Targeting a drug on hydroxyapatite (HA) could be a promising way for selective drug delivery to bone, because HA, an inorganic component in hard tissues (bone and teeth), does not exist in soft tissues. Several bone noncollagenous proteins, which bind to HA, have repeating sequences of acidic amino acids in their structures as possible HA-binding sites. Thus, we think that a small peptide of repetitive acidic amino acid could work as a carrier for selective drug delivery to the bone. To test this hypothesis, we conjugated (Asp) 6 to fluorescein isothiocyanate (FITC), evaluated its affinity to HA in vitro, and examined its tissue distribution after injection into rats. Although fluorescein itself did not bind to HA, (Asp) 6 -FITC bound to HA as well as calceine and tetracycline. Twenty-four hours after intravenous injection of (Asp) 6 -FITC to rats, animals were killed, and ground sections of hard tissues and cryosections of soft tissues were made. Under a confocal laser scanning microscope, clear labeling lines were observed in bones and teeth, whereas no labeling was detected in soft tissues. In the rats administered with fluorescein alone, the fluorescent labeling was detected in neither hard nor soft tissues. Fluorescent analysis of blood, urine, and bones after (Asp) 6 -FITC administration revealed that biological half-life of FITC in blood was short (60 minutes) and that within 24 h, 95% of the administered FITC was excreted as urine whereas 2% of the FITC accumulated in bones. After subcutaneous administration of (Asp) 6 -FITC to mice, fluorescent intensity remaining in the femurs was measured periodically. In these mice the biological half-life of FITC in the femur was 14 days. Present results indicate that (Asp) 6 is effective as a carrier for selective drug delivery to bone. (J Bone Miner Res 2000;15:936 -943)
Hard tissues, such as bone and teeth, consist of hydroxyapatite (HAP), collagenous proteins and noncollagenous proteins. Osteopontin and bone sialoprotein are two major noncollagenous proteins in bone and have many L-Asp and L-Glu repetitive sequences, respectively, as possible hydroxyapatite (HAP)binding sites. Fluorescence-labeled acidic amino acid (L-Asp or L-Glu) homopeptides containing six or more residues bound strongly to HAP and were selectively delivered to and retained in bone after systemic administration. This result stimulated the development of bone-targeting drugs by tagging with acidic oligopeptides. Three model drugs, estradiol, quinolone antibiotics and tissue-nonspecific alkaline phosphatase (TNSALP), tagged with aspartic acid hexapeptide were examined the clinical feasibility of the acidic oligopeptide strategy for selective drug delivery to bone. In vivo experiments confirmed the long acting effects of L-Asp hexapeptide-tagged estradiol and levofloxacin on animal models of osteoporosis and osteomyelitis, respectively, although there was loss of in vitro bioactivity, suggesting that the acidic hexapeptide was removed by hydrolysis in the body after delivery to bone. The adverse effect of estradiol on the uterus was greatly reduced by conjugation to the hexapeptide. The L-Asp hexapeptide-tagged TNSALP has the enzyme activity similar to untagged enzyme and was selectively delivered to bone and retained for long time in comparison with the untagged enzyme. These results support the usefulness of acidic oligopeptides as bone-targeting carriers for therapeutic agents.
We have reported that denbufylline, a phosphodiesterase 4 (PDE4) inhibitor, inhibits bone loss in Walker256/S tumor-bearing rats, suggesting therapeutic potentiality of a PDE4 inhibitor in osteopenia. In the present study, effects of a new PDE4 inhibitor, 1-n-butyl-3-n-propylxanthine (XT-44), in bone were evaluated in cell cultures and animal experiments. In rat bone marrow culture, XT-44 stimulated mineralized-nodule formation, whereas it inhibited osteoclast-like cell formation in mouse bone marrow culture. In Walker256/S-bearing rats (6-week-old female Wistar Imamichi rats), rapid decrease in bone mineral density (BMD) was prominent, and oral administration of XT-44 (0.3 mg/kg, every 2 days) inhibited the decrease in BMD. In the second animal experiment, female Wistar rats (6-week-old) were sciatic neurectomized, and XT-44 was orally administered to these rats every 2 days for 4 weeks. XT-44 administration (0.3 mg/kg) recovered BMD in these neurectomized animals. Furthermore, 19-week-old, female Wistar rats were ovariectomized (OVX), and 15 weeks after surgery, these rats were orally administered XT-44 every 2 days for 8 weeks. XT-44 treatment (1 mg/kg) increased the BMD of OVX rats. These results indicate that XT-44 could be a candidate as a therapeutic drug for treating osteopenia including osteoporosis.
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