尖吻蝮蛇无出血活性纤溶酶药物代谢动力学及毒理学的研究
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摘要
目的：研究从蝮亚科尖吻蝮蛇(Agkistrodon acutus)分离纯化出的无出血活性纤溶酶(Non-hemorrhagic fibrinolytic enzyme，NHFLE)的一般药理学、药物代谢动力学及毒理学，了解该药在体内的变化规律，评价其安全性，为进一步临床研究提供实验依据。
方法：
1．小鼠尾静脉注射2.0、1.0、0.5mg／kg高、中、低三组剂量的NHFLE后，观察一般行为、协调活动、对阈下戊巴比妥钠的催眠作用。尾静脉注射1.4、0.7、0.35mg／kg高、中、低三组剂量的NHFLE，观察大鼠的心率、心电、平均动脉压、呼吸频率和深度以及大鼠的凝血时间等指标。对照组给予生理盐水。
2．药物代谢动力学采用氯胺T法对NHFLE进行~(125)I标记，经Sephadex G-50柱分离纯化，测定其放化纯度。通过静脉注射~(125)I-NHFLE，应用放射性核素示踪技术研究~(125)I-NHFLE在大鼠体内的血药浓度、组织器官的分布以及在尿、粪、胆汁中的排泄。
3．用昆明种小鼠尾静脉注射给药观察NHFLE的急性毒性。
4．Wistar大鼠，连续尾静脉注射9.0、4.5、2.25mg／kg高、中、低三种不同剂量的NHFLE 4周，对照组给等容积的生理盐水，观察NHFLE的长期毒性。
结果：
1．静脉注射NHFLE，对小鼠的一般行为、协调活动和阈下戊巴比妥钠的催眠作用无影响；各组剂量NHFLE对大鼠心率、心电电压以及呼吸的频率和深度也无影响。在大鼠凝血试验中，1.4mg／kg的高剂量组在10-30min内显著延长大鼠的凝血时间(P＜0.05)，在60min后恢复正常，其余剂量组对大鼠凝血时间没有影响。
2．~(125)I-NHFLE的标记率比为7.318MBq／mg NHFLE。放射性浓度25.8GSq／L。大鼠静脉注射NHFLE1.4、0.7、0.35mg／kg三个剂量后，主要药代动力学参数：t_(1／2α)为0.626～0.672h，t_(1／2β)为10.384～13.818h，t_(1／2α)及t_(1／2β)在三组剂量间无显著性差异(P＞0.05)。AUC分别为7073.03±124.14、3920.00±549.64、3315.78±125.30μg·h·L~(-1)，与剂量成正比(P＜0.05)。血药浓度-时间
曲线经拟合符合二房室开放模型。给药24h，在尿、粪、胆汁中的累积排泄率
分别占注射剂量的75 .31%、3.29%和6.54%。给药48h，在尿、粪中的累积排
泄率分别达到86 22%及4.98%。给药lh内，各脏器中放射活性达到峰值，
‘251一NH于LE在大鼠体内的分布以肾脏中放射活性为最高，其次为甲状腺、肝、
脾、胃，在脑、脂肪、肌肉中的含量很低。
3.小鼠急性毒性:NHFLE尾静脉给药小鼠的LDS。为28.84mg瓜g。
4.大鼠长期毒性:大鼠连续静脉给药4周，高剂量组大鼠出现多动、烦
躁不安、轻微腹泻以及体重增长缓慢(P<0 .05)，以上改变在停药恢复期得到
改善，其余动物体重增长、外观及一般行为未见异常。血液学及血生化检查、
脏器系数、病理检查均未见异常。
结论:一般药理学研究表明NHFLE对动物的神经系统、心血管系统、呼
吸系统均无影响。药代动力学实验发现两个时相的半衰期在各剂量组之间无
差异，AUC与剂量成正比，排泄以肾脏为主。毒性试验证实该药毒性低，临
床应用安全性好。
Objective: To understand the change of metabolism in rats and evaluate safety of Non-hemorrhagic Fibrinolytic Enzyme (NHFLE) from Agkistrodon Acutus by general pharmacology, pharmacokinetics and toxicology research, and provide foundation for clinical trials and application.
Methods: 1. NHFLE was injected intravenously at the tail into mice with the doses of 2.0, 1.0 and 0.5mg/kg respectively. The same administration method was used to rats but with the different doses of 1.4, 0.7 and 0.5mg/kg. The saline was gave as the control group. We observed the changes in general behavior, coordinate activity and synergism with sodium pentobarbital at subthreshold dose of mice. To rats, we observed the heart rate, cardiac electric voltage, mean arterial pressure, respiratory rate and depth and clotting time. 2. During pharmacokinetics research, NHFLE was labeled with 125I by the method of Chloramine-T. After purification
using Sephadex G-50, 125I-NHFLE was separated and then the radiochemical
purity of I-NHFLE was assayed by y -counter. After intravenous administration of l25I-NHFLE to rats, the plasma samples of 125I-NHFLE, the distribution in tissues or organs and the excretion in urine, feces and bile were studied with via radioactive tracer technique respectively. 3. The acute toxicity of NHFLE was studied on Kunming species mice. 4. Experimental animals of long-term toxicity were healthy Wistar rats. Route of medication was intravenous injection with 9.0, 4.5 and 2.25mg/kg by three different dosages of high dose group, middle dose group and low dose group, while the saline was gave as the control group.
Results: 1.A11 the animal groups of mice showed that NHFLE had no influence on their general behavior, coordinate activity and synergism with sodium pentobarbital at subthreshold dose after intravenous injection. The nervous, cardiovascular and respiratory systems in rats after administrated at different doses of NHFLE were normal. The blood clotting time of the rats at high dose of NHFLE was delayed obviously after injection 10-30 min (P<0.05 ) but changed normal at 60 min, meanwhile there was no significant difference of blood clotting time in
other groups. 2. Radiospecific activity of 125I-NHFLE was 25.8GBq/L. The labelled ratio was 7.318MBq per milligram NHFLE. After intravenous injection at doses of 1.4 0.7 0.35mg/kg to rats, the main pharmacokinetic parameters of I-NHFLE were as follows: T1/2a was between 0.626~0.672h and t1/2B was between 10.384~13.818h. AUC were 7073.03 + 124.14, 3920.00 + 549.64 and 3315.78+125.30 ug hL-1. There were no significant differences of t1/2a and t1/2B among the three dose groups (P>0.05) , but the AUC increased markedly with the dose increase (P<0.05) .The concentration-time curves of 125I-NHFLE were conformed to two-compartment model. The amount of radioactivity excreted in urine, feces and bile within 24h after intravenous administration of I-NHFLE to rats was 75.31%, 3.29% and 6.45% of the dose respectively. Within 48h after administration drug 86.22% of 125I-NHFLE was excreted in urine and 4.98% in feces. The peak concentration of 125I-NHFLE in tissues or organs appeared at Ih. Following intravenous injectio
n to rats, the radioactivity was selectively distributed to kidney, thyroid, liver, spleen and stomach, while the levels in brain, adipose and muscle were low. 3. Acute toxicity experiment showed that LD50 of NHFLE was 28.84mg/kg in mice. 4. Long-term toxicity experiment showed that during the 4 weeks of intravenous administration, the rats of high dose group had more activity, dysphoria, slight diarrhea and less weight increase than the control group ( P<0.05) and were recovered in 2 weeks withdrawal. Rats' weight increase, appearance and behavior were normal in other groups. There was no significant abnormality in hematology and laboratory test. The organ coefficients and pathologic examination were normal in all groups.
Conclusions: The drug has no effects on nervous, cardiovascular and respiratory systems. The half-lives of two phases were no statistically differenc
方法：
1．小鼠尾静脉注射2.0、1.0、0.5mg／kg高、中、低三组剂量的NHFLE后，观察一般行为、协调活动、对阈下戊巴比妥钠的催眠作用。尾静脉注射1.4、0.7、0.35mg／kg高、中、低三组剂量的NHFLE，观察大鼠的心率、心电、平均动脉压、呼吸频率和深度以及大鼠的凝血时间等指标。对照组给予生理盐水。
2．药物代谢动力学采用氯胺T法对NHFLE进行~(125)I标记，经Sephadex G-50柱分离纯化，测定其放化纯度。通过静脉注射~(125)I-NHFLE，应用放射性核素示踪技术研究~(125)I-NHFLE在大鼠体内的血药浓度、组织器官的分布以及在尿、粪、胆汁中的排泄。
3．用昆明种小鼠尾静脉注射给药观察NHFLE的急性毒性。
4．Wistar大鼠，连续尾静脉注射9.0、4.5、2.25mg／kg高、中、低三种不同剂量的NHFLE 4周，对照组给等容积的生理盐水，观察NHFLE的长期毒性。
结果：
1．静脉注射NHFLE，对小鼠的一般行为、协调活动和阈下戊巴比妥钠的催眠作用无影响；各组剂量NHFLE对大鼠心率、心电电压以及呼吸的频率和深度也无影响。在大鼠凝血试验中，1.4mg／kg的高剂量组在10-30min内显著延长大鼠的凝血时间(P＜0.05)，在60min后恢复正常，其余剂量组对大鼠凝血时间没有影响。
2．~(125)I-NHFLE的标记率比为7.318MBq／mg NHFLE。放射性浓度25.8GSq／L。大鼠静脉注射NHFLE1.4、0.7、0.35mg／kg三个剂量后，主要药代动力学参数：t_(1／2α)为0.626～0.672h，t_(1／2β)为10.384～13.818h，t_(1／2α)及t_(1／2β)在三组剂量间无显著性差异(P＞0.05)。AUC分别为7073.03±124.14、3920.00±549.64、3315.78±125.30μg·h·L~(-1)，与剂量成正比(P＜0.05)。血药浓度-时间
曲线经拟合符合二房室开放模型。给药24h，在尿、粪、胆汁中的累积排泄率
分别占注射剂量的75 .31%、3.29%和6.54%。给药48h，在尿、粪中的累积排
泄率分别达到86 22%及4.98%。给药lh内，各脏器中放射活性达到峰值，
‘251一NH于LE在大鼠体内的分布以肾脏中放射活性为最高，其次为甲状腺、肝、
脾、胃，在脑、脂肪、肌肉中的含量很低。
3.小鼠急性毒性:NHFLE尾静脉给药小鼠的LDS。为28.84mg瓜g。
4.大鼠长期毒性:大鼠连续静脉给药4周，高剂量组大鼠出现多动、烦
躁不安、轻微腹泻以及体重增长缓慢(P<0 .05)，以上改变在停药恢复期得到
改善，其余动物体重增长、外观及一般行为未见异常。血液学及血生化检查、
脏器系数、病理检查均未见异常。
结论:一般药理学研究表明NHFLE对动物的神经系统、心血管系统、呼
吸系统均无影响。药代动力学实验发现两个时相的半衰期在各剂量组之间无
差异，AUC与剂量成正比，排泄以肾脏为主。毒性试验证实该药毒性低，临
床应用安全性好。
Objective: To understand the change of metabolism in rats and evaluate safety of Non-hemorrhagic Fibrinolytic Enzyme (NHFLE) from Agkistrodon Acutus by general pharmacology, pharmacokinetics and toxicology research, and provide foundation for clinical trials and application.
Methods: 1. NHFLE was injected intravenously at the tail into mice with the doses of 2.0, 1.0 and 0.5mg/kg respectively. The same administration method was used to rats but with the different doses of 1.4, 0.7 and 0.5mg/kg. The saline was gave as the control group. We observed the changes in general behavior, coordinate activity and synergism with sodium pentobarbital at subthreshold dose of mice. To rats, we observed the heart rate, cardiac electric voltage, mean arterial pressure, respiratory rate and depth and clotting time. 2. During pharmacokinetics research, NHFLE was labeled with 125I by the method of Chloramine-T. After purification
using Sephadex G-50, 125I-NHFLE was separated and then the radiochemical
purity of I-NHFLE was assayed by y -counter. After intravenous administration of l25I-NHFLE to rats, the plasma samples of 125I-NHFLE, the distribution in tissues or organs and the excretion in urine, feces and bile were studied with via radioactive tracer technique respectively. 3. The acute toxicity of NHFLE was studied on Kunming species mice. 4. Experimental animals of long-term toxicity were healthy Wistar rats. Route of medication was intravenous injection with 9.0, 4.5 and 2.25mg/kg by three different dosages of high dose group, middle dose group and low dose group, while the saline was gave as the control group.
Results: 1.A11 the animal groups of mice showed that NHFLE had no influence on their general behavior, coordinate activity and synergism with sodium pentobarbital at subthreshold dose after intravenous injection. The nervous, cardiovascular and respiratory systems in rats after administrated at different doses of NHFLE were normal. The blood clotting time of the rats at high dose of NHFLE was delayed obviously after injection 10-30 min (P<0.05 ) but changed normal at 60 min, meanwhile there was no significant difference of blood clotting time in
other groups. 2. Radiospecific activity of 125I-NHFLE was 25.8GBq/L. The labelled ratio was 7.318MBq per milligram NHFLE. After intravenous injection at doses of 1.4 0.7 0.35mg/kg to rats, the main pharmacokinetic parameters of I-NHFLE were as follows: T1/2a was between 0.626~0.672h and t1/2B was between 10.384~13.818h. AUC were 7073.03 + 124.14, 3920.00 + 549.64 and 3315.78+125.30 ug hL-1. There were no significant differences of t1/2a and t1/2B among the three dose groups (P>0.05) , but the AUC increased markedly with the dose increase (P<0.05) .The concentration-time curves of 125I-NHFLE were conformed to two-compartment model. The amount of radioactivity excreted in urine, feces and bile within 24h after intravenous administration of I-NHFLE to rats was 75.31%, 3.29% and 6.45% of the dose respectively. Within 48h after administration drug 86.22% of 125I-NHFLE was excreted in urine and 4.98% in feces. The peak concentration of 125I-NHFLE in tissues or organs appeared at Ih. Following intravenous injectio
n to rats, the radioactivity was selectively distributed to kidney, thyroid, liver, spleen and stomach, while the levels in brain, adipose and muscle were low. 3. Acute toxicity experiment showed that LD50 of NHFLE was 28.84mg/kg in mice. 4. Long-term toxicity experiment showed that during the 4 weeks of intravenous administration, the rats of high dose group had more activity, dysphoria, slight diarrhea and less weight increase than the control group ( P<0.05) and were recovered in 2 weeks withdrawal. Rats' weight increase, appearance and behavior were normal in other groups. There was no significant abnormality in hematology and laboratory test. The organ coefficients and pathologic examination were normal in all groups.
Conclusions: The drug has no effects on nervous, cardiovascular and respiratory systems. The half-lives of two phases were no statistically differenc
引文
1. Cheng Heisn-Chang, Ouyang Chaoho. Isolation of coagulant and anticoagulant principles from the venom of Agkistrodon acutus. Toxicon, 1967; 4: 235～242
2. Ouyang Chaoho, Huang Tur-Fu. Platelet aggregation inhibitors from Agkistrodon acutus snake venom. Toxicon, 1986; 24: 1099～1106
3．刘广芬．尖吻蝮蛇毒的柱层析分离及各组分生化药理特性的测定．两栖爬行动物学报，1982；1：69～75
4. Ouyang C, Huang TF. Purification and characterization of fibrinolytic principle of Agkistrodon acutus venom. Biochimica and Biophysica Acta, 1976, Jul, 19.439(1): 146-153
5．钱小红，马立人，张庆宇．尖吻蝮蛇毒纤维蛋白溶解活性组分的纤溶作用．中华血液学杂志，1993；14(10)：512-515
6．陈家树，孙家钧，梁陆光．五步蛇蛇毒纤溶组分Ⅱ的分离和若干药效学的特征．中国药理学通报，1993；9(1)：22～23
7．陈家树，俞励平，梁秀霞等．五步蛇蛇毒纤溶组分Ⅱ对大鼠动脉血栓的溶栓作用．中国药理学通报，1998；14(5)：463～466
8. Siigur E, Siigur J. Purification and characterization of lebetase, a fibfinolytic enzyme from Vipera lebetina (snake) venom. Biochimica and Biophysica Acta, 1991; Jul 8,1074(2): 223-229
9. Guan AL, Retzios AD, Henderson GN, et al. Purification and characterization of a fibrinolytic enzyme from venom of the southern copperhead snake(Agkistrodon contortrix contortrix). Archives of Biochemistry & Biophysics, 1991 Sep; 289(2): 197-207
10. Datta G, Dong A, Witt J, et al. Biochemical characterization of basilase, a fibrinolytic enzyme from Crotalus basiliscus basiliscus. Archives of Biochemistry & Biophysics, 1995 Mar 10; 317(2): 365-373
11. Lee JW, Seu JH, Rhee IK, et al. Purification and characterization of brevinase, from the venom of Korean snake, Agkistrodon blomhoffii brevicaudus. Biochemical & Biophysical Research Communications, 1999 Jul 14; 260(3): 665-670
12．黎肇炎，雷丹青，杨斌等．尖吻蝮蛇毒无出血活性纤溶酶的纯化及其理化性质的研究．广西医科大学学报，2003；20(2)：157-160
13．黎肇炎，杨斌，唐双意等．尖吻蝮蛇毒无出血活性纤溶酶对动物血栓的溶栓作用．中国药理学通报，2003 Jul；19(7)：830-832
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30．汤仲明，刘秀文，柴彪新等．蛋白质多肽类药物药代动力学研究的方法和实验设计．中国药理学和毒理学杂志，1996；10(3)：161～168
2. Ouyang Chaoho, Huang Tur-Fu. Platelet aggregation inhibitors from Agkistrodon acutus snake venom. Toxicon, 1986; 24: 1099～1106
3．刘广芬．尖吻蝮蛇毒的柱层析分离及各组分生化药理特性的测定．两栖爬行动物学报，1982；1：69～75
4. Ouyang C, Huang TF. Purification and characterization of fibrinolytic principle of Agkistrodon acutus venom. Biochimica and Biophysica Acta, 1976, Jul, 19.439(1): 146-153
5．钱小红，马立人，张庆宇．尖吻蝮蛇毒纤维蛋白溶解活性组分的纤溶作用．中华血液学杂志，1993；14(10)：512-515
6．陈家树，孙家钧，梁陆光．五步蛇蛇毒纤溶组分Ⅱ的分离和若干药效学的特征．中国药理学通报，1993；9(1)：22～23
7．陈家树，俞励平，梁秀霞等．五步蛇蛇毒纤溶组分Ⅱ对大鼠动脉血栓的溶栓作用．中国药理学通报，1998；14(5)：463～466
8. Siigur E, Siigur J. Purification and characterization of lebetase, a fibfinolytic enzyme from Vipera lebetina (snake) venom. Biochimica and Biophysica Acta, 1991; Jul 8,1074(2): 223-229
9. Guan AL, Retzios AD, Henderson GN, et al. Purification and characterization of a fibrinolytic enzyme from venom of the southern copperhead snake(Agkistrodon contortrix contortrix). Archives of Biochemistry & Biophysics, 1991 Sep; 289(2): 197-207
10. Datta G, Dong A, Witt J, et al. Biochemical characterization of basilase, a fibrinolytic enzyme from Crotalus basiliscus basiliscus. Archives of Biochemistry & Biophysics, 1995 Mar 10; 317(2): 365-373
11. Lee JW, Seu JH, Rhee IK, et al. Purification and characterization of brevinase, from the venom of Korean snake, Agkistrodon blomhoffii brevicaudus. Biochemical & Biophysical Research Communications, 1999 Jul 14; 260(3): 665-670
12．黎肇炎，雷丹青，杨斌等．尖吻蝮蛇毒无出血活性纤溶酶的纯化及其理化性质的研究．广西医科大学学报，2003；20(2)：157-160
13．黎肇炎，杨斌，唐双意等．尖吻蝮蛇毒无出血活性纤溶酶对动物血栓的溶栓作用．中国药理学通报，2003 Jul；19(7)：830-832
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