title: Cobalt(III)-induced hexamerization of PEGylated insulin.
abstract: Insulin conjugates in which the B1Phe residue has been chemically modified often exhibit a reduced tendency to associate into hexamers due to weakened interactions between subunits. The purpose of this study was to prepare a hexamer formulation for such insulin conjugates by using Co(III) as a coordinating metal ion. PEGylated insulin in which monomethoxypoly(ethylene glycol) (mPEG, M(r) 5000 or 20,000) had been site-specifically attached to B1Phe was chosen as a model conjugate. Hexamerization of mPEG-insulin upon H(2)O(2)-mediated oxidation of Co(II) was kinetically and quantitatively analysed by visible spectrometry and size-exclusion HPLC. Co(III) mPEG-insulin hexamers thus obtained were extremely stable, existing mostly as a hexameric form even at nanomolar concentrations. A remarkable increase in hydrodynamic volumes was observed for Co(III) mPEG(20k)-insulin hexamers (1600 kDa), as well as Co(III) mPEG(5k)-insulin hexamers (300 kDa). Our results demonstrate the potential benefits of Co(III) hexamer formulation for weakly associating insulin conjugates in the treatment of diabetes.
authors.affiliation: Research Center, Hanmi Pharm. Co., Ltd., Dongtan-myeon, Hwaseong-si, Gyeonggi-do, Republic of Korea. vvinhyok@naver.com
authors.forename: Sung In
authors.initials: SI
authors.lastname: Lim
authors.forename: Myung Hyun
authors.initials: MH
authors.lastname: Jang
authors.forename: Dae Jin
authors.initials: DJ
authors.lastname: Kim
authors.forename: Sung Min
authors.initials: SM
authors.lastname: Bae
authors.forename: Se Chang
authors.initials: SC
authors.lastname: Kwon
mesh_terms: D002138:Calibration; D002851:Chromatography, High Pressure Liquid; D003035:Cobalt / Q000494:pharmacology*; D006801:Humans; D007328:Insulin / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000302:isolation & purification / Q000378:metabolism*; D008970:Molecular Weight; D011092:Polyethylene Glycols / Q000138:chemical synthesis / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D055503:Protein Multimerization / Q000187:drug effects*; D013057:Spectrum Analysis
chemical_list: D007328:Insulin; C039441:insulin, polyethylene glycol(B1)-; D003035:Cobalt; D011092:Polyethylene Glycols; C028210:monomethoxypolyethylene glycol