{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,18]],"date-time":"2026-08-18T13:37:03Z","timestamp":1787060223646,"version":"build-2736575974"},"reference-count":35,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2023,7,27]],"date-time":"2023-07-27T00:00:00Z","timestamp":1690416000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000027","name":"National Institute on Alcohol Abuse and Alcoholism of the National Institutes of Health","doi-asserted-by":"publisher","award":["R01AA030155"],"award-info":[{"award-number":["R01AA030155"]}],"id":[{"id":"10.13039\/100000027","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Biology"],"abstract":"<jats:p>In a search for a reliable, inexpensive, and versatile technique for high-throughput kinetic assays of drug metabolism, we elected to rehire an old-school approach based on the determination of formaldehyde (FA) formed in cytochrome P450-dependent demethylation reactions. After evaluating several fluorometric techniques for FA detection, we chose the method based on the Hantzsch reaction with acetoacetanilide as the most sensitive, robust, and adaptable to high-throughput implementation. Here we provide a detailed protocol for using our new technique for automatized assays of cytochrome P450-dependent drug demethylations and discuss its applicability for high-throughput scanning of drug metabolism pathways in the human liver. To probe our method further, we applied it to re-evaluating the pathways of metabolism of ketamine, a dissociative anesthetic and potent antidepressant increasingly used in the treatment of alcohol withdrawal syndrome. Probing the kinetic parameters of ketamine demethylation by ten major cytochrome P450 (CYP) enzymes, we demonstrate that in addition to CYP2B6 and CYP3A enzymes, which were initially recognized as the primary metabolizers of ketamine, an important role is also played by CYP2C19 and CYP2D6. At the same time, the involvement of CYP2C9 suggested in the previous reports was deemed insignificant.<\/jats:p>","DOI":"10.3390\/biology12081055","type":"journal-article","created":{"date-parts":[[2023,7,28]],"date-time":"2023-07-28T01:51:47Z","timestamp":1690509107000},"page":"1055","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["High-Throughput Assay of Cytochrome P450-Dependent Drug Demethylation Reactions and Its Use to Re-Evaluate the Pathways of Ketamine Metabolism"],"prefix":"10.3390","volume":"12","author":[{"given":"Nadezhda Y.","family":"Davydova","sequence":"first","affiliation":[{"name":"Department of Chemistry, Washington State University, Pullman, WA 99164, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"David A.","family":"Hutner","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Washington State University, Pullman, WA 99164, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kari A.","family":"Gaither","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Sciences, Washington State University, Spokane, WA 99202, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7248-6714","authenticated-orcid":false,"given":"Dilip Kumar","family":"Singh","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Sciences, Washington State University, Spokane, WA 99202, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Bhagwat","family":"Prasad","sequence":"additional","affiliation":[{"name":"Department of Pharmaceutical Sciences, Washington State University, Spokane, WA 99202, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8520-0033","authenticated-orcid":false,"given":"Dmitri R.","family":"Davydov","sequence":"additional","affiliation":[{"name":"Department of Chemistry, Washington State University, Pullman, WA 99164, USA"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2023,7,27]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"705","DOI":"10.1016\/j.drudis.2021.11.005","article-title":"The role of DMPK science in improving pharmaceutical research and development efficiency","volume":"27","author":"Mak","year":"2022","journal-title":"Drug Discov. 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