大豆苷元抗骨质疏松及其壳聚糖长效缓释微球制剂的研究
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摘要
大豆苷元(daidzein)来源于天然植物大豆，作为一种异黄酮成分，具有弱雌激素样和抗雌激素样作用，广泛用于心脑血管疾病、癌症、骨质疏松等疾病的治疗和预防，具有疗效好、副作用小等特点。为了进一步了解大豆苷元抗骨质疏松的作用，并制成合适给药的新剂型，本文从细胞学和整体动物两个方面进行大豆苷元抗骨质疏松的实验研究，并以壳聚糖为载体材料，设计和研制了大豆苷元-壳聚糖长效缓释微球，并采用体内外不同方法对其进行了科学的评价。
在培养基中添加β-甘油磷酸二钠、L-抗坏血酸和培养时间10、20、30天条件下，采用MTT比色法、碱性磷酸酶活性测定、Ca和P含量测定、茜素红染色和von Kossa染色，考察不同大豆苷元浓度(10~(-9)～10~(-5)M)对MC3T3-E1成骨样细胞增殖、分化和矿化的影响，结果表明大豆苷元能促进MC3T3-E1细胞的增殖和分化特别是促进矿化的发生和增加矿化的量；建造去卵巢小鼠骨质疏松模型，选择小鼠进行去卵巢手术后1、3和5周模型，与阳性对照药17β-雌二醇比较，考察腹腔注射大豆苷元溶液对小鼠子宫重量系数、股骨病理切片HE染色的骨组织结构和骨组织中细胞因子OPG、RANKL mRNA相对表达水平的影响，从而初步探讨大豆苷元抗骨质疏松的机理，大豆苷元能改善去卵巢导致的小鼠皮毛蓬松、行动迟缓、反应迟钝和精神不振，能增加去卵巢小鼠的子宫重量系数、骨小梁分布密度和骨皮质厚度，并呈现剂量和时间效应，此外大豆苷元能通过调节OPG／RANKL比例从而调节骨重建过程中破骨细胞和成骨细胞的平衡，说明大豆苷元具有抗骨质疏松的作用，这对于了解植物雌激素抗骨质疏松的作用机制具有重要意义。
在制剂处方研究前，建立大豆苷元原药的高效液相分析方法，对原料药的理化性质进行了考察。大豆苷元溶液在强光、温度、不同pH介质中72h内的稳定性研究表明药物含量无明显变化，稳定性较好；测定了大豆苷元在H_20、0.1mol／LHCl、pH6.8PBS和pH7.4PBS中的平衡溶解度以及不同pH下正辛醇／水系统中，大豆苷元的表观油水分配系数P_(app)；原料药的稳定性影响因素实验表明，在露置空气、高湿及40℃、60℃条件下，大豆苷元含量无明显变化，在光照条件下含量略有降低。采用Caco-2细胞体外吸收模型，考察了大豆苷元在不同浓度和不同pH条件下穿单分子层细胞膜的渗透速率和吸收百分数，并对其在Caco-2细胞转运实验中产生的代谢物进行了初步测定，大豆苷元跨单分子层细胞膜的转运不受介质pH和浓度的影响，与仅通过经细胞通道转运的标记化合物普萘诺尔的P_(app)值相近，预测大豆苷元体内吸收良好，通过β-葡萄糖醛酸酶水解，在转运介质和Caco-2单分子层细胞膜中均能检测到少量代谢物，证明存在葡萄糖醛酸化、硫酸酯化代谢物等。
采用乳化／戊二醛交联法制备载药壳聚糖微球，以司盘-80为乳化剂和液体石蜡为油相，大豆苷元经微粉化后以微晶状态混悬于壳聚糖水溶液中为水相，乳化后包裹入乳滴继而交联，形成的微球外形圆整，分散性好，载药量高。经单因素考察筛选出影响微球粒径、粒径分布、载药量及释放快慢的处方因素和工艺条件包括聚合物浓度、药物与聚合物投料比和乳化转速，进一步采用球面对称设计-效应面优化法对其进行优化，经专业统计Statistica进行数据处理。优化的微球中位径、跨距、载药量分别为70.40μm、0.526、30.48％，这些实际测定值与模型方程拟和值偏差较小说明设计和优化结果是成功可靠的。透析袋法释药缓慢，符合表观零级动力学过程。三批样品的重现性试验及稳定性加速试验结果表明，制备的大豆苷元-壳聚糖微球处方稳定、质量可控，达到制剂的设计目的与要求。
建立测定肌肉内注射微球后药物残留量的分析方法，小鼠肌肉内注射大豆苷元-壳聚糖微球后缓慢释药长达35天，突释效应不明显，回收肌肉内注射微球的扫描电镜和注射部位肌肉病理切片表明微球生物组织相容性较好，注射于体内21～35天内发生轻微降解。采用大豆苷元时间分辨荧光免疫分析试剂盒测定血浆内总大豆苷元的浓度，测定大鼠静脉注射大豆苷元溶液和肌肉内注射大豆苷元-壳聚糖微球后的体内血浆药时曲线，大豆苷元的体内过程符合二室模型，药物动力学参数k_(10)、t_(1／2(α))、t_(1／2(β))、V_c分别为1.17 h~(-1)、0.30h、18.94h、0.46 L／kg。大鼠肌肉内注射微球后血浆药物浓度持久保持35天，其绝对生物利用度为39.02％，较口服大豆苷元有显著性提高。体内外相关性研究表明，微球体内吸收百分数与透析袋释药法测定的体外累积释药百分数有较好的相关性(r=0.9975)，微球中药物主要以药物结晶溶解和扩散方式释放，在后期伴有体系降解溶蚀释药特征。
建立去卵巢小鼠骨质疏松模型，进行大豆苷元-壳聚糖长效缓释微球肌肉内注射给药。股骨病理切片HE染色表明，与灌胃大豆苷元混悬液相比，微球组特别是高剂量具有明显改善骨小梁和皮质骨组织结构疏松的作用，提示其在体内药效得到提高。
Daidzein is a natural isoflavone found in Leguminosae with structural similarities to natural and synthetic estrogens and anti-estrogens. Studies suggest that daidzein exhibits a variety of biological effects on human health in cardiovascular disease, cancer chemoprevention and especially in estrogen replacement therapy (ERT) to prevent and treat osteoporosis with good therapy and low side effect. In order to further learn isoflavones, the effect of daidzein on osteoporosis was studied in cells and animals. Moreover, daidzein loading microspheres were prepared to obtain long sustained release using chitosan as carrier and evaluated in vitro and in vivo.MC3T3-E1 osteoblastic-like cells were cultured for 10, 20 or 30 days in the presence of P-GP and L-AA. The effect of daidzein on the differentiation and mineralization in MC3T3-E1 cells was investigated. The MTT assay, the measurement of alkaline phosphatase (ALP) activity, the measurement of calcium (Ca) and phosphorus (P) concentrations, the alizarin red S and von Kossa staining were used to investigate the effect of different concentrations of daidzein from 10~(-9) M to 10~(-5)M on the cells. The results showed that daidzein prompted the proliferation, differentiation and mineralization of MC3T3-E1 cells. In the study, ovariectomized mice osteoporosis model was built to investigate the effect of daidzein solution through intraperitoneal administration (i.p.) on osteoporosis. Compared with 17β-estradiol, daidzein meliorated the appearance of shaggy coat, tardive action, slow response and hyponoia, due to ovariectomization and increased the uterus index, density of trabecular bone and thickness of cortical bone. Moreover, dadizein regulated the ratio of osteoprotegerin (OPG) to receptor activator of nuclear factor kB ligand (RANKL) mRNA expression level to modulate the balance between osteoblasts and osteoclasts in the bone remodeling process, which showed significance to learn the action of daizein on osteoporosis.In the pre-formulation study, high performance liquid chromatography (HPLC) was used to determine the content of daidzein. The stability of daidzein solution, solubility, partition coefficient in oil/water, and stability of daidzein powder were investigated by measuring the relative change of daidzein content. Using Caco-2 cells model, transport characteristics of daidzein across the monolayer in the different pH medium and the different concentration were studied. The apparent permeability coefficient (P_(app)) of daidzein, independent to the pH medium and concentration, was similar to that of transcellcular marker--propranolol, suggesting the good absorption of daidzein in vivo. By hydrolysis withβ-glucuronidase, low metabolites were detected in monolayer and transport medium, verifying the existence of glucuronides and sulfates.
The emulsification/glutaraldehyde crosslinking method was used to prepare daidzein loading chitosan micropsheres. Micronized daidzein was dispersed in chitosan solution by ultrasonication and dropped into liquid paraffin containing span-80 to form emulsion droplets under stirring. The following crosslink with glutaraldehyde produced the spherical appearance, good dispersing and high drug loading microspheres. After the single factor investigation for the effe, ct on the particle size, particle size distribution, drug loading and release rate, a spherical symmetric design-response surface methodology was applied to optimize the formulation and preparing condition of microspheres. The medium size, span and drug loading of the optimized microspheres were 70.40μm, 0.526, 30.48％, respectively. The release of daidzein from microspheres was long sustained with dialysis bag release method. The experimental values were close to the predicted values stimulated by Statistics softare Statistica version 6.0, with low percentage bias, suggesting that the optimized formulation was reliable and reasonable. The repeatability of three batches and stability experiments of daidzein-loaded chitosan microspheres suggested that the quality of preparation was stable and the purposed was obtained.
The assay method of determining the residue amount of daidzein after intramuscular injection of microspheres was built to study drug release in vivo. The results showed that the release of drug was sustained for 35 days and the burst effect was not clearly in the release profile. Micrography of the retrived microspheres in muscle using a scanning electron microscope and pathological section of muscle by HE staining showed good biocompatibility and weak degradation in the upper release period. Plasma concentrations of daidzein were determined using time-resolved fluoroimmunoassay. The intravenous injection of a daidzein solution in rats was best fitted to two-compartment model. The pharmacokinetic parameters k_(10)、t_(1/2(α))、t_(1/2(β)) and V_c were 1.17h~(-1)、0.30h、18.94h、0.46 L/kg, respectively. The mean plasma concentration-time profile of daidzein after intramuscular injection of the daidzein-loaded chitosan microspheres showed that the total daidzein concentration showed fluctuation and lasted for about 35 days in plasma. The absolute bioavailability (F) of the daidzein-loaded chitosan microspheres was 39.02％. Compared with the low absolute bioavailability of oral formulations, our result indicated that intramuscular injection of the daidzein-loaded chitosan microspheres significantly improved the bioavailability of daidzein. The relation between the absorption percent in vivo and the accumulative release percent with dialysis bag release method in vitro showed linear (the relative coefficient was 0.9975). The release of daidzein from the chitoan microspheres was the diffused manner, companied with degradable erosion in the end.
In the study, ovariectomized mice osteoporosis model was built to investigate the effect of daidzein loading chitosan microspheres through i.m. route on osteoporosis. The pathological section of femur by HE staining showed that compared with daidzein i.g. group, daidzein microspheres i.m. groups, especially high dose microspheres i.m. group, increased the uterus index, density of and thickness of cortical bone, suggesting the increase of bioavailability in vivo.
在培养基中添加β-甘油磷酸二钠、L-抗坏血酸和培养时间10、20、30天条件下，采用MTT比色法、碱性磷酸酶活性测定、Ca和P含量测定、茜素红染色和von Kossa染色，考察不同大豆苷元浓度(10~(-9)～10~(-5)M)对MC3T3-E1成骨样细胞增殖、分化和矿化的影响，结果表明大豆苷元能促进MC3T3-E1细胞的增殖和分化特别是促进矿化的发生和增加矿化的量；建造去卵巢小鼠骨质疏松模型，选择小鼠进行去卵巢手术后1、3和5周模型，与阳性对照药17β-雌二醇比较，考察腹腔注射大豆苷元溶液对小鼠子宫重量系数、股骨病理切片HE染色的骨组织结构和骨组织中细胞因子OPG、RANKL mRNA相对表达水平的影响，从而初步探讨大豆苷元抗骨质疏松的机理，大豆苷元能改善去卵巢导致的小鼠皮毛蓬松、行动迟缓、反应迟钝和精神不振，能增加去卵巢小鼠的子宫重量系数、骨小梁分布密度和骨皮质厚度，并呈现剂量和时间效应，此外大豆苷元能通过调节OPG／RANKL比例从而调节骨重建过程中破骨细胞和成骨细胞的平衡，说明大豆苷元具有抗骨质疏松的作用，这对于了解植物雌激素抗骨质疏松的作用机制具有重要意义。
在制剂处方研究前，建立大豆苷元原药的高效液相分析方法，对原料药的理化性质进行了考察。大豆苷元溶液在强光、温度、不同pH介质中72h内的稳定性研究表明药物含量无明显变化，稳定性较好；测定了大豆苷元在H_20、0.1mol／LHCl、pH6.8PBS和pH7.4PBS中的平衡溶解度以及不同pH下正辛醇／水系统中，大豆苷元的表观油水分配系数P_(app)；原料药的稳定性影响因素实验表明，在露置空气、高湿及40℃、60℃条件下，大豆苷元含量无明显变化，在光照条件下含量略有降低。采用Caco-2细胞体外吸收模型，考察了大豆苷元在不同浓度和不同pH条件下穿单分子层细胞膜的渗透速率和吸收百分数，并对其在Caco-2细胞转运实验中产生的代谢物进行了初步测定，大豆苷元跨单分子层细胞膜的转运不受介质pH和浓度的影响，与仅通过经细胞通道转运的标记化合物普萘诺尔的P_(app)值相近，预测大豆苷元体内吸收良好，通过β-葡萄糖醛酸酶水解，在转运介质和Caco-2单分子层细胞膜中均能检测到少量代谢物，证明存在葡萄糖醛酸化、硫酸酯化代谢物等。
采用乳化／戊二醛交联法制备载药壳聚糖微球，以司盘-80为乳化剂和液体石蜡为油相，大豆苷元经微粉化后以微晶状态混悬于壳聚糖水溶液中为水相，乳化后包裹入乳滴继而交联，形成的微球外形圆整，分散性好，载药量高。经单因素考察筛选出影响微球粒径、粒径分布、载药量及释放快慢的处方因素和工艺条件包括聚合物浓度、药物与聚合物投料比和乳化转速，进一步采用球面对称设计-效应面优化法对其进行优化，经专业统计Statistica进行数据处理。优化的微球中位径、跨距、载药量分别为70.40μm、0.526、30.48％，这些实际测定值与模型方程拟和值偏差较小说明设计和优化结果是成功可靠的。透析袋法释药缓慢，符合表观零级动力学过程。三批样品的重现性试验及稳定性加速试验结果表明，制备的大豆苷元-壳聚糖微球处方稳定、质量可控，达到制剂的设计目的与要求。
建立测定肌肉内注射微球后药物残留量的分析方法，小鼠肌肉内注射大豆苷元-壳聚糖微球后缓慢释药长达35天，突释效应不明显，回收肌肉内注射微球的扫描电镜和注射部位肌肉病理切片表明微球生物组织相容性较好，注射于体内21～35天内发生轻微降解。采用大豆苷元时间分辨荧光免疫分析试剂盒测定血浆内总大豆苷元的浓度，测定大鼠静脉注射大豆苷元溶液和肌肉内注射大豆苷元-壳聚糖微球后的体内血浆药时曲线，大豆苷元的体内过程符合二室模型，药物动力学参数k_(10)、t_(1／2(α))、t_(1／2(β))、V_c分别为1.17 h~(-1)、0.30h、18.94h、0.46 L／kg。大鼠肌肉内注射微球后血浆药物浓度持久保持35天，其绝对生物利用度为39.02％，较口服大豆苷元有显著性提高。体内外相关性研究表明，微球体内吸收百分数与透析袋释药法测定的体外累积释药百分数有较好的相关性(r=0.9975)，微球中药物主要以药物结晶溶解和扩散方式释放，在后期伴有体系降解溶蚀释药特征。
建立去卵巢小鼠骨质疏松模型，进行大豆苷元-壳聚糖长效缓释微球肌肉内注射给药。股骨病理切片HE染色表明，与灌胃大豆苷元混悬液相比，微球组特别是高剂量具有明显改善骨小梁和皮质骨组织结构疏松的作用，提示其在体内药效得到提高。
Daidzein is a natural isoflavone found in Leguminosae with structural similarities to natural and synthetic estrogens and anti-estrogens. Studies suggest that daidzein exhibits a variety of biological effects on human health in cardiovascular disease, cancer chemoprevention and especially in estrogen replacement therapy (ERT) to prevent and treat osteoporosis with good therapy and low side effect. In order to further learn isoflavones, the effect of daidzein on osteoporosis was studied in cells and animals. Moreover, daidzein loading microspheres were prepared to obtain long sustained release using chitosan as carrier and evaluated in vitro and in vivo.MC3T3-E1 osteoblastic-like cells were cultured for 10, 20 or 30 days in the presence of P-GP and L-AA. The effect of daidzein on the differentiation and mineralization in MC3T3-E1 cells was investigated. The MTT assay, the measurement of alkaline phosphatase (ALP) activity, the measurement of calcium (Ca) and phosphorus (P) concentrations, the alizarin red S and von Kossa staining were used to investigate the effect of different concentrations of daidzein from 10~(-9) M to 10~(-5)M on the cells. The results showed that daidzein prompted the proliferation, differentiation and mineralization of MC3T3-E1 cells. In the study, ovariectomized mice osteoporosis model was built to investigate the effect of daidzein solution through intraperitoneal administration (i.p.) on osteoporosis. Compared with 17β-estradiol, daidzein meliorated the appearance of shaggy coat, tardive action, slow response and hyponoia, due to ovariectomization and increased the uterus index, density of trabecular bone and thickness of cortical bone. Moreover, dadizein regulated the ratio of osteoprotegerin (OPG) to receptor activator of nuclear factor kB ligand (RANKL) mRNA expression level to modulate the balance between osteoblasts and osteoclasts in the bone remodeling process, which showed significance to learn the action of daizein on osteoporosis.In the pre-formulation study, high performance liquid chromatography (HPLC) was used to determine the content of daidzein. The stability of daidzein solution, solubility, partition coefficient in oil/water, and stability of daidzein powder were investigated by measuring the relative change of daidzein content. Using Caco-2 cells model, transport characteristics of daidzein across the monolayer in the different pH medium and the different concentration were studied. The apparent permeability coefficient (P_(app)) of daidzein, independent to the pH medium and concentration, was similar to that of transcellcular marker--propranolol, suggesting the good absorption of daidzein in vivo. By hydrolysis withβ-glucuronidase, low metabolites were detected in monolayer and transport medium, verifying the existence of glucuronides and sulfates.
The emulsification/glutaraldehyde crosslinking method was used to prepare daidzein loading chitosan micropsheres. Micronized daidzein was dispersed in chitosan solution by ultrasonication and dropped into liquid paraffin containing span-80 to form emulsion droplets under stirring. The following crosslink with glutaraldehyde produced the spherical appearance, good dispersing and high drug loading microspheres. After the single factor investigation for the effe, ct on the particle size, particle size distribution, drug loading and release rate, a spherical symmetric design-response surface methodology was applied to optimize the formulation and preparing condition of microspheres. The medium size, span and drug loading of the optimized microspheres were 70.40μm, 0.526, 30.48％, respectively. The release of daidzein from microspheres was long sustained with dialysis bag release method. The experimental values were close to the predicted values stimulated by Statistics softare Statistica version 6.0, with low percentage bias, suggesting that the optimized formulation was reliable and reasonable. The repeatability of three batches and stability experiments of daidzein-loaded chitosan microspheres suggested that the quality of preparation was stable and the purposed was obtained.
The assay method of determining the residue amount of daidzein after intramuscular injection of microspheres was built to study drug release in vivo. The results showed that the release of drug was sustained for 35 days and the burst effect was not clearly in the release profile. Micrography of the retrived microspheres in muscle using a scanning electron microscope and pathological section of muscle by HE staining showed good biocompatibility and weak degradation in the upper release period. Plasma concentrations of daidzein were determined using time-resolved fluoroimmunoassay. The intravenous injection of a daidzein solution in rats was best fitted to two-compartment model. The pharmacokinetic parameters k_(10)、t_(1/2(α))、t_(1/2(β)) and V_c were 1.17h~(-1)、0.30h、18.94h、0.46 L/kg, respectively. The mean plasma concentration-time profile of daidzein after intramuscular injection of the daidzein-loaded chitosan microspheres showed that the total daidzein concentration showed fluctuation and lasted for about 35 days in plasma. The absolute bioavailability (F) of the daidzein-loaded chitosan microspheres was 39.02％. Compared with the low absolute bioavailability of oral formulations, our result indicated that intramuscular injection of the daidzein-loaded chitosan microspheres significantly improved the bioavailability of daidzein. The relation between the absorption percent in vivo and the accumulative release percent with dialysis bag release method in vitro showed linear (the relative coefficient was 0.9975). The release of daidzein from the chitoan microspheres was the diffused manner, companied with degradable erosion in the end.
In the study, ovariectomized mice osteoporosis model was built to investigate the effect of daidzein loading chitosan microspheres through i.m. route on osteoporosis. The pathological section of femur by HE staining showed that compared with daidzein i.g. group, daidzein microspheres i.m. groups, especially high dose microspheres i.m. group, increased the uterus index, density of and thickness of cortical bone, suggesting the increase of bioavailability in vivo.
引文
[1] Gregory, R., Mundy, M.D., 2000. Pathogenesis of osteoporosis and challenges for drug delivery. Ad. Drug Deliv. Rev., 42, 165-173.
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