重组人干扰素α2b微球在大鼠体内的药动学研究
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摘要
干扰素α(Interferon- alpha, IFN-α)是一种多功能的细胞因子,除具有抗病毒活性外,还有抑制肿瘤细胞生长、调节机体免疫功能的作用,临床上已被广泛应用于病毒性疾病和肿瘤的治疗,是乙肝和丙肝抗病毒治疗的一线用药。但是,干扰素α半衰期短,需要长期频繁注射给药,导致血药浓度波动大、疗效受限而且副作用相对较多。因而,干扰素α长效注射剂的开发倍受学者们的关注。
本文采用本实验室专利方法(一种复乳(W/O/W)溶剂挥发法)制备了基因重组人IFNα-2b可注射缓释微球,考察了不同处方IFNα-2b微球的体外释药特性,研究了IFNα-2b微球在大鼠体内药动学特征,并通过主要药动学参数比较,对微球的制备处方进行了评价。
首先按照本实验室已有的微球制备工艺专利,制备了不同黏度和浓度的PLGA IFNα-2b微球,对所制备微球的体外释药特性进行了考察。结果显示,黏度0.89dL/g、浓度15%的PLGA制备的IFNα-2b微球突释效应较小、可缓释30天,具有较好的体外释药特性。
在大鼠体内药动学研究中,第一、对照组大鼠肌内注射三种剂量(0.5 MIU、1 MIU、2MIU)IFNα-2b市售粉针的药动学研究结果表明:IFNα-2b在大鼠体内药动学特性符合二室模型。第二、比较了0.89dL/g-15%、0.89dL/g-20%、1.13dL/g-15% PLGA的IFNα-2b粉针微球在大鼠体内缓释效果。结果表明:主要药动学参数——血药浓度达峰时间(T_(max))、峰浓度(C_(max))、平均滞留时间(MRT)和药时曲线下面积(AUC)均无显著性差异(P>0.05)。第三、比较了肌内注射0.5MIU IFNα-2b粉针和IFNα-2b粉针微球、原液微球(两种微球处方都为0.89dL/g-15%的PLGA)在大鼠体内的缓释效果。结果显示:微球组间两两有显著性差异(P<0.05),微球和粉针组有非常显著性差异(P<0.01)。第四、比较0.89dL/g-15% PLGA的IFNα-2b原液微球0.5MIU、1MIU、2MIU三种剂量在大鼠体内的药动学特性。结果显示:C_(max)与注射剂量呈线性相关(r=0.9997),其中单次肌内注射2MIU IFNα-2b微球制剂后T_(max)为12h、C_(max)为5329.88pg/ml、AUC为192084(pg/ml)*h、MRT为146.40h,血药浓度可维持21天。大鼠单次肌内注射0.5MIU IFNα-2b粉针和两种微球后, T_(max)分别为0.75、1.5、12.0h;C_(max)为5889.82、1275.34、1404.09pg/ml;MRT为2.19、38.49、122.33h;AUC为11294.70、22278.6、71096.14 (pg/ml)*h。
研究结果显示,采用本实验室专利方法制备的0.89dL/g-15%PLGA IFNα-2b原液微球在大鼠体内具有较好的缓释效果。本研究将为多肽、蛋白类药物微球制剂的研发提供有意义的参考和借鉴。
Interferon-alpha (IFN-α) is a multifunctional cytokine. In addition to anti-viral activity, it also has effects of inhibiting the growth of tumor cells and regulating the immune function, is widely used in clinical treatment of viral diseases and cancers, and is the first drug for anti-virus therapy of hepatitis B and C. But as the half-life of IFN-αis short, long-term frequent injection is needed, resulting in large fluctuation of plasma concentration, limited effects and relatively more side effects. Therefore, development of a kind of sustained release injection of IFN-αis urgently needed.
IFNα-2b injectabe microspheres were prepared by the double emulsion(W/O/W) solvent evaporation method with Poly(lactic acid-co-glycolic acid) (PLGA) as carriers in this study. First, in vitro release of IFNα-2b microspheres of different formulations were studied. Second, the pharmacokinetics of IFNα-2b microspheres following intramuscular administration in rats was studied to evaluate the formulations of IFNα-2b microspheres.
According to the IFN microspheres preparation process of our patent, IFNα-2b microspheres were obtained by using different viscosities and concentrations PLGA along with IFNα-2b, and their In vitro release were studied. IFNα-2b microspheres of 0.89dL/g -15% PLGA had good release profiles within 30 days.
In pharmacokinetics study on IFNα-2b microspheres after intramuscular administration in rats, following characteristics were revealed: The pharmacokinetics of three doses of IFNα-2b injection resembles two-compartment model. The pharmacokinetics of IFNα-2b injection (market-approved injectable IFNα-2b lyophilized powder) microspheres with different viscosities and concentrations of PLGA (0.89dL/g -15%, 0.89dL/g -20% and 1.13dL/g -15% PLGA) showed that there was no significant differences of peak time (T_(max)), peak concentration (C_(max)), area under curve (AUC), and mean residence time (MRT) (P>0.05). The pharmacokinetics of three doses (0.5MIU, 1MIU, 2MIU) of microspheres loading IFNα-2b stock solution were compared. Results showed the C_(max) were linear related with the injected doses(r=0.9997). Following single administration of 2MIU IFNα-2b microspheres, T_(max), C_(max), AUC, and MRT were 12h, 5329.88pg/ml, 192084(pg/ml)*h, and 146.40h, respectively. The microspheres had sustained–release characteristics with a long release term of 21 days in vivo. Following single administration of 0.5MIU IFNα-2b injection, injection microspheres and stock solution microspheres (both microspheres with 0.89dL/g -15% PLGA), the pharmacokinetic parameters were as follows: T_(max) were 0.75, 1.5, 8.0 and 12.0h; C_(max) were 5889.82, 1275.34 and 1404.09pg/ml; MRT were 2.19, 38.49 and 122.33h; AUC were 1294.70, 22278.6 and 71096.14 (pg/ml)*h, respectively.
In short, our IFNα-2b microspheres with 0.89dL/g -15% PLGA loading stock solution had good pharmacokinetics in rats. The study would be helpful references for other scholars.
本文采用本实验室专利方法(一种复乳(W/O/W)溶剂挥发法)制备了基因重组人IFNα-2b可注射缓释微球,考察了不同处方IFNα-2b微球的体外释药特性,研究了IFNα-2b微球在大鼠体内药动学特征,并通过主要药动学参数比较,对微球的制备处方进行了评价。
首先按照本实验室已有的微球制备工艺专利,制备了不同黏度和浓度的PLGA IFNα-2b微球,对所制备微球的体外释药特性进行了考察。结果显示,黏度0.89dL/g、浓度15%的PLGA制备的IFNα-2b微球突释效应较小、可缓释30天,具有较好的体外释药特性。
在大鼠体内药动学研究中,第一、对照组大鼠肌内注射三种剂量(0.5 MIU、1 MIU、2MIU)IFNα-2b市售粉针的药动学研究结果表明:IFNα-2b在大鼠体内药动学特性符合二室模型。第二、比较了0.89dL/g-15%、0.89dL/g-20%、1.13dL/g-15% PLGA的IFNα-2b粉针微球在大鼠体内缓释效果。结果表明:主要药动学参数——血药浓度达峰时间(T_(max))、峰浓度(C_(max))、平均滞留时间(MRT)和药时曲线下面积(AUC)均无显著性差异(P>0.05)。第三、比较了肌内注射0.5MIU IFNα-2b粉针和IFNα-2b粉针微球、原液微球(两种微球处方都为0.89dL/g-15%的PLGA)在大鼠体内的缓释效果。结果显示:微球组间两两有显著性差异(P<0.05),微球和粉针组有非常显著性差异(P<0.01)。第四、比较0.89dL/g-15% PLGA的IFNα-2b原液微球0.5MIU、1MIU、2MIU三种剂量在大鼠体内的药动学特性。结果显示:C_(max)与注射剂量呈线性相关(r=0.9997),其中单次肌内注射2MIU IFNα-2b微球制剂后T_(max)为12h、C_(max)为5329.88pg/ml、AUC为192084(pg/ml)*h、MRT为146.40h,血药浓度可维持21天。大鼠单次肌内注射0.5MIU IFNα-2b粉针和两种微球后, T_(max)分别为0.75、1.5、12.0h;C_(max)为5889.82、1275.34、1404.09pg/ml;MRT为2.19、38.49、122.33h;AUC为11294.70、22278.6、71096.14 (pg/ml)*h。
研究结果显示,采用本实验室专利方法制备的0.89dL/g-15%PLGA IFNα-2b原液微球在大鼠体内具有较好的缓释效果。本研究将为多肽、蛋白类药物微球制剂的研发提供有意义的参考和借鉴。
Interferon-alpha (IFN-α) is a multifunctional cytokine. In addition to anti-viral activity, it also has effects of inhibiting the growth of tumor cells and regulating the immune function, is widely used in clinical treatment of viral diseases and cancers, and is the first drug for anti-virus therapy of hepatitis B and C. But as the half-life of IFN-αis short, long-term frequent injection is needed, resulting in large fluctuation of plasma concentration, limited effects and relatively more side effects. Therefore, development of a kind of sustained release injection of IFN-αis urgently needed.
IFNα-2b injectabe microspheres were prepared by the double emulsion(W/O/W) solvent evaporation method with Poly(lactic acid-co-glycolic acid) (PLGA) as carriers in this study. First, in vitro release of IFNα-2b microspheres of different formulations were studied. Second, the pharmacokinetics of IFNα-2b microspheres following intramuscular administration in rats was studied to evaluate the formulations of IFNα-2b microspheres.
According to the IFN microspheres preparation process of our patent, IFNα-2b microspheres were obtained by using different viscosities and concentrations PLGA along with IFNα-2b, and their In vitro release were studied. IFNα-2b microspheres of 0.89dL/g -15% PLGA had good release profiles within 30 days.
In pharmacokinetics study on IFNα-2b microspheres after intramuscular administration in rats, following characteristics were revealed: The pharmacokinetics of three doses of IFNα-2b injection resembles two-compartment model. The pharmacokinetics of IFNα-2b injection (market-approved injectable IFNα-2b lyophilized powder) microspheres with different viscosities and concentrations of PLGA (0.89dL/g -15%, 0.89dL/g -20% and 1.13dL/g -15% PLGA) showed that there was no significant differences of peak time (T_(max)), peak concentration (C_(max)), area under curve (AUC), and mean residence time (MRT) (P>0.05). The pharmacokinetics of three doses (0.5MIU, 1MIU, 2MIU) of microspheres loading IFNα-2b stock solution were compared. Results showed the C_(max) were linear related with the injected doses(r=0.9997). Following single administration of 2MIU IFNα-2b microspheres, T_(max), C_(max), AUC, and MRT were 12h, 5329.88pg/ml, 192084(pg/ml)*h, and 146.40h, respectively. The microspheres had sustained–release characteristics with a long release term of 21 days in vivo. Following single administration of 0.5MIU IFNα-2b injection, injection microspheres and stock solution microspheres (both microspheres with 0.89dL/g -15% PLGA), the pharmacokinetic parameters were as follows: T_(max) were 0.75, 1.5, 8.0 and 12.0h; C_(max) were 5889.82, 1275.34 and 1404.09pg/ml; MRT were 2.19, 38.49 and 122.33h; AUC were 1294.70, 22278.6 and 71096.14 (pg/ml)*h, respectively.
In short, our IFNα-2b microspheres with 0.89dL/g -15% PLGA loading stock solution had good pharmacokinetics in rats. The study would be helpful references for other scholars.
引文
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2. Finter NB. The classification and biological functions of the interferons. A review. J Hepatol. 1986; 3 Suppl 2: S157-160
3. Alexenko AP, Ealy AD, Bixby JA, et al. A classification for the interferon-tau. J Interferon Cytokine Res. 2000; 20(9): 817-822
4. Fritz E, Ludwig H. Interferon-alpha treatment in multiple myeloma: meta-analysis of 30 randomised trials among 3948 patients. Ann Oncol. 2000; 11(11): 1427-1436
5. Vilcek J. Novel interferons. Nat Immunol. 2003; 4(1): 8-9
6. Kalvakolanu DV. Interferons and cell growth control. Histol Histopathol. 2000; 15(2): 523-537
7. Bailon P, Palleroni A, Schaffer CA, et al. Rational design of a potent, long-lasting form of interferon: a 40 kDa branched polyethylene glycol-conjugated interferon alpha-2a for the treatment of hepatitis C. Bioconjug Chem. 2001; 12(2): 195-202
8. Castellanos IJ, Crespo R, Griebenow K. Poly(ethylene glycol) as stabilizer and emulsifying agent: a novel stabilization approach preventing aggregation and inactivation of proteins upon encapsulation in bioerodible polyester microspheres. J Control Release. 2003; 88(1): 135-145
9. Cleland JL, Daugherty A, Mrsny R. Emerging protein delivery methods. Curr Opin Biotechnol. 2001; 12(2): 212-219
10. Burgess DJ, Hussain AS, Ingallinera TS, et al. Assuring quality and performance of sustained and controlled release parenterals: workshop report. AAPS PharmSci. 2002; 4(2): E7
11 Lee ES, Kwon MJ, Na K, et al. Protein release behavior from porous microparticle with lysozyme/hyaluronate ionic complex. Colloids Surf BBiointerfaces. 2007; 55(1): 125-130
12. Geze A, Venier-Julienne MC, Saulnier P, et al. Modulated release of IdUrd from poly (D,L-lactide-co-glycolide) microspheres by addition of poly (D,L-lactide) oligomers. J Control Release. 1999; 58(3): 311-322
13. Orienti I, Gianasi E, Aiedeh K, et al. Microparticles of BSA substituted with deoxycholic acid and triethylene glycol glutarate. Correlations between the physico-chemical properties of the matrix and the release kinetics. J Pharm Belg. 1996; 51(3): 125-130
14. Meager A. Biological assays for interferons. J Immunol Methods. 2002; 261(1-2): 21-36.
15. 吴 玉 厚 , 吴 冰 洁 , 周 国 利 , 等 . 干 扰 素 研 究 进 展 . 生 物 学 教学.2007,32(7):2-4
16. 杨帆,张永明,赵耀明,等.干扰素 α 微球的制备及其体外释药性能研究[J].中国医院药学杂志.2005,25(9):807
17. 宋凤兰,杨帆,张永明,等.基因重组人干扰素 α 微球的理化性质研究[J].中国药房,2007,18(10):752
18 徐安龙, 杨帆, 高建萍, 等. 一种干扰素聚乳酸-羟乙酸共聚物 PLGA 微球的制备方. [P]. 中国专利:200410077622.0
19. 国家药典委员会编. 中华人民共和国药典, 第三部. 化学工业出版社
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21. Paredes-Lopez O, Guevara-Lara F, Schevenin-Pinedo ML, et al. Comparison of procedures to determine protein content of developing bean seeds (Phaseolus vulgaris). Plant Foods Hum Nutr. 1989; 39(2): 137-148
22. Schooley RT, Spino C, Kuritzkes D, et al. Two double-blinded, randomized, comparative trials of 4 human immunodeficiency virus type 1 (HIV-1) envelope vaccines in HIV-1-infected individuals across a spectrum of disease severity: AIDS Clinical Trials Groups 209 and 214. J Infect Dis. 2000; 182(5): 1357-1364
23. King TW, Patrick CW. Development and in vitro characterization of vascularendothelial growth factor (VEGF)-loaded poly(DL-lactic-co-glycolic acid)/poly(ethylene glycol) microspheres using a solid encapsulation/single emulsion/solvent extraction technique. J Biomed Mater Res. 2000; 51(3): 383-390
24 Makino K, Arakawa M, Kondo T. Preparation and in vitro degradation properties of polylactide microcapsules. Chem Pharm Bull (Tokyo). 1985; 33(3): 1195-1201
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2. Finter NB. The classification and biological functions of the interferons. A review. J Hepatol. 1986; 3 Suppl 2: S157-160
3. Alexenko AP, Ealy AD, Bixby JA, et al. A classification for the interferon-tau. J Interferon Cytokine Res. 2000; 20(9): 817-822
4. Fritz E, Ludwig H. Interferon-alpha treatment in multiple myeloma: meta-analysis of 30 randomised trials among 3948 patients. Ann Oncol. 2000; 11(11): 1427-1436
5. Vilcek J. Novel interferons. Nat Immunol. 2003; 4(1): 8-9
6. Kalvakolanu DV. Interferons and cell growth control. Histol Histopathol. 2000; 15(2): 523-537
7. Bailon P, Palleroni A, Schaffer CA, et al. Rational design of a potent, long-lasting form of interferon: a 40 kDa branched polyethylene glycol-conjugated interferon alpha-2a for the treatment of hepatitis C. Bioconjug Chem. 2001; 12(2): 195-202
8. Castellanos IJ, Crespo R, Griebenow K. Poly(ethylene glycol) as stabilizer and emulsifying agent: a novel stabilization approach preventing aggregation and inactivation of proteins upon encapsulation in bioerodible polyester microspheres. J Control Release. 2003; 88(1): 135-145
9. Cleland JL, Daugherty A, Mrsny R. Emerging protein delivery methods. Curr Opin Biotechnol. 2001; 12(2): 212-219
10. Burgess DJ, Hussain AS, Ingallinera TS, et al. Assuring quality and performance of sustained and controlled release parenterals: workshop report. AAPS PharmSci. 2002; 4(2): E7
11 Lee ES, Kwon MJ, Na K, et al. Protein release behavior from porous microparticle with lysozyme/hyaluronate ionic complex. Colloids Surf BBiointerfaces. 2007; 55(1): 125-130
12. Geze A, Venier-Julienne MC, Saulnier P, et al. Modulated release of IdUrd from poly (D,L-lactide-co-glycolide) microspheres by addition of poly (D,L-lactide) oligomers. J Control Release. 1999; 58(3): 311-322
13. Orienti I, Gianasi E, Aiedeh K, et al. Microparticles of BSA substituted with deoxycholic acid and triethylene glycol glutarate. Correlations between the physico-chemical properties of the matrix and the release kinetics. J Pharm Belg. 1996; 51(3): 125-130
14. Meager A. Biological assays for interferons. J Immunol Methods. 2002; 261(1-2): 21-36.
15. 吴 玉 厚 , 吴 冰 洁 , 周 国 利 , 等 . 干 扰 素 研 究 进 展 . 生 物 学 教学.2007,32(7):2-4
16. 杨帆,张永明,赵耀明,等.干扰素 α 微球的制备及其体外释药性能研究[J].中国医院药学杂志.2005,25(9):807
17. 宋凤兰,杨帆,张永明,等.基因重组人干扰素 α 微球的理化性质研究[J].中国药房,2007,18(10):752
18 徐安龙, 杨帆, 高建萍, 等. 一种干扰素聚乳酸-羟乙酸共聚物 PLGA 微球的制备方. [P]. 中国专利:200410077622.0
19. 国家药典委员会编. 中华人民共和国药典, 第三部. 化学工业出版社
20. 杨帆. 重组人干扰素 α-2b 缓释微球给药系统的研究.[D]. 广州: 中山大学生命科学院. 2006: 56-79
21. Paredes-Lopez O, Guevara-Lara F, Schevenin-Pinedo ML, et al. Comparison of procedures to determine protein content of developing bean seeds (Phaseolus vulgaris). Plant Foods Hum Nutr. 1989; 39(2): 137-148
22. Schooley RT, Spino C, Kuritzkes D, et al. Two double-blinded, randomized, comparative trials of 4 human immunodeficiency virus type 1 (HIV-1) envelope vaccines in HIV-1-infected individuals across a spectrum of disease severity: AIDS Clinical Trials Groups 209 and 214. J Infect Dis. 2000; 182(5): 1357-1364
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