Bisphosphonate Conjugation to Proteins as a Means To Impart Bone Affinity

Abstract

Growth factors are endogenous proteins capable of stimulating new bone formation, but their clinical benefit for systemic stimulation of bone mass has not been demonstrated. The critical challenge is to deliver a significant dose of the proteins to bone after intravenous injection. This challenge may be overcome by derivatizing proteins with ligands that exhibit a high bone affinity (e.g., bisphosphonates). To demonstrate the feasibility of this approach, 1-amino-1,1-diphosphonate methane (aminoBP) was conjugated to a model protein, albumin. The conjugation was performed by (1) converting the amino group of aminoBP to a thiol group using 2-iminothiolane, (2) derivatizing the albumin amino groups with a thiol-reactive sulfosuccinimidyl-4-(N-maleimidomethyl)-1-cyclohexane carboxylate, and (3) reacting the derivatized albumin with thiolated aminoBP. Typically, 1-4 aminoBP molecules per albumin were obtained. The conjugated albumin exhibited a high affinity to hydroxyapatite that was proportional to the extent of conjugation. The conjugates were shown to exhibit a high affinity to bone matrix in vitro in a serum-containing medium. Once bound to bone matrix, the conjugates were found to desorb more slowly than the unmodified albumin, especially from bone whose organic matrix was removed by ashing. In conclusion, conjugation of bisphosphonates to albumin was shown to impart a high bone affinity to the protein, and such conjugates can be potentially targeted to bone.

Imparting mineral affinity to proteins with thiol–labile disulfide linkages

“…The feasibility of this approach has been shown by conjugating BPs to several proteins, including albumin, 5 lysozyme, 6 nonspecific IgG, 7 and fetuin, 8 which were all shown to exhibit increased hydroxyapatite binding upon BP conjugation. The fetuin-BP conjugates were recently shown to exhibit an increased mineral affinity in vivo when they were implanted subcutaneously by using coralline apatite Pro-Osteon™ implants that are used for spinal surgery.…”

“…Stability of the conjugate linkage was beneficial for retaining the high mineral affinity of proteins under a variety of conditions, such as the medium pHs, osmolarity, as well as the presence of serum in binding medium. 5 For in vivo applications, however, conjugates prepared with cleavable linkages might be desirable because this type of linkage will eventually release the protein in free form in situ, which will facilitate the interactions of proteins with their intended biological targets on cellular surfaces.…”

“…The unreacted materials were thoroughly extracted with hexane (3 ϫ 15 mL). The aqueous layer was vacuum concentrated to give a yellowcolored oily product, thiolBP (5).…”

Enhancement of the Regenerative Potential of Anorganic Bovine Bone Graft Utilizing a Polyglutamate-Modified BMP2 Peptide with Improved Binding to Calcium-Containing Materials

“…[34][35][36][37][38] As an additional approach, bioactive molecules can be anchored onto calcium-containing substrates through the use of negatively charged domains that bind to positively charged calcium ions. Examples of such domains include bisphosphonates [39][40][41][42] as well as sequences comprised of negatively charged amino acids, including glutamate, aspartate, or the noncanonical amino acids, gcarboxyglutamate and phosphoserine. [15][16][17]19,23,25,[43][44][45] For delivery of bioactive peptides, polyglutamate or polyaspartate domains offer some advantages over other types of calciumbinding modules.…”

Bifosfonatos (BFs) como transportadores osteotrópicos no planejamento de fármacos dirigidos

“…A idéia de BFs conjugados a proteínas foi divulgada através de uma patente utilizando polietilenoglicol (PEG) hidrofílico como ligante. Entretanto, a afinidade aos ossos não foi comprovada 43 . Uludag e colaboradores 44 , utilizando albumina sérica bovina (ASB) como proteína modelo, demonstraram in vitro que a afinidade da proteína modificada é diretamente proporcional à extensão de conjugação (5 BFs por proteína).…”