This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor 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.
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