KINETIN IN CARRIERS OF THE FAMILIAL DYSAUTONOMIA MIS-SPLICING MUTATION

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 激动素是一种植物细胞分裂素，也是一种营养药物，可能成为家族性植物神经功能异常（FD）的重要治疗药物。为了测试这一点，首先必须确定口服激动素的吸收率和达到假设治疗剂量所需的剂量，以及通过测量白色血细胞中野生型IKBKAP的表达来确定激动素是否确实影响遗传错误剪接。 家族性自主神经功能障碍（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。IKAP作为人类延长子复合体的成员，有助于转录，因此作为IKAP减少的结果，其他几个靶基因被下调。许多下调的基因参与细胞迁移，这可以解释FD神经病理学，其与导致不同程度的感觉和自主神经功能障碍的小纤维神经元发育停滞和进行性神经元损失一致。 作为NINDS赞助的神经生成药物筛选联盟的一部分，发现激动素，一种作为抗氧化剂销售的植物细胞分裂素和营养药，增强了FD细胞中20号外显子的包含，并显着增加了野生型IKBKAP mRNA和IKAP蛋白的表达，表明激动素是这种遗传性疾病的潜在重要治疗剂。此外，最近完成的初步研究表明，野生型IKBKAP mRNA的水平在FD患者的白色血细胞、携带者的白色血细胞和非携带者的白色血细胞中显著不同，这表明 在这些容易获得的细胞中野生型IKBKAPmRNA的水平可能是药物功效的潜在有用的分子标记。 大鼠和小鼠的动物研究表明，激动素口服吸收良好，易于代谢并分布到血浆和中枢神经系统中。根据啮齿动物研究，雄性动物的无明显不良作用水平（NOAEL）为200 mg/kg/d，雌性动物为400 mg/kg/d。 口服毒性研究（5天）和更长时间的毒性研究（35和90天）表明，激动素在给予极高剂量（雄性动物800 mg/kg/d，雌性动物1000 mg/kg/d，高于预期临床剂量的数量级）之前没有毒性。 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中的药代动力学。将使用6.2作为转换因子，将大鼠中的NOAEL转换为人体等效剂量（HED）。对于队列1中的志愿者，该HED（32.25 mg/kg/d）将减少10倍，然后根据改良的Fibonacci剂量递增方案确定后续队列的剂量。 主要目的是确定激动素在人体中的口服剂量，以达到推测的治疗组织浓度10 μ M（即215.2 μ g/100 cc或2.15 μ g/ml）。 次要目的如下：1）评估口服施用的激动素在FD剪接突变的健康携带者中的吸收曲线。 2)确定FD剪接突变的健康携带者口服激动素是否有任何短期不良反应。 3)确定给予何种剂量的激动素显著增加健康携带者的白色血细胞中野生型IKBKAP mRNA的水平。 研究者将对FD突变强制性携带者的父母每天口服激动素，持续1周。 对于每个队列中的志愿者，首次单次口服激动素后，将通过重复采血（30 min、1 h、2 h、6 h、12 h、24 h）监测吸收和清除时间。 第一个队列的剂量为计算的人等效剂量（HED）的1/10。 [根据啮齿动物研究，雄性和雌性动物的无明显不良作用水平（NOAEL）分别为200 mg/kg/d和400 mg/kg/d。将使用6.2作为转换因子，将大鼠中的NOAEL转换为人体等效剂量（HED）。该HED为32.25 mg/kg/d。] 后续组群的剂量将根据修改的斐波那契剂量递增方案（例如，x、2x、3.3x、5x、7x）。在后续队列中进行剂量递增，直至激动素血浆水平达到预计的治疗目标10 μ M（即215.2 μ g/100 cc或2.15 μ g/ml），前提是剂量递增未按照剂量递增方案中的规定缩减。 所有受试者都将进行基线和研究结束时的血象和全面代谢筛查，以检测肝或肾功能不全。 血清激动素水平分析将在ADMET Laboratories进行。白色血细胞中野生型IKBKAP mRNA水平的分析将在马萨诸塞州综合医院Slaugenhaupt博士的实验室进行。将由GCRC实验室采集血样进行血象和综合代谢检查。 研究人员将招募大约35名FD剪接突变的强制性携带者。FD携带者将被分为5组;每组将由5至7人组成。 本研究的总时长为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.