Search

20-hydroxyeicosatetraenoic acid (20-HETE) is a vasoconstrictive metabolite of arachidonic acid by cytochrome P450 (CYP) 4A11 and 4F2 in humans. Inhibition of 20-HETE formation produces neuroprotective effects in cardiac arrest and stroke preclinical models. Clinical evidence shows that high CSF 20-HETE level is associated with three-fold increased mortality and unfavorable outcomes in subarachnoid hemorrhage patients. These findings suggest that 20-HETE synthase inhibition is a potential therapeutic strategy for neuroprotection after brain injury. HET0016, a 20-HETE inhibitor, did not enter clinical development due to poor solubility and short half-life. Currently, a clinically relevant 20-HETE inhibitor is not available to be evaluated as a therapeutic intervention. The primary goal of this dissertation is to develop novel 20-HETE formation inhibitors that have strong potency and favorable physicochemical, metabolic stability, selectivity, solubility, CYP inhibition, and blood-brain barrier (BBB) permeability. We developed analytical methods to facilitate metabolic stability, CYP inhibition evaluation, and quantify the novel compound in a pharmacokinetic study. After iterations of screening and in vitro evaluation for 143 novel compounds, UPMP107 was identified as a preclinical candidate that had strong potency and advantage of highly metabolically stable, wide selectivity window, and good brain penetration in vitro. Intravenous administration of 107 to postnatal day 17 rats showed that it could penetrate BBB and reduce brain 20-HETE levels. The 20-HETE inhibitory effect was maintained up to 9 hrs after IV administration at 20 mg/kg. UPMP107 exhibited a biphasic plasma concentration-time profile after IV administration. It has a low clearance, an intermediate volume of distribution, and a relatively short half-life. The secondary goal was to optimize marketed CNS therapy, propofol, to improve the neurological outcome. The pharmacokinetics of propofol in an ICU population was described using a population pharmacokinetic model. A two-compartment, zero-order infusion, first-order elimination model best described the profile. This model could be used to explore the relationship between propofol exposure and sedative response to optimize the therapy in the ICU population. Collectively, the work identified a novel compound that inhibits 20-HETE formation and can be used in proof of concept animal studies. Propofol pharmacokinetics was characterized in the ICU population, and the model could potentially be used to explore an exposure-response relationship in the future.

20-hydroxyeicosatetraenoic acid (20-HETE) is a metabolite of arachidonic acid (AA) formed by cytochrome P450 (CYP) 4A11 and CYP4F2 in humans, with potent microvascular constriction activity. Inhibition of 20-HETE formation is neuroprotective in subarachnoid hemorrhage, cardiac arrest and thromboembolic stroke preclinical models. These findings suggest that inhibition of 20-HETE formation is a potential therapeutic strategy for neuroprotection after brain injury. At this point, a clinically relevant 20-HETE inhibitor is not available to be evaluated as a therapeutic intervention. Our goal is to identify a selective, metabolically stable, and potent 20-HETE inhibitor. Test compounds were obtained either via virtual screening against a CYP4F2 homology model or from scaffold hopping from structures of known inhibitors. Four different types of microsomes including human liver microsome (HLM), recombinant CYP4F2 (rCYP4F2), rat liver microsome (RLM) and rat kidney microsome (RKM) were used. AA was incubated in microsomes with/without compound for 20 min. 20-HETE formation rate was quantified using a validated UPLC-MS/MS assay and normalized by vehicle control group. Other eicosanoids including 15-, 12-HETEs, epoxyeicosatrienoic acids (EETs),and dihydroxyeicosatrienoic acids (DiHETs) were monitored simultaneously. Selected compounds were tested in HLM for metabolic stability over 60 minutes. Remaining amount of compound was quantified using UPLC-MS/MS and normalized to corresponding 0-min values. Among 26 compounds, compounds 10 and 26 both inhibited 20-HETE formation in a dosedependent manner. At 2500nM, compound 10 reduced 20-HETE formation to 19.9±1.8%, 24.0±5.5% in rCYP4F2 and HLM compared with control; compound 26 decreased 20-HETE formation to 32.4±6.5%, 34.8±5.1% in rCYP4F2 and HLM, respectively. After structure modification, compound 19 and its hydrochloride salt compound 18 were the most potent and dose-dependently inhibited 20-HETE formation. At 2500nM, compound 19 decreased 20-HETE formation to 4.4±0.4%, 8.6±1.3% in rCYP4F2 and HLM, respectively without inhibitory effects on 15-, 12-HETE, EETs or DiHETS formation. Compounds 10 and 19 were more stable with 91.4±11.0% and 100.4±1.7% remaining compound at 30min in HLM compared to 35.1±5.7% of 3-(4-n-butoxyphenyl)pyrazole. These results suggested that compounds 10, 19 and 26 are potent 20-HETE formation inhibitors, compounds 10 and 19 have improved microsomal stability, and these three compounds can serve as lead compounds for further structure modifications that may lead to novel 20-HETE formation inhibitors.