KINETIN IN CARRIERS OF THE FAMILIAL DYSAUTONOMIA MIS-SPLICING MUTATION

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 激动素是一种植物细胞分裂素和营养物质，可能成为治疗遗传性疾病家族性自主神经障碍(FD)的重要药物。要测试这一点，首先必须确定口服激动素的吸收速度和达到假设治疗剂量所需的剂量，以及通过测量野生型IKBKAP在白细胞中的表达来确定激动素是否确实影响遗传错接。 家族性自主神经障碍(FD)是一种罕见的遗传性疾病，由IKBKAP基因突变引起。最常见的突变导致剪接改变，导致所有组织中蛋白质(IKAP)表达减少，但这种减少是可变的，在神经元组织中最严重。作为人类Elongator复合体的一员，IKAP协助转录，因此由于IKAP降低，其他几个靶基因下调。许多下调的基因参与了细胞迁移，这可能解释了FD的神经病理，这与小纤维神经元发育受阻和进行性神经元丢失相一致，导致不同程度的感觉和自主神经功能障碍。 作为NINDS赞助的神经生成药物筛选联盟的一部分，研究发现激动素，一种植物细胞分裂素和营养食品，作为一种抗氧化剂，在FD细胞中增加了外显子20的包涵体，并显著增加了野生型IKBKAP mRNA和IKAP蛋白的表达，表明激动素是一种潜在的重要的治疗这种遗传性疾病的药物。此外，最近完成的一项初步研究表明，FD患者的白细胞与携带者和非携带者的白细胞中野生型IKBKAP mRNA的水平存在显著差异，这表明这些容易获得的细胞中的野生型IKBKAP mRNA水平可能是一个潜在的有用的药物疗效的分子标志物。 对大鼠和小鼠的动物研究表明，激动素在口服中吸收良好，很容易代谢并分布到血浆和中枢神经系统中。根据对啮齿动物的研究，未观察到的不良反应水平(NOAEL)在雄性为200 mg/kg/d，在雌性为400 mg/kg/d。口服毒性研究(5天)和长期毒性研究(35~90天)表明，激动素没有毒性，直到动物给予极高剂量的激动素，雄性为800 mg/kg/d，雌性为1000 mg/kg/d，比预计的临床剂量高出一个数量级。 研究人员计划评估口服激动素的吸收曲线，以确定激动素在人体内的药代动力学。大鼠的NOAEL将被换算为人体当量剂量(Hed)，使用6.2作为换算因子。对于第1组中的志愿者，这一剂量(32.25 mg/kg/d)将减少10倍，然后后续队列的剂量将根据修改后的费波纳奇剂量递增方案确定。 主要目标是确定激动素在人体内的口服剂量，以达到推测的治疗组织浓度10M(即215.2？g/100cc或2.15g/ml)。次要目标如下：1)评估口服激动素在健康FD剪接突变携带者中的吸收曲线。2)确定口服激动素对FD剪接突变健康携带者是否有短期不良反应。3)研究激动素对健康携带者外周血白细胞野生型IKBKAP基因表达水平的影响。 研究人员将每天给FD突变携带者的父母口服激动素，为期一(1)周。对于每个队列中的志愿者，在口服第一剂激动素后，将通过重复采血(30分钟、1小时、2小时、6小时、12小时、24小时)监测吸收和清除时间。第一组的剂量将是计算的人体当量剂量(HED)的十分之一。[根据啮齿动物研究，未观察到的不良反应水平(NOAEL)在雄性为200 mg/kg/d，在雌性为400 mg/kg/d。大鼠的NOAEL将被换算为人体当量剂量(Hed)，使用6.2作为换算因子。该HED为32.25毫克/公斤/天。]后续队列的剂量将根据修改后的斐波纳契剂量递增方案确定(例如，x、2x、3.3x、5x、7x)。后续队列中的剂量递增将被执行，直到激动素血浆水平达到10？M的计划治疗目标(即215.2？g/100cc或2.15？g/ml)，前提是剂量递增方案中规定的剂量递增没有减少。所有受试者都将有基线和研究结束时的血象和全面的代谢筛查，以检测肝或肾功能障碍。 血清激动素水平的分析将在ADMET实验室进行。对白细胞中野生型IKBKAP信使核糖核酸水平的分析将在马萨诸塞州总医院斯劳根豪特博士的实验室进行。血象和综合代谢测试的血液样本将由GCRC实验室进行。 研究人员将招募大约35名FD剪接突变的强制性携带者。FD携带者将被分成5组，每组由5至7人组成。这项研究的总时间为15个月。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Kinetin, a plant cytokinin and nutraceutical, may be a potential important therapeutic agent for the genetic disorder familial dysautonomia (FD). To test this one first must determine the absorption rate of oral kinetin and the dose necessary to achieve hypothesized therapeutic doses, as well as determine if kinetin does affect genetic mis-splicing by measuring expression of wild-type IKBKAP in white blood cells. Familial dysautonomia (FD) is a rare genetic disease that is caused by mutations in the IKBKAP gene. The most common mutation causes a splicing alteration that leads to reduced protein (IKAP) expression in all tissues, but this reduction is variable and is most severe in neuronal tissue. IKAP, as a member of the human Elongator complex, aids in transcription, so that as a result of decreased IKAP, several other target genes are down-regulated. Many of the down regulated genes are involved in cell migration which may explain FD neuropathology that is consistent with arrested small fiber neuronal development and progressive neuronal loss resulting in variable degrees of sensory and autonomic dysfunction. As part of the NINDS sponsored Neurogeneration Drug Screening Consortium, it was found that kinetin, a plant cytokinin and nutraceutical, that is marketed as an antioxidant, enhanced exon 20 inclusion and dramatically increased expression of wild-type IKBKAP mRNA and IKAP protein in FD cells, suggesting that kinetin is a potential important therapeutic agent for this genetic disorder. Furthermore, a recently completed pilot study has demonstrated that levels of wild-type IKBKAP mRNA are significantly different in white blood cells of FD patients vs. white blood cells of carriers vs. white blood cells of non-carriers suggesting that levels of wild-type IKBKAP mRNA in these easily accessible cells could be a potentially useful molecular marker of drug efficacy. Animal studies in rats and mice indicate that kinetin is well absorbed orally and is readily metabolized and distributed into plasma and into the central nervous system. Based on rodent studies, the no observed adverse effect level (NOAEL) was found to be 200 mg/kg/d in males and 400 mg/kg/d in females. Oral toxicity study (5 day) and longer toxicity studies (35 & 90 day) indicate that kinetin was not toxic until animals were given extremely high doses of 800 mg/kg/d in male and 1000 mg/kg/d in female animals which are orders of magnitude above projected clinical doses. The investigators plan to assess absorption curves of orally administered kinetin to determine pharmacokinetics of kinetin in humans. The NOAEL in rats will be converted to a human equivalent dose (HED) using 6.2 as a conversion factor. This HED (32.25 mg/kg/d) will be reduced by a factor of 10 for the volunteers in cohort 1 and then doses for subsequent cohorts will be determined from the modified Fibonacci dose escalation scheme. The primary objective is to determine the oral dose of kinetin in humans to reach the speculated therapeutic tissue concentration of 10 ¿M (i.e. 215.2 ¿g/100 cc or 2.15¿g/ml). Secondary objectives are as follows: 1) To assess absorption curves of orally administered kinetin in healthy carriers of the FD splicing mutation. 2) To determine if there are any short term adverse effects of oral kinetin administration to healthy carriers of the FD splicing mutation. 3) To determine what dose of administered kinetin significantly increases levels of wild type IKBKAP mRNA in white blood cells of healthy carriers. The investigators will administer oral kinetin daily to parents who are obligatory carriers of the FD mutation for a one (1) week period. For volunteers in each cohort, after the first single dose of orally administered kinetin, absorption and clearance times will be monitored by repeated blood sampling (at 30 min, 1 hr, 2 hr, 6 hr, 12 hr, 24 hr). Dosing for the first cohort will be 1/10th of the calculated human equivalent dose (HED). [Based on rodent studies, the no observed adverse effect level (NOAEL) was found to be 200 mg/kg/d in males and 400 mg/kg/d in females. The NOAEL in rats will be converted to a human equivalent dose (HED) using 6.2 as a conversion factor. This HED is 32.25 mg/kg/d.] Doses for subsequent cohorts will be determined from the modified Fibonacci dose escalation scheme (e.g., x, 2x, 3.3x, 5 x, 7 x). Dose escalation in subsequent cohorts will be performed until kinetin plasma levels reach projected therapeutic goal of 10 ¿M (i.e. 215.2 ¿g/100 cc or 2.15¿g/ml), providing that the dose escalation is not curtailed as specified in the dose escalation scheme. All subjects will have baseline and end of study hemogram and comprehensive metabolic screen to detect liver or renal dysfunction. Analysis of serum kinetin levels will be performed at ADMET Laboratories. Analysis of levels of wild type IKBKAP mRNA in white blood cells will be performed at Massachusetts General Hospital in Dr. Slaugenhaupt's laboratory. Blood samples for hemogram and comprehensive metabolic tests will be performed by GCRC laboratories. The investigators will enroll approximately 35 obligatory carriers of the FD splicing mutation. The FD carriers will be divided into 5 groups; each group will consist of five to seven individuals. The total length of this study will be 15 months.