Metabolism and toxicity of NRTIs in non-replicating tissues

说明(申请人提供)：核苷逆转录酶抑制剂(NRTI)是长期艾滋病治疗的重要组成部分，与许多轻微到致命的副作用有关，这些副作用是由药物的线粒体毒性引起的，通常局限于成人非复制组织。这些药物必须被细胞酶转化为三磷酸，才能作为病毒逆转录酶的抑制剂发挥作用。已经提出了两种毒性机制。类似的三磷酸盐抑制线粒体DNA聚合酶，导致线粒体DNA耗竭；或者，对于至少一个NRTI，AZT，毒性可能是由于支持mtDNA复制的dNTP前体池的改变而抑制胸苷磷酸化而引起的。虽然已经在培养细胞分裂时测量了磷酸化NRTI类似物的速率和水平，但这些药物在成年非复制组织中被修饰或磷酸化的程度尚未被研究，在这些组织中，挽救酶的表达预计会有所不同。这项建议的目的是通过研究NRTI在非复制组织和分离的线粒体中的代谢，更好地了解最近释放和积极使用的NRTI的毒性。这一目标将通过以下方式实现：1)在从各种大鼠组织、灌流的心脏和体内的各种大鼠组织中分离的线粒体中，确定放射性NRTI到其三磷酸盐的转化；2)使用NRTI和(1)中确定的高水平NRTI-三磷酸的组织，进行长期治疗研究，以确定NRTI三磷酸盐是否与mtDNA耗竭有关，如果是的话；3)研究外源添加竞争脱氧核苷和/或尿苷对NRTI磷酸化和mtDNA耗竭的影响；4)确定是否有任何NRTI抑制线粒体、灌流心脏或体内其他非复制组织中典型脱氧核苷的磷酸化；5)使用(4)中确定的NRTI，确定dNTP池是否被破坏，这种破坏是否导致mtDNA水平下降，如果是，则研究添加天然存在的竞争脱氧核苷和/或尿苷对纠正dNTP缺陷的影响。为了扩大这一分析范围，将通过RT-PCR测量脱氧核苷回收和合成途径中酶的mRNA水平，以捕捉可能因长期药物治疗而导致的基因表达变化。此外，还将包括微阵列和蛋白质组分析，以与RT-PCR结果相关联，并捕捉可能提供与毒性相关的重要信息的全基因组基因表达变化。最后，据报道，在人类和啮齿动物中，NRTI的毒性在雄性和雌性之间是不同的，而且这些差异可能与性别特定的新陈代谢差异有关，上述所有研究都将在雄性和雌性队列中进行。这项提案的目标的成功完成，不仅将为了解NRTI的毒性提供重要的基本数据，而且还将解决性别差异和潜在的治疗方法，包括补充尿苷，这只能改善合理的治疗，以限制毒性。

DESCRIPTION (provided by applicant): Nucleoside reverse transcriptase inhibitors (NRTIs) are an important component of long-term AIDS therapy and are associated with a host of mild to lethal side effects that are caused by mitochondrial toxicity of the drugs, often localized to adult non-replicating tissues. These drugs must be converted by cellular enzymes to the triphosphate to be active as inhibitors of the viral reverse transcriptase. Two mechanisms of toxicity have been proposed. The analog triphosphates inhibit the mitochondrial DNA polymerase leading to mtDNA depletion; or for at least one NRTI, AZT, toxicity may be caused by the inhibition of thymidine phosphorylation with alterations of the dNTP precursor pools supporting mtDNA replication. While the rates and levels of phosphorylated NRTI analogs have been measured in dividing cells in culture, the degree to which these drugs are modified or phosphorylated in adult non-replicating tissues in which the expression of salvage enzymes are expected to be different has not been studied. The goal of this proposal is to obtain a better understanding of the toxicities of the recently released and actively used NRTIs by studying their metabolism in non-replicating tissues and isolated mitochondria. This goal will be accomplished by: 1) Determining the conversion of radioactive NRTIs to their tri-phosphates in mitochondria isolated from a variety of rat tissues, in the perfused heart, and in vivo in a variety of rat tissues; 2) Using NRTIs and tissues identified in (1) that have high levels of NRTI-triphosphate, perform long term treatment studies to determine if NRTI triphosphates are associated with mtDNA depletion, and if so; 3) study the effect of exogenous addition of competing deoxynucleosides and/or uridine on NRTI phosphorylation and mtDNA depletion; 4) Determine if any of NRTIs inhibit phosphorylation of the typical deoxynucleosides in mitochondria, the perfused heart, or in vivo in other non-replicating rat tissues; 5) Using NRTIs identified in (4), determine if dNTP pools are disrupted and if this disruption leads to decreases in mtDNA level, and if so; 6) Study the effect of addition of the naturally occurring competing deoxynucleosides and/or uridine on correcting the dNTP defect. To broaden this analysis, mRNA levels of enzymes of the deoxynucleoside salvage and synthesis pathways will be measured by RT-PCR to capture changes in gene expression that may be caused by long-term drug therapy. Further, microarray and proteomic analysis will be included to correlate with the RT-PCR results and to capture genome wide gene expression changes that may provide important information relative to toxicity. Finally, as NRTI toxicities are reported to be different between males and females in both humans and rodents, and because these differences may be related to sex specific differences in metabolism, all of the above studies will be done in male versus female cohorts. The successful completion of the aims of this proposal will not only provide significant essential data in understanding NRTI toxicity, but will also address sex specific differences and potential treatments including uridine supplementation, which can only improve rational therapy to limit toxicity.