other embodiments, 10 dimethylaminopropyl)carbodiimide hydrochloride, EDC). water and methanol combinations are suitable for conducting Alternatively, N-hydroxysuccinimide esters (NHS) can be polymerization reactions. The reaction may also be con employed to prepare amides. Reaction to prepare amides ducted in Supercritical solvents such as CO. employing NHS esters are typically conducted near neutral As noted above, in some embodiments it is desirable to pH in phosphate, bicarbonate, borate, HEPES or other non include water in the polymerization mixture in an amount 15 amine containing buffers at 4° to 25°C. In some embodi from about 10% to about 100% by weight based on the weight ments, reactions employing EDC as a dehydrating agent, a pH of monomers to be polymerized. In other embodiments the of 4.5-7.5 can be employed; in other embodiments, a pH of total non-polymerizable solvent is from about 1% to about 4.5 to 5 can be employed. Morpholinoethanesulfonic acid, 500% by weight, based on the weight of the monomers MES, is an effective carbodiimide reaction buffer. present in the reaction mixture. In other embodiments, the Thiol groups can be reacted under a variety of conditions to total non-polymerizable solvent is from about 10% to about prepare different products. Where a thiol is reacted with a 500% by weight or alternatively from 20% to 400%, based on maleimide to form a thioether bond, the reaction is typically the weight of the monomers present in the reaction mixture. It carried out at a pH of 6.5-7.5. Excess maleimide groups can is also desirable in some cases to manipulate the solubility of be quenched by adding free thiol reagents such as mercapto an input reagent, such as initiator or monomer, for example by 25 ethanol. Where disulfide bonds are present as a linkage, they modifying temperature or solvent or other method so as to can be prepared by thiol-disulfide interchange between a modify the reaction conditions in a dynamic fashion. Sulfhydryl present in the bioactive group and an X function In some embodiments, contact time of the Zwitterionic ality which is a disulfide such as a pyridyl disulfide. Reactions monomer and water prior to contact with the initiator and involving pyridyl disulfides can be conducted at pH 4-pH 5 catalyst are minimized by forming a premix comprising all 30 and the reaction can be monitored at 343 nm to detect the components other than the Zwitterionic monomer and for the released pyridine-2-thione. Thiol groups may also be reacted Zwitterionic monomer to be added to the premix last. with epoxides in aqueous solution to yield hydroxy thioet The polymerization reactions can be carried out at any hers. Suitable temperature. In some embodiments the temperature The reaction of guanido groups (e.g., those of anarginine in can be from about ambient (room temperature) to about 120° 35 a protein or polypeptide of interest) with a glyoxal can be C. In other embodiments the polymerizations can be carried carried out at pH 7.0-8.0. The reaction typically proceeds at out at a temperature elevated from ambient temperature in the 25° C. The derivative, which contains two phenylglyoxal range of about 60° to 80°C. In other embodiments the reac moieties per guanido group, is more stable under mildly tion is carried out at ambient (room temperature). acidic conditions (below pH4) than at neutral or alkaline pHs, In some embodiments, the compounds of the invention 40 and permits isolation of the linked materials. At neutral or have a polydispersity (of molecular weight) of less than 1.5, alkaline pH values, the linkage decomposes slowly. Where an as judged by gel permeation chromatography. In other arginine residue of a protein or polypeptide is reacted with a embodiments the polydispersities can be in the range of 1.2 to phenylglyoxal reagent, about 80% of the linkage will hydro 1.4. lyze to regenerate the original arginine residue (in the absence A number of workup procedures can be used to purify the 45 of excess reagent) in approximately 48 hours at 37° at about polymer of interest Such as precipitation, fractionation, repre pH 7. cipitation, membrane separation and freeze-drying of the Imidoester reactions with amines are typically conducted polymers. at pH of 8-10, and preferably at about pH 10. The amidine Non-Halogenated Polymer Terminus linkage formed from the reaction of an imidoester with an In some embodiments, it can be desirable to replace the 50 amine is reversible, particularly at high pH. halogen, or other initiator fragment I', with another function Haloacetals can be reacted with sulfhydryl groups over a ality. A variety of reactions can be employed for the conver broad pH range. To avoid side reactions between histidine sion of the aliphatic halogen. In some embodiments, the con residues that can be present, particularly where the sulfhydryl version of the aliphatic halogen can include reaction to group is present on a protein or polypeptide, the reaction can prepare an alkyl, alkoxy, cycloalkyl, aryl, heteroaryl or 55 be conducted at about pH 8.3. hydroxy group. Halogens can also be subject to an elimina Aldehydes can be reacted with amines under a variety of tion reaction to give rise to an alkene (double bond). Other conditions to form imines. Where either the aldehyde or the methods of modifying the halogenated terminus are amine is immediately adjacent to an aryl group the product is described in Matyjaszewski et al. Prog. Polym. Sci. 2001, 26, a Schiffbase that tends to be more stable than where no aryl 337, incorporated by reference in its entirety herein. 60 group is present. Conditions for the reaction of amines with Attachment of Functional Agents aldehydes to forman imine bond include the use of a basic pH The coupling of functional agents to the random copoly from about pH 9 to about pH 11 and a temperature from about mers of the present invention can be conducted employing 0°C. to room temperature, over 1 to 24 hours. Buffers includ chemical conditions and reagents applicable to the reactions ing borohydride and tertiary amine containing buffers are being conducted. Exemplary methods are described in Bio 65 often employed for the preparation of imines. Where it is conjugate Techniques, Greg T. Hermanson, Academic Press, desired imine conjugates, which are hydrolytically suscep 2d ed., 2008 (incorporated in its entirety herein). Other bio tible, can be reduced to form an amine bond which is not US 8,765,432 B2 51 52 hydrolytically susceptible. Reduction can be conducted with ety of routes including injection directly into tumors, the a variety of Suitable reducing agents including sodium boro blood stream, or body cavities. hydride or sodium cyanoborohydride. While the pharmaceutical compositions may be liquid Solutions, Suspensions, or powders that can be reconstituted The reaction conditions provided above are intended to immediately prior to administration, they may also take other provide general guidance to the artisan. The skilled artisan forms. In some embodiments, the pharmaceutical composi will recognize that reaction conditions can be varied as nec tions may be prepared as syrups, drenches, boluses, granules, essary to promote the attachment of the functional agent to the pastes, Suspensions, creams, ointments, tablets, capsules random copolymers of the present invention and that guid (hard or Soft) sprays, emulsions, microemulsions, patches, ance for modification of the reactions can be obtained from Suppositories, powders, and the like. The compositions may standard texts in organic chemistry. Additional guidance can 10 also be prepared for routes of administration other than be obtained from texts such as Wong, S. S., “Chemistry of parenteral administration including, but not limited to, topical Protein Conjugation and Cross-Linking (CRC Press 1991), (including buccal and Sublingual), pulmonary, rectal, trans which discuss related chemical reactions. dermal, transmucosal, oral, ocular, and so forth. In some embodiments, the pharmaceutical compositions of V. Compositions 15 the present invention comprise one or more random copoly mers of the present invention. Other pharmaceutical compositions of the present inven The present invention includes and provides for pharma tion may comprise one or more random copolymers of the ceutical compositions comprising one or more compounds of present invention that function as biological ligands that are the invention and one or more pharmaceutically acceptable specific to an antigen or target molecule. Such compositions excipients. The compounds of the invention may be present as may comprise a random copolymer of the present invention, a pharmaceutically acceptable salt, prodrug, metabolite, ana where the bioactive agent is a polypeptide that comprises the log or derivative thereof, in the pharmaceutical compositions amino acid sequence of an antibody, or an antibody fragment of the invention. As used herein, "pharmaceutically accept such as a FAb or FAb' fragment or an antibody variable able excipient’ or “pharmaceutically acceptable carrier is 25 region. Alternatively, the compound may be a random intended to include any and all solvents, dispersion media, copolymer and the polypeptide may comprise the antigen coatings, antibacterial and antifungal agents, isotonic and binding sequence of a single chain antibody. Where a bioac absorption delaying agents, and the like, compatible with tive agent present in a random copolymer of the present pharmaceutical administration. invention functions as a ligand specific to an antigen or target Pharmaceutically acceptable carriers for use in formulat 30 molecule, those compounds may also be employed as diag ing the random copolymers of the present invention include, nostic and/or imaging reagents and/or in diagnostic assays. but are not limited to: Solid carriers such as lactose, terra alba, The amount of a compound in a pharmaceutical composi Sucrose, talc, gelatin, agar, pectin, acacia, magnesium Stear tion will vary depending on a number of factors. In one ate, Stearic acid and the like; and liquid carriers such as embodiment, it may be a therapeutically effective dose that is syrups, Saline, phosphate buffered saline, water and the like. 35 Suitable for a single dose container (e.g., a vial). In one Carriers may include any time-delay material known in the embodiment, the amount of the compound is an amount Suit art, such as glyceryl monostearate or glyceryl distearate, able for a single use Syringe. In yet another embodiment, the alone or with a wax, ethylcellulose, hydroxypropylmethyl amount is suitable for multi-use dispensers (e.g., containers cellulose, methylmethacrylate or the like. suitable for delivery of drops of formulations when used to Other fillers, excipients, flavorants, and other additives 40 deliver topical formulations). A skilled artisan will be able to Such as are known in the art may also be included in a phar determine the amount a compound that produces a therapeu maceutical composition according to this invention. The use tically effective dose experimentally by repeated administra of Such media and agents for pharmaceutically active Sub tion of increasing amounts of a pharmaceutical composition stances is well known in the art. Except insofar as any con to achieve a clinically desired endpoint. ventional media or agentis incompatible with the active com 45 Generally, a pharmaceutically acceptable excipient will be pound, use thereof in the compositions of the invention is present in the composition in an amount of about 0.01% to contemplated. Supplementary active compounds can also be about 99.999% by weight, or about 1% to about 99% by incorporated into the compositions of the present invention. weight. Pharmaceutical compositions may contain from The pharmaceutical preparations encompass all types of about 5% to about 10%, or from about 10% to about 20%, or formulations. In some embodiments they are parenteral (in 50 from about 20% to about 30%, or from about 30% to about cluding Subcutaneous, intramuscular, intravenous, intrader 40%, or from about 40% to about 50%, or from about 50% to mal, intraperitoneal, intrathecal, intraventricular, intracra about 60%, or from about 60% to about 70%, or from about nial, intraspinal, intracapsular, and intraosseous) 70% to about 80%, or from about 80% to about 90% excipient formulations Suited for injection or infusion (e.g., powders or by weight. Other suitable ranges of excipients include from concentrated Solutions that can be reconstituted or diluted as 55 about 5% to about 98%, from about from about 15 to about well as Suspensions and Solutions). Where the composition is 95%, or from about 20% to about 80% by weight. a solid that requires reconstitution or a concentrate that Pharmaceutically acceptable excipients are described in a requires dilution with liquid media, any Suitable liquid media variety of well known sources, including but not limited to may be employed. Preferred examples of liquid media “Remington: The Science & Practice of Pharmacy”, 19' ed., include, but are not limited to, water, saline, phosphate buff 60 Williams & Williams, (1995) and Kibbe, A. H., Handbook of ered saline, Ringer's Solution, Hank’s solution, dextrose solu Pharmaceutical Excipients, 3" Edition, American Pharma tion, and 5% human serum albumin. ceutical Association, Washington, D.C., 2000. Where a compound or pharmaceutical composition com prising a random copolymer of the present invention is Suit VI. Methods able for the treatment of cell proliferative disorders, including 65 but not limited to cancers, the compound or pharmaceutical The random copolymers of the present invention are useful composition can be administered to a subject through a vari for treating any disease state or condition. By combining US 8,765,432 B2 53 54 appropriate targeting agents, drugs and therapeutic proteins, Hyaluronidase Deficiency, Maroteaux-Lamy, Sly Syndrome, along with the phosphorylcholine, the random copolymers of Mucolipidosis I/Sialidosis, Mucolipidosis, and Mucolipido the present invention can be used to address the panoply of sis, Multiple sulfatase deficiency, Niemann-Pick Disease, mechanisms provided by any one disease state or condition. Neuronal Ceroid Lipofuscinoses, Pompe disease/Glycogen For example, the disease state or condition can be acute or storage disease type II, Pycnodysostosis, Sandhoff disease, chronic. Schindler disease, Salla disease/Sialic Acid Storage Disease, Disease states and conditions that can be treated using the Tay-Sachs/GM2 gangliosidosis and Wolman disease. random copolymers of the present invention include, but are Conjugates of the invention and compositions (e.g., phar not limited to, cancer, autoimmune disorders, genetic disor maceutical compositions) containing conjugates of the inven 10 tion can be used to treat a variety of conditions. For example, ders, infections, inflammation, and metabolic disorders. there are many conditions for which treatment therapies are Cancers that can be treated using the random copolymers known to practitioners of skill in the art in which functional of the present invention include, but are not limited to, ovarian agents, as disclosed herein, are employed. The invention con cancer, breast cancer, lung cancer, bladder cancer, thyroid templates that the conjugates of the invention (e.g., phospho cancer, liver cancer, pleural cancer, pancreatic cancer, cervi rylcholine containing polymers conjugated to a variety of cal cancer, testicular cancer, colon cancer, anal cancer, bile 15 duct cancer, gastrointestinal carcinoid tumors, esophageal functional agents) and compositions containing the conju cancer, gall bladder cancer, rectal cancer, appendix cancer, gates of the invention can be employed to treat Such condi Small intestine cancer, stomach (gastric) cancer, renal cancer, tions and that Such conjugates provide for an enhanced treat cancer of the central nervous system, skin cancer, choriocar ment therapy relative to the same functional agent not cinomas; head and neck cancers, osteogenic sarcomas, fibro coupled to a phosphorylcholine containing polymer. sarcoma, neuroblastoma, glioma, melanoma, leukemia, and Therefore, the invention contemplates the treatment of a lymphoma. condition known to be treatable by a certain bioactive agent In some embodiments, the present invention provides a by treating the condition using the same certain bioactive method of treating cancer, comprising administering a thera agent conjugated to a phosphorylcholine containing polymer. peutically effective amount of the random copolymer of for Another aspect of the present invention relates to methods 25 of treating a condition responsive to a biological agent com mula I to a Subject in need thereof, thereby treating cancer. In prising administering to a Subject in need thereofatherapeu other embodiments, the random copolymer has formula: tically effective amount of a compound of the invention or of a pharmaceutically acceptable composition of the invention as described above. Dosage and administration are adjusted 30 to provide sufficient levels of the bioactive agent(s) to main R-I , tain the desired effect. The appropriate dosage and/or admin istration protocol for any given Subject may vary depending on various factors including the severity of the disease state, O O O general health of the Subject, age, weight, and gender of the L2 Subject, diet, time and frequency of administration, drug com 35 bination(s), reaction sensitivities, and tolerance/response to A2 therapy. Therapeutically effective amounts for a given situa PC tion can be determined by routine experimentation that is within the skill and judgment of the clinician. wherein A is camptothecin. The pharmaceutical compositions described herein may be Autoimmune diseases that can be treated using the random 40 administered singly. Alternatively, two or more pharmaceu copolymers of the present invention include, but are not lim tical compositions may be administered sequentially, or in a ited to, multiple Sclerosis, myasthenia gravis, Crohn's dis cocktail or combination containing two random copolymers ease, ulcerative colitis, primary biliary cirrhosis, type 1 dia of the present invention or one random copolymer of the betes mellitus (insulin dependent diabetes mellitus or present invention and another bioactive agent. Other uses of IDDM), Grave's disease, autoimmune hemolytic anemia, 45 bioactive agents set forth herein may be found in standard pernicious anemia, autoimmune thrombocytopenia, Vascu reference texts such as the Merck Manual of Diagnosis and litides such as Wegener's granulomatosis, Behcet’s disease, Therapy, Merck & Co., Inc., Whitehouse Station, N.J. and rheumatoid arthritis, Systemic lupus erythematosus (lupus), Goodman and Gilman's The Pharmacological Basis of Scleroderma, systemic Sclerosis, Guillain-Barre Syndromes, Therapeutics, Pergamon Press, Inc., Elmsford, N.Y., (1990). Hashimoto's thyroiditis spondyloarthropathies such as anky 50 The random copolymers of the present invention are useful losing spondylitis, psoriasis, dermatitis herpetiformis, for treating, detecting and imaging a variety of disease states inflammatory bowel diseases, pemphigus Vulgaris and viti and conditions. The random copolymers can be used as a ligo. chemotherapy agent in the treatment of cancer where the Some metabolic disorders treatable by the random copoly initiator fragment I is not functionalized and R includes a mers of the present invention include lysosomal storage dis cancer chemotherapeutic agent A that is loaded onto the orders. Such as mucopolysaccharidosis IV or Morquio Syn 55 random copolymer via click chemistry: drome, Activator Deficiency/GM2 Gangliosidosis, Alpha mannosidosis, Aspartylglucosaminuria, Cholesteryl ester storage disease, Chronic Hexosaminidase A Deficiency, Cys tinosis, Danon disease, Fabry disease, Farber disease, Fucosi H-I y-rey-a-y 60 L2 dosis, Galactosialidosis, Gaucher Disease, GM1 gangliosi s dosis, hypophosphatasia, I-Cell disease/Mucolipidosis II, A2 Infantile Free Sialic Acid Storage Disease/ISSD. Juvenile PC Hexosaminidase A Deficiency, Krabbe disease, Metachro matic Leukodystrophy, Mucopolysaccharidoses disorders such as Pseudo-Hurler polydystrophy/Mucolipidosis IIIA, 65 Additional cancer treatment agents using the random Hurler Syndrome, Scheie Syndrome, Hurler-Scheie Syn copolymers can include a targeting agent of an anti-angio drome, Hunter syndrome, Sanfilippo syndrome, Morquio, genic protein such as an anti-VEGF sclv fragment A' conju US 8,765,432 B2 55 56 gated via a C-terminal cysteine to a maleimide initiator I. The sia, can include recombinant alkaline phosphatase enzyme A' random copolymer can also include a cancer chemotherapeu conjugated via aldehyde-modified initiator I through a stable tic agent A* that is linked to the polymer backbone via a linkage L'. The random copolymer can include phosphoryl cleavable linker. Moreover, the cancer chemotherapeutic is choline stably attached to the polymer, and a comonomer loaded onto the random copolymer via click chemistry. For useful for targeting via a stably attached bone targeting moi example, in Ewing's sarcoma: the targeting agent can be an ety A such as an aspartate rich peptide sequence or a bispho anti-cancer antibody fragment Such as a Fab' or scFv frag sphonate such that more than five targeting moieties are ment that binds to an angiogenic growth factor Such as VEGF. present (y' is greater than 5). These copolymers are useful for In addition, bone targeting comonomer A can include an Subcutaneous delivery: aspartate rich peptide or a bisphosphonate. Other comono 10 mers. A can include Vincristine, Doxorubicin, and/or cyclo phosphamide attached via a cleavable linker: A-L-I M!---M? t t y - it M2c t 15 L2a L2b L2c S A.l, PC Other random copolymers are useful as a targeted platform for bone enzyme replacement therapies, specifically Morquio Random copolymers for more efficacious and longer resi Syndrome (MPS type IVa). These types of random copoly dence time therapy for wet or dry macular degeneration can mers include a recombinant N-Acetylgalactosamine-6-sul include an anti-inflammatory oranti-angiogenic protein Such fate sulfatase enzyme A' conjugated via site specific chemis as anti-VEGF or anti-IL-6 scFv fragment A' conjugated via a 25 try initiator I through a cleavable linker L'. The random C-terminal cysteine to a maleimide initiator I. The random copolymer can include phosphorylcholine stably attached to copolymer prepared can be either a homopolymer of phos the polymer, and a targeting comonomer containing a bone phorylcholine or a copolymer of phosphorylcholine stably targeting moiety A such as an aspartate rich peptide attached to the polymer backbone, in combination with an sequence or a bisphosphonate linked via a non-cleavable anti-inflammatory Small molecule or an anti-angiogenic 30 small molecule A linked to the polymer backbone via a linker L, such that more than five targeting moieties are cleavable linker L. Alternatively, the random copolymer can present. These copolymers are useful for Subcutaneous deliv include another comonomer having a vitreous extracellular ery: matrix (hyaluronic acid) binding moiety A attached via a non-cleavable linker L' such as choline or a positively 35 charged amino acid: Al-L-I-H-M- ey in I L2 Al-L-I yl-, -yr-e- 40 l, J. L2 PC s A2 PC Random copolymers for targeted platforms for safer, more efficacious treatment of Rheumatoid Arthritis can include Random copolymers for real-time diagnostic estimate of 45 several different drugs, including an anti-TNFC. biopharma tumor burden and imaging for oncology can include an anti ceutical such as an antibody fragment A' that is linked to the tumor-associated protein such as an anti-Carcino Embryonic initiator I via a linker L, or an anti-VEGFR2, a small mol Antigen (CEA) scrv fragment A' conjugated via a C-terminal ecule A", as a kinase inhibitor, and methotrexate A', an cysteine to a maleimide initiator I. The random copolymer antineoplastic antimetabolite with immunosuppressant prop can include phosphorylcholine stably attached and an imag 50 erties: ing reagent A* Such as a fluorescent dye (fluorescent probe detection) orgadolinium (for whole body imaging detection). Additional comonomers can be added having Small molecule chemotherapy agents A* to add atherapeutic element. These structures provide both therapeutic and diagnostic functions, 55 A-L-I M!-- y t t y I I and are commonly referred to as theranostics: L2a L2b A2a A2b S. PC Al-L-I y-r-ty-ty-on 60 L2a L2b A2a A2b S Similar random copolymers to those above can be prepared PC by replacing the anti-TNFC. biopharmaceutical of A" with a 65 Small protein dual domain inhibitor Such as an avimer or a Random copolymers for use as a targeted platform for bone sch V dimer that inhibits two proteins, for example TNFC. and enzyme replacement therapies, specifically hypophosphata also VEGF, but without the small molecule inhibitor. In addi US 8,765,432 B2 57 58 tion, the methotrexate A can be substituted for cyclophos magnetic field for therapeutic delivery and imaging are also phamide: Suitable. First, our approach conceptually can be combined with all of the below: Synthetic biodegradable polymer-based nanoparticles 5 encapsulating a therapeutic gene, a gadolinium contrast L2 agent for MRI analysis, and functionalized with anti A2 bodies to target specific disease sites. rt PC Liposomes encapsulating Small or large drug molecules, labeled with Fluorine for PET analysis, and function Finally, a random copolymer for targeted and protected 10 alized with antibodies to target specific disease sites. RNAi can be prepared without a functionalized initiator I. Polyplexes containing a siRNA molecule, an iron-oxide The random copolymer can include phosphorylcholine stably contrast agent for MRI analysis, and modified with cell attached to the polymer, and a comonomer having an siRNA binding ligands and cell-penetrating peptides for tar A linked to the polymer via a cleavable bond L', and geted cellular and intracellular delivery respectively. another comonomer having a cell- or tissue-targeting group 15 Fluorescent quantum dots intercalated with a drug mol A’ attached via a non-cleavable linker L’. The siRNA con ecule for optical imaging and sensing of the delivery and taining comonomer can be prepared using a monomer having functionalized with an RNA aptamer to target specific a linking group Suitable for click chemistry wherein the diseases. siRNA is linked to the linking group following polymeriza Inorganic or organic nanoparticles containing an antisense tion. The comonomer having the targeting moiety can either oligonucleotide for gene therapy, a gadolinium contrast already contain the targeting moiety, or link to the targeting agent for MRI analysis, a fluorophore for optical imag moiety via a comonomer having a linking group suitable for ing, and Surface modified to target specific diseases. click chemistry via a different chemistry than for attachment pH sensitive polymeric nanocomposites with a drug mol of the siRNA. The cleavable linker is preferably a pH sensi ecule that is released as a function of pH, an iron oxide tive linker. The random copolymer can be prepared with a 25 contrast agent for MRI imaging, CdTe quantum dots for target Stoichiometry of approximately five oligonucleotide optical imaging, and functionalized with antibodies to moieties per drug A and five targeting moieties per drug target specific diseases. (such that the ratio of y':y:y' is about 5:5:1). Moreover, Nanoparticle-DNA aptamer conjugates containing a drug the phosphorylcholine polymer backbone can be optimized and a radiotracer such as '''In for SPECT imaging and not for half-life, but to protect the siRNA in its journey from 30 functionalized with disease-specific membrane antibod injection site to the targeted tissues. The siRNA can be 1CS replaced with microRNA: Second, the polymers of the present invention can be spe cifically combined with the above: Phosphorylcholine polymer-based construct containing a therapeutic gene (bioactive 1), a gadolinium contrast H-I Y', fly-ory-tity of agent for MRI analysis (functional 1), and a small pro L2a L2b L2c tein (Such as an antibody fragment) to target specific S. disease sites. A2a A2b A2c Imaging agent Fluorine for PET analysis, and function PC alized with Small protein (such as an antibody fragment) 40 to target specific disease sites. Phosphorylcholine polymers containing one or more In addition, the initiator I can optionally be linked to a bioac siRNA molecules, an iron-oxide contrast agent for MRI tive moiety A' such as an antibody fragment for targeting and analysis, and modified with cell binding ligands and therapy: cell-penetrating peptides for targeted cellular and intra 45 cellular delivery respectively. Phosphorylcholine polymers containing fluorescent quan A-L-I MI t ye t t y t t y t I tum dots(functional agent) intercalated with a drug mol ecule (functional agent) for optical imaging and sensing L2a L2b L2c of the delivery and functionalized with an RNA aptamer s or a small protein (such as an antibody fragment or A2a A2b A2c Scaffold derived protein) to target specific diseases. PC Phosphorylcholine containing polymers containing an antisense oligonucleotide for gene therapy, a gado In Some other embodiments, the engineering of novel mul linium contrast agent for MRI analysis, a fluorophore for tifunctional therapeutic systems can combine phosphoryl optical imaging, and an additional functional agent for choline polymers with drug or gene targeting agent with 55 targeting specific diseases such as folate for tumor or imaging and/or sensing capabilities. Systems can have at least choline for electrostatic interactions for targeting extra 3 components: (1) a targeting moiety or molecular signatures cellular matrix. that can target delivery to specific sites, (2) the appropriate pH sensitive phosphorylcholine polymer with a drug mol imaging agent/probe/tags for visualization or monitoring of ecule that is released as a function of pH, an iron oxide the systems, and (3) a therapeutic agent to effectively treat a 60 contrast agent for MRI imaging, CdTe quantum dots for particular disease or disorder. optical imaging, and functionalized with antibodies or The following are examples of multifunctional systems other protein or aptamer to target and treat specific dis that contain targeting, imaging, and drug/gene moieties. This CaSCS. list is not intended to be exclusive of a phosphorylcholine Phosphorylcholine polymer with aptamer functional agent containing polymer system. Targeted systems that can be 65 conjugates containing a drug and a radiotracer Such as activated by internal processes such as pH, enzyme cleavage 'In for SPECT imaging and further functionalized or external stimuli such as near IR light, ultrasound, heat, or with disease-specific membrane antibodies. US 8,765,432 B2 59 60 VII. Examples J–7.2 Hz, 2H), 2.13-2.32 (complex m, 2H), 2.45 (t, J=7.6 Hz, 2H), 2.51-2.65 (complex m, 2H), 3.35 (t, J=5 Hz, 2H), 3.63 Example 1 3.68 (m, 4H), 4.21-4.25 (m, 2H), 5.30 (brs, 2H), 5.41 (d. J=17.2 Hz, 1H), 5.68 (d. J=17.2 Hz, 1H), 7.21 (s, 1H), 7.68 (t, Preparation of Camptothecin PC-Copolymer J=6.8 Hz, 1H), 7.84 (app t, J=8.4 Hz, 1H), 7.95 (d. J=8 Hz, 1H), 8.23 (d. J=8 Hz, 1H), 8.40 (s, 1H). Synthesis of 2-(2-Azidoethoxy)ethanol Synthesis of Copolymer of Methacryloyloxyethyl Phosphoryl Choline and Trimethylsilyl Ho-1N1 'N-1- 10 (TMS)-Protected Propargyl Methacrylate Ethyl C.-bromoisobutyrate (18.84 mg. 0.096 mmol), bipy A solution of 10.0 grams of 2-(2-chloroethoxy)ethanol in ridine (30.1 mg 0.192 mmol) and 450 mg of DMSO were 50 mL of deionized water was treated with 10.4 grams (2 eq) initially loaded into a Schlenk tube. The mixture was care of sodium azide, and the reaction mixture was heated at 80°C. fully degassed and the tube filled with nitrogen. CuBr was for 48 hours. The solution was cooled to room temperature, 15 then added to the tube under inert conditions (13.8 mg, 0.096 saturated with sodium chloride and extracted with 3x50 mL mmol). The reaction mixture was sealed and cooled at -78 C. of ether. The combined organics were dried over anhydrous A mixture of trimethylsilyl (TMS)-protected propargyl meth Sodium sulfate, filtered and concentrated to give 7.25 grams acrylate (66 mg, 0.336 mmol) and methacryloyloxyethyl (69%) of the desired product as a clear, colorless oil. HNMR phosphorylcholine (0.9, 3.04 mmol) were dissolved in 4 mL (400 MHz, CDC1): 8–2.05 (t, J=6.4 Hz, 1H, OH), 3.42 (t, J=5 of degassed 200 proof ethanol. The solution was added drop Hz, 2H), 3.63 (dd, J–44, 5.6 Hz), 3.71 (dd, J=44, 4.8 Hz, wise under inert conditions to the cooled reaction vessel. The 2H), 3.77 (dt, J=4.4, 6 Hz, 2H). mixture was thoroughly degassed under vacuum for 15 minat 0° C. and filled with inert gas. Polymerization was allowed to Synthesis of proceed for 15 hours. 5-2-(2-AZidoethoxy)ethoxy-4-OXopentanoic acid 25 O CuBr/Bpy O O ~ x B r DMSO, EtOH --~~~ 30 A solution of 3.0 grams of 2-(2-azidoethoxy)ethanol in 50 mL of dichloromethane was treated with 280 mg of 4-(dim D Nuav ethylamino)pyridine and 64 mL (2 eq) of triethylamine, and the solution was cooled with an ice bath. A solution of 2.61 grams (1.0 eq) of glutaric anhydride in 5 mL of dichlo 35 romethane was then added dropwise over a few minutes. The o=-o reaction was stirred, then heated at gentle reflux overnight. The reaction was cooled to room temperature, washed with 2x25 mL of 1N HC1 and 25 mL of HO, then dried over NN1) Sodium sulfate. Filtration and concentration gave 4.66 grams 40 (83%) of the desired product as a clear, colorless oil. HNMR / V (400 MHz, CDC1): 8=1.97 (quintet, J–7.2 Hz, 2H), 2.45 (t, O J–7.2 Hz, 4H), 3.39 (t, J=4.8 Hz, 2H), 3.66-3.72 (m, 4H), 4.26 (app t, J=4.6 Hz, 2H). 1No pi Br Synthesis of Camptothecin azide Conjugate 45 O Onu-2Siv 50 o=-0 O O O o1n 1 n1n N, -- 55 ) O A solution of 70 mg of 5-2-(2-azidoethoxy)ethoxy-4- oXopentanoic acid in 10 mL of dichloromethane was cooled After 15 hours, the reaction mixture was found to be very in an ice-water bath, and treated with 55 mg of EDC, followed homogeneous with no apparent crosslinking. The reaction by 35 mg of DMAP and 50 mg of camptothecin. The reaction 60 was quenched by exposure to air and the mixture turned from was then allowed to warm to room temperature and stirred dark brown to green. overnight as the Solution slowly became homogeneous. The GPC analysis of a crude sample before purification per reaction mixture was then concentrated and applied to a silica formed on a Shodex column (OH806) calibrated with poly gel column, which was eluted first with 1-2% methanol in ethylene oxide standards indicated the formation of a poly dichloromethane. The appropriate fractions were then con 65 meras a single peak of narrow distribution (molecular weight centrated to give the desired conjugate as a yellow solid. "H at peak Mp was found to be 13200 g/mol). Analysis by light NMR (400 MHz, CDC1): 8–0.98 (t, J=7.6H), 1.98 (quintet, scattering showed a Mn of 22900 g/mol, Mp of 25000 g/mol US 8,765,432 B2 61 62 and PDi of 1.14. The crude reaction was passed through silica ance of the free camptothecin peak and a high molecular gel, concentrated and precipitated carefully into diethyl ether. weight peak which corresponded to the camptothecin copoly The solid was isolated by filtration and washed several times mer conjugate. with diethyl ether. Copolymer was dried inside an oven at 50 Covernight, yielding 0.9 g of copolymer. The reaction mixture was exposed to air, concentrated to Analysis by 'HNMR spectroscopy showed no TMS group. half its Volume, passed through silica gel to remove the cop As a precautionary step, 0.5g of the copolymer was further per catalyst and then precipitated carefully into diethyl ether. treated by 100 mg of tetrabutyl ammonium fluoride trihydate The polymer was washed with an excess of diethyl ether. The and purified by precipitation. solid was isolated by filtration and washed several times with Grafting of camptothecin azide conjugate onto the alkyne 10 diethyl ether. The polymer was dried in an oven at 50 C functionalized copolymer. CuBr (13 mg) was loaded inside a overnight and was isolated as a light-brown powder. "H NMR degassed Schlenk tube followed by the addition of 15 mg of spectroscopy analysis performed on the camptothecin grafted N.N.N',N',N'-pentamethyl diethylenetriamine. 240 mg of copolymers (CDOD) showed weak and broad aromatic sig copolymer was dissolved into 2 g of 200 proof degassed 15 nals in the 7-9 ppm area, characteristic of protons from the ethanol and 50 mg of camptothecin azide conjugate (CPT-L- incorporated camptothecin. N3) were dissolved into 1.5g of DMF. The solution of CPT L-N3 was added dropwise under inert conditions to the Schlenk tube while stirring, followed by the addition of the Example 2 solution of alkyne-functionalized copolymer. The mixture was degassed by three cycles of vacuum-nitrogen and was allowed to react at room temperature for 3 hours. Camptothecin Release Study from Camptothecin Grafted Copolymer O 25 Samples of camptothecin grafted copolymer were pre 1no pi - Br pared at approximately 10 mg/mL in Tris Buffer, pH-8.0. Liver esterase from rabbit liver (Sigma-Aldrich E0887-IKU, O OSO OSu-2. CPTLN3 Lot #061K74451) was added to the sample and the sample -e- 30 was incubated at 37° C. for up to 65 hours. D PMDETACuBr, GPC analysis of the samples was made using an HPLC system consisting of a Waters Alliance 2995 with Waters o--o 2410 Refractive Index Detector, Waters 2996 Photodiode O 35 Array Detector, and a Shodex Protein KW-803 column. The mobile phase used for the elution was phosphate buffered -- D saline containing 10% absolute ethanol. The flow rate was set /\ to 1 mL/min and the presence of camptothecin monitored at O 370 nm. Ten microliter injections of the samples were made at 40 each time point. 1No pi Br mg/mL O S "Y-1S 45 Time (h) Camptothecin Released N.See N.Yn O N L NCPT O.059 O.O79 o--o O.132 O 50 O.130 -- D O.128 17 O.208 /\ 26 O.251 N O 41 O.335 N 55 4. 65 O.427 \ / O Although the foregoing invention has been described in O Q 60 Some detail by way of illustration and example for purposes o1N1 N of clarity of understanding, one of skill in the art will appre --~~ ciate that certain changes and modifications can be practiced CPT-L-N3 within the scope of the appended claims. In addition, each 65 reference provided herein is incorporated by reference in its After 3 hours, an aliquot was taken from the crude mixture entirety to the same extent as if each reference was individu and analyzed by GPC at 370 nm which showed the disappear ally incorporated by reference. US 8,765,432 B2 63 64 What is claimed is: -continued 1. A random copolymer of Formula I: N-9 O NSN wherein n and mare each independently from 1-1000. - O \-CN-11-n \e 2. A method of treating cancer, comprising administering O-1-P V an ameliorative amount of the random copolymer of claim 1 to a Subject in need thereof, wherein the cancer is colon cancer, lung cancer, breast cancer, Stomach cancer, ovarian cancer, or malignant melanoma. k k k k k UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. : 8,765,432 B2 Page 1 of 2 APPLICATION NO. 13/515913 DATED : July 1, 2014 INVENTOR(S) : Charles et al. It is certified that error appears in the above-identified patent and that said Letters Patent is hereby corrected as shown below: In the specification In column 61, lines 40-54, replace the following chemical structure: 66 /N With O 1n f B O ro1 ) N S. N -N- NCPT o-i-o- O MND Signed and Sealed this Thirteenth Day of September, 2016 74-4-04- 2% 4 Michelle K. Lee Director of the United States Patent and Trademark Office CERTIFICATE OF CORRECTION (continued) Page 2 of 2 U.S. Pat. No. 8,765,432 B2 In the claims In claim 1, in column 63, lines 5-20, and column 64, lines 2-10, replace the following chemical Structure: 66 With