Bisphosphonate conjugation to proteins as a means to impart bone affinity

Growth factors are endogenous proteins capable of stimulating new bone form ation, 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 ma y be overcome by derivatizing proteins with ligands that exhibit a high bon e affinity (e.g., bisphosphonates). To demonstrate the feasibility of this approach, l-amino-l,l-diphosphonate methane (aminoBP) was conjugated to a m odel protein, albumin. The conjugation was performed by (1) converting the amino group of aminoBP to a thiol group using 2-iminothiolane, (2) derivati zing the albumin amino groups with a thiol-reactive sulfosuccinimidyl-4-(N- maleimidamethyl)-1-cyclohexane carboxylate, and (3) reacting the derivatize d albumin with thiolated aminoBP. Typically, 1-4 aminoBP molecules per albu min were obtained The conjugated albumin exhibited a high affinity to hydro xyapatite that was proportional to the extent of conjugation. The conjugate s 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 bisphos phonates to albumin was shown to impart a high bone affinity to the protein , and such conjugates can be potentially targeted to bone.