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Tang, Chenxiao (2016) SCREENING 20-HETE INHIBITORS IN MICROSOMAL INCUBATES USING UPLC-MS/MS. Master's Thesis, University of Pittsburgh. (Unpublished)

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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.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Tang, ChenxiaoCHT86@pitt.eduCHT86
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Thesis AdvisorPoloyac,
Committee MemberMcDermott,
Committee MemberNolin,
Committee MemberXie,
Date: 31 July 2016
Date Type: Publication
Defense Date: 31 May 2016
Approval Date: 31 July 2016
Submission Date: 22 July 2016
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Number of Pages: 65
Institution: University of Pittsburgh
Schools and Programs: School of Pharmacy > Pharmaceutical Sciences
Degree: MS - Master of Science
Thesis Type: Master's Thesis
Refereed: Yes
Uncontrolled Keywords: 20-hydroxyeicosatetraenoic acid; epoxyeicosatrienoic acid; UPLC-MS/MS; microsome incubation; inhibitor
Date Deposited: 31 Jul 2016 21:12
Last Modified: 01 Aug 2016 05:15


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