USOO8741832B2 (12) United States Patent (10) Patent No.: US 8,741,832 B2 Acharya et al. (45) Date of Patent: Jun. 3, 2014 (54) PEGYLATED ALBUMIN AND USES 6,875.423 B1 4/2005 Intaglietta et al. THEREOF 6,887,952 B1* 5/2005 Buechler et al. .............. 525,532 7,019,117 B2 3/2006 Acharya et al. 7,037,895 B2 * 5/2006 Assaly et al. ................ 514f13.5 (75) Inventors: Seetharama A. Acharya, Cresskill, NJ 7,038,016 B2 5/2006 TE. et al. (US); Belur N. Manjula, Cresskill, NJ 7,144.989 B2 12/2006 Acharya et al. (US) 7,169,900 B2 1/2007 Acharya et al. 7,271,145 B2 9, 2007 Winslow et al. (73) Assignee: Albert Einstein College of Medicine of 7,501,499 B2 3/2009 Acharya et al. Yeshiva University, Bronx, NY (US) 7,521, 174 B2 4/2009 Acharya et al. y, s 8,071,546 B2 * 12/2011 Cabrales et al. ............. 514f15.2 - 2003/0004 105 A1* 1/2003 Assaly et al. ................... 514/12 (*) Notice: Subject to any disclaimer, the term of this 2003/0009145 A1 1/2003 Struijker-Boudier et al. patent is extended or adjusted under 35 2003/0161809 A1 8/2003 Houston et al. U.S.C. 154(b) by 1377 days. 2004.0002443 A1 1/2004 Acharya et al. 2005, 0026816 A1 2/2005 Winslow et al. 2005, 0037966 A1 2/2005 Ruben et al. (21) Appl. No.: 11/921,064 2005/0159339 A1 7/2005 Acharya et al. 2005/02O1988 A1 9/2005 Acharya et al. (22) PCT Filed: Jun. 9, 2006 2006/01 11275 A1 5/2006 Acharya et al. 2009/0215670 A1 8/2009 Acharya et al. (86). PCT No.: PCT/US2O06/022463 2010/0216695 A1 8/2010 Acharya et al. 2010/0222260 A1 9/2010 Cabrales et al. S371 (c)(1), (2), (4) Date: Jun. 12, 2009 FOREIGN PATENT DOCUMENTS (87) PCT Pub. No.: WO2006/135740 JP 11-271306 * 8, 1999 ........ GO1N 33,532 WO WO2005/0972O3 * 10/2005 ............. A61K 47/48 PCT Pub. Date: Dec. 21, 2006 WO WO 2007/05O121 A2 5/2007 WO WO 2007/058678 A2 5/2007 (65) Prior Publication Data OTHER PUBLICATIONS US 2009/02987.46A1 Dec. 3, 2009 eC. 3, Winslow etal. Comparison of PEG-modified albuminand hemoglo bin in extreme hemodilution in the rat. J Appl Physiol. Published Related U.S. Application Data online Jun. 18, 2004. vol. 97, pp. 1527-1534.* Pendri et al. Poly(ethylene glycol) Fluorescent Linkers. (60) Provisional application No. 60/689,175, filed on Jun. Bioconjugate Chem. 1995, vol. 6, pp. 596-598.* 10, 2005. Manjula et al. Site-Specific PEGylation of Hemoglobin at Cys 93(beta): Correlation between the Colligative Properties of the (51) Int. Cl. PEGylated Protein and the Length of the Conjugated PEG Chain. Bioconjugate Chem. 2003, vol. 14, pp. 464–472.* g ise R Vandergriff et al. MP4, a new nonvasoactive PEG-Hb conjugate. ( .01) Blood Components. Transfusion. Apr. 2003. vol.43, pp. 509-516.* C07K I4/00 (2006.01) Fujiwara et al. Novel enzyme immunoassay forthyrotropin-releasing C07K 16/00 (2006.01) hormone using N-(4-diazophenyl)maleimide as a coupling agent. C07K 17/00 (2006.01) FEBS. 1986, vol. 202, No. 2, pp. 197-201.* CO7K 4/76 (2006.01) CO7K 4/805 (2006.01) (Continued)Continued (52) U.S. Cl. Primary Examiner — Marcela M Cordero Garcia CPC ................. A61 K38/00 (2013.01); C07K 14/76 (74) Attorney, Agent, or Firm — Amster, Rothstein & (2013.01); C07K 14/805 (2013.01) Ebenstein LLP USPC ............................................ 514/1.1; 530/362 (58) Field of Classification Search (57) ABSTRACT None The present invention provides PEGylated hemoglobins and See application file for complete search history. PEGylated albumins comprising polyethylene glycol (PEG) conjugated to hemoglobin or to albumin, wherein the PEG is (56) References Cited a maleimide PEG, an alkylamide PEG, an iodoacetamide PEG, a p-nitro thio-phenyl PEG, a vinyl sulfone PEG, or a mixed disulfide PEG; and PEGylated albumins and PEGy U.S. PATENT DOCUMENTS lated hemoglobins comprising polyethylene glycol (PEG) 4,970,298 A * 1 1/1990 Silver et al. ................... 530,356 attached to a thiolated amino group of albumin or hemoglo 5,585,484 A 12/1996 Acharya et al. bin, wherein the amino group is thiolated using dithio Sulfo 5,585,489 A * 12/1996 Russ et al. ...................... 544f76 succinimidyl propionate (DTSSP) or dithiosuccinimidyl pro 5,741,893 A * 4, 1998 Hsia .............................. 530,385 pionate (DTSP) or dithiobispropionimidate. The invention 5,750,725 A 5/1998 Acharya et al. also provides methods of preparing PEGylated hemoglobins 6,017,943 A 1/2000 Acharya et al. 6,310,039 B1 * 10/2001 Kratz ........................... 514, 19.5 and PEGylated albumins comprising a) reacting hemoglobin 6,371,975 B2 4/2002 Cruise et al. or albumin with a thiolating agent and with a PEGylating 6,458,147 B1 10/2002 Cruise et al. agent, and b) capping unPEGylated reactive thiols of hemo 6,670,323 B1 12/2003 Lee et al. globin or albumin with N-ethyl maleimide. 6,773,613 B1 8, 2004 Winslow et al. 6,844,317 B2 1/2005 Winslow et al. 7 Claims, 11 Drawing Sheets US 8,741,832 B2 Page 2 (56) References Cited Veronese et al. Peptide and protein PEGylation: a review of problems and solutions. Biomaterials, 2001, vol. 22, pp. 405-417.* OTHER PUBLICATIONS Manjula et al. Conjugation of Multiple Copies of Polyethylene Gly Nacharaju et al. 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Polarity. Bioconjugate Chemistry, 2001. vol. 12, pp. 470-475.* Wettstein Ret al., entitled “Resuscitation From Hemorrhagic Shock Manjula et al. Cys-93-BBSuccinidophenyl Polyethylene Glycol With MaIPEG-Albumin: Comparison With MalPEG-Hemoglobin.” 2000 Hemoglobin A. The Jornal of Biological Chemistry, 2000. vol. Shock, vol. 22, No. 4, pp. 351-357, 2004, pp. 351-356. 275, Nol. 8, pp. 5527-5534.* International Searching Authority, “International Search Report of Swain et al. Unexpected Products from the Reaction of the Synthetic the International Searching Authority.” for International Application Cross-Linker 3,3'-Dithibis(Sulfosuccinimidyl Propionate), DTSSP No. PCT/US2006/22463, 6 pages, Nov. 3, 2006. with Peptides. Published online Mar. 17, 2004, vol. 15, pp. 736-749.* International Searching Authority, “Written Opinion of the Interna Cabralesetal. Microvascular PO2 during extreme hemodilution with tional Searching Authority.” for International Application No. PCT/ hemoglobin site specifically PEGylated at Cys-93(B) in hamster US2006/22463, 3 pages, Nov. 3, 2006. window chamber. Am J Physiol Heart Circ Physiol. First published Jun. 10, 2004, vol. 287, pp. H1609-H1617.* * cited by examiner U.S. Patent Jun. 3, 2014 Sheet 1 of 11 US 8,741,832 B2 O O- NH-C-O-PEG9 ty - s Male?midophenyl Carbamate of PEG (otel)-NH + H.C.-C-NH.CF + O S O 2-liminothiolane |-CH-NH-PEG y lodoacetamide PEG ExtensionAim - - - - - - - - (o)-NHetch-chs:NH, Thiolated Protein (Intermediato species) op/ N- PEG Extension Ann Confugating Extension Arm Conjugating - - - - - - - Group - - - - - - - Group NH d s like Group NH, To O t Protein NH-clic-cy - N H--olPEG Protein Ho-CCHCH-sché-NH-PEG Figure 1 U.S. Patent Jun. 3, 2014 Sheet 2 of 11 US 8,741,832 B2 A B Coomassie blue stain Iodine stain Figure 2A-2B U.S. Patent Jun. 3, 2014 Sheet 3 of 11 US 8,741,832 B2 (Propyl-PEG5K)-aa-Hb (Propyl-PEG5K)-Hb O 20 40 60 80 Time (min) Figure 3 U.S. Patent Jun. 3, 2014 Sheet 4 of 11 US 8,741,832 B2 s 1 2 8 E E (Propyl-PEG2OKaa-Hb 4 O al w w 2 (Propyl-PEG2OK)-Hbpy s as O us O 5 10 15 20 3 (Propyl-PEG5Kar-Hb t E12000 O 9 s k C (Propyl-PEG2K)-aa-Hb 8000 C o (Propyl-PEG2K)-Hb 5 4000 O 20 40 60 80 o O S 10 15 20 Time (min) Length of PEG chains (kDa) Figure 4A-4B U.S. Patent Jun. 3, 2014 Sheet 5 of 11 US 8,741,832 B2 7s Xe e O 9 l D 1 2 3 4. Protein concentration (g/dL) Figure 5 U.S. Patent Jun. 3, 2014 Sheet 6 of 11 US 8,741,832 B2 S 20,w Protein concentration (uM) Figure 6 U.S. Patent Jun. 3, 2014 Sheet 7 of 11 US 8,741,832 B2 2 - HibA 2 oot-Hb o (Propyl-PEG5K)-Hb N (Propyl-PEG5K)-aa-Hb E O C O) g9 -2 O ve ap P 4 200 210 220 230 240 250 - HbA 8 Co-Hb -(Propyl-PEG5K)-Hb 6 : (Propyl-PEG5K)-aa-Hb O4.2 280 320 360 400 440 480 Wavelength (nm) Figure 7A-7B U.S. Patent Jun. 3, 2014 Sheet 8 of 11 US 8,741,832 B2 2.0 - HbA - oto-Hb orrorman (Propyl-PEG5K)-Hb 1 5 -(Propyl-PEG5Kao-Hb 0.1 5O 0.0 300 320 340 360 380 400 Wavelength (nm) Figure 8 U.S.