关节腔注射用甲氨蝶呤温敏凝胶的药效学及体内滞留性评价

Pharmacodynamics and in vivo retention of methotrexate thermosensitive hydrogel for intra-articular injection

目的:制备关节腔注射用甲氨蝶呤(methotrexate,MTX)温敏凝胶,并研究其在大鼠炎症模型中的治疗效果及体内滞留性。方法:以物理混合法制备甲氨蝶呤温敏凝胶,27只健康的SD大鼠随机分为3组,于皮下囊内注射含青霉素钠的羧甲基纤维素钠和脂多糖溶液建立类风湿关节炎模型,并分别注射生理盐水、MTX溶液及MTX温敏凝胶,并于给药后d 3,d 6,d 9采用酶联免疫吸附法检测各组大鼠渗出液中肿瘤坏死因子水平,同时取皮下囊皮肤制组织切片观察。将6只健康的小鼠随机分为2组后,分别经小鼠皮下囊注射混有近红外染料新吲哚菁绿的MTX溶液和MTX凝胶后,定时观察皮下囊处的荧光强度,考察载体的局部滞留性。结果:给药后d 3,MTX溶液组和MTX凝胶组的肿瘤坏死因子水平与生理盐水组相比分别下调了(27.31±4.37)%和(24.28±3.05)%;给药后d 6,2组分别下调了(42.70±3.92)%和(52.45±3.22)%;给药后d 9 2组分别下调了(52.28±4.00)%和(68.96±2.85)%。组织切片显示,MTX溶液组和MTX凝胶组相对生理盐水组炎症均有改善,而MTX凝胶组在给药后9 d时炎性细胞仍保持在较低水平,与溶液组具有显著性差异。在滞留性考察中,MTX溶液组在注射后1 d已基本消除,而MTX凝胶组在9 d时的滞留量为17.11%。结论:MTX温敏凝胶对该关节炎模型具有良好的治疗作用,且可增加药物在体内的滞留时间,长期治疗效果优于MTX溶液。

Objective： To prepare methotrexate thermosensitive hydrogel for intra-articular injection and study its treatment effect and in vivo retention in inflammation model of rats. Methods： Methotrexate thermosensitive hydrogel was prepared by physical mixing method. Twenty-seven rats were randomly divided into three groups. The model of rheumatoid arthritis was established by subcutaneous injecting carboxymethylcellulose sodium solution containing penicillin and lipopoly saccharide solution. At the same time stroke-physiological saline solution,methotrexate solution,and methotrexate thermosensitive hydrogel were injected,respectively. The level of tumor necrosis factor-α（TNF-α） in the exudate of the rats was determined by ELISA on the 3rd,6th and 9th day after the injection. And then the paraffin section of subcutaneous tissue was prepared. Six mice were divided randomly into two groups and injected with methotrexate solution and methotrexate thermosensitive hydrogel mixed with near infrared absorbing dyes IR-820 through the air-pouch of the rats. The fluorescence intensity in the air-pouch wasobserved to inspect the topical retention of the vector. Results： On the 3rd,6th,and 9th day after injecting the drugs,the levels of TNF-α in the groups of methotrexate solution and methotrexate thermosensitive hydrogel were lowered by（27. 31 ± 4. 37） % and（24. 28 ± 3. 05） %,（42. 70 ± 3. 92） % and（52. 45 ± 3. 22） %,（52. 28 ±4. 00） % and（68. 96 ± 2. 85） %,respectively,compared to the group of stroke-physiological saline solution.Observation of the paraffin sections showed that the inflammation in the groups of methotrexate solution and methotrexate thermsoensitive hydrogel both ameliorated compared with the group of stroke-physiological saline solution. And the inflammatory cells of the group of methotrexate thermosensitive hydrogel still retained at significantly lower level on the 9th day after injecting the drug compared with the group of stroke-physiological saline solution. In the retention observation,methotrexate solution was almost eliminated on the next day after the injection,while methotrexate thermosensitive hydrogel still remained at 17. 11% on the 9th day after the injection.Conclusion： Methotrexate thermosensitive hydrogel has satisfying therapeutic effect for rheumatoid arthritis model and can extend the drug residence time in vivo,which suggesting that its long-run treatment outcome would be better than methotrexate solution.