重组水蛭素在大鼠体内的生物分析及其药代动力学研究
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摘要
随着基因工程技术的发展,重组多肽类药物已被广泛应用于临床医疗实践。这类药物的新药开发及临床合理应用必须首先阐明其药代动力学(Pharmacokinetics,PK)特点.。然而,多肽类药物PK研究一直是药物PK研究中的一大难点。本文针对这一难点问题进行了深入的研究。首先通过对凝血酶特异性抑制剂重组水蛭素(Recombinant Hirudin,rH)PK研究,建立多肽类药物PK研究的通用方法和适用于rH PK研究的个性方法；其次通过对rH在大鼠尿中代谢产物的分析建立多肽类药物在动物体内代谢产物研究的通用方法,同时研究rH在大鼠体内的代谢过程和代谢产物,为国产rH的研发和临床应用奠定基础。
实验方法1、建立一种双抗体夹心酶联免疫吸附分析(Enzyme-Linked Immunosorbent Assay,ELISA)技术,测定国产rH(N-Ile1-Thr-63-脱硫酸基-r-重组水蛭素)在大鼠血浆等生物样品中的浓度／含量,并全面研究静脉注射给药(intravenous injection,iv)rH后,rH在大鼠体内的PK和组织分布；2、根据rH具有抗凝血酶的生物特性,建立用生色底物法测定rH在大鼠血浆和尿中的浓度,并研究iv给予大鼠后,rH在大鼠血浆的PK及尿排泄：3、用125I标记rH得125I-rH,iv给予大鼠后,研究125I-rH在大鼠体内的PK,并同时研究rH在大鼠体内的代谢过程；4、将凝胶色谱和Y-计数仪相结合,通过测定血浆和尿中的放射性活性,研究rH在大鼠血浆中的代谢过程以及尿中的代谢产物,用凝胶色谱和反相高效液相色谱(RP-HPLC)对尿中代谢产物进行分离纯化,采用飞行时间质谱以及氨基酸序列测定仪分析其代谢产物的分子量和结构序列。
实验结果1.ELISA法能有效的测定大鼠体液和组织中rH的浓度/含量,最低检出量为0.25ng·mL-1,样品浓度在0.25ng·mL-1-3.0ng·mL-1之间线性关系良好(r=0.999)。其血药浓度用3p97程序软件拟合,均符合开放二室模型一级动力学过程。静脉注射rH剂量为0.5、1.0、2.0mg·kg-1时其各剂量主要动力学参数分别为：t1/2α(min):6.408±2.603,5.568±1.106,5.695±0.682; t1/2β(min):47.28±13.14,45.41±6.207,46.82±5.562;AUC(μg·mL-1×min):127.8±27.81,330.8±115.92,575.2±39.42.除AUC外,各浓度间的药代动力学参数均很接近,经统计处理无显著差异(P>0.05),其中t1/2α为5-6min,说明药物自中央室向外周室分布很快；t1,2β为45-47min,说明药物从血浆中消除也较快。三种剂量的AUC与剂量成正比(经回归计算r=0.991),说明在此范围内,药物在大鼠体内的消除为线性动力学。静脉注射给药1.0mg·kg-1,大鼠尿、粪便和胆汁中几乎检测不到原型的rH。组织分布实验表明,iv1.0mg·kg-1,呈快速分布,15分钟各组织中药物含量最高；以后逐渐减少。15分钟各组织分布以肺部含量最高为78.88±32.30ng·g-1,1h以肾脏含量最高,为24.39±39.7ng-g-1；3h以肝脏含量最高,仅为7.55±11.33ng·g-1.2.生色底物法能有效的测定大鼠血浆中rH的含量,rH的浓度在3.125-40ng·mL-1范围内呈线性关系(r=-0.995)。用该方法研究iv给药rH2.0mg·kg-1大鼠血浆药动学,除AUC外,其它药动学参数均与ELISA法一致；生色底物法能有效的测定大鼠尿中rH的含量,rH在6.25-75ng·mL-1浓度范围内呈线性关系(r=-0.997)。用该方法研究大鼠iv0.5、1.0和2.0mg·kg-1rH后尿药浓度及排泄动力学,0-12h的累积排泄率为分别给药量的22.30%、23.23%和23.29%,不随剂量改变而显著改变,表明rH在体内遵循一级线性动力学方式从尿中排泄。3、同位素示踪法研究125I-rH在大鼠体内的药动学结果显示,rH在大鼠体内的消除半衰期t1/2β(min)为701.9±198.0(RA)和724.9±81.2(TCA-RA);按总放射性活性计算,0-12h累计排泄率为给药量的62.1%,96h累计排泄率为给药量的90.8%。4、rH在血浆中被代谢,经由尿中排泄；其代谢产物具有抗凝血酶的生物活性,其中抗凝血酶活性最强的代谢产物的分子量为5162Da,由51-52个氨基酸组成。
结论1、用ELISA法和生色底物法测定给药后大鼠生物样品中rH浓度/含量,经方法学验证表明灵敏度高、特异性强,符合多肽类药物药动学研究要求。用于rH的PK研究表明,国产rH在大鼠体内的PK行为符合开放二室模型一级动力学过程,其体内消除快速(半衰期在46min左右)。2、用同位素示踪法研究rH在大鼠体内的PK所得到的PK参数与所建立的ELISA以及生色底物法所得到的PK参数之间差异很大,很可能由于同位素示踪法缺乏选择性所致,提示这种方法不宜用于rH以及相关多肽PK研究。3、rH在大鼠体内经受广泛的代谢降解,尿中仅测得极微量原形rH,尿中排泄的部分代谢产物具有抗凝血酶活性；并首次发现并查明抗凝血酶活性最强的代谢产物的分子量为5162Da,由51/或52个氨基酸组成；首次发现血液是rH的主要代谢部位,而不是像国外文献报导那样在肝肾代谢。
With rapid development of gene engineering techniques, the recombinant polypeptide drugs have been widely used in clinical therapeutic practices. The R&D and clinical rational application of this kind of drugs need first to elucidate their pharmacokinetics (PK) properties. However, the PK study of polypeptide drugs continues to be a great difficult problem, with no ideal method available to date, the combination of different methods are usually required. In this paper, PK of the recombinant hirudin (rH), an highly specific thrombin inhibitor, has been studied in order to develop a routine methodology of polypeptide drug PK study and an individual character method suitable for rH PK study, and to develop a routine methodology for study of metabolic products in animals by means of analysis of urinary metabolites of rH in rats, and thereby to build up a basis for R&D and clinical application of newly made domestic rH.(N-Ile'-Thr-63-desulfato-r-hirudin).
Methods1. A double antibody sandwich ELISA method was established for determination of the rH in rat plasma and tissue homogenate samples, and thereby to study the rH PK and tissue distribution after intravenous(iv) of rH.2. Based on antithrombin activity of rH, a chromogenic substrate assay (CSA) was established for determination of the rH in rat plasma and urine samples, and thereby to study the rH plasma PK and urinary excretion after iv of rH.3. rH was labeled with125I, the resultant125I-rH was employed to study the rH PK and metabolic process after iv of125I-rH.4. The125I-rH radioactivity in rat plasma and urine samples was measured by means of the gel chromatography and the gamma-counter so as to study the metabolic processes in rat plasma and metabolic products in rat urine. Urinary metabolic products were isolated and purified by the gel chromatography and the reversed-phase HPLC, and subsequently subjected to MW measurement using TOF-MS and identification of chemical structure as well as amino acid sequence by the amino acid analyzer.
Results1. The rH concentration/content in body fluids and tissues of rats was measured by ELISA assay with the limit of detection of0.25ng·mL-1and good linearity ranged from0.25ng·mL-1to3.0ng·mL-1(r=0.999). The rH plasma concentration over time after iv injection at doses of0.5,1.0and2.0mg·kg-1was characterized by a rapid distribution phase followed by the relatively slow elimination phase and could be described by the bi-exponential equation for two-compartment open model and followed the first-order kinetics; the main PK parameters t1/2α=5-6min, t1/2β=45-47min, AUC (μg·mL-1·min) were 127.8±27.8,330.8±115.9,575.1±39.4proportional to doses; demonstrating that the PK behaviour of the tested drug complies with the linear kinetics. After iv of1.0mg·kg-1, parent rH were hardly detected in urine, feces and bile samples; Tissue distribution experiment showed that the rH distributed rapidly into tissue after iv dosing at1.0mg·kg-1and the rH contents in tissues reached a peak at15min postdose and afterwards decreased gradually, the rH content was found to be highest in lung (78.88±32.3ng·g-1) at15minutes, in kidney (24.39±9.71ng·g-1) at1hour and in liver (7.55±11.33ng·g-1) at3hours.2. The established CSA showed good linearity in the range from3.125ng·mL-1to40ng·mL-1(r=0.995) for plasma and from6.25ng·mL-1to75ng·mL-1(r=0.997) for urine. The main plasma kinetic parameters based on the CSA at dose of2.0mg·kg-1were similar to that based on the ELISA except for AUC. After iv rH at0.5,1.0and2.0mg·kg-1the percent urinary cumulative excretion in relation to dose between0-12h was found22.3%,23.2%and23.3%, respectively. This dose-independent excretion manner indicated that rH urinary excretion followed the first-order i.e. linear kinetics.3. The PK of rH in rats was also studied by the125I-rH isotope tracer technology. The parameter t1/20was701.8±198.7min on a total radio activity (RA) basis, and724.8±81.2on a TCA precipitation-radio activity (TCA-RA) basis. The percent cumulative excretion of rH in urine was62.1%between0-12h based on the RA detection.4. The rH was shown to be metabolized in plasma, followed by being excreted in urine as metabolites. One of these metabolites showed potent anti-thrombin activity and was found to be composed of51-52amino acids with molecular weight being5162Da,.
Conclusion1. By validation of methodology it has been shown that the established ELISA and the CSA are highly sensitive and specific for rH bio-analysis in rats and, therefore, completely meet requirements for rH PK study. The rH PK in the rat can be described by the open two-compartment model and follows the first-order kinetics with rapid elimination from body (t1/246min or so).2. The PK parameters obtained by the RA and TCA-RA are quite different from that by the ELISA assay and by the chromogenic substrate method.3. The rH undergoes extensive metabolic degradation in rats, with only extremely minute amount of unchanged rH recovered in urine of rats. Some metabolic products excreted into urine have anti-thrombin activity. Among them, the strongest in activity is the metabolite having molecular weight of5162Da and composed of51-52amino acids. rH has been found for the first time by us to be metabolized mainly in plasma but not in liver and kidneys as described in foreign literature.
实验方法1、建立一种双抗体夹心酶联免疫吸附分析(Enzyme-Linked Immunosorbent Assay,ELISA)技术,测定国产rH(N-Ile1-Thr-63-脱硫酸基-r-重组水蛭素)在大鼠血浆等生物样品中的浓度／含量,并全面研究静脉注射给药(intravenous injection,iv)rH后,rH在大鼠体内的PK和组织分布；2、根据rH具有抗凝血酶的生物特性,建立用生色底物法测定rH在大鼠血浆和尿中的浓度,并研究iv给予大鼠后,rH在大鼠血浆的PK及尿排泄：3、用125I标记rH得125I-rH,iv给予大鼠后,研究125I-rH在大鼠体内的PK,并同时研究rH在大鼠体内的代谢过程；4、将凝胶色谱和Y-计数仪相结合,通过测定血浆和尿中的放射性活性,研究rH在大鼠血浆中的代谢过程以及尿中的代谢产物,用凝胶色谱和反相高效液相色谱(RP-HPLC)对尿中代谢产物进行分离纯化,采用飞行时间质谱以及氨基酸序列测定仪分析其代谢产物的分子量和结构序列。
实验结果1.ELISA法能有效的测定大鼠体液和组织中rH的浓度/含量,最低检出量为0.25ng·mL-1,样品浓度在0.25ng·mL-1-3.0ng·mL-1之间线性关系良好(r=0.999)。其血药浓度用3p97程序软件拟合,均符合开放二室模型一级动力学过程。静脉注射rH剂量为0.5、1.0、2.0mg·kg-1时其各剂量主要动力学参数分别为：t1/2α(min):6.408±2.603,5.568±1.106,5.695±0.682; t1/2β(min):47.28±13.14,45.41±6.207,46.82±5.562;AUC(μg·mL-1×min):127.8±27.81,330.8±115.92,575.2±39.42.除AUC外,各浓度间的药代动力学参数均很接近,经统计处理无显著差异(P>0.05),其中t1/2α为5-6min,说明药物自中央室向外周室分布很快；t1,2β为45-47min,说明药物从血浆中消除也较快。三种剂量的AUC与剂量成正比(经回归计算r=0.991),说明在此范围内,药物在大鼠体内的消除为线性动力学。静脉注射给药1.0mg·kg-1,大鼠尿、粪便和胆汁中几乎检测不到原型的rH。组织分布实验表明,iv1.0mg·kg-1,呈快速分布,15分钟各组织中药物含量最高；以后逐渐减少。15分钟各组织分布以肺部含量最高为78.88±32.30ng·g-1,1h以肾脏含量最高,为24.39±39.7ng-g-1；3h以肝脏含量最高,仅为7.55±11.33ng·g-1.2.生色底物法能有效的测定大鼠血浆中rH的含量,rH的浓度在3.125-40ng·mL-1范围内呈线性关系(r=-0.995)。用该方法研究iv给药rH2.0mg·kg-1大鼠血浆药动学,除AUC外,其它药动学参数均与ELISA法一致；生色底物法能有效的测定大鼠尿中rH的含量,rH在6.25-75ng·mL-1浓度范围内呈线性关系(r=-0.997)。用该方法研究大鼠iv0.5、1.0和2.0mg·kg-1rH后尿药浓度及排泄动力学,0-12h的累积排泄率为分别给药量的22.30%、23.23%和23.29%,不随剂量改变而显著改变,表明rH在体内遵循一级线性动力学方式从尿中排泄。3、同位素示踪法研究125I-rH在大鼠体内的药动学结果显示,rH在大鼠体内的消除半衰期t1/2β(min)为701.9±198.0(RA)和724.9±81.2(TCA-RA);按总放射性活性计算,0-12h累计排泄率为给药量的62.1%,96h累计排泄率为给药量的90.8%。4、rH在血浆中被代谢,经由尿中排泄；其代谢产物具有抗凝血酶的生物活性,其中抗凝血酶活性最强的代谢产物的分子量为5162Da,由51-52个氨基酸组成。
结论1、用ELISA法和生色底物法测定给药后大鼠生物样品中rH浓度/含量,经方法学验证表明灵敏度高、特异性强,符合多肽类药物药动学研究要求。用于rH的PK研究表明,国产rH在大鼠体内的PK行为符合开放二室模型一级动力学过程,其体内消除快速(半衰期在46min左右)。2、用同位素示踪法研究rH在大鼠体内的PK所得到的PK参数与所建立的ELISA以及生色底物法所得到的PK参数之间差异很大,很可能由于同位素示踪法缺乏选择性所致,提示这种方法不宜用于rH以及相关多肽PK研究。3、rH在大鼠体内经受广泛的代谢降解,尿中仅测得极微量原形rH,尿中排泄的部分代谢产物具有抗凝血酶活性；并首次发现并查明抗凝血酶活性最强的代谢产物的分子量为5162Da,由51/或52个氨基酸组成；首次发现血液是rH的主要代谢部位,而不是像国外文献报导那样在肝肾代谢。
With rapid development of gene engineering techniques, the recombinant polypeptide drugs have been widely used in clinical therapeutic practices. The R&D and clinical rational application of this kind of drugs need first to elucidate their pharmacokinetics (PK) properties. However, the PK study of polypeptide drugs continues to be a great difficult problem, with no ideal method available to date, the combination of different methods are usually required. In this paper, PK of the recombinant hirudin (rH), an highly specific thrombin inhibitor, has been studied in order to develop a routine methodology of polypeptide drug PK study and an individual character method suitable for rH PK study, and to develop a routine methodology for study of metabolic products in animals by means of analysis of urinary metabolites of rH in rats, and thereby to build up a basis for R&D and clinical application of newly made domestic rH.(N-Ile'-Thr-63-desulfato-r-hirudin).
Methods1. A double antibody sandwich ELISA method was established for determination of the rH in rat plasma and tissue homogenate samples, and thereby to study the rH PK and tissue distribution after intravenous(iv) of rH.2. Based on antithrombin activity of rH, a chromogenic substrate assay (CSA) was established for determination of the rH in rat plasma and urine samples, and thereby to study the rH plasma PK and urinary excretion after iv of rH.3. rH was labeled with125I, the resultant125I-rH was employed to study the rH PK and metabolic process after iv of125I-rH.4. The125I-rH radioactivity in rat plasma and urine samples was measured by means of the gel chromatography and the gamma-counter so as to study the metabolic processes in rat plasma and metabolic products in rat urine. Urinary metabolic products were isolated and purified by the gel chromatography and the reversed-phase HPLC, and subsequently subjected to MW measurement using TOF-MS and identification of chemical structure as well as amino acid sequence by the amino acid analyzer.
Results1. The rH concentration/content in body fluids and tissues of rats was measured by ELISA assay with the limit of detection of0.25ng·mL-1and good linearity ranged from0.25ng·mL-1to3.0ng·mL-1(r=0.999). The rH plasma concentration over time after iv injection at doses of0.5,1.0and2.0mg·kg-1was characterized by a rapid distribution phase followed by the relatively slow elimination phase and could be described by the bi-exponential equation for two-compartment open model and followed the first-order kinetics; the main PK parameters t1/2α=5-6min, t1/2β=45-47min, AUC (μg·mL-1·min) were 127.8±27.8,330.8±115.9,575.1±39.4proportional to doses; demonstrating that the PK behaviour of the tested drug complies with the linear kinetics. After iv of1.0mg·kg-1, parent rH were hardly detected in urine, feces and bile samples; Tissue distribution experiment showed that the rH distributed rapidly into tissue after iv dosing at1.0mg·kg-1and the rH contents in tissues reached a peak at15min postdose and afterwards decreased gradually, the rH content was found to be highest in lung (78.88±32.3ng·g-1) at15minutes, in kidney (24.39±9.71ng·g-1) at1hour and in liver (7.55±11.33ng·g-1) at3hours.2. The established CSA showed good linearity in the range from3.125ng·mL-1to40ng·mL-1(r=0.995) for plasma and from6.25ng·mL-1to75ng·mL-1(r=0.997) for urine. The main plasma kinetic parameters based on the CSA at dose of2.0mg·kg-1were similar to that based on the ELISA except for AUC. After iv rH at0.5,1.0and2.0mg·kg-1the percent urinary cumulative excretion in relation to dose between0-12h was found22.3%,23.2%and23.3%, respectively. This dose-independent excretion manner indicated that rH urinary excretion followed the first-order i.e. linear kinetics.3. The PK of rH in rats was also studied by the125I-rH isotope tracer technology. The parameter t1/20was701.8±198.7min on a total radio activity (RA) basis, and724.8±81.2on a TCA precipitation-radio activity (TCA-RA) basis. The percent cumulative excretion of rH in urine was62.1%between0-12h based on the RA detection.4. The rH was shown to be metabolized in plasma, followed by being excreted in urine as metabolites. One of these metabolites showed potent anti-thrombin activity and was found to be composed of51-52amino acids with molecular weight being5162Da,.
Conclusion1. By validation of methodology it has been shown that the established ELISA and the CSA are highly sensitive and specific for rH bio-analysis in rats and, therefore, completely meet requirements for rH PK study. The rH PK in the rat can be described by the open two-compartment model and follows the first-order kinetics with rapid elimination from body (t1/246min or so).2. The PK parameters obtained by the RA and TCA-RA are quite different from that by the ELISA assay and by the chromogenic substrate method.3. The rH undergoes extensive metabolic degradation in rats, with only extremely minute amount of unchanged rH recovered in urine of rats. Some metabolic products excreted into urine have anti-thrombin activity. Among them, the strongest in activity is the metabolite having molecular weight of5162Da and composed of51-52amino acids. rH has been found for the first time by us to be metabolized mainly in plasma but not in liver and kidneys as described in foreign literature.
引文
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