(12) Patent Application Publication (10) Pub. No.: US 2004/0167287 A1 Kozlowski Et Al
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US 2004O167287A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2004/0167287 A1 Kozlowski et al. (43) Pub. Date: Aug. 26, 2004 (54) MALEAMIC ACID POLYMER DERIVATIVES Related U.S. Application Data AND THEIR BIOCONJUGATES (60) Provisional application No. 60/437,251, filed on Dec. (76) Inventors: Antoni Kozlowski, Huntsville, AL 31, 2002. Provisional application No. 60/468,340, (US); Remy F. Gross III, Huntsville, filed on May 5, 2003. AL (US); Samuel P. McManus, Brevard, NC (US) Publication Classification (51) Int. Cl. ................................................. C08L 39/04 Correspondence Address: (52) U.S. Cl. .............................................................. 525/205 NEKTAR THERAPEUTICS 150 INDUSTRIAL ROAD (57) ABSTRACT SAN CARLOS, CA 94070 (US) The present invention is directed to maleamic acid deriva tives of water Soluble polymers, to chemically stable water (21) Appl. No.: 10/750,996 Soluble polymer Succinamic acid-active agent conjugates, and to methods for reproducibly preparing, characterizing (22) Filed: Dec. 31, 2003 and using Such polymer reagents and their conjugates. Patent Application Publication Aug. 26, 2004 Sheet 1 of 3 US 2004/0167287 A1 O Reaction Scheme O Protein-SH Sn Protein Poly-L- N -O- Poly-L-N pH - 6.5-7.5 O O Polymer maleimide, I Conjugated polymer succinimide, III-A pH - 7-9 HO pH - 7.0-9.5 HO O O Poly-L Protein-SH Poly-L S Y -Op- h YProtein H \ H O p H - 6.5 - 7.5 O very slow HO HO Polymer maleamic acid, I yer succinamic acid, O O Poly-l Poly =water soluble polymer N L=optional linker O s1 Protein HO V-A F.G. 1 Patent Application Publication Aug. 26, 2004 Sheet 2 of 3 US 2004/0167287 A1 O Reaction Scheme O H N N Protein-NH2 Protein Poly-L-N -O- Poly-L-N pH > 8 O O Polymer maleimide, I Conjugated polymer succinimide, III-B pH~7-9 H2O pH - 7.5-9.5 HO O O Poly-l Protein-NH Poly-l N N -o- N Protein H - 8 - 9 N O \ p H O very slow HO HO Polymer maleamic acid, II Polymer succinamic acid, IV-B O O Poly-l N H O N1 Protein H - V-B HO FG. 2 Patent Application Publication Aug. 26, 2004 Sheet 3 of 3 US 2004/0167287 A1 m PEG2-MAL-40K Hydrolysis to Maleamic Acid in HBPES Buffered Aqueous Solution .998 y = -0.0000143x + 1.997.4083 YN R. O.9920917 1998 YS ... S 1988 .984 O 100 200 3OO 400 soo GOO 7oo 800 Time (minutes) FG. 3 US 2004/0167287 A1 Aug. 26, 2004 MALEAMIC ACID POLYMER DERVATIVES AND Prepr. (Am. Chem. Soc., Div. Polym. Chem), 32(1), 154-155 THEIR BIOCONJUGATES (1991), mixed anhydrides, N-hydroxysuccinimide esters, carbonylimadazolides, and chlorocyanurates (Herman, S., et CROSS REFERENCE TO RELATED al., Macromol. Chem. Phys. 195, 203-209 (1994)). Although APPLICATIONS many proteins have been shown to retain activity during PEG modification, in Some instances, polymer attachment 0001. This application claims the benefit of priority to through protein amino groups can be undesirable, Such as U.S. provisional application Serial No. 60/437,251, filed when derivatization of Specific lysine residues inactivates Dec. 31, 2002, and to U.S. provisional application Serial No. the protein (Suzuki, T., et al., Biochimica et Biophysica Acta 60/468,340, filed May 05, 2003, which are both incorporated 788, 248-255 (1984)). Moreover, since most proteins pos herein by reference in their entirety. SeSS Several available/accessible amino groups, the polymer conjugates formed are typically mixtures of mono-pegy FIELD OF THE INVENTION lated, di-pegylated, tri-pegylated Species and So on, which 0002 This invention relates generally to the field of can be difficult and also time-consuming to characterize and polymer chemistry, and more Specifically to chemically Separate. Further, Such mixtures are often not reproducibly Stable active agent conjugates prepared from maleimide- or prepared, which can create problems during Scale-up for maleamic acid functionalized water-Soluble polymerS Such regulatory approval and Subsequent commercialization. as polyethylene glycol, and to methods for Synthesizing, characterizing, and using Such polymer reagents and conju 0006. One method for avoiding these problems is to gateS. employ a Site-Selective polymer reagent that targets func tional groups other than amines. One particularly attractive BACKGROUND OF THE INVENTION target is the thiol group, which in proteins in present in the amino acid, cysteine. CySteines are typically leSS abundant 0003. Due to recent advances in biotechnology, therapeu in proteins than lysines, thus reducing the likelihood of tic proteins and other biomolecules, e.g. antibodies and protein deactivation upon conjugation to these thiol-contain antibody fragments, can now be prepared on a large Scale, ing amino acids. Moreoever, conjugation to cysteine Sites making Such biomolecules more widely available. Unfortu can often be carried out in a well-defined manner, leading to nately, the clinical usefulness of potential therapeutic bio the formation of Single species polymer-conjugates. molecules is often hampered by their rapid proteolytic degradation, low bioavailability, instability upon manufac 0007 Polyethylene glycol derivatives having a thiol ture, Storage or administration, or by their immunogenicity. Selective reactive end group include maleimides, Vinyl Sul Due to the continued interest in administering proteins and fones, iodoacetamides, thiols, and disulfides, with maleim other biomolecules for therapeutic use, various approaches ides being the most popular. These derivatives have all been to overcoming these deficiencies have been explored. used for coupling to the cysteine Side chains of proteins (Zalipsky, S. Bioconjug. Chem. 6, 150-165 (1995); Green 0004 One approach that has been widely explored is the wald, R. B. et al. Crit. Rev. Ther. Drug Carrier Syst. 17, modification of proteins and other potentially therapeutic 101-161 (2000); Herman, S., et al., Macromol. Chem. Phys. molecules by covalent attachment of a water-Soluble poly 195, 203-209 (1994)). However, many of these reagents mer such as polyethylene glycolor “PEG” (Abuchowski, A., have not been widely exploited due to the difficulty in their et al., J. Biol.Chem. 252 (11), 3579 (1977); Davis, S., et al., Synthesis and purification. Clin.Exp Immunol., 46, 649-652 (1981). The biological properties of PEG-modified proteins, also referred to as 0008 Polyethylene glycol derivatives having a terminal PEG-conjugates or pegylated proteins, have been shown, in maleimide group are one of the most popular types of many cases, to be considerably improved over those of their Sulfhydryl-Selective reagents, and are commercially avail non-pegylated counterparts (Herman, et al., Macromol. able from a number of Sources. Although not widely appre Chem. Phys., 195, 203-209 (1994). Polyethylene glycol ciated or recognized, the Applicants have recognized that modified proteins have been shown to possess longer cir many PEG-maleimides unfortunately exhibit hydrolytic culatory times in the body due to increased resistance to instability during Storage and/or conjugation to a drug proteolytic degradation, and also to possess increased ther candidate. More particularly, a Substantial degree of mostability (Abuchowski, A., et al., J. Biol. Chem., 252, hydrolysis of the maleimide ring has been observed, both 3582-3586 (1977). A similar increase in bioefficacy is prior to and after conjugation. This instability can result in observed with other biomolecules, e.g. antibodies and anti the formation of multiple species of drug conjugates within body fragments (Chapman, A., Adv. Drug Del. Rev. 54, a drug-conjugate composition. The various drug conjugate 531-545 (2002)). Species are likely to possess Similar biological activities, but may differ in their pharmacokinetic properties. This is 0005 Typically, attachment of polyethylene glycol to a particularly disadvantageous for compositions intended for drug or other Surface is accomplished using an activated patient administration, Since the resulting drug compositions PEG derivative, that is to say, a PEG having at least one can be ill-defined mixtures of drug conjugate Species whose activated terminus Suitable for reaction with a nucleophilic particular Safety and accumulation profiles are unknown. center of a biomolecule (e.g., lysine, cysteine and Similar Moreover, due to different factors impacting hydrolysis residues of proteins). Most commonly employed are meth rates, inconsistency between drug conjugate batch compo ods based upon the reaction of an activated PEG with protein Sitions can present an additional problem. amino groups, Such as those present in the lysine Side chains of proteins. Polyethylene glycol having activated end groups 0009. Another potential problem that has been observed Suitable for reaction with the amino groups of proteins by the applicants is the de-pegylation of conjugates prepared include PEG-aldehydes (Harris, J. M., Herati, R. S., Polym from PEG maleimides to yield mixtures of altered drug and US 2004/0167287 A1 Aug. 26, 2004 detached PEG impurity. For these reasons, the Applicants 0016. In a preferred embodiment, the hydrolysis is car have found that PEG maleimides can be undesirable ried out under conditions effective to provide a chemically reagents for coupling to thiol groups on target drugs or other Stable polymer-Succinamic acid-conjugate composition. active agents. Previous attempts to address this problem 0017. In one embodiment of the method, the hydrolysis have focused on increasing the Stability of a polymer male reaction is carried out until at least about 15% of the polymer imide