小分子化合物J2缓释剂型药代动力学及药效学的实验研究
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摘要
目的：研究和比较小分子化合物J2两种缓释剂型-海藻酸钠微球(Sodium alginate microspheres,SAM)及纳米混悬液(nano-suspension,NS)在体外释放情况和体内眼部药代动力学行为,观察J2-NS结膜下注射对大鼠角膜移植排斥反应的作用并验证其作用机理。
方法：①体外释药性能研究：光学显微镜观察J2-SAM的形态及大小；激光粒度扫描仪测量J2-NS的粒径；高效液相色谱法测定J2-SAM和J2-NS的载药量以及在体外的累积释放量。②体内药代动力学研究：实验兔结膜下注射J2-SAM和J2-NS,分别于注药后1d、3d、1w、2w、3w、4w后取角膜、房水、虹膜以及外周血,使用高效液相色谱仪检测组织内的药物浓度,并使用WinNonlin药代动力学软件计算主要的药代动力学参数。③药效学研究：成年雌性Wistar大鼠为供体,SD大鼠为受体,建立同种异体穿透性角膜移植模型。手术大鼠随机分成4组：A组为SD大鼠自体角膜移植结膜下空白溶剂0.05ml注射组,16只；B组为同种异体角膜移植结膜下空白溶剂0.05ml注射组16只；C组为同种异体角膜移植结膜下空白溶剂0.05ml注射和1%CsA点眼组每日5次,16只；D组同种异体角膜移植结膜下注J2-NS 0.05mL,16只。术后记录植片排斥指数(RI),比较各组大鼠角膜植片生存时间。角膜移植后7d、14d分别对各组大鼠术眼及同侧颌下淋巴结行病理学检查。④机理研究：以成年雌性Wistar大鼠作为供体,SD大鼠作为受体,建立同种异体穿透性角膜移植模型。A组为正常SD大鼠结膜下空白溶剂0.05ml注射组,6只。手术大鼠随机分成3组：B组为SD大鼠自体角膜移植结膜下空白溶剂0.05ml注射组,6只；C组为同种异体角膜移植结膜下空白溶剂0.05ml注射组6只；D组同种异体角膜移植结膜下注射1%J2-NS纳米混悬液0.05ml,6只。各组动物分别于术后3d、1w、2w、3w,使用流式细胞仪检测外周血中T细胞亚群分布情况并比较。
结果：①体外释药性能研究：J2-SAM和J2-NS平均粒径分别为165.61±37.943μm和170nm；载药量分别为0.12%和0.1%,两种缓释剂型在体外均有显著的缓释效果,释药量平稳,基本上没有突释,缓释时间持续30d以上；30d时累计释放分别39.19%和32.91%。②体内药代动力学研究：J2-SAM和J2-NS兔结膜下注射后,在各组织内缓慢均匀释放,在角膜药物浓度最高,在房水和虹膜内的药物浓度较低,全血中可以检测到药物浓度,在眼局部可以达到长期缓释效果。③药效学研究：A组在观察期内未发生排斥；B、C、D组生存时间分别为10.75±1.60d、16.00±3.79d和14.33±2.93d,C、D两组间比较无显著性差异,与A、B组比较均有显著性差异。角膜同种异体移植术后7d角膜基质主要由多形核白细胞浸润为主,14d时则以淋巴细胞浸润为主,D组术后角膜植片各组织层中细胞成分和新生血管均较同期B组少。角膜同种异体移植术后移植同侧的颌下淋巴结增生明显,但用药组弱于未用药组。免疫组化A组角膜未见CD4+、CD8+T细胞浸润,角膜移植术后角膜基质层可见核膜棕黄色物质沉积的T细胞,同侧颌下淋巴结内淋巴结副皮质区及滤泡间区阳性细胞增多,以B组最为明显。④机理研究：B组各时间点外周血中淋巴细胞总CD3+T淋巴细胞、CD4+T淋巴细胞、CD8+T淋巴细胞以及CD4+／CD8+T淋巴细胞无显著性差异；C组术后3d和1w总CD3+T淋巴细胞、CD4+T淋巴细胞、CD8+T淋巴细胞无显著性差异,1w和2w总CD3+T淋巴细胞、CD4+T淋巴细胞、CD8+T淋巴细胞数目增多,差异有显著性；D组中,1w和2w时CD4+T淋巴细胞和CD8+T淋巴细胞无显著增生。同一时间点横向比较：3d、1w、2w时D组总CD3+T淋巴细胞明显少于C组,有显著性差异,而在第3w时D组与C组无明显差别；CD4+T淋巴细胞在术后3d和1w时D组较C组比较数目少,但无显著性差异。CD4+／CD8+比值,在3d、1w、3w时D组与C药组比较均无显著性差异。
结论：①J2-SAM和J2-NS粒径均匀是良好的药物载体,体外有良好的缓释效应。②结膜下单次注射J2-SAM和J2-NS可以在眼部缓慢均匀的释放,长期维持稳定的药物浓度,两者药代动力学参数无显著性差异,但J2-NS较J2-SAM更方便注药和更易控制给药剂量。③J2-NS结膜下注射有抗大鼠角膜移植排斥作用,效果与CsA相似。J2 J2-NS结膜下注射可见减轻角膜移植术后角膜植片和同侧颌下淋巴结的细胞增生,抑制免疫应答。④大鼠自体角膜移植并不引起外周血T淋巴细胞增生；当异体角膜移植排斥反应发生时,外周血中T淋巴细胞明显增多。J2通过抑制T淋巴细胞增生,抑制T细胞介导的角膜移植排斥反应。
Objective:To investigate and evaluate the pharmacokinetic behavior of two sustained-release formulations-sodium alginate microspheres(SAM) and nano-suspension (NS) of micromolecular compound J2 in vitro and in vivo; To examine the effect of nano-suspension J2 on preventing rat allograft rejection via subconjunctival injection and its influence on rat T cell subsets as well as to investigate its pharmacodynamics and mechanism.
Method:①Release behavior in vitro:the shape and size of J2-SAM was observed with optical microscope; Laser Particle Size Analyzer was used to scan the particle size of J2-NS; the drug loading of J2-SAM and J2-NS and its cumulative release in vitro were measured by HPLC.②Pharmacokinetic study in vivo:J2-SAM and J2-NS were injected in rabbits'conjunctiva and drug concentration of the cornea, aqueous humor, iris, and peripheral blood was detected by HPLC on Id,3d, 1w,2w,3w,4w respectively after injection. The main pharmacokinetic parameters were calculated by pharmacokinetic software-WinNonlin.③Pharmacodynamic study:allogeneic penetrating keratoplasty model was established using the adult female Wistar rats as donors and SD rats as recipients. Rats were divided into 4 groups randomly(n=16). Group A:SD rats were injected 0.05ml placebo subconjunctivally after autograft. Group B:allograft rats were injected 0.05ml placebo subconjunctivally after surgery. Group C:allograft rats were injected 0.05ml placebo subconjunctivally and administered 1% CsA 5 times a day after surgery. Group D:allograft rats were injected 0.1% J2-NS 0.05ml subconjunctivally after surgery. The graft rejection index (RI) and the survival time of animals in each group were recorded. The cornea and ipsilateral submandibular lymph nodes were examined pathologically on 7d,14d respectively after surgery.④Mechanism study:allogeneic penetrating keratoplasty model was established using the adult female Wistar rats as donors and SD rats as recipients. Group A:normal SD rats were injected 0.05ml placebo subconjunctivally, n=6. Group B:autograft rats were injected 0.05ml placebo subconjunctivally after surgery, n=6. Group C:allograft rats were injected 0.05ml placebo subconjunctivally after surgery, n=6. Group D:allograft rats were injected J2-NS 0.05ml subconjunctivally after surgery, n=6. Peripheral blood T lymphocyte subsets of each group were tested and compared by Flow cytometry Technology on 3d, 1w,2w,3w after operation.
Results:①Release behavior in vitro:the mean diameters of J2-SAM and J2-NS were 165.61±37.943μm and 170nm respectively; drug loading was 0.12% and 0.1%.Two kinds of sustained release formulation showed significant release effect, steady release and no burst in vitro. Sustained release time all maintained more than 30d and cumulative release was 39.19% and 32.91% respectively in the 30th day.②Pharmacokinetic study in vivo: J2-SAM and J2-NS were injected in rabbit subconjunctiva and the drugs were released in ocular organizations slowly. The drug concentration was higher in cornea and lower in aqueous humor and iris, it also could be detected in blood. The sustained release formulation had long-term sustained-release effect in the eye.③Pharmacodynamic study: there was no rejection happened in group A in the observation period; the survival time of group B, C, D was 10.75±1.60d,16.00±3.79d and 14.33±2.93d respectively; Group C and D showed no significant difference, but all showed significant difference with group A and B. There were mainly infiltrated by polymorphonuclear leukocytes in corneal stroma of allograft groups at 7d after operation, lymphocytes at 14d. The cellular components and angiogenesis in all layers of cornea of group D were less compared with group B at the same period. The ipsilateral lymph nodes were hyperplasia after the allograft. However, the treatment groups were weaker than untreated group B. By immunohistochemistry dye, there was no CD4+, CD8+T cell infiltration in group A and there showed brown material depositing in nuclear membrane of T cells in stroma after transplantation. There were more positive cells in paracortex and interfollicular areas of ipsilateral submandibular lymph nodes, especially in group B.④Mechanism study:There were no significant difference of the total CD3+T cells, CD4+T cells, CD8+T cells and CD4+/CD8+T cells ratio of peripheral blood lymphocytes in group B during the observed time. There were no significant difference of the total CD3+T cells, CD4+T cells, CD8+T cells in group C at 3d and 1w after operation and significant difference at 2w and 3w whose cell numbers all increased. There were no significant difference of the total CD3+T cells, CD4+T cells, CD8+T cells in group B at 1w and 2w after operation in group D. The horizontal comparison of the same time:The total CD3+T lymphocytes of group D was significantly less than group C at 3d, 1w,2w after operation whereas there was no significant difference at 3w. There were smaller numbers of CD4+T lymphocytes in group D than group C at 3d and 1w but with no significant difference. The ratio of CD4+/CD8+ had no significant difference in group D compared with group C at 3d, 1w,3w.
Conclusion:①J2-SAM and J2-NS exhibit uniform size that can be good drug carriers and have wonderful sustained release potency in vitro.②J2-SAM and J2-NS can be released slowly in eyes and sustain long-term stable drug concentration by single subconjunctival injection. There is no significant difference of pharmacokinetic parameters between the two slow-release formulations. However, J2-NS is more convenient to inject and it is easier to control the injection dosage of J2-NS than J2-SAM.③Subconjunctival injection of J2-NS can inhibit rat corneal allograft rejection, which has similar pharmacodynamic action with CsA. It also can reduce cell proliferation and inhibit the immune response in cornea and ipsilateral submandibular lymph nodes.④Rat corneal autograft does not irritate proliferation of peripheral blood T lymphocytes whereas it increases significantly in corneal allograft rejection. J2 inhibits T lymphocyte proliferation and then inhibits the T cell-mediated corneal allograft rejection.
方法：①体外释药性能研究：光学显微镜观察J2-SAM的形态及大小；激光粒度扫描仪测量J2-NS的粒径；高效液相色谱法测定J2-SAM和J2-NS的载药量以及在体外的累积释放量。②体内药代动力学研究：实验兔结膜下注射J2-SAM和J2-NS,分别于注药后1d、3d、1w、2w、3w、4w后取角膜、房水、虹膜以及外周血,使用高效液相色谱仪检测组织内的药物浓度,并使用WinNonlin药代动力学软件计算主要的药代动力学参数。③药效学研究：成年雌性Wistar大鼠为供体,SD大鼠为受体,建立同种异体穿透性角膜移植模型。手术大鼠随机分成4组：A组为SD大鼠自体角膜移植结膜下空白溶剂0.05ml注射组,16只；B组为同种异体角膜移植结膜下空白溶剂0.05ml注射组16只；C组为同种异体角膜移植结膜下空白溶剂0.05ml注射和1%CsA点眼组每日5次,16只；D组同种异体角膜移植结膜下注J2-NS 0.05mL,16只。术后记录植片排斥指数(RI),比较各组大鼠角膜植片生存时间。角膜移植后7d、14d分别对各组大鼠术眼及同侧颌下淋巴结行病理学检查。④机理研究：以成年雌性Wistar大鼠作为供体,SD大鼠作为受体,建立同种异体穿透性角膜移植模型。A组为正常SD大鼠结膜下空白溶剂0.05ml注射组,6只。手术大鼠随机分成3组：B组为SD大鼠自体角膜移植结膜下空白溶剂0.05ml注射组,6只；C组为同种异体角膜移植结膜下空白溶剂0.05ml注射组6只；D组同种异体角膜移植结膜下注射1%J2-NS纳米混悬液0.05ml,6只。各组动物分别于术后3d、1w、2w、3w,使用流式细胞仪检测外周血中T细胞亚群分布情况并比较。
结果：①体外释药性能研究：J2-SAM和J2-NS平均粒径分别为165.61±37.943μm和170nm；载药量分别为0.12%和0.1%,两种缓释剂型在体外均有显著的缓释效果,释药量平稳,基本上没有突释,缓释时间持续30d以上；30d时累计释放分别39.19%和32.91%。②体内药代动力学研究：J2-SAM和J2-NS兔结膜下注射后,在各组织内缓慢均匀释放,在角膜药物浓度最高,在房水和虹膜内的药物浓度较低,全血中可以检测到药物浓度,在眼局部可以达到长期缓释效果。③药效学研究：A组在观察期内未发生排斥；B、C、D组生存时间分别为10.75±1.60d、16.00±3.79d和14.33±2.93d,C、D两组间比较无显著性差异,与A、B组比较均有显著性差异。角膜同种异体移植术后7d角膜基质主要由多形核白细胞浸润为主,14d时则以淋巴细胞浸润为主,D组术后角膜植片各组织层中细胞成分和新生血管均较同期B组少。角膜同种异体移植术后移植同侧的颌下淋巴结增生明显,但用药组弱于未用药组。免疫组化A组角膜未见CD4+、CD8+T细胞浸润,角膜移植术后角膜基质层可见核膜棕黄色物质沉积的T细胞,同侧颌下淋巴结内淋巴结副皮质区及滤泡间区阳性细胞增多,以B组最为明显。④机理研究：B组各时间点外周血中淋巴细胞总CD3+T淋巴细胞、CD4+T淋巴细胞、CD8+T淋巴细胞以及CD4+／CD8+T淋巴细胞无显著性差异；C组术后3d和1w总CD3+T淋巴细胞、CD4+T淋巴细胞、CD8+T淋巴细胞无显著性差异,1w和2w总CD3+T淋巴细胞、CD4+T淋巴细胞、CD8+T淋巴细胞数目增多,差异有显著性；D组中,1w和2w时CD4+T淋巴细胞和CD8+T淋巴细胞无显著增生。同一时间点横向比较：3d、1w、2w时D组总CD3+T淋巴细胞明显少于C组,有显著性差异,而在第3w时D组与C组无明显差别；CD4+T淋巴细胞在术后3d和1w时D组较C组比较数目少,但无显著性差异。CD4+／CD8+比值,在3d、1w、3w时D组与C药组比较均无显著性差异。
结论：①J2-SAM和J2-NS粒径均匀是良好的药物载体,体外有良好的缓释效应。②结膜下单次注射J2-SAM和J2-NS可以在眼部缓慢均匀的释放,长期维持稳定的药物浓度,两者药代动力学参数无显著性差异,但J2-NS较J2-SAM更方便注药和更易控制给药剂量。③J2-NS结膜下注射有抗大鼠角膜移植排斥作用,效果与CsA相似。J2 J2-NS结膜下注射可见减轻角膜移植术后角膜植片和同侧颌下淋巴结的细胞增生,抑制免疫应答。④大鼠自体角膜移植并不引起外周血T淋巴细胞增生；当异体角膜移植排斥反应发生时,外周血中T淋巴细胞明显增多。J2通过抑制T淋巴细胞增生,抑制T细胞介导的角膜移植排斥反应。
Objective:To investigate and evaluate the pharmacokinetic behavior of two sustained-release formulations-sodium alginate microspheres(SAM) and nano-suspension (NS) of micromolecular compound J2 in vitro and in vivo; To examine the effect of nano-suspension J2 on preventing rat allograft rejection via subconjunctival injection and its influence on rat T cell subsets as well as to investigate its pharmacodynamics and mechanism.
Method:①Release behavior in vitro:the shape and size of J2-SAM was observed with optical microscope; Laser Particle Size Analyzer was used to scan the particle size of J2-NS; the drug loading of J2-SAM and J2-NS and its cumulative release in vitro were measured by HPLC.②Pharmacokinetic study in vivo:J2-SAM and J2-NS were injected in rabbits'conjunctiva and drug concentration of the cornea, aqueous humor, iris, and peripheral blood was detected by HPLC on Id,3d, 1w,2w,3w,4w respectively after injection. The main pharmacokinetic parameters were calculated by pharmacokinetic software-WinNonlin.③Pharmacodynamic study:allogeneic penetrating keratoplasty model was established using the adult female Wistar rats as donors and SD rats as recipients. Rats were divided into 4 groups randomly(n=16). Group A:SD rats were injected 0.05ml placebo subconjunctivally after autograft. Group B:allograft rats were injected 0.05ml placebo subconjunctivally after surgery. Group C:allograft rats were injected 0.05ml placebo subconjunctivally and administered 1% CsA 5 times a day after surgery. Group D:allograft rats were injected 0.1% J2-NS 0.05ml subconjunctivally after surgery. The graft rejection index (RI) and the survival time of animals in each group were recorded. The cornea and ipsilateral submandibular lymph nodes were examined pathologically on 7d,14d respectively after surgery.④Mechanism study:allogeneic penetrating keratoplasty model was established using the adult female Wistar rats as donors and SD rats as recipients. Group A:normal SD rats were injected 0.05ml placebo subconjunctivally, n=6. Group B:autograft rats were injected 0.05ml placebo subconjunctivally after surgery, n=6. Group C:allograft rats were injected 0.05ml placebo subconjunctivally after surgery, n=6. Group D:allograft rats were injected J2-NS 0.05ml subconjunctivally after surgery, n=6. Peripheral blood T lymphocyte subsets of each group were tested and compared by Flow cytometry Technology on 3d, 1w,2w,3w after operation.
Results:①Release behavior in vitro:the mean diameters of J2-SAM and J2-NS were 165.61±37.943μm and 170nm respectively; drug loading was 0.12% and 0.1%.Two kinds of sustained release formulation showed significant release effect, steady release and no burst in vitro. Sustained release time all maintained more than 30d and cumulative release was 39.19% and 32.91% respectively in the 30th day.②Pharmacokinetic study in vivo: J2-SAM and J2-NS were injected in rabbit subconjunctiva and the drugs were released in ocular organizations slowly. The drug concentration was higher in cornea and lower in aqueous humor and iris, it also could be detected in blood. The sustained release formulation had long-term sustained-release effect in the eye.③Pharmacodynamic study: there was no rejection happened in group A in the observation period; the survival time of group B, C, D was 10.75±1.60d,16.00±3.79d and 14.33±2.93d respectively; Group C and D showed no significant difference, but all showed significant difference with group A and B. There were mainly infiltrated by polymorphonuclear leukocytes in corneal stroma of allograft groups at 7d after operation, lymphocytes at 14d. The cellular components and angiogenesis in all layers of cornea of group D were less compared with group B at the same period. The ipsilateral lymph nodes were hyperplasia after the allograft. However, the treatment groups were weaker than untreated group B. By immunohistochemistry dye, there was no CD4+, CD8+T cell infiltration in group A and there showed brown material depositing in nuclear membrane of T cells in stroma after transplantation. There were more positive cells in paracortex and interfollicular areas of ipsilateral submandibular lymph nodes, especially in group B.④Mechanism study:There were no significant difference of the total CD3+T cells, CD4+T cells, CD8+T cells and CD4+/CD8+T cells ratio of peripheral blood lymphocytes in group B during the observed time. There were no significant difference of the total CD3+T cells, CD4+T cells, CD8+T cells in group C at 3d and 1w after operation and significant difference at 2w and 3w whose cell numbers all increased. There were no significant difference of the total CD3+T cells, CD4+T cells, CD8+T cells in group B at 1w and 2w after operation in group D. The horizontal comparison of the same time:The total CD3+T lymphocytes of group D was significantly less than group C at 3d, 1w,2w after operation whereas there was no significant difference at 3w. There were smaller numbers of CD4+T lymphocytes in group D than group C at 3d and 1w but with no significant difference. The ratio of CD4+/CD8+ had no significant difference in group D compared with group C at 3d, 1w,3w.
Conclusion:①J2-SAM and J2-NS exhibit uniform size that can be good drug carriers and have wonderful sustained release potency in vitro.②J2-SAM and J2-NS can be released slowly in eyes and sustain long-term stable drug concentration by single subconjunctival injection. There is no significant difference of pharmacokinetic parameters between the two slow-release formulations. However, J2-NS is more convenient to inject and it is easier to control the injection dosage of J2-NS than J2-SAM.③Subconjunctival injection of J2-NS can inhibit rat corneal allograft rejection, which has similar pharmacodynamic action with CsA. It also can reduce cell proliferation and inhibit the immune response in cornea and ipsilateral submandibular lymph nodes.④Rat corneal autograft does not irritate proliferation of peripheral blood T lymphocytes whereas it increases significantly in corneal allograft rejection. J2 inhibits T lymphocyte proliferation and then inhibits the T cell-mediated corneal allograft rejection.
引文
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