Selective Drug Delivery to Bone

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Targeting a drug on hydroxyapatite (HA) could be a promising way for selective drug delivery to bone because HA does not exists in soft tissues. Some of non-collagenous proteins in bone have repeating sequence of acidic amino acids in their structures as possible HA-binding sites. The purpose of this study is. to examine whether a small peptide of repetitive Asp could work as a carrier for selective drug delivery to bone. Fluorescein (Flu) were conjugated with (Asp)_6 peptide and affinities of this compound and bone-seeking compounds (tetracycline, calcein, alizarin red S and tiludronate) to HA were spectrophotometrically measured after incubation with HA bead solution and centrifugation. Flu or (Asp)_6-Flu (1mg or 3mg, respectively, in 200mu was intravenously injected and fluorescent intensity in blood plasma was periodically measured. Twenty-four hours after injection, ground sections of bones and teeth and cryosections of soft tissues were prepared and then examined under confocal l … More aser scanning microscope. Flu did not bind to HA, however, (Asp)_6-Flu showed high affinities to HA, which were comparable to the bone-seeking compounds. Fluorescence measurement in the plasma revealed rapid excretion of both Flu and (Asp)_6-Flu from the blood. Biological half lives of Flu and (Asp)_6-Flu were 39 and 60 minutes, respectively. In the rats injected with (Asp)_6-Flu systemically, clear fluorescent lines were observed in bones and teeth whereas no fluorescence was detected in soft tissues (brain, muscle, kidney, liver, spleen, thymus, heart, intestine, dermal connective tissue). In the animals injected with Flu systemically, the fluorescence was detected in neither haul nor connective tissues. We also examined biological half life of Flu in femurs after injecting (Asp)_6-Flu into mice and it was 14 days. Furthermore, we conjugated estradiol with (Asp)_6 and (Asp)_6-estradiol showed high affinity to HA whereas estradiol did not. (Asp)_6-estradiol inhibited bone loss in ovariectomized mice without increasing uterine weight. These results indicate that (Asp)6 conjugation increases drug affinity to HA and could be effective as a carrier for drug delivery to bone.In this research project we also demonstrated that rolipram, a phosphodiesterase 4 (PDE4) inhibitor which inhibits cAMP degradation specifically, exerts anabolic effect in bone and that XT-44, which is a new PDE4 inhibitor developed by Dr. Miyamoto, one of the investigators in this project, is therapeutically effective in three osteopenia models : carcinoma bearing rats, ovariectomized rats and nurolectomized rats. Furthermore we clarified active site of fibroblast growth factor (FGF) 4, and produced a recombinant protein of N-terminal truncated FGF4 containing its active site. Systemic administration with this short FGF4 to normal mice increased femur BMD.PDE4 inhibitors and this short FGF4 could be new candidates for therapeutic drugs of osteopenia, however systemic administration of these molecules might also exert unfavorable effects in other tissues. Targeting of these molecules to bone by conjugation with (Asp) 6 could open a new avenue for treatment of osteopenia including osteoporosis. Less