Preclinical dosing optimization: Dosing schedule, tissue

成功的药物开发需要安全且最有效的治疗方案。吗啉代介导的外显子跳读已被证明在动物模型（小鼠和狗）和DMD男孩的早期临床试验中实现肌营养不良蛋白的恢复。除了疗效外，在优化AO治疗的剂量选择时必须考虑毒性。PMO-AO的经验表明，虽然这些化合物与其他AO化学物质相比具有更好的治疗指数，但预计肾脏效应是主要的剂量限制性毒性。建立可靠的和可重复的方法用于临床前和临床 监测吗啉代AO的肾效应对于最佳剂量选择和“类”批准管理策略是关键的。初步GLP毒理学研究和使用针对外显子51的高剂量吗啉代AO的非GLP研究（12周，960 mg/kg/周IV小鼠）显示药物在肾近端小管细胞中显著蓄积。然而，一旦药物治疗停止，肾脏蓄积消退，表明优化给药可以最大限度地减少肾毒性，或者在方案中可能需要药物“假期”。在这里，我们假设在DMD患者中建立适当的治疗窗口将涉及AO药物的肌肉保留（疗效）与肾脏保留（毒性）的平衡。因此，本项目的目标是系统地研究通过以下两个目标实现这种平衡的给药方案。目标1将使用iPRECIO遥测泵（颈静脉插管）在大鼠中进行6个月的吗啉代药物长期比较给药，终点为尿液和上皮细胞生物化学（核心B）、肾脏组织学和新生物标志物（项目2）。在目标2中，我们将在小鼠疾病模型（mdx）中进行类似的IV给药方案，目的是检测药物疗效、肌肉中的药物生物利用度（RT-PCR和肌营养不良蛋白量）和肾脏蓄积（核心B和项目2）。这种在多个物种中对多种药物进行的系统比较将提供对优化治疗指数的评估。使用吗啉代AO纠正遗传缺陷正在迅速发展到其他几种人类疾病，如FSHD，囊性纤维化，强直性肌营养不良症，LGMD等，因此，从这个建议产生的疗效和毒性数据比治疗杜氏肌营养不良症有更广泛的意义。

Successful drug development requires a therapeutic regimen that is safe and maximally effective. Morpholino-mediated exon skipping has been demonstrated to achieve restoration of dystrophin in both animal models (mice and dogs) and in DMD boys in early clinical trials. In addition to efficacy, toxicity must be considered in optimizing dose selection for AO therapy. Experience with PMO-AO suggests that while these compounds have a better therapeutic index compared to other AO chemistries, renal effects are anticipated to be the primary dose limiting toxicity. Establishment of reliable and reproductive methods for pre-clinical and clinical monitoring of renal effects of morpholino AO is critical for optimal dose selection and a "class" approval regulatory strategy. Preliminary GLP toxicology studies, and non-GLP studies using high dose morpholino AO directed at exon 51 (12 wk, 960 mg/kg/wk IV murine) showed significant accumulation of the drug in kidney proximal tubule cells. However, kidney accumulation resolved once drug treatment was stopped, suggesting optimized dosing could minimize renal toxicity or that a drug "holiday" may be needed in the regimen. Here we hypothesize that establishing an appropriate therapeutic window in DMD patients will involve a balance of muscle retention of AO drug (efficacy) vs. kidney retention (toxicity). Therefore, the goal of this project is to systematically investigate the dosing schedules that achieve this balance through the following two aims. Aim 1 will carry out 6 month chronic comparative dosing of morpholino drug in rats using iPRECIO telemetry pumps (jugular vein cannulation), with endpoints of urine and epithelial cell biochemistry (Core B), kidney histology and novel biomarkers (Project 2). In Aim 2, we will carry out a similar IV dosing schedule in the mouse disease model (mdx), with the goal of testing drug efficacy, drug bioavailability in muscle (RT-PCR and dystrophin quantity), and kidney accumulation (Core B, and Project 2). This systematic comparison of multiple drugs in multiple species will provide an assessment of the optimized therapeutic Index. Use of morpholino AO to correct genetic defects is rapidly advancing to several other human diseases such as FSHD, Cystic fibrosis, myotonic dystrophy, LGMD etc. Therefore the efficacy and toxicity data generated from this proposal have much broader implications than treatment for Duchenne muscular dystrophy.