{"doi":"10.17615/zzta-na83","title":"Impact of Organic Solvents on Cytochrome P450 Probe Reactions: Filling the Gap with (S)-Warfarin and Midazolam Hydroxylation","abstract":"(S)-Warfarin 7-hydroxylation and midazolam 1′-hydroxylation are among the preferred probe substrate reactions for CYP2C9 and CYP3A4/5, respectively. The impact of solvents on enzyme activity, kinetic parameters, and predicted in vivo hepatic clearance (ClH) associated with each reaction has not been evaluated. The effects of increasing concentrations [0.1–2% (v/v)] of six organic solvents (acetonitrile, methanol, ethanol, dimethyl sulfoxide, acetone, isopropanol) were first tested on each reaction using human liver microsomes (HLMs), human intestinal microsomes (midazolam 1′-hydroxylation only), and recombinant enzymes. Across enzyme sources, relative to water, acetonitrile and methanol had the least inhibitory effect on (S)-warfarin 7-hydroxylation (0–58 and 9–96%, respectively); acetonitrile, methanol, and ethanol had the least inhibitory effect on midazolam 1′-hydroxylation (0–29, 0–22, and 0–20%, respectively). Using HLMs, both acetonitrile and methanol (0.1–2%) decreased the Vmax (32–60 and 24–65%, respectively) whereas methanol (2%) increased the Km (100%) of (S)-warfarin-hydroxylation. (S)-Warfarin ClH was underpredicted by 21–65% (acetonitrile) and 13–84% (methanol). Acetonitrile, methanol, and ethanol had minimal to modest impact on both the kinetics of midazolam 1′-hydroxylation (10–24%) and predicted midazolam ClH (2–20%). In conclusion, either acetonitrile or methanol at ≤0.1% is recommended as the primary organic solvent for the (S)-warfarin 7-hydroxylation reaction; acetonitrile is preferred if higher solvent concentrations are required. Acetonitrile, methanol, and ethanol at ≤2% are recommended as primary organic solvents for the midazolam 1′-hydroxylation reaction. This information should facilitate optimization of experimental conditions and improve the interpretation and accuracy of in vitro-in vivo predictions involving these two preferred cytochrome P450 probe substrate reactions.","journal":"UNC Libraries","year":2020,"id":112981,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":2,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9562,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2020-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":534383,"name":"L. C. Wienkers","orcid":null,"position":1,"is_corresponding":false},{"id":532352,"name":"A. S. Bridges","orcid":null,"position":2,"is_corresponding":false},{"id":534384,"name":"E. A. Connolly","orcid":null,"position":3,"is_corresponding":false},{"id":534385,"name":"V. Gonzalez-Perez","orcid":null,"position":4,"is_corresponding":false},{"id":435184,"name":"M. F. Paine","orcid":null,"position":0,"is_corresponding":true}],"reference_count":0,"raw_metadata":null,"created_at":"2026-07-18T23:13:17.845093Z","pmid":null,"pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}