title: A comparison of mineral affinity of bisphosphonate-protein conjugates constructed with disulfide and thioether linkages.
abstract: Chemical conjugation of bisphosphonates (BPs) to therapeutic proteins is an effective means to impart mineral affinity to proteins. Such conjugates can be implanted with mineral-based matrices to control the local delivery kinetics of the proteins. BPs linked to proteins with reversible (i.e., cleavable) linkages are desirable over conjugates with stable linkages to release the protein in free form. This study conducted a direct comparison of mineral affinity of BP-protein conjugates linked together with cleavable disulfide and non-cleavable thioether linkages. Bovine serum albumin (BSA) was used as a model protein and the desired conjugates were created with N-succinimidyl-3-(2-pyridyldithio)propionate (disulfide) and succinimidyl-4-(N-maleimido-methyl)cyclohexane-1-carboxylate (thioether) linkers. The disulfide-linked conjugates were cleaved in the presence of a major thiol constituent of serum, cysteine. The imparted mineral affinity, as assessed by hydroxyapatite binding in vitro, was lost upon the cleavage of the disulfide-linked aminoBP. The presence of the serum did not accelerate the cleavage of disulfide-linked conjugates. The aminoBP-BSA conjugates formed with disulfide and thioether linkages were subcutaneously implanted in rats with two different mineral-based matrices to assess protein loss from the matrices. All conjugates exhibited a higher retention in mineral matrices as compared to unmodified BSA. However, no significant differences in in situ pharmacokinetics of the disulfide- and thioether-linked conjugates were observed. We conclude that disulfide-linked BP conjugates were readily cleavable by the amino acid cysteine in vitro, but in vivo cleavage of the disulfide-linked conjugates was not evident when the proteins were implanted adsorbed to mineral-based matrices. BP-protein conjugates with faster-cleaving tethers might be required to significantly influence the release of the BP conjugates from the mineral matrices.
authors.affiliation: Department of Chemical & Materials Engineering, Faculty of Engineering, University of Alberta, Edmonton, Alta., Canada.
authors.forename: Jennifer E I
authors.initials: JE
authors.lastname: Wright
authors.forename: Sebastien A
authors.initials: SA
authors.lastname: Gittens
authors.forename: Geeti
authors.initials: G
authors.lastname: Bansal
authors.forename: Pavel I
authors.initials: PI
authors.lastname: Kitov
authors.forename: Dennis
authors.initials: D
authors.lastname: Sindrey
authors.forename: Cezary
authors.initials: C
authors.lastname: Kucharski
authors.forename: Hasan
authors.initials: H
authors.lastname: Uludağ
mesh_terms: D000577:Amides / Q000737:chemistry; D000818:Animals; D002417:Cattle; D003432:Cross-Linking Reagents / Q000737:chemistry*; D003545:Cysteine / Q000737:chemistry; D004164:Diphosphonates / Q000737:chemistry*; D004220:Disulfides / Q000737:chemistry*; D005260:Female; D008903:Minerals / Q000737:chemistry*; D015394:Molecular Structure; D051381:Rats; D017207:Rats, Sprague-Dawley; D012710:Serum Albumin, Bovine / Q000737:chemistry*; D013440:Sulfides / Q000737:chemistry*
chemical_list: D000577:Amides; D003432:Cross-Linking Reagents; D004164:Diphosphonates; D004220:Disulfides; D008903:Minerals; D013440:Sulfides; D012710:Serum Albumin, Bovine; D003545:Cysteine