Clinical pharmacokinetics study of phenytoin in epilepsy patients & expression of oxysterol 7 Alpha - hydroxylase (hCYP7B1) in E.coli

Abstract

The determination of pharmacokinetic parameters is crucial both for clinical studies and early in the drug discovery and development. This thesis describes a clinical pharmacokinetics study of one anticonvulsant drug, i.e. phenytoin (PHT) and the early screening study of potential chemopreventive agent for prostate cancer. PHT is extensively bound to plasma proteins, is excreted from the body as oxidative metabolites in the urine, and exhibits a non-linear pharmacokinetics profile. In this study, stable-labeled PHT was given either intravenously or intramuscularly. The simultaneous administration of oral and IV PHT enables a direct determination of the pharmacokinetic parameters of clearance, volume, half-life, and absolute oral bioavailability. Urine samples from epilepsy patients who were on maintenance therapy of PHT were collected from 0-12 hours and 12-24 hours after a single daily dose to measure the two principal PHT urinary metabolites, 5-(4-hydroxyphenyl)-5-phenylhydantoin (p-HPPH) and 5-(3,4-dihydroxy-1,5-cyclohexadien-1-yl)-5-phenylhydantoin (DHD). An isocratic HPLC-NI-APCI-MS method was used to quantify metabolites in urine. A weak, but significant, Spearman Rank Correlation was observed between the total urinary metabolites recovered and the oral bioavailability (p-value = 0.00924, r2= 0.166). The percent of dose recovered in urine ranged from 35.4% ± 15.7% in young adult patients (age 21-49 years old) and 32.9% ± 15.0% in patients of age 65-93 years indicating highly variable absorption. In contrast, absolute bioavailability was 0.864 ± 0.194 and 0.925 ± 0.252 for the two groups, as determined by the stable-isotope technique. Unaccounted biliary-fecal excretion of p-HPPH glucuronide, subjects’ noncompliance, and incomplete urine collection are possible explanations. Consequently, bioavailability is best determined by stable-isotope method. Chapter 2 of this thesis illustrates the early screening study of chemopreventive agent for prostate cancer. The rationale of this study was attempting to inhibit the metabolism of 5-androstane-3,17β-diol (3-Adiol). A metabolite of dihydrotestosterone, 3-Adiol, inhibits LNCaP prostate cell proliferation in the presence of Estrogen Receptor . CYP7B1 is the enzyme responsible for catalyzing the 6 and 7 hydroxylations of 3-Adiol in prostate. In this study, expression and purification of human CYP7B1 in E.coli was attempted. Despite spectroscopic evidence of P450 expression, the enzyme failed to turn over its endogenous substrate, DHEA to 7-hydroxy DHEA.