高效液相色谱—荧光检测法研究阿德福韦前药药代动力学及组织分布
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摘要
阿德福韦酯(adefovir dipivoxil,ADV)是新一代核苷类抗病毒药物,ADV在体内迅速代谢为活性物质阿德福韦(adefovir,PMEA),为PMEA的前体药物。体内外试验表明阿德福韦不仅对野生乙肝病毒(hepatitis B virus,HBV)有效,对拉米夫定耐药的HBV病毒株也有显著的抑制作用,现已广泛应用于慢性乙型肝炎(chronic hepatitis B,CHB)的抗病毒治疗。然而,动物和人体试验研究表明口服ADV具有潜在的剂量依赖肾脏毒性,其毒性作用和严重程度与剂量及用药时间呈正相关,一定程度上限制了其临床应用,因此,寻求新的ADV替代药物成为必要。
M1和M2是一类新合成的阿德福韦前体药物,其结构与ADV类似,为考察二者的早期药代动力学及组织分布,本研究选用beagle犬和wister大鼠为研究对象,以ADV作为参比药物,比较M1和M2与ADV的相对生物利用度。由于M1,M2在体内迅速代谢为活性物质PMEA,因此只需要通过测定PMEA的浓度,即可反映M1和M2在动物体内的血药浓度和组织分布变化情况。
本论文分别建立了高效液相色谱-荧光检测法测定了PMEA在beagle犬血浆及wister大鼠组织中的分布情况,并进行了系统的方法学考察。通过动物实验筛选并比较阿德福韦酯类前药M1和M2与ADV在beagle犬血浆及wister大鼠组织中的PMEA分布情况,为抗HBV新药的进一步开发提供一定的理论基础。
第一部分:建立PMEA在beagle犬血浆中的HPLC-荧光测定方法。研究ADV与M1,M2在beagle犬体内的药代动力学,比较M1,M2与ADV的相对生物利用度。以PMEA为测定药物,替诺福韦(tenofovir,PMPA)为内标,PMEA经柱前衍生化使之与氯乙醛反应生成高荧光物质1,N6-乙烯基腺嘌呤衍生物,建立了HPLC-荧光检测的测定方法,并进行了系统的方法学验证。药代动力学研究表明:灌服给药后的平均血浆药物浓度为ADV>M1>M2,相对生物利用度比较结果:以ADV的AUC值为标准计算后二者相对生物利用度F分别为56.79%,42.90%。
第二部分:建立PMEA在wister大鼠组织中HPLC-荧光测定方法。研究ADV与M1,M2在wister大鼠组织肝、肾内分布情况。以PMEA为测定药物,PMPA为内标,PMEA经柱前衍生化使之与氯乙醛反应生成高荧光物质1,N6-乙烯基腺嘌呤衍生物,建立了HPLC-荧光检测的测定方法,并进行了系统的方法学验证。组织分布研究表明:肝中M2>M1>ADV,肾中药物含量ADV>M1>M2,表明前药M2提高PMEA在肝中的分布,比M1,ADV具有更强的肝靶向性和更低肾毒性。
Adefovir dipivoxil (ADV) is new generation nucleoside anti-virus drug.ADV rapidly metabolizes to the active substance adeofvir (PMEA) in vivo,which is a prodrug of PMEA.Studies in vitro and vivo have shown that PMEA may not only fight effectively against wild hepatitis B virus (HBV)but that it may obviously restrain HBV resistant to lamivudine.It was widely used to anti-virus in the therapy of chronic hepatitis B (CHB). In the studies on animals and human, the oral prodrug of PMEA,ADV shows potential dose-limiting kidney toxicity.Toxic effects and severity of the drug have a positive correlation with dose and time,which has limited the clinical application to a certain extent.It is necessary to explore new drugs to substitute ADV.
M1, M2 are the newly synthetic prodrugs of adefovir,whose structures are similar to ADV.To study the early pharmacokinetics and tissue distribution of the two,we compared the relative bioavailability of M1, M2 with ADV as the beagle dogs and wister rats for the objects of research,using ADV for the reference standard drug. Because M1, M2 rapidly metabolize to the active substance PMEA in vivo, we can reflect the alteration of concentrations of M1 and M2 in the animals blood and tissue distribution through determining the concentration of PMEA.
This thesis established method separately to determinate the concentration of PMEA in plasma of beagle dogs and tissues of wister rats by HPLC with fluorescence detection and validated the method systematically.Through animal experiments,the thesis screened and compared the analogs M1 and M2 with ADV of PMEA distribution in plasma of beagle dogs and tissues of wister rats and provided a theoretical foundation for further exploitation of new dugs anti-HBV.
Part I:establishment of HPLC with fluorescence detection for the determination of PMEA in the plasma of beagle dogs.We studied the pharmacokinetics of ADV, M1 and M2 in beagle dogs and compared the relative bioavailability of the three drugs,verified the method of HPLC with fluorescence detection systematically as PMEA for the determination drug,tenofovir(PMPA) for the internal standard.PMEA was treated with chloroacetaldehyde to yield the corresponding highly fluorescent 1,N6-ethenoadenine derivative.Pharmacokinetic study showed that: after drench administration,the average drug concentration in plasma: ADV> M1>M2.The comparison on the results of relative bioavailability: AUCM1/AUCADV=56.79%, AUCM2/AUCADV=42.90%.
Part II:establishment of HPLC with fluorescence detection for the determination of PMEA in rat tissues.We researched the distribution of ADV,M1 and M2 in liver,kidney of rats and verified the method of HPLC with fluorescence detection systematically as PMEA for the determination drug,PMPA for the internal standard. PMEA was treated with chloroacetaldehyde to yield the corresponding highly fluorescent 1,N6-ethenoadenine derivative.Tissue distribution study shows that: in the liver: M2>M1>ADV,in the kidney: ADV>M1>M2, M2 improved the distribution of PMEA in the liver,it has better targeting to the liver than M1 and ADV and lower nephrotoxicity.
M1和M2是一类新合成的阿德福韦前体药物,其结构与ADV类似,为考察二者的早期药代动力学及组织分布,本研究选用beagle犬和wister大鼠为研究对象,以ADV作为参比药物,比较M1和M2与ADV的相对生物利用度。由于M1,M2在体内迅速代谢为活性物质PMEA,因此只需要通过测定PMEA的浓度,即可反映M1和M2在动物体内的血药浓度和组织分布变化情况。
本论文分别建立了高效液相色谱-荧光检测法测定了PMEA在beagle犬血浆及wister大鼠组织中的分布情况,并进行了系统的方法学考察。通过动物实验筛选并比较阿德福韦酯类前药M1和M2与ADV在beagle犬血浆及wister大鼠组织中的PMEA分布情况,为抗HBV新药的进一步开发提供一定的理论基础。
第一部分:建立PMEA在beagle犬血浆中的HPLC-荧光测定方法。研究ADV与M1,M2在beagle犬体内的药代动力学,比较M1,M2与ADV的相对生物利用度。以PMEA为测定药物,替诺福韦(tenofovir,PMPA)为内标,PMEA经柱前衍生化使之与氯乙醛反应生成高荧光物质1,N6-乙烯基腺嘌呤衍生物,建立了HPLC-荧光检测的测定方法,并进行了系统的方法学验证。药代动力学研究表明:灌服给药后的平均血浆药物浓度为ADV>M1>M2,相对生物利用度比较结果:以ADV的AUC值为标准计算后二者相对生物利用度F分别为56.79%,42.90%。
第二部分:建立PMEA在wister大鼠组织中HPLC-荧光测定方法。研究ADV与M1,M2在wister大鼠组织肝、肾内分布情况。以PMEA为测定药物,PMPA为内标,PMEA经柱前衍生化使之与氯乙醛反应生成高荧光物质1,N6-乙烯基腺嘌呤衍生物,建立了HPLC-荧光检测的测定方法,并进行了系统的方法学验证。组织分布研究表明:肝中M2>M1>ADV,肾中药物含量ADV>M1>M2,表明前药M2提高PMEA在肝中的分布,比M1,ADV具有更强的肝靶向性和更低肾毒性。
Adefovir dipivoxil (ADV) is new generation nucleoside anti-virus drug.ADV rapidly metabolizes to the active substance adeofvir (PMEA) in vivo,which is a prodrug of PMEA.Studies in vitro and vivo have shown that PMEA may not only fight effectively against wild hepatitis B virus (HBV)but that it may obviously restrain HBV resistant to lamivudine.It was widely used to anti-virus in the therapy of chronic hepatitis B (CHB). In the studies on animals and human, the oral prodrug of PMEA,ADV shows potential dose-limiting kidney toxicity.Toxic effects and severity of the drug have a positive correlation with dose and time,which has limited the clinical application to a certain extent.It is necessary to explore new drugs to substitute ADV.
M1, M2 are the newly synthetic prodrugs of adefovir,whose structures are similar to ADV.To study the early pharmacokinetics and tissue distribution of the two,we compared the relative bioavailability of M1, M2 with ADV as the beagle dogs and wister rats for the objects of research,using ADV for the reference standard drug. Because M1, M2 rapidly metabolize to the active substance PMEA in vivo, we can reflect the alteration of concentrations of M1 and M2 in the animals blood and tissue distribution through determining the concentration of PMEA.
This thesis established method separately to determinate the concentration of PMEA in plasma of beagle dogs and tissues of wister rats by HPLC with fluorescence detection and validated the method systematically.Through animal experiments,the thesis screened and compared the analogs M1 and M2 with ADV of PMEA distribution in plasma of beagle dogs and tissues of wister rats and provided a theoretical foundation for further exploitation of new dugs anti-HBV.
Part I:establishment of HPLC with fluorescence detection for the determination of PMEA in the plasma of beagle dogs.We studied the pharmacokinetics of ADV, M1 and M2 in beagle dogs and compared the relative bioavailability of the three drugs,verified the method of HPLC with fluorescence detection systematically as PMEA for the determination drug,tenofovir(PMPA) for the internal standard.PMEA was treated with chloroacetaldehyde to yield the corresponding highly fluorescent 1,N6-ethenoadenine derivative.Pharmacokinetic study showed that: after drench administration,the average drug concentration in plasma: ADV> M1>M2.The comparison on the results of relative bioavailability: AUCM1/AUCADV=56.79%, AUCM2/AUCADV=42.90%.
Part II:establishment of HPLC with fluorescence detection for the determination of PMEA in rat tissues.We researched the distribution of ADV,M1 and M2 in liver,kidney of rats and verified the method of HPLC with fluorescence detection systematically as PMEA for the determination drug,PMPA for the internal standard. PMEA was treated with chloroacetaldehyde to yield the corresponding highly fluorescent 1,N6-ethenoadenine derivative.Tissue distribution study shows that: in the liver: M2>M1>ADV,in the kidney: ADV>M1>M2, M2 improved the distribution of PMEA in the liver,it has better targeting to the liver than M1 and ADV and lower nephrotoxicity.
引文
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