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.

Boi-ogi-to (Fang-ji-huang-qi-tang) is a traditional prescription in Kampo medicine and consists of Stephania tetrandra Radix (Stephania), Astragalus membranaceus BUNGE Radix (Astragali), Atractylodes Lancea Rhizoma, Glycyrrhizae Radix, Zingiberis Rhizoma and Zizyphi Fructus. Boi-ogi-to has long been used clinically in the treatment of arthritis and edema in China and Japan. It also improves abnormal glucose and lipid metabolism in obese diabetic patients. 1) We have reported that Boi-ogi-to increases blood insulin and decreases blood glucose levels in streptozotocin (STZ)-diabetic mice. The anti-hyperglycemic and anti-hypoinsulinemic actions of Boi-ogi-to depend on the combination of Stephania and Astragali.2) Astragali does not have a direct anti-hyperglycemic effect, but potentiates the actions of Stephania on blood levels of both glucose and insulin in STZ-induced diabetes. 3,4) In addition, Stephania suppresses abnormal choroidal and retinal neovascularization in STZ-induced diabetes in vitro and in vivo. 5)Stephania contains many bis-benzylisoquinoline-type constituents, such as tetrandrine and fangchinoline.6,7) Fangchinoline significantly improves hyperglycemia of STZ-diabetic mice.4) Tetrandrine inhibits precapillary formation of vascular endothelial cells and neovascularization of cultured choroidal explants in STZ-diabetic rats, 8,9) but does not affect hyperglycemia in diabetic mice.4) Fangchinoline and tetrandrine show different anti-inflammatory actions via inhibition of cyclooxygenase and interleukin-5 activities.10) In other studies, fangchinoline and tetrandrine were found to have similar inhibitory activities on both angiotensin I converting enzyme, 6,7) and induction of proinflammatory cytokines, interleukin-1 and tumor necrosis factor-alpha by Staphylococcus aureus Cowan 1-stimulated human peripheral blood mononuclear cells. 11)Astragali contains many isoflavones and isoflavonoids, such as formononetin, calycosin and ononin, and many saponins, such as astragaloside IV, astragaloside II, astragaloside I, and acetylastragaloside I.12) Formononetin significantly reduces arachidonic acid release and production of nitric oxide in lipopolysaccharide activated RAW 264.7 macrophages.13) It also shows estrogen receptor agonistic activity in human breast cell line MCF-7.14,15) Formononetin and calycosin activate the peroxisome proliferator-activated receptors (PPAR) alpha and gamma. The action of formononetin is more potent than that of calycosin.16) Calycosin is a vasorelaxant and is a noncompetitive Ca 2ϩ channel blocker, whose action is endothelium-independent and unrelated to intracellular Ca 2ϩ release. The effect of calycosin on Ca 2ϩ channel blockade may be different from that of dihydropyridines.17) In addition, formononetin, calycosin and ononin all inhibit glutamate-induced cell damage by increasing endogenous antioxidant and stabilizing cell membrane structures. 18)In the present study, the combined effects of fangchinoline with formononetin, calycosin and ononin on the blood levels of glucos...

Site-specific drug delivery to bone is considered to be achievable by utilizing acidic amino acid homopeptides. We found that fluorescence-labeled acidic amino acid (L-Asp or L-Glu) homopeptides containing six or more residues bound strongly to hydroxyapatite, which is a major component of bone, and were selectively delivered to and retained in bone after systemic administration. We explored the applicability of this result for drug delivery by conjugation of estradiol and levofloxacin with an L-Asp hexapeptide. We also similarly tagged an enzyme, tissue-nonspecific alkaline phosphatase, to see whether this would improve the efficacy of enzyme replacement therapy. The L-Asp hexapeptide-tagged drugs, including the enzyme, were selectively delivered to bone in comparison with the untagged drugs. It was expected that the ester linkage to the hexapeptide would be susceptible to hydrolysis in situ, releasing the drug or enzyme from the acidic oligopeptide. An in vivo experiment confirmed the efficacy of L-Asp hexapeptide-tagged estradiol and levofloxacin, although there was some loss of bioactivity of estradiol and levofloxacin in vitro, suggesting that the acidic hexapeptide was partly 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. These results support the usefulness of acidic oligopeptides as bone-targeting carriers for therapeutic agents. We present some pharmacokinetic and pharmacological properties of the L-Asp hexapeptide-tagged drugs and enzyme.