Selective Drug Delivery to Bone

由于羟基磷灰石（HA）不存在于软组织中，因此将药物靶向到羟基磷灰石（HA）上可能是一种有前途的选择性药物递送到骨的方法。骨中的一些非胶原蛋白在其结构中具有重复的酸性氨基酸序列作为可能的HA结合位点。本研究的目的是。研究重复的天冬氨酸小肽是否可以作为选择性药物递送到骨的载体。将（Asp）_6肽段与流感黄绿素（Flu）偶联，经HA微球溶液孵育、离心后，用比色法测定该化合物和趋骨化合物（四环素、钙黄绿素、茜素红S和替鲁膦酸盐）与HA的亲和力。静脉注射Flu或（Asp）_6-Flu（1 mg或3 mg）200 μ l，定期测定血浆荧光强度。注射后24小时，制备骨骼和牙齿的研磨切片以及软组织的冷冻切片，然后在共聚焦激光显微镜下进行检查。 ...更多信息 激光扫描显微镜Flu不与HA结合，而（Asp）_6-Flu与HA有很高的亲和力，与趋骨化合物相当。血浆中的荧光测量显示Flu和（Asp）_6-Flu从血液中快速排出。Flu和（Asp）_6-Flu的生物半衰期分别为39和60分钟。在全身注射（Asp）_6-Flu的大鼠中，骨骼和牙齿中观察到清晰的荧光线，而软组织（脑、肌肉、肾、肝、脾、胸腺、心脏、肠、真皮结缔组织）中未检测到荧光。在全身注射流感病毒的动物中，在牵引组织和结缔组织中均未检测到荧光。同时测定了注射（Asp）_6-Flu后小鼠股骨中Flu的生物半衰期为14天。此外，我们用（Asp）_6和（Asp）_6-雌二醇偶联的雌二醇对HA表现出高亲和力，而雌二醇则没有。(Asp)6-雌二醇抑制去卵巢小鼠的骨丢失，但不增加子宫重量。这些结果表明，（Asp）6结合增加了药物对HA的亲和力，可以作为药物递送到骨的有效载体。在本研究项目中，我们还证明了rolipram，一种磷酸二酯酶4（PDE 4）抑制剂，特异性抑制cAMP降解，在骨中发挥合成代谢作用，XT-44，这是Miyamoto博士开发的一种新的PDE 4抑制剂，本项目的研究者之一，在三种骨质减少模型中治疗有效：荷癌大鼠、卵巢切除大鼠和神经切除大鼠。进一步阐明了成纤维细胞生长因子（FGF）4的活性位点，并制备了含其活性位点的N端截短型FGF 4重组蛋白。对正常小鼠全身给予这种短FGF 4增加了股骨BMD. PDE 4抑制剂，并且这种短FGF 4可能是骨质减少症治疗药物的新候选物，然而这些分子的全身给予也可能在其他组织中产生不利影响。通过与（Asp）6缀合将这些分子靶向骨，可以为治疗骨质减少症（包括骨质疏松症）开辟新的途径。少

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