【作者】 李程；

【导师】 裴福兴；

【作者基本信息】 四川大学 ， 外科学， 2007， 博士

【副题名】依替二膦酸锶的合成及药效研究

【摘要】 目的：（1）改进锶盐母体设计，寻找并合成一种具有毒副作用小、一定的溶解度、能够有效地改善锶的生物利用度同时具有骨靶向性抗骨质疏松效应的化合物。（2）检测该化合物体外对成骨样细胞株的毒性和对其骨形成生理功能的影响。（3）进一步检测该化合物对绝经期后骨质疏松动物模型的治疗作用。方法：（1）改进锶盐的母体：以依替二膦酸根作为锶盐的母体，将锶与依替二膦酸两者拼合起来合成新化合依替二膦酸锶——一种新的锶盐。（2）进行体外羟磷灰石吸附实验，以检测依替二膦酸锶的体外骨靶向性。（3）通过细胞形态学观察、台盼蓝排斥实验和MTT比色，检测10^-5M～10^-3M浓度的依替二膦酸二钠、二氯化锶和依替二膦酸锶对ROS17／2.8成骨样细胞的毒性。（4）通过碱性磷酸酶和Ⅰ型胶原，检测10^-5M～10^-3M浓度的依替二膦酸二钠、二氯化锶和依替二膦酸锶对ROS17／2.8成骨样细胞成骨活性的影响。（5）通过对不同时点绝经期后骨质疏松大鼠血清中BGP和TRAP-5b的测定，检测依替二膦酸二钠（80mg／Kg. day）、二氯化锯（60mg／Kg. day）和依替二膦酸锶（50mg／Kg. day, 100mg／Kg. day和150mg／Kg. day）对绝经期后骨质疏松大鼠骨代谢的影响。（6）通过对不同时点绝经期后骨质疏松大鼠股骨及第二腰椎BMD的测定，检测依替二膦酸二钠（80mg／Kg. day）、二氯化锶（60mg／Kg. day）和依替二膦酸锶（50mg／Kg. day, 100mg／Kg. day和150mg／Kg. day）对绝经期后骨质疏松大鼠股骨和椎骨BMD的影响。（7）通过对不同时点绝经期后骨质疏松大鼠股骨及第二腰椎生物力学特性的测定，检测依替二膦酸二钠（80mg／Kg. day）、二氯化锶（60mg／Kg. day）和依替二膦酸锶（50mg／Kg. day, 100mg／Kg. day和150mg／Kg. day）对绝经期后骨质疏松大鼠股骨和椎骨生物力学性质的影响。（8）通过对不同时点绝经期后骨质疏松大鼠股骨及第一腰椎组织形态学的观察，检察依替二膦酸二钠（80mg／Kg. day）、二氯化锶（60mg／Kg. day）和依替二膦酸锶（50mg／Kg. day, 100mg／Kg. day和150mg／Kg. day）对绝经期后骨质疏松大鼠股骨和椎骨组织形态学的影响。结果：（1）运用冰乙酸和三氯化磷，于实验室可得到较高纯度的依替膦酸锶。（2）进行体外羟磷灰石吸附实验证实：氯化锶溶液中的锶离子对羟基磷灰石无亲和性；依替二膦酸锶中的锶离子对羟基磷灰石的有明显亲和性。（3）实验未发现依替二膦酸锶在10^-5～10^-3M浓度对ROS17／2.8成骨样细胞的明显毒性。本实验发现，依替二膦酸锶对ROS17／2.8成骨样细胞的生长和增殖具有明显的促进作用，该促进作用与二氯化锶相比具有明显差异，该差异具有统计学意义。（4）本实验发现：不同药物干预48h和72h后，二氯化锶组与依替二膦酸锶组其成骨样细胞内碱性磷酸酶和Ⅰ型胶原着色程度均深于同摩尔浓度的依替二膦酸二钠；二氯化锶及依替二膦酸锶增强体外培养ROS17／2.8成骨样细胞的碱性磷酸酶活性和Ⅰ型胶原的表达能力可能较相同摩尔浓度的依替磷酸二钠强。（5）通过对不同时点绝经期后骨质疏松大鼠血清中BGP和TRAP-5b的测定发现：依替二膦酸二钠（80mg／Kg. day）、二氯化锶（60mg／Kg. day）和依替二膦酸锶（00mg／Kg. day）治疗后，依替二膦酸锶干预组大鼠血清BGP基本与去势对照组相同；依替二膦酸二钠治疗组大鼠血清BGP表现为持续下降；二氯化锶治疗组的大鼠血清BGP基本维持于相对较高的水平。依替二膦酸二钠组大鼠血清TRAP-5b迅速下降，其差异表现出统计学意义；依替二膦酸锶治疗组大鼠血清TRAP-5b逐渐下降，到治疗8周后基本与依替二膦酸二钠干预组相同水平，两者均明显低于周期去势对照组，并且两者的差异均表现出明显的统计学意义；二氯化锶干预组的大鼠血清依替二膦酸二钠基本维持于略高于去势对照组的水平。依替二膦酸锶50mg／Kg. d干预组和100mg／Kg. d干预组与同期的去势组相比其血清BGP较未见明显变化；150mg／Kg. d干预组其血清BGP有缓慢上升的趋势，于12周末时与去势对照组相比，其差异表现出显著的统计学意义。依替二膦酸锶50 mg／Kg. d、100mg／Kg. d和150mg／Kg. d干预组其血清TRAP-5b均逐渐下降；于用药12周末，三个不同剂量干预组的血清TRAP-5b浓度趋向一致，均明显低于去势对照组，且表现出明显统计学差异。（6）通过对不同时点去势后骨质疏松大鼠股骨及第二腰椎BMD的测定发现：依替二膦酸二钠、二氯化锶（60mg／Kg. day）和依替二膦酸锶依替二膦酸二钠（80mg／Kg. day）、依替二膦酸锶（00mg／Kg. day）治疗后，大鼠椎体BMD逐渐上升，其与去势对照组的差距逐渐加大；二氯化锶干预组的大鼠椎体BMD基本维持于原有水平，并表现出略有增加。依替二膦酸锶、依替二膦酸二钠及二氯化锶干预组大鼠股骨BMD与去势组的缓慢下降相比，无下降现象，表现为平稳之中略有上升。依替二膦酸锶50mg／Kg. day干预组、100mg／Kg. day干预组和150mg／Kg. day组其大鼠椎体BMD不断升高；在治疗12周后三组BDM值，与为去势对照组相比其差异显著性具有统计学意义。依替二膦酸锶50mg／Kg. day干预组、100mg／Kg. day干预组和150mg／Kg. day组其大鼠股骨BMD有所升高；且在150mg／Kg. day干预组其股骨BMD与为去势对照组相比，其差异显著性具有统计学意义。（7）通过对不同时点绝经期后骨质疏松大鼠股骨及第二腰椎生物力学特性的测定发现：依替二膦酸二钠（80mg／Kg. day）、二氯化锶（60mg／Kg. day）治疗后，在治疗后在4～12周内，大鼠股骨于三点弯曲试验下的最大负荷（Max Load），二氯化锶干预组其Max Load变化亦不明显；依替二膦酸二钠治疗12周后其股骨的Max Load与去势对照组相比升高，并且其差异表现出统计学意义；依替二膦酸锶（100mg／Kg. day）干预组8周、12周后，其股骨的Max Load分别升高21.9％和22.3％，且两者均表现出统计学差异。在大鼠椎体于压缩试验下的最大负荷（Max Load），所有干预组在治疗4周后其大鼠椎体的抗压缩强度（Max Load）均明显高于去势对照组；且随着治疗时间的延长，均表现为逐渐下降。其中，依替二膦酸二钠治疗后其大鼠椎体的Max Load下降较为缓慢；而依替二膦酸锶（100mg／Kg. day）干预组前8周其椎体（Max Load）表现为逐渐下降，而12周后，其股骨的Max Load明显升高，并表现出统计学差异。依替二膦酸锶（50mg／Kg. day, 100mg／Kg. day和150mg／Kg. day）治疗后，50mg／Kg. day干预组股骨Max Load变未观察到明显变化；100mg／Kg. day和150mg／Kg. day干预组干预8周后，其股骨Max Load升高，但并表现出统计学意义；50mg／Kg. day, 100mg／Kg. day和150mg／Kg. day干预组干预后，其股骨于Max Load下变形与去势对照组相似，并未表现出明显差异。50mg／Kg. day, 100mg／Kg. day和150mg／Kg. day干预组于治疗前其椎体的抗压缩强度明显高于去势对照组，并具有统计学意义；干预后，其椎体Max Load均表现为在治疗的前8周逐渐降低，而12周后明显升高；其中100mg／Kg. day和150mg／Kg. day干预组明显高于去势对照组，并表现出统计学意义。50mg／Kg. day, 100mg／Kg. day和150mg／Kg. day干预组于治疗前其椎体的于Max Load下的变形高于去势对照组，并具有统计学意义；干预后，其椎体变形均有所增加，于12周后100mg／Kg. day和150mg／Kg. day干预组明显高于去势对照组，并表现出统计学意义。（8）通过对不同时点绝经期后骨质疏松大鼠股骨及第一腰椎组织形态学的观察发现：80mg／Kg. day依替二膦酸二钠治疗4周后，骨小梁间的显微骨折明显增多；破骨细胞的骨吸收现象被明显抑制，成骨细胞的骨形成也受到明显影响；8～12周后，破骨细胞的骨吸收现象被明显抑制；骨形成虽也受到明显抑制，骨小梁的数目和体积仍有所恢复。60mg／Kg. day二氯化锶治疗4周后，骨小梁间的显微骨折明显增多；破骨细胞的骨吸收现象明显，成骨细胞的骨形成也明显；8～12周后，骨吸收及骨形成现象仍明显；但骨小梁的数目和体积未见到明显恢复。100mg／Kg. day依替二膦酸锶治疗4周后，骨小梁间的显微骨折无明显增多；破骨细胞的骨吸收现象被明显抑制，成骨细胞的骨形成未受到明显影响；8～12周后，破骨细胞的骨吸收现象被明显抑制；骨形成活跃，骨小梁的数目和体积仍有明显恢复。50mg／Kg. day剂量的依替二膦酸锶治疗4周后，即可明显抑制绝经期后大鼠骨组织内活跃的骨吸收现象；而成骨细胞的骨形成受到的影响较少；8～12周后，可减少骨组织的骨量丢失、修复部分骨组织内的显微骨折。而100mg／Kg. day和150mg／Kg. day剂量组两组其骨形成现象明显多于50mg／Kg. day剂量组，8～12周后，骨形成活跃，骨小梁的数目和体积仍有明显恢复，绝大部分显微骨折均被修复。结论：（1）运用冰乙酸与三氯化磷通过化学缩合等步骤，可于实验室得到较高纯度的依替膦酸锶。（2）氯化锶溶液中的锶离子对羟基磷灰石无亲和性；依替二膦酸根能够显著提高锶原子对羟基磷灰石的亲和性。（3）本实验发现新化合物依替二膦酸锶在10^-5～10^-3M浓度对ROS17／2.8成骨样细胞的无明显毒性作用。依替二膦酸锶对ROS17／2.8成骨样细胞的生长和增殖的促进作用。（4）二氯化锶（60mg／Kg. day）和依替二膦酸锶（100mg／Kg. day）能增强体外培养ROS17／2.8成骨样细胞的碱性磷酸酶活性和细胞内Ⅰ型胶原的表达。（5）依替二膦酸锶对去势后大鼠成骨细胞分泌BGP水平无抑制作用。三种不同治疗剂量该物质均能有效抑制破骨细胞分泌TRAP-5b的能力。（6）三种不同剂量的依替二膦酸锶均能有效地增加去势大鼠椎体和股骨的BMD，其中以150mg／Kg.d干预组为明显。（7）依替二膦酸锶（100mg／Kg. day）干预8周后即可增强股骨的抗弯曲强度。依替二膦酸锶干预4周后，可起到防止去势所致的大鼠腰椎抗压缩强度迅速下降；依替二磷酸100mg／Kg和150 mg／Kg治疗剂量治疗12周后，能够明显改善大鼠腰椎的抗压缩力学强度。（8）三种不同依替二膦酸锶治疗，能明显明显抑制绝经期后大鼠骨吸收活动，减少骨质丢失。依替二膦酸锶治疗，能明显增强骨组织的骨形成活动，增加骨组织的骨量；该作用与药物剂量呈正相关。该课题显示了依替二膦酸锶具有体外骨靶向性、良好的细胞相容性和对成骨样细胞生长的促进作用；血清酶学实验显示该耦联物在降低血清TRAP-5b水平的同时，能明显升高BGP水平；骨密度结果证明耦联物能明显增加大鼠椎体及股骨的BMD；生物力学结果显示耦联物能增加大鼠股骨和椎体的生物力学性能。体外实验和动物实验结果均提示：耦联物在显著抑制骨吸收的同时，明显刺激骨形成。该论文将骨吸收抑制物与骨形成促进物进行互补性耦联，利用骨代谢“解耦剂”调节骨代谢平衡的观点具有较强的创新性，为抗骨质疏松药物的研发提供了新思路；为进一步开发具有自主知识产权的新型抗骨质疏松药物，提供了前期实验基础。更多还原

【Abstract】 Objective: （1）To explore a new strontium compound with little toxicity and side effects, with considerable solubility, with upgrading bioavalibility and bone target, we designed the compound with improved organic principle. （2）We investigate the toxicity and physiological effects of the compound to ROS17/2.8 cells. （3）To ivestegate the therapeutical effects to the SD rat model of postmenopausal osteoporosis.Methords. （1）Improving the organic principle of strontium: Taking the etidronate as the organic principle of strontium, we combined the both and got a new compound—strontium etidronate. （2）In order to study the vitro bone-targeted characteristic, we did the hydroxylapatite absorption experiment. （3）We studied the cell morphology, typan blue exclude ecperiment, and MTT reduction assay experiment of ROS17/2.8 cells, with 10-5M～10-3M disodium etidronate, strontium chloride and strontium etidronate, to investigat the cell toxicity. （4）We studied the ALP activity, I collagen express of ROS17/2.8 cells, with 10-5M～10-3M disodium etidronate, strontium chloride and strontium etidronate, to investigat the cell physiological effects. （5）We studied the serum BGP and TRAP-5b of SD rats model of postmenopausal osteoporosis model at different times, to investegat the drug effects of bone metabolism involving disodium etidronate （80mg/Kg.day）, strontium chloride （60mg/Kg.day） and strontium etidronate （50mg/Kg.day, 100mg/Kg.day and 150mg/Kg.day）. （6）We studied the femoral and second lumbar vertebral BMD of SD rats model of postmenopausal osteoporosis model at different times, to investegat the drug effects of BMD involving disodium etidronate （80mg/Kg.day）, strontium chloride （60mg/Kg.day） and strontium etidronate （50mg/Kg.day, 100mg/Kg.day and 150mg/Kg.day）. （7）We studied the femoral and second lumbar vertebral biomechanical characteristics of SD rats model of postmenopausal osteoporosis model at different times, to investegat the drug effects of biomechanical characteristics involving disodium etidronate （80mg/Kg.day）, strontium chloride （60mg/Kg.day） and strontium etidronate （50mg/Kg.day, 100mg/Kg.day and 150mg/Kg.day）. （8）We studied the femoral and second lumbar vertebral histomorphology of SD rats model of postmenopausal osteoporosis model at different times, to investegat the drug effects of femoral and second lumbar vertebral histomorphology involving disodium etidronate （80mg/Kg.day）, strontium chloride （60mg/Kg.day） and strontium etidronate （50mg/Kg.day, 100mg/Kg.day and 150mg/Kg.day）.Rsults. （1）Condensed phosphorus trichloride with glacial acetic acid, then counteracted by NaOH, we could get disodium etidronate. Added strontium chloride into the solution of disodium etidronate, we got highly purified strontium etidronate. （2）In the vitro hydroxylapatite absorption experiment, we found the strontium iron in strontium etidronate showed obvious affinity with the hydroxylapatite. （3）In experiment, we didn’t find the cell toxicity form 10-5M～10-3M strontium etidronate. We found strontium etidronate could evidently prompt the growth and proliferation of ROS 17/2.8 cells, and the effects was more obvious than strontium chloride. （4）In experiment, we found there was more colour of ALP and I collagen express in ROS17/2.8 cells cultivated with strontium chloride and strontium etidronate. The strontium chloride and strontium etidronate had more ability on improving ALP activity and I collagen express in ROS17/2.8 cells than disodium etidronate. （5）We didn’t find the obvious changes of serum BGP level, after being treated with strontium etidronate （100mg/Kg.day） and strontium chloride （60mg/Kg.day）, and it continually decrease in involving disodium etidronate （80mg/Kg.day） rats, but it continually decrease in involving strontium chloride （60mg/Kg.day）rats. We found the quickly decrease of serum TRAP-5b level, after being treated with disodium etidronate （80mg/Kg.day）, it decrease gradually in strontium etidronate （100mg/Kg.day）, and it showed little higher in strontium chloride （60mg/Kg.day） rats. We found there no obvious changes of serum BGP level, after being treated with strontium etidronate （50mg/Kg.day, and 100mg/Kg.day）, but it increased gradually in strontium etidronate （150mg/Kg.day） rats, and show obviously different. Being treated with strontium etidronate （50mg/Kg.day, 100mg/Kg.day and 150mg/Kg.day）, serum TRAP-5b level of all rats decrease apparently. （6）We found the second lumbar vertebral BMD increased gradually in the rats treated by disodium etidronate （80mg/Kg.day） and strontium etidronate （100mg/Kg.day）, and there no obvious changes of those treated by strontium chloride （60mg/Kg.day）, there no obvious changes on femoral BMD in all rats above. We ound the second lumbar vertebral BMD increased gradually in the rats treated by strontium etidronate （50mg/Kg.day, 100mg/Kg.day and 150mg/Kg.day）, and the obvious increase on femoral BMD in the rats treated by strontium etidronate （150mg/Kg.day）. （7）We found no obvious changes in femoral Max Load and Flexure at Max Load under three point bending test in rats treated by disodium etidronate （80mg/Kg.day）, strontium chloride （60mg/Kg.day） and strontium etidronate （50mg/Kg.day, 100mg/Kg.day and 150mg/Kg.day）. we found the apparently increase in second lumbar vertebral Max Load under compress test in rats treated by strontium etidronate （50mg/Kg.day, 100mg/Kg.day and 150mg/Kg.day）, and Flexure at Max Load in rats treated by strontium etidronate （100mg/Kg.day and 150mg/Kg.day）. （8）We found there obviously increased bone trabecular micro-crack, inhibition of both bone resorption and formation, and increase of bone trabecular on number and volume after 8～12 weeks treated by disodium etidronate （80mg/Kg.day）. We also found there obviously increased bone trabecular micro-crack, activity of both bone resorption and formation, and no obvious increase of bone trabecular on number and volume after 8～12 weeks treated by strontium chloride （60mg/Kg.day）. We found there no obviously increased bone trabecular micro-crack, inhibition of both bone resorption and activity of bone formation, and obvious increase of bone trabecular on number and volume after 8～12 weeks treated by strontium etidronate （100mg/Kg.）, and these phenomemons also could be found in rats treated by strontium etidronate （50mg/Kg.day）. We found above phenomemons were more obvious in 100mg/Kg.day and 150mg/Kg.day groups than 50mg/Kg.day group, and most micro-cracks had been repaired.Conclusions: （1） we could get highly purified strontium etidronate in lab. （2）The etidronate could improve strontium iron affinity with the hydroxylapatite. （3）10^-5M～10^-3M strontium etidronate shows no toxicity to ROS 17/2.8 cells. Strontium etidronate could evidently prompt the growth and proliferation of ROS17/2.8 cells. （4）Strontium etidronate could improve the ALP activity and I collagen express in ROS17/2.8 cells. （5）Strontium etidronate （100mg/Kg.day） shows no obvious effect on the serum BGP level, and decrease the serum TRAP-5b level. Strontium etidronate （50mg/Kg.day, 100mg/Kg.day and 150mg/Kg.day） could decrese serum TRAP-5b level of all rats apparently. （6）Strontiurn etidronate （100mg/Kg.day） could increase the second lumbar vertebral BMD in the osteoporosis rats, and prevent the decrease of femoral BMD. Strontium etidronate （50mg/Kg.day, 100mg/Kg.day andlS0mg/Kg.day）, could obviously increase the lumbar vertebral BMD in osteoporosis rats. （7）Strontium etidronate （100mg/Kg.day） could improve the osteoporosis rats on femoral Max Load after 8 weeks. Strontium etidronate （100mg/Kg.day and 150mg/Kg.day） could improve the osteoporosis lumbar vertebral Max Load and the deformation ability under compress test. （8）Strontium etidronate （50mg/Kg.day, 100mg/Kg.day,and150mg/Kg） could increase the number and volume bone trabecular and repair activity, increase the bone mass, and the effect shows positive relation with the dose.更多还原