Effects of Immunosuppressants on Cell Metabolism

描述（由申请人提供）：钙调磷酸酶抑制剂、环孢素和他克莫司是器官移植后大多数免疫抑制方案的基础。人们一直认识到，这些药物最终会损害它们想要保护的器官，尤其是肾脏，但由于它们改善短期预后的惊人能力，它们在很大程度上是可以容忍的。随着雷帕霉素靶点（TOR）抑制剂西罗莫司，一种同样有效的免疫抑制剂，它本身没有Cls最重要的副作用，如肾毒性和神经毒性，已经可用。西罗莫司与CIs的联合应用具有协同免疫抑制作用。虽然单独使用西罗莫司没有肾毒性，但在临床研究中意外地增强了环孢素的肾毒性。免疫抑制剂单独或联合产生毒性的确切生化机制尚不清楚。基于我们之前的工作，我们假设CI诱导的血管内皮线粒体功能障碍导致血管收缩和对线粒体肾脏能量代谢的直接负面影响都导致CI肾毒性，TOR抑制剂通过增强CIs对线粒体能量代谢的负面影响来增强CI肾毒性。为了验证我们的假设，我们建议使用磁共振波谱（MRS）系统地研究钙调磷酸酶抑制剂和/或西罗莫司对大鼠体内肾脏和动脉内皮细胞代谢的影响，并将这些影响与已知CI肾毒性典型的组织学、功能和分子变化联系起来。我们将利用MRS识别生化机制，将评估亲环蛋白和钙调磷酸酶在观察到的变化中的作用，将比较不同的体内肾毒性模型。将评估钙调磷酸酶和TOR抑制剂联合使用时药物相互作用的药效学和药代动力学贡献，并将评估研究药物及其联合使用对移植肾脏（暴露于缺血/再灌注）与非移植肾脏的负面影响的差异。我们的研究结果(a)将为免疫抑制剂及其组合的潜在毒性的生化机制提供重要的新见解，(b)将确定免疫抑制剂毒性的替代标记（例如异前列腺素用于CI毒性的临床药效学监测的潜在用途），(c)将提出在临床前开发过程中测试免疫抑制剂相互作用毒性的分析方法。

DESCRIPTION (provided by applicant): The calcineurin inhibitors, (CIs) cyclosporinen, and tacrolimus are the basis of most immunosuppressive protocols after organ transplantation. The propensity of these agents to ultimately damage the very organs they were intended to protect, especially the kidney, was always recognized, but largely tolerated due to their impressive ability to improve short-term outcomes. With the target-of-rapamycin (TOR) inhibitor sirolimus, an equally potent immunosuppressant that itself is lacking the most important side effects of Cls, such as nephrotoxicity and neurotoxicity, has become available. The combination of sirolimus with CIs is attractive since it results in synergistic immunosuppressive activity. Although devoid of nephrotoxicity when administered alone, sirolimus unexpectedly enhanced cyclosporine nephrotoxicity in clinical studies. The exact biochemical mechanisms underlying immunosuppressant toxicity alone and in combination is still poorly understood. Based on our previous work, we hypothesize that both, CI-induced mitochondrial dysfunction in blood vessel endothelium leading to vasoconstriction and direct negative effects on mitochondrial kidney energy metabolism, cause CI nephrotoxicity and that TOR inhibitors enhance CI nephrotoxicity by enhancing the negative effects of CIs on mitochondrial energy metabolism. To test our hypothesis, we propose to systematically study the effect of calcineurin inhibitors and/or sirolimus on kidney and arterial endothelial cell metabolism in the rat in vivo using magnetic resonance spectroscopy (MRS) and to correlate those with histological, functional and molecular changes known to be typical for CI nephrotoxicity. We will identify the biochemical mechanisms using MRS, will evaluate the role of cyclophilin and calcineurin in the observed changes, will compare different in vivo kidney toxicity models, will evaluate the contribution of pharmacodynamic and pharmacokinetic drug interactions when calcineurin and TOR inhibitors are combined and will evaluate differences in the negative effects of the study drugs and their combinations on transplant kidneys (exposed to ischemia/reperfusion) versus non-transplant kidneys. The results of our studies (a) will give important new insights in the biochemical mechanisms underlying toxicity of immunosuppressants and their combinations, (b) will identify surrogate markers for immunosuppressant toxicity (e.g. the potential use of isoprostanes for clinical pharmacodynamic monitoring of CI toxicity) and (c) will propose assays to test interactions of immunosuppressants in terms of toxicity during pre-clinical development.