Metabolism and toxicity of nucleoside reverse transcriptase inhibitors in non-rep

描述（由申请人提供）：核苷逆转录酶抑制剂（NRTI）是长期AIDS治疗的重要组成部分，并与由药物的线粒体毒性引起的许多轻度至致死性副作用相关，通常局限于成人非复制组织。这些药物必须通过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活性。已经提出了两种毒性机制。类似物三磷酸盐抑制线粒体DNA聚合酶，导致mtDNA耗竭;或者对于至少一种NRTI、AZT，毒性可能是由于胸苷磷酸化的抑制以及支持mtDNA复制的dNTP前体池的改变引起的。虽然磷酸化NRTI类似物的速率和水平已在培养物中的分裂细胞中测量，但尚未研究这些药物在其中补救酶的表达预期不同的成体非复制组织中被修饰或磷酸化的程度。该提案的目标是通过研究其在非复制组织和分离的线粒体中的代谢，更好地了解最近发布和积极使用的NRTI的毒性。1）测定从多种大鼠组织分离的线粒体中、灌注心脏中和体内多种大鼠组织中放射性NRTI向其三磷酸盐的转化; 2）使用（1）中鉴定的具有高水平NRTI-三磷酸的NRTI和组织，进行长期治疗研究以确定NRTI三磷酸是否与mtDNA耗竭相关，如果是，3）研究外源性添加竞争性脱氧核苷和/或尿苷对NRTI磷酸化和mtDNA消耗的影响; 4）确定是否有任何NRTI抑制线粒体、灌注心脏或体内其他非复制大鼠组织中典型脱氧核苷的磷酸化; 5）使用（4）中鉴定的NRTI，确定dNTP池是否被破坏，以及这种破坏是否导致mtDNA水平降低，如果是这样; 6）研究添加天然存在的竞争性脱氧核苷和/或尿苷对校正dNTP缺陷的影响。为了拓宽该分析，将通过RT-PCR测量脱氧核苷补救和合成途径的酶的mRNA水平，以捕获可能由长期药物治疗引起的基因表达变化。此外，将包括微阵列和蛋白质组学分析，以与RT-PCR结果相关联，并捕获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，由于在人类和啮齿类动物中，雄性和雌性之间的NRTI毒性报告不同，并且由于这些差异可能与代谢的性别特异性差异有关，因此所有上述研究都将在雄性与雌性队列中进行。本提案目标的成功完成不仅将为理解NRTI毒性提供重要的基本数据，而且还将解决性别特异性差异和潜在的治疗方法，包括补充尿苷，这只能改善合理治疗以限制毒性。 公共卫生相关性：该资助的主要目的是更好地了解最近发布的核苷逆转录酶抑制剂（NRTI）在非复制大鼠组织中的毒性，并比较其在雄性和雌性队列中的代谢和毒性。由于NRTI被确定为具有毒性，我们建议在大鼠模型的长期研究中测试竞争性外源脱氧核苷和/或尿苷在减轻毒性方面的作用。本提案目标的成功完成不仅将为理解NRTI毒性（包括性别差异）提供必要的数据，还将解决潜在的治疗方法，包括尿苷补充（目前处于III期试验），这只能改善合理治疗以限制毒性。

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. PUBLIC HEALTH RELEVANCE: The major aim of this grant is to obtain a better understanding of the toxicities of the more recently released nucleoside reverse transcriptase inhibitors (NRTIs) in non-replicating rat tissues and compare their metabolism and toxicities in both male and female cohorts. As NRTIs are identified with toxicities, we propose to test the effect of the competing exogenous deoxynucleoside and/or uridine in relieving the toxicity in long-term studies in a rat model. The successful completion of the aims of this proposal will not only provide essential data in understanding NRTI toxicity, including sex differences, but will also address potential treatments including uridine supplementation (now in Phase III trials), which can only improve rational therapy to limit toxicity.