A771726的临床前大鼠药代动力学研究
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摘要
A771726是免疫调节药来氟米特(leflunomide,LEF)在机体内的活性代谢物,后者在临床上主要用于治疗类风湿性关节炎、系统性红斑狼疮、抗移植排斥反应和银屑病。A771726是原形药物,将其制成口服制剂在体内直接吸收起作用,省略了前体药物转化的环节,目前正在申报一类新药。本实验旨在观察A771726在大鼠体内的吸收、血浆蛋白结合、分布、排泄特点,并与来氟米特的相关药动学参数进行比较,进行合理评价,提供新药临床前研究资料,以支持人体药代动力学研究并为临床用药提供参考依据。
1.生物样品中A771726分析方法的建立
本文采用沉淀蛋白处理血浆样品和液液萃取处理生物样品,建立A771726的HPLC分析方法。色谱分离条件为:色谱柱:Kromasil ODS C_(18)(150×4.6mm);流动相为0.01mol/L磷酸盐缓冲液(pH4.5)和甲醇(43:57);检测波长288nm;柱温:30℃。采用本文建立的生物样品预处理方法和反相高效液相色谱法分离测定生物样品中A771726的含量,生物样品预处理方法回收率高,色谱分离选择性好。本法的准确度、精密度、灵敏度、专属性和定量线性范围均达到了体内药物分析的要求。该方法成功运用于A771726的大鼠药代动力学研究。
2.A771726在大鼠体内的药代动力学研究
SD大鼠单次口服给药(3、6、12mg/kg)后不同时间点取血,用上述建立的HPLC法测定血浆中A771726的浓度,根据所得的血浆药物浓度—时间曲线,计算药动学参数,T_(1/2Ke)(h):16.71±2.34、14.69±1.94、16.45±2.49;AUC_((0-tn))(mg/L·h):271.38±78.87、785.57±170.92、994.13±165.43;C_(max)(mg/L):11.42±2.59、29.38±5.41、42.10±5.73;T_(max)(h):4.00±0.82、4.10±0.74、4.20±1.03;MRT_((0-tn))(h):22.07±1.80、21.91±1.60、20.71±1.88。单剂量口服A771726三个剂量组的药物吸收均成线性关系,其血药浓度—时间曲线符合一级吸收的一房室模型。
3.A771726的血浆蛋白结合率研究
血浆中加入A771726使最终浓度分别达到3,6,12μg/ml,每份加药血浆样品用平衡透析法在4℃条件下透析108h,透析结束后,用HPLC法分别测定血浆及缓冲液样品中的药物浓度,计算药物的血浆蛋白结合率,结果表明在上述范围内A771726与人的血浆蛋白结合率约97.7%,无浓度依赖性。
4.A771726在大鼠体内的组织分布研究
SD大鼠口服给药A771726(6mg/kg)后,分别于给药后2h、12h、24h股动脉放血处死,立即取出各脏器组织,滤纸吸干浮血,称重后用生理盐水制成匀浆,取匀浆液处理,用HPLC法测定各组织中药物浓度。结果显示,给药后A771726分布于主要组织,各脏器组织中的药物含量均低于血浆,肝肾组织中A771726浓度较高,大多数组织在12h达高峰,脑、骨髓、胸腺、睾丸中含量较低。
5.A771726在大鼠体内的排泄研究
SD大鼠口服给药A771726(6mg/kg)后收集胆汁、粪便、尿液样品,记录体积和重量,用HPLC法分析样品中A771726的浓度,将胆汁、粪便、尿液中的药物浓度乘以相应的胆汁排泄量、粪便量、尿量,并累加求和,计算药物的累积排泄量。结果显示大鼠单次灌胃给药后,粪便的排泄结果显示,0～48h排泄量占总给药量的1.61%;尿液中的排泄结果显示,0～48h排泄量占总给药量的0.87%;0～24h内从胆汁排泄的A771726量占总给药量的13.88%。
A771726 is the active metabolic product of leflunomide,the later is a type of immunoregulatory agents.Leflunomide educes the pharmacological action by metabolized into A771726,and it has been extensively exerted on rheumatoid arthritis, systemic lupus erythematosus,anti-rejection organ transplantation and psoriasis. A771726 is an active compound,which is directly absorbed in vivo,abbreviates the transformation of prodrug.The works of this paper were designed to evaluate the pharmacokinetics features of A771726 in rats,compare to the parameters of leflunomide,so as to support the pharmacokinetics study in human and offer the reference for clinical dosage and dosage form changing.
Part one:The establishment of analytical method of A771726 in biological matrix
A reversed-phase high-performance liquid chromatographic(RP-HPLC) method has been developed for the determination of A771726 in rat plasma,bile,urine,feces and tissue homogenates.The sample pretreatment included deproteinization for plasma samples and a liquid-liquid extraction for bile,urine,feces and tissue homogenates. Separation was obtained on a Kromasil C18 column(150×4.6mm),using an excitation wavelength of 288nm.The isocratic mobile phase consisted of methanol-0.01mol/L potassium dihydrogen phosphate buffer(pH 4.5)(57:43,v/v) was run at a flow rate of 1 ml/min for different biological matrix.The chromatographic system used provided good separation of the compound without interfering peaks from endogenous substance.The calibration curves were linear in each sample range with correlation coefficient above 0.999.The precision of intra-day and inter-day were evaluated by analysis of variance with the result of 0.7～2.3%and 0.7～6.4%,respectively.The method has been successfully used to support the pharmacokinetics study of A771726.
Part two:The pharmacokineties study of A771726 in rats
Following a single oral administration with different doses of A771726 to SD rats (3,6,12mg/kg),the blood samples were collected at different time after dosing.All collected blood samples were centrifuged to obtain plasma and the concentrations of A771726 in plasma were determined by HPLC method described as above,then calculated corresponding pharmacokinetic parameters based on the time process of blood drug concentration.The blood plasma Cot curve of A771726 conformed to one compartment model of the first order absorption.The main pharmacokinetic parameters of A771726(3,6,12mg/kg) were T_(1/2K2)(h):16.71±2.34,14.69±1.94,16.45±2.49, AUC_((0.tn))(mg/L·h):271.38±78.87,785.57±170.92,994.13±165.43,C_(max)(mg/L): 11.42±2.59,29.38±5.41,42.10±5.73,T_(max)(h):4.00±0.82,4.10±0.74,4.20±1.03, MRT_((0-tn))(h):22.07±1.80,21.91±1.60,20.71±1.88.The metabolism of A771726 following oral administration accords with the linear relation,and its blood plasma Cot curve consists with the first order kinetics of one compartment model.
Part three:In vitro binding of A771726 to human plasma proteins
Plasma protein binding was determined by equilibrium dialysis(108h) at 4℃with final concentrations of 3,6 and 12μg/ml.After the dialysis,the concentrations of A771726 were determined by HPLC method.The binding ratio was calculated as percent of total concentration.The results indicated that the mean extent of binding to plasma proteins in human appeared to be in dependent of concentration used.The binding percentage of the drug was about 97.7%.
Part four:The tissue distribution study of A771726 in rats
SD rats were killed by exsanguination at 2h,12h and 24h after oral administration (6mg/kg).Tissues were taken out and made into homogenates preparation with normal sodium and the concentrations of A771726 in the tissues were determined by HPLC method.It was shown that A771726 was distributed to the major organism after oral administration.The A771726 concentrations were.high in liver,kidney,and low in brain, bone marrow,thymus and testis/uterus.The drug seems not accumulate in rats.
Part five:Urinary,fecal and biliary excretion of A771726 after oral administration
Bile,urine and feces of rats were collected after oral administration(6mg/kg),and then the concentrations of A771726 were determined by HPLC method.Accumulative excretion amount of A771726 were 13.88%in bile,0.87%in urine and 1.61%in feces, respectively.
1.生物样品中A771726分析方法的建立
本文采用沉淀蛋白处理血浆样品和液液萃取处理生物样品,建立A771726的HPLC分析方法。色谱分离条件为:色谱柱:Kromasil ODS C_(18)(150×4.6mm);流动相为0.01mol/L磷酸盐缓冲液(pH4.5)和甲醇(43:57);检测波长288nm;柱温:30℃。采用本文建立的生物样品预处理方法和反相高效液相色谱法分离测定生物样品中A771726的含量,生物样品预处理方法回收率高,色谱分离选择性好。本法的准确度、精密度、灵敏度、专属性和定量线性范围均达到了体内药物分析的要求。该方法成功运用于A771726的大鼠药代动力学研究。
2.A771726在大鼠体内的药代动力学研究
SD大鼠单次口服给药(3、6、12mg/kg)后不同时间点取血,用上述建立的HPLC法测定血浆中A771726的浓度,根据所得的血浆药物浓度—时间曲线,计算药动学参数,T_(1/2Ke)(h):16.71±2.34、14.69±1.94、16.45±2.49;AUC_((0-tn))(mg/L·h):271.38±78.87、785.57±170.92、994.13±165.43;C_(max)(mg/L):11.42±2.59、29.38±5.41、42.10±5.73;T_(max)(h):4.00±0.82、4.10±0.74、4.20±1.03;MRT_((0-tn))(h):22.07±1.80、21.91±1.60、20.71±1.88。单剂量口服A771726三个剂量组的药物吸收均成线性关系,其血药浓度—时间曲线符合一级吸收的一房室模型。
3.A771726的血浆蛋白结合率研究
血浆中加入A771726使最终浓度分别达到3,6,12μg/ml,每份加药血浆样品用平衡透析法在4℃条件下透析108h,透析结束后,用HPLC法分别测定血浆及缓冲液样品中的药物浓度,计算药物的血浆蛋白结合率,结果表明在上述范围内A771726与人的血浆蛋白结合率约97.7%,无浓度依赖性。
4.A771726在大鼠体内的组织分布研究
SD大鼠口服给药A771726(6mg/kg)后,分别于给药后2h、12h、24h股动脉放血处死,立即取出各脏器组织,滤纸吸干浮血,称重后用生理盐水制成匀浆,取匀浆液处理,用HPLC法测定各组织中药物浓度。结果显示,给药后A771726分布于主要组织,各脏器组织中的药物含量均低于血浆,肝肾组织中A771726浓度较高,大多数组织在12h达高峰,脑、骨髓、胸腺、睾丸中含量较低。
5.A771726在大鼠体内的排泄研究
SD大鼠口服给药A771726(6mg/kg)后收集胆汁、粪便、尿液样品,记录体积和重量,用HPLC法分析样品中A771726的浓度,将胆汁、粪便、尿液中的药物浓度乘以相应的胆汁排泄量、粪便量、尿量,并累加求和,计算药物的累积排泄量。结果显示大鼠单次灌胃给药后,粪便的排泄结果显示,0～48h排泄量占总给药量的1.61%;尿液中的排泄结果显示,0～48h排泄量占总给药量的0.87%;0～24h内从胆汁排泄的A771726量占总给药量的13.88%。
A771726 is the active metabolic product of leflunomide,the later is a type of immunoregulatory agents.Leflunomide educes the pharmacological action by metabolized into A771726,and it has been extensively exerted on rheumatoid arthritis, systemic lupus erythematosus,anti-rejection organ transplantation and psoriasis. A771726 is an active compound,which is directly absorbed in vivo,abbreviates the transformation of prodrug.The works of this paper were designed to evaluate the pharmacokinetics features of A771726 in rats,compare to the parameters of leflunomide,so as to support the pharmacokinetics study in human and offer the reference for clinical dosage and dosage form changing.
Part one:The establishment of analytical method of A771726 in biological matrix
A reversed-phase high-performance liquid chromatographic(RP-HPLC) method has been developed for the determination of A771726 in rat plasma,bile,urine,feces and tissue homogenates.The sample pretreatment included deproteinization for plasma samples and a liquid-liquid extraction for bile,urine,feces and tissue homogenates. Separation was obtained on a Kromasil C18 column(150×4.6mm),using an excitation wavelength of 288nm.The isocratic mobile phase consisted of methanol-0.01mol/L potassium dihydrogen phosphate buffer(pH 4.5)(57:43,v/v) was run at a flow rate of 1 ml/min for different biological matrix.The chromatographic system used provided good separation of the compound without interfering peaks from endogenous substance.The calibration curves were linear in each sample range with correlation coefficient above 0.999.The precision of intra-day and inter-day were evaluated by analysis of variance with the result of 0.7～2.3%and 0.7～6.4%,respectively.The method has been successfully used to support the pharmacokinetics study of A771726.
Part two:The pharmacokineties study of A771726 in rats
Following a single oral administration with different doses of A771726 to SD rats (3,6,12mg/kg),the blood samples were collected at different time after dosing.All collected blood samples were centrifuged to obtain plasma and the concentrations of A771726 in plasma were determined by HPLC method described as above,then calculated corresponding pharmacokinetic parameters based on the time process of blood drug concentration.The blood plasma Cot curve of A771726 conformed to one compartment model of the first order absorption.The main pharmacokinetic parameters of A771726(3,6,12mg/kg) were T_(1/2K2)(h):16.71±2.34,14.69±1.94,16.45±2.49, AUC_((0.tn))(mg/L·h):271.38±78.87,785.57±170.92,994.13±165.43,C_(max)(mg/L): 11.42±2.59,29.38±5.41,42.10±5.73,T_(max)(h):4.00±0.82,4.10±0.74,4.20±1.03, MRT_((0-tn))(h):22.07±1.80,21.91±1.60,20.71±1.88.The metabolism of A771726 following oral administration accords with the linear relation,and its blood plasma Cot curve consists with the first order kinetics of one compartment model.
Part three:In vitro binding of A771726 to human plasma proteins
Plasma protein binding was determined by equilibrium dialysis(108h) at 4℃with final concentrations of 3,6 and 12μg/ml.After the dialysis,the concentrations of A771726 were determined by HPLC method.The binding ratio was calculated as percent of total concentration.The results indicated that the mean extent of binding to plasma proteins in human appeared to be in dependent of concentration used.The binding percentage of the drug was about 97.7%.
Part four:The tissue distribution study of A771726 in rats
SD rats were killed by exsanguination at 2h,12h and 24h after oral administration (6mg/kg).Tissues were taken out and made into homogenates preparation with normal sodium and the concentrations of A771726 in the tissues were determined by HPLC method.It was shown that A771726 was distributed to the major organism after oral administration.The A771726 concentrations were.high in liver,kidney,and low in brain, bone marrow,thymus and testis/uterus.The drug seems not accumulate in rats.
Part five:Urinary,fecal and biliary excretion of A771726 after oral administration
Bile,urine and feces of rats were collected after oral administration(6mg/kg),and then the concentrations of A771726 were determined by HPLC method.Accumulative excretion amount of A771726 were 13.88%in bile,0.87%in urine and 1.61%in feces, respectively.
引文
1.杨秀伟,杨晓达,蒲小平,等.创新药物研究中的吸收、分布、代谢、排泄/毒性平台建设[J].北京大学学报(医学版),2004,36(1):5
2.梁文权主编,生物药剂学与药代动力学[M].北京:人民卫生出版社,2003,155
3.史国兵,马艳,于洋.液相色谱技术在体内药物分析中的应用[J].中国药师,2003,6(2):105
4.吴小曼,蔡梅,陈玉英.毛细管电泳应用于体内药物分析的新技术[J].药学进展,1999,23(6):325
5.童珊珊,余江南,刘文英,等.色谱技术在体内药物分析中的进展[J].国外医学药学分册,2000,27(6):360
6.曾苏,程冀宇.药物分析学研究进展[J].浙江大学学报,2004,33(1):5
7.王洪允,江骥,胡蓓,等.固相萃取技术进展及在生物药物分析中的应用[J].药物分析杂志,2003,23(3):236
8.汪维鹏,丁黎.柱切换HPLC技术及其在体内药物分析中的应用[J].药学进展,2003,27(5):274
9.周婷婷,范国荣,吴玉田.超临界流体萃法在生物样品前处理中的应用[J].药学学报,2004,39(4):317
10.安登魁主编.现代药物分析选论[M].北京:中国医药科技出版社,2001,575
11.刘文英主编.药物分析[M].北京:人民卫生出版社,2003,16
12.Bertolini G,Aquino M,Biffi M,et al.Leflunomide,an immunorestoring drug for the therapy of autoimmune disorders,especially rheumatoid arthritis[J],Transplant Proc,1996,28:3074-3078
13.Pinto P,Douqados M.Leflunomide in clinical practice[J].Acta Reumatol Port.2006 Jul-Sep;31(3):215-24
14.Chong A,Finnegan A,Jiang XL,et al.Leflunomide,a novel immunosuppression agent:the mechanism of inhibition of T cell proliferation[J].Transplantation 1993,55:1361
15.Siemasko KF,Chong A,Wiliams JW,et al.Regulation of B cell function by the immunosuppressive agent leflunomide[J].Transplantation.1996,61:635
16.Chong A,Rezaik,Gebel H,et al.Effects of Leflunomide and other immunosuppressive agent on T cell proliferation in vitro[J].Transplantation.1996,61:140-145
17.Paul WE,Seder RA.Lymphocyte responses and cytokines[J],Cell,2004,76:241
18.Weiqmann B,Jarman ER,Sudowe S,et al.Induction of regulatory T cells by leflunomide in a murine model of contact allergen sensitivity[J].Invest Dermatol.2006Jul;126(7):1524-33
19.曾经泽主编.生物药物分析[M].北京:北京医科大学,中国协和医科大学联合出版社,1998,26
20.国家药品监督管理局药品审评中心.化学药品临床前药代动力学研究指导原则(2001)
21.乔建忠,袁淑兰,王伟欣,等.HPLC法测定生物样品中来氟米特活性代谢产物A771726[J].解放军药学学报,2004,20(1):49-51
22.LI Jun,Yao Hong-wei,Jin Yong,et al.Pharmacokinetics of leflunomide in Chinese healthy volunteers[J].Acta Pharmacol Sin,2002 Jun,23(6):551-555
23.朱全刚,胡晋红,孙华君,等.RP-HPLC法同时测定来氟米特及A.771726[J].药物分析杂志,2000,20(5):299-301
24.金涌,李俊,姚宏伟,等.反相高效液相色谱法测定血清中来氟米特活性代谢物A771726的浓度[J].中国医院药学杂志,2002,22(11):679-680
25.姚宏伟,李俊,金涌,等.单剂量口服来氟米特的人体药动学[J].中国药学杂志,2003,38(1):45-47
26.徐叔云主编.临床药理学(第三版)[M].北京:人民卫生出版社,2002,13-24
27.梁文权主编.生物药剂学与药物动力学(第二版)[M].北京:人民卫生出版社,2004,155-160
28.姚宏伟,金涌,李常玉,等来氟米特人体耐受性试验研究[J].安徽医药,2001,5(2):93
29.Pinto P,Douqados M.Leflunomide in clinical practice.Acta Reumatol Port.2006Jul-Sep,31(3):215-24
30.彭磊,张茜,金涌,等.来氟米特的活性代谢物A771726在小鼠体内的药代动力学研究[J].安徽医药,2006,10(10):729-730
31.Schmidt A,Schwind B,Gillich M,et al.Simultaneous determination of leflunomide and its active metabolite,A771726,in human plasma by high-performance liquid chromatograph[J].Biomed Chromatography,2003,17:276
32.曾经泽主编.生物药物分析[M].北京:北京医科大学,中国协和医科大学联合出版社,1998,236
33.徐叔云主编.临床药理学(第三版)[M].北京:人民卫生出版社,2002,723-726
34.姜红,任耘,罗粤.新型免疫调节剂——来氟米特[J].中国新药与临床杂志,2004,23(7):451-454
35.梁文权主编.生物药剂学与药物动力学(第二版)[M].北京:人民卫生出版社,2004,89-92
36.梁文权主编.生物药剂学与药物动力学(第二版)[M].北京:人民卫生出版社,2004,142-153
1.Schmidt A,Schwind B,Gillich M,et al.Simultaneous determination of leflunomide and its active metabolite,A771726,in human plasma by high-performance liquid chromatograph[J].Biomed Chromatography,2003,17:276
2.Smolen JS,Kalden JR,Scott DI,et al.Efficacy and safety of leflunomide compared with placebo and sulphasalazine in active rheumatoid arthritis:a double-blind,randomised,multicentre trial[J].Lancet,1999,353(9149):259
3.Grisar J,Arinqer M,Koller MD,et al.Leflunomide inhibits transendothelial migration of peripheral blood mononuclear cells[J].Ann Rheum Dis.2004 Dec,63(12):1632-7
4.Loffier M,Klein A,Hayek-Ouassini M,et al.Dihydroorotate dehydro genase mRNA and protein expression analysis in normal and drug-resistant cells[J].Nucleic Acids.2004 Oct,23(8-9):1281-5
5.Kim TH,Na HS,Loffier M,et al.Synthesis of beta-hydroxy-propenamide derivatives and the inhibition of human dihydroorotate dehydrogenase[J].Arch Pharm Res.2003Mar,26(3):197-201
6.Manna SK,A ggarwal BB.Immuno suppressive leflunomide metabolite(A771726)blocks TNF-dependent nuclear factor-κB activation and gene expression[J].J Immunol,1999,162(4):2095
7.Feng H,Li xy Zheng JR.Inhibition of the nuclear factor-kappaB signaling pathway by leflunomide or triptolide also inhibits the anthralin-induced inflammatory response but does not affect keratinocyte growth inhibition[J].Biol Pharm Bull.2005 Sep,28(9):1597-602
8.Herrmann ML,Schleybach R,Kirschbaum BJ.Leflunomide:an immunomodulatory drug for the treatment of rtheumatoid arthritis and other autoimmune disease[J].Immunopharmacology,2000,47(2-3):273-289
9.Paul WE,Seder RA.Lymphocyte responses and cytokines[J],Cell,2004,76:241
10.Weiqmann B,Jarman ER,Sudowe S,et al.Induction of regulatory T cells by leflunomide in a murine model of contact allergen sensitivity[J].J Invest Dermatol.2006 Jul,126(7):1524-33
11.Petya Dimitrova,Alla Skapenko,Matthias L,et al.Restriction of de novo pyrimidine biosynthesis inhibits Th1 cell activation and promotes Th2 Cell differentiation[J].J Immunol,2002,169(6):3392
12.Waldman WJ,Knight DA,Blinder L,et al.Inhibition of cytomegalovirus in vitro and in vivo by the experimental immunosuppressiveagent leflunomide[J].Intervirology,2000,42:412
13.姚宏伟,金涌,李常玉,等.来氟米特人体耐受性试验研究[J].安徽医药,2001,5(2):93
14.LI J,YAO HW,JIN Y,et al.Pharmacokinetics of leflunomide in Chinese healthy volunteers[J].Acta Pharmacol Sin,2002,23(6):551-555
15.Rozman B.Clinical pharmacokinetics of leflunomide[J].Clin Pharmacokinet,2002,41(6):421-430
16.劳志英,倪立青,张立澧,等.来氟米特治疗类风湿关节炎双盲试验[J].中国新药与临床杂志,2001,20(2):94-97
17.鲍春德,陈顺乐,劳志英,等.来氟米特治疗类风湿关节炎的Ⅱ期临床试验[J].中国新药与临床杂志,2002,21(6):325-328
18.Xiao F,Lv SY,Xu SY,et al.Leflunomide,a new disease modifying drugs for treating active rheumatoid arthritis in Phase Ⅱ methotrexate controlled clinical trial[J].Chinese Medicine,2002,impress
19.Remer CF,Weisman MH,Wallace DJ.Benefits of leflunomide in systemic lupus erythematosus:apilot observational study[J].Lupus,2001,10(7):480
20.Baltimore M.High dose arava in lupus(HAIL)[J].Arthritis Rheum,2001,44(9,Suppl):S280
21.张凤山,赵阴环,孙铀,等.LEF治疗31例狼疮肾炎的临床研究[J].中华风湿病学,2002,6(4):282
22.Wallace DJ.Management of lupus erythematosus:recent insights.Curr Opin Rheumatol,2002,14(3):212
23.王宏智,李永伟.甲氨蝶呤合并LEF治疗强直性脊柱炎疗效观察[J].浙江预防医学,2002,14(8):75
24.Williams JW,Mital D,Chong A,et al.Experiences with leflunomide in solid organ transplantation[J].Transplantation,2002,73(3):358
25.Pan F,Ebbs A,Wynn C,et al.FK778,a powerful new immunosuppressant,effectively reduces functional and histologic changes of chronic rejection in rat renal allografts[J].Transplantation,2003,75(8):1110
26.Hardinger KL,Wang CD,Schnitzler MA,et al.Prospective,pilot,openlable,short term study of conversion to leflunomide reverse chronic renal allografi dysfunction[J].Am J Transplant,2002,2:867
27. John GT, Manivannan J, Chandy S, et al. A prospective evaluation of lefiunomide therapy for cytomegalovirus disease in renal transplant recipients [J]. Transplant Proc. 2005 Dec, 37(10): 4303-5
28. Knight DA, Hejmanow ski AQ, Dierk sheide JE, et al. Inhibition of herpes simplex virus type I by the experimental immunosuppressive agent leflunomide[J]. Transplantation, 2001, 71(1):170
29. Seqoloni GP, Quaqlia M. New immunosuppressive drugs for prevention and treatment of rejection in renal transplant[J]. J Nephrol, 2006 Sep-Oct, 19(5): 578-86
30. Pan F, Ebbs A, Wynn C, et al. FK778, a powerful new immunosuppressant, effectively reduces functional and histologic changes of chronic rejection in rat renal allografts[J]. Transplantation, 2003, 75(8): 1110
31. Scarpa R, Manguso F, O riente A, et al. Lefiunomide in psoriatic polyarthritis: an Italian pilot study[J]. Arthritis Rheum, 2001,44 (9, Suppl): S92
32. Liang GC, Barr WG. Long term follow-up of the use of lefiunomide (LEF) in recalcitrant psoriatic arthritis (PA) and psoriasis (PS)[J]. Arthritis Rheum, 2001, 44 (9, Suppl): S121
33. Kaltwasser P, Nash P, Gladman D, et al. Efficacy and safety of lefiunomide in the treatment of psoriatic arthritis: results from the TOPAS study. Program and abstracts of the American College of Rheumatology 66th Annual Scientific Meeting, 2002. New Orleans, Louisiana, Abstract LB04
34. Reich K, Hummel KM, Beckmann I, et al. Treatment of severe psoriasis and psoriatic arthritis with lefiunomide [J]. Br J Dermatol, 2002, 146: 335
35. Fox RI, Sjogren. Syndrome: current therapies remain inadequate for a common disease[J]. Expert Opin Investig Drugs, 2002, 9: 2007
36. Van Woerkom J, Kruize AA, Geenen R. Safety and efficacy of Lefiunomide in primary Sjogren's syndrome - A phase II pilot study[J]. Ann Rheum Dis, 2007 Jan 12
37. Nousari HC, Anhait GJ. Bullous pemphigoid treated with lefiunomide: a novel immunomodulatory agent[J].Arch Dermatol,2000,136(10):1204-1205
38.Metzler C,Fink C,Lamprecht P,et al.Maintenance of remission with leflunomide in Wegeners granulomatosis[J].Rheumatology,2004,43(3):315-320
39.Petera P,Manger B,Manger K,et al.A pilot study of leflunomide in systemic lupus erythematosus[J].Arthritis Rheum,2000,43(suppl):241-245
40.Tai Gen Cui,Zhao Hui Ni,Hang Liu,et al.Efficacy and Safety of leflunomide in the treatment of proliferative lupus nephritis:preliminary results from an randomized,Cyclophosphamide-controlled study[J].American Society of Nephrology renal week 2003,Poster Board Numbe:SA-P0697
41.张凤山,赵阴环,孙铀,等.来氟米特治疗31例狼疮肾炎的临床研究[J].中华风湿病学杂志,2002,6(4):282-284
42.Zhao Hui Ni,Jia Qi Qian,Ai Wu Li,et al.A controlled,prospective study of efficacy of leflunomide in patients with nephrotic syndrome[J].American Society of Nephrology renal week 2003,Poster Board Number:SA-P01025
43.周厚永,郭明阳.来氟米特治疗类风湿关节炎的临床应用[J].西南军医,2005,13(5):24
44.Pinto P,Douqados M.Leflunomide in clinical practice[J].Acta Reumatol Port,2006Jul-Sep,31(3):215-24
45.Balakrishnan C,Dasgupta B.Pulmonary adverse events with leflunomide-myth or reality?[J].Rheumatology(Oxford),2007,Feb,46(2):372
46.陆家明.来氟米特和间质性肺炎[J].中国新药与临床杂志,2004,23(4):245
47.Richards BL,Spies J,Mc Gill N,et al.Effect of leflunomide on the peripheral nerves in rheumatoid arthritis[J].Intern Med J,2007,Feb,37(2):101-7
48.Gaffo A,Saaq KG,CurtisJR,et al.Treatment of rheumatoid arthritis[J].Am J Health Syst Pharm,2006,Dec 15,63(24):2451-65
49.张运芳,金涌,李俊,等.来氟米特对实验性肝纤维化的预防作用及部分机 制研究[J].中国药理学通报, 2001, 17: 203
50. Si HF, Li J, Lu XW, et al. Suppressive effects of leflunomide on leptin-induced collagen I production involved in hepatic stellate cell proliferation[J]. Exp Biol Med (Maywood), 2007, Mar, 232(3): 427-36
51. Knight DA. Hejinanewski AQ, Dierksheide JF, et al. Inhibition of herpes simplex virus type I by the experimental immunosuppressive agent leflunomide[J]. T ransplantation, 2001, 71: 170
52. Affolter VK, Moore PF. Cannine cutaneous and systemic histiocytosis: reactive histiocytosis of dendritic cells[J]. Am J Dermatopathol, 2000, 22: 40
2.梁文权主编,生物药剂学与药代动力学[M].北京:人民卫生出版社,2003,155
3.史国兵,马艳,于洋.液相色谱技术在体内药物分析中的应用[J].中国药师,2003,6(2):105
4.吴小曼,蔡梅,陈玉英.毛细管电泳应用于体内药物分析的新技术[J].药学进展,1999,23(6):325
5.童珊珊,余江南,刘文英,等.色谱技术在体内药物分析中的进展[J].国外医学药学分册,2000,27(6):360
6.曾苏,程冀宇.药物分析学研究进展[J].浙江大学学报,2004,33(1):5
7.王洪允,江骥,胡蓓,等.固相萃取技术进展及在生物药物分析中的应用[J].药物分析杂志,2003,23(3):236
8.汪维鹏,丁黎.柱切换HPLC技术及其在体内药物分析中的应用[J].药学进展,2003,27(5):274
9.周婷婷,范国荣,吴玉田.超临界流体萃法在生物样品前处理中的应用[J].药学学报,2004,39(4):317
10.安登魁主编.现代药物分析选论[M].北京:中国医药科技出版社,2001,575
11.刘文英主编.药物分析[M].北京:人民卫生出版社,2003,16
12.Bertolini G,Aquino M,Biffi M,et al.Leflunomide,an immunorestoring drug for the therapy of autoimmune disorders,especially rheumatoid arthritis[J],Transplant Proc,1996,28:3074-3078
13.Pinto P,Douqados M.Leflunomide in clinical practice[J].Acta Reumatol Port.2006 Jul-Sep;31(3):215-24
14.Chong A,Finnegan A,Jiang XL,et al.Leflunomide,a novel immunosuppression agent:the mechanism of inhibition of T cell proliferation[J].Transplantation 1993,55:1361
15.Siemasko KF,Chong A,Wiliams JW,et al.Regulation of B cell function by the immunosuppressive agent leflunomide[J].Transplantation.1996,61:635
16.Chong A,Rezaik,Gebel H,et al.Effects of Leflunomide and other immunosuppressive agent on T cell proliferation in vitro[J].Transplantation.1996,61:140-145
17.Paul WE,Seder RA.Lymphocyte responses and cytokines[J],Cell,2004,76:241
18.Weiqmann B,Jarman ER,Sudowe S,et al.Induction of regulatory T cells by leflunomide in a murine model of contact allergen sensitivity[J].Invest Dermatol.2006Jul;126(7):1524-33
19.曾经泽主编.生物药物分析[M].北京:北京医科大学,中国协和医科大学联合出版社,1998,26
20.国家药品监督管理局药品审评中心.化学药品临床前药代动力学研究指导原则(2001)
21.乔建忠,袁淑兰,王伟欣,等.HPLC法测定生物样品中来氟米特活性代谢产物A771726[J].解放军药学学报,2004,20(1):49-51
22.LI Jun,Yao Hong-wei,Jin Yong,et al.Pharmacokinetics of leflunomide in Chinese healthy volunteers[J].Acta Pharmacol Sin,2002 Jun,23(6):551-555
23.朱全刚,胡晋红,孙华君,等.RP-HPLC法同时测定来氟米特及A.771726[J].药物分析杂志,2000,20(5):299-301
24.金涌,李俊,姚宏伟,等.反相高效液相色谱法测定血清中来氟米特活性代谢物A771726的浓度[J].中国医院药学杂志,2002,22(11):679-680
25.姚宏伟,李俊,金涌,等.单剂量口服来氟米特的人体药动学[J].中国药学杂志,2003,38(1):45-47
26.徐叔云主编.临床药理学(第三版)[M].北京:人民卫生出版社,2002,13-24
27.梁文权主编.生物药剂学与药物动力学(第二版)[M].北京:人民卫生出版社,2004,155-160
28.姚宏伟,金涌,李常玉,等来氟米特人体耐受性试验研究[J].安徽医药,2001,5(2):93
29.Pinto P,Douqados M.Leflunomide in clinical practice.Acta Reumatol Port.2006Jul-Sep,31(3):215-24
30.彭磊,张茜,金涌,等.来氟米特的活性代谢物A771726在小鼠体内的药代动力学研究[J].安徽医药,2006,10(10):729-730
31.Schmidt A,Schwind B,Gillich M,et al.Simultaneous determination of leflunomide and its active metabolite,A771726,in human plasma by high-performance liquid chromatograph[J].Biomed Chromatography,2003,17:276
32.曾经泽主编.生物药物分析[M].北京:北京医科大学,中国协和医科大学联合出版社,1998,236
33.徐叔云主编.临床药理学(第三版)[M].北京:人民卫生出版社,2002,723-726
34.姜红,任耘,罗粤.新型免疫调节剂——来氟米特[J].中国新药与临床杂志,2004,23(7):451-454
35.梁文权主编.生物药剂学与药物动力学(第二版)[M].北京:人民卫生出版社,2004,89-92
36.梁文权主编.生物药剂学与药物动力学(第二版)[M].北京:人民卫生出版社,2004,142-153
1.Schmidt A,Schwind B,Gillich M,et al.Simultaneous determination of leflunomide and its active metabolite,A771726,in human plasma by high-performance liquid chromatograph[J].Biomed Chromatography,2003,17:276
2.Smolen JS,Kalden JR,Scott DI,et al.Efficacy and safety of leflunomide compared with placebo and sulphasalazine in active rheumatoid arthritis:a double-blind,randomised,multicentre trial[J].Lancet,1999,353(9149):259
3.Grisar J,Arinqer M,Koller MD,et al.Leflunomide inhibits transendothelial migration of peripheral blood mononuclear cells[J].Ann Rheum Dis.2004 Dec,63(12):1632-7
4.Loffier M,Klein A,Hayek-Ouassini M,et al.Dihydroorotate dehydro genase mRNA and protein expression analysis in normal and drug-resistant cells[J].Nucleic Acids.2004 Oct,23(8-9):1281-5
5.Kim TH,Na HS,Loffier M,et al.Synthesis of beta-hydroxy-propenamide derivatives and the inhibition of human dihydroorotate dehydrogenase[J].Arch Pharm Res.2003Mar,26(3):197-201
6.Manna SK,A ggarwal BB.Immuno suppressive leflunomide metabolite(A771726)blocks TNF-dependent nuclear factor-κB activation and gene expression[J].J Immunol,1999,162(4):2095
7.Feng H,Li xy Zheng JR.Inhibition of the nuclear factor-kappaB signaling pathway by leflunomide or triptolide also inhibits the anthralin-induced inflammatory response but does not affect keratinocyte growth inhibition[J].Biol Pharm Bull.2005 Sep,28(9):1597-602
8.Herrmann ML,Schleybach R,Kirschbaum BJ.Leflunomide:an immunomodulatory drug for the treatment of rtheumatoid arthritis and other autoimmune disease[J].Immunopharmacology,2000,47(2-3):273-289
9.Paul WE,Seder RA.Lymphocyte responses and cytokines[J],Cell,2004,76:241
10.Weiqmann B,Jarman ER,Sudowe S,et al.Induction of regulatory T cells by leflunomide in a murine model of contact allergen sensitivity[J].J Invest Dermatol.2006 Jul,126(7):1524-33
11.Petya Dimitrova,Alla Skapenko,Matthias L,et al.Restriction of de novo pyrimidine biosynthesis inhibits Th1 cell activation and promotes Th2 Cell differentiation[J].J Immunol,2002,169(6):3392
12.Waldman WJ,Knight DA,Blinder L,et al.Inhibition of cytomegalovirus in vitro and in vivo by the experimental immunosuppressiveagent leflunomide[J].Intervirology,2000,42:412
13.姚宏伟,金涌,李常玉,等.来氟米特人体耐受性试验研究[J].安徽医药,2001,5(2):93
14.LI J,YAO HW,JIN Y,et al.Pharmacokinetics of leflunomide in Chinese healthy volunteers[J].Acta Pharmacol Sin,2002,23(6):551-555
15.Rozman B.Clinical pharmacokinetics of leflunomide[J].Clin Pharmacokinet,2002,41(6):421-430
16.劳志英,倪立青,张立澧,等.来氟米特治疗类风湿关节炎双盲试验[J].中国新药与临床杂志,2001,20(2):94-97
17.鲍春德,陈顺乐,劳志英,等.来氟米特治疗类风湿关节炎的Ⅱ期临床试验[J].中国新药与临床杂志,2002,21(6):325-328
18.Xiao F,Lv SY,Xu SY,et al.Leflunomide,a new disease modifying drugs for treating active rheumatoid arthritis in Phase Ⅱ methotrexate controlled clinical trial[J].Chinese Medicine,2002,impress
19.Remer CF,Weisman MH,Wallace DJ.Benefits of leflunomide in systemic lupus erythematosus:apilot observational study[J].Lupus,2001,10(7):480
20.Baltimore M.High dose arava in lupus(HAIL)[J].Arthritis Rheum,2001,44(9,Suppl):S280
21.张凤山,赵阴环,孙铀,等.LEF治疗31例狼疮肾炎的临床研究[J].中华风湿病学,2002,6(4):282
22.Wallace DJ.Management of lupus erythematosus:recent insights.Curr Opin Rheumatol,2002,14(3):212
23.王宏智,李永伟.甲氨蝶呤合并LEF治疗强直性脊柱炎疗效观察[J].浙江预防医学,2002,14(8):75
24.Williams JW,Mital D,Chong A,et al.Experiences with leflunomide in solid organ transplantation[J].Transplantation,2002,73(3):358
25.Pan F,Ebbs A,Wynn C,et al.FK778,a powerful new immunosuppressant,effectively reduces functional and histologic changes of chronic rejection in rat renal allografts[J].Transplantation,2003,75(8):1110
26.Hardinger KL,Wang CD,Schnitzler MA,et al.Prospective,pilot,openlable,short term study of conversion to leflunomide reverse chronic renal allografi dysfunction[J].Am J Transplant,2002,2:867
27. John GT, Manivannan J, Chandy S, et al. A prospective evaluation of lefiunomide therapy for cytomegalovirus disease in renal transplant recipients [J]. Transplant Proc. 2005 Dec, 37(10): 4303-5
28. Knight DA, Hejmanow ski AQ, Dierk sheide JE, et al. Inhibition of herpes simplex virus type I by the experimental immunosuppressive agent leflunomide[J]. Transplantation, 2001, 71(1):170
29. Seqoloni GP, Quaqlia M. New immunosuppressive drugs for prevention and treatment of rejection in renal transplant[J]. J Nephrol, 2006 Sep-Oct, 19(5): 578-86
30. Pan F, Ebbs A, Wynn C, et al. FK778, a powerful new immunosuppressant, effectively reduces functional and histologic changes of chronic rejection in rat renal allografts[J]. Transplantation, 2003, 75(8): 1110
31. Scarpa R, Manguso F, O riente A, et al. Lefiunomide in psoriatic polyarthritis: an Italian pilot study[J]. Arthritis Rheum, 2001,44 (9, Suppl): S92
32. Liang GC, Barr WG. Long term follow-up of the use of lefiunomide (LEF) in recalcitrant psoriatic arthritis (PA) and psoriasis (PS)[J]. Arthritis Rheum, 2001, 44 (9, Suppl): S121
33. Kaltwasser P, Nash P, Gladman D, et al. Efficacy and safety of lefiunomide in the treatment of psoriatic arthritis: results from the TOPAS study. Program and abstracts of the American College of Rheumatology 66th Annual Scientific Meeting, 2002. New Orleans, Louisiana, Abstract LB04
34. Reich K, Hummel KM, Beckmann I, et al. Treatment of severe psoriasis and psoriatic arthritis with lefiunomide [J]. Br J Dermatol, 2002, 146: 335
35. Fox RI, Sjogren. Syndrome: current therapies remain inadequate for a common disease[J]. Expert Opin Investig Drugs, 2002, 9: 2007
36. Van Woerkom J, Kruize AA, Geenen R. Safety and efficacy of Lefiunomide in primary Sjogren's syndrome - A phase II pilot study[J]. Ann Rheum Dis, 2007 Jan 12
37. Nousari HC, Anhait GJ. Bullous pemphigoid treated with lefiunomide: a novel immunomodulatory agent[J].Arch Dermatol,2000,136(10):1204-1205
38.Metzler C,Fink C,Lamprecht P,et al.Maintenance of remission with leflunomide in Wegeners granulomatosis[J].Rheumatology,2004,43(3):315-320
39.Petera P,Manger B,Manger K,et al.A pilot study of leflunomide in systemic lupus erythematosus[J].Arthritis Rheum,2000,43(suppl):241-245
40.Tai Gen Cui,Zhao Hui Ni,Hang Liu,et al.Efficacy and Safety of leflunomide in the treatment of proliferative lupus nephritis:preliminary results from an randomized,Cyclophosphamide-controlled study[J].American Society of Nephrology renal week 2003,Poster Board Numbe:SA-P0697
41.张凤山,赵阴环,孙铀,等.来氟米特治疗31例狼疮肾炎的临床研究[J].中华风湿病学杂志,2002,6(4):282-284
42.Zhao Hui Ni,Jia Qi Qian,Ai Wu Li,et al.A controlled,prospective study of efficacy of leflunomide in patients with nephrotic syndrome[J].American Society of Nephrology renal week 2003,Poster Board Number:SA-P01025
43.周厚永,郭明阳.来氟米特治疗类风湿关节炎的临床应用[J].西南军医,2005,13(5):24
44.Pinto P,Douqados M.Leflunomide in clinical practice[J].Acta Reumatol Port,2006Jul-Sep,31(3):215-24
45.Balakrishnan C,Dasgupta B.Pulmonary adverse events with leflunomide-myth or reality?[J].Rheumatology(Oxford),2007,Feb,46(2):372
46.陆家明.来氟米特和间质性肺炎[J].中国新药与临床杂志,2004,23(4):245
47.Richards BL,Spies J,Mc Gill N,et al.Effect of leflunomide on the peripheral nerves in rheumatoid arthritis[J].Intern Med J,2007,Feb,37(2):101-7
48.Gaffo A,Saaq KG,CurtisJR,et al.Treatment of rheumatoid arthritis[J].Am J Health Syst Pharm,2006,Dec 15,63(24):2451-65
49.张运芳,金涌,李俊,等.来氟米特对实验性肝纤维化的预防作用及部分机 制研究[J].中国药理学通报, 2001, 17: 203
50. Si HF, Li J, Lu XW, et al. Suppressive effects of leflunomide on leptin-induced collagen I production involved in hepatic stellate cell proliferation[J]. Exp Biol Med (Maywood), 2007, Mar, 232(3): 427-36
51. Knight DA. Hejinanewski AQ, Dierksheide JF, et al. Inhibition of herpes simplex virus type I by the experimental immunosuppressive agent leflunomide[J]. T ransplantation, 2001, 71: 170
52. Affolter VK, Moore PF. Cannine cutaneous and systemic histiocytosis: reactive histiocytosis of dendritic cells[J]. Am J Dermatopathol, 2000, 22: 40