Effects of Immunosuppressants on Cell Metabolism

说明(申请人提供)：钙调神经磷酸酶抑制剂，(顺式)环孢菌素和他克莫司是器官移植后大多数免疫抑制方案的基础。这些药物最终损害它们原本打算保护的器官，特别是肾脏的倾向总是被认识到，但由于它们有令人印象深刻的改善短期结果的能力，因此在很大程度上是可以容忍的。随着雷帕霉素靶标(TOR)抑制剂西罗莫司的问世，一种同样有效的免疫抑制剂本身就没有CLS最重要的副作用，如肾毒性和神经毒性，已经可以获得。西罗莫司与顺铂的联合应用具有协同免疫抑制活性，因而具有很大的吸引力。虽然单独使用西罗莫司没有肾毒性，但在临床研究中，西罗莫司出人意料地增加了环孢素的肾毒性。免疫抑制药物单独毒性和联合毒性的确切生化机制仍然知之甚少。在我们前期工作的基础上，我们推测CI引起的血管内皮细胞线粒体功能障碍和对线粒体肾脏能量代谢的直接负效应均可导致CI肾毒性，TOR抑制剂可能通过加强顺式作用对线粒体能量代谢的负效应而增强CI肾毒性。为了验证我们的假设，我们建议利用磁共振波谱(MRS)系统地研究钙调神经磷酸酶抑制剂和/或西罗莫司对在体大鼠肾脏和动脉内皮细胞代谢的影响，并将这些变化与已知的脑梗塞肾毒性的组织学、功能和分子变化相关联。我们将使用MRS确定生化机制，评估亲环素和钙调神经磷酸酶在观察到的变化中的作用，比较不同的体内肾脏毒性模型，评估钙调神经磷酸酶和TOR抑制剂联合使用时药效学和药代动力学药物相互作用的贡献，并评估研究药物及其组合对移植肾脏(暴露于缺血/再灌注)和非移植肾脏的负面影响的差异。我们的研究结果(A)将对免疫抑制剂及其组合的毒性潜在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物(例如，异前列腺素可能用于CI毒性的临床药效学监测)，以及(C)将提出在临床前开发期间根据毒性来测试免疫抑制剂的相互作用的方法。

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.