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Use of Phenotypically Poor Metabolizer Individual Donor Human Liver Microsomes To Identify Selective Substrates of UGT2B10.
Drug Metab Dispos. 2020 03; 48(3):176-186.DM

Abstract

UDP-glucuronosyltransferase (UGT)1A4 and UGT2B10 are the human UGT isoforms most frequently involved in N-glucuronidation of drugs. UGT2B10 exhibits higher affinity than UGT1A4 for numerous substrates, making it potentially the more important enzyme for metabolism of these compounds in vivo. Clinically relevant UGT2B10 polymorphisms, including a null activity splice site mutation common in African populations, can lead to large exposure differences for UGT2B10 substrates that may limit their developability as marketed drugs. UGT phenotyping approaches using recombinantly expressed UGTs are limited by low enzyme activity and lack of validation of scaling to in vivo. In this study, we describe the use of an efficient experimental protocol for identification of UGT2B10-selective substrates (i.e., those with high fraction metabolized by UGT2B10), which exploits the activity difference between pooled human liver microsomes (HLM) and HLM from a phenotypically UGT2B10 poor metabolizer donor. Following characterization of the approach with eight known UGT2B10 substrates, we used ligand-based virtual screening and literature precedents to select 24 potential UGT2B10 substrates of 140 UGT-metabolized drugs for testing. Of these, dothiepin, cidoxepin, cyclobenzaprine, azatadine, cyproheptadine, bifonazole, and asenapine were indicated to be selective UGT2B10 substrates that have not previously been described. UGT phenotyping experiments and tests comparing conjugative and oxidative clearance were then used to confirm these findings. These approaches provide rapid and sensitive ways to evaluate whether a potential drug candidate cleared via glucuronidation will be sensitive to UGT2B10 polymorphisms in vivo. SIGNIFICANCE STATEMENT: The role of highly polymorphic UDP-glucuronosyltransferase (UGT)2B10 is likely to be underestimated currently for many compounds cleared via N-glucuronidation due to high test concentrations often used in vitro and low activity of UGT2B10 preparations. The methodology described in this study can be combined with the assessment of UGT versus oxidative in vitro metabolism to rapidly identify compounds likely to be sensitive to UGT2B10 polymorphism (high fraction metabolized by UGT2B10), enabling either chemical modification or polymorphism risk assessment before candidate selection.

Authors+Show Affiliations

Pharmaceutical Sciences, Roche Pharma Research and Early Development, Roche Innovation Centre Basel, Basel, Switzerland (N.M., N.Q., B.M., S.F.); Department of Chemistry, Biology, and Biotechnology, University of Perugia, Perugia, Italy (N.M., G.C.); and Department of Pharmaceutics, Center for Pharmacometrics and Systems Pharmacology, University of Florida at Lake Nona, Orlando, Florida (J.B.).Pharmaceutical Sciences, Roche Pharma Research and Early Development, Roche Innovation Centre Basel, Basel, Switzerland (N.M., N.Q., B.M., S.F.); Department of Chemistry, Biology, and Biotechnology, University of Perugia, Perugia, Italy (N.M., G.C.); and Department of Pharmaceutics, Center for Pharmacometrics and Systems Pharmacology, University of Florida at Lake Nona, Orlando, Florida (J.B.).Pharmaceutical Sciences, Roche Pharma Research and Early Development, Roche Innovation Centre Basel, Basel, Switzerland (N.M., N.Q., B.M., S.F.); Department of Chemistry, Biology, and Biotechnology, University of Perugia, Perugia, Italy (N.M., G.C.); and Department of Pharmaceutics, Center for Pharmacometrics and Systems Pharmacology, University of Florida at Lake Nona, Orlando, Florida (J.B.).Pharmaceutical Sciences, Roche Pharma Research and Early Development, Roche Innovation Centre Basel, Basel, Switzerland (N.M., N.Q., B.M., S.F.); Department of Chemistry, Biology, and Biotechnology, University of Perugia, Perugia, Italy (N.M., G.C.); and Department of Pharmaceutics, Center for Pharmacometrics and Systems Pharmacology, University of Florida at Lake Nona, Orlando, Florida (J.B.).Pharmaceutical Sciences, Roche Pharma Research and Early Development, Roche Innovation Centre Basel, Basel, Switzerland (N.M., N.Q., B.M., S.F.); Department of Chemistry, Biology, and Biotechnology, University of Perugia, Perugia, Italy (N.M., G.C.); and Department of Pharmaceutics, Center for Pharmacometrics and Systems Pharmacology, University of Florida at Lake Nona, Orlando, Florida (J.B.).Pharmaceutical Sciences, Roche Pharma Research and Early Development, Roche Innovation Centre Basel, Basel, Switzerland (N.M., N.Q., B.M., S.F.); Department of Chemistry, Biology, and Biotechnology, University of Perugia, Perugia, Italy (N.M., G.C.); and Department of Pharmaceutics, Center for Pharmacometrics and Systems Pharmacology, University of Florida at Lake Nona, Orlando, Florida (J.B.) stephen.fowler@roche.com.

Pub Type(s)

Journal Article
Research Support, N.I.H., Extramural
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

31839590

Citation

Milani, Nicolo, et al. "Use of Phenotypically Poor Metabolizer Individual Donor Human Liver Microsomes to Identify Selective Substrates of UGT2B10." Drug Metabolism and Disposition: the Biological Fate of Chemicals, vol. 48, no. 3, 2020, pp. 176-186.
Milani N, Qiu N, Molitor B, et al. Use of Phenotypically Poor Metabolizer Individual Donor Human Liver Microsomes To Identify Selective Substrates of UGT2B10. Drug Metab Dispos. 2020;48(3):176-186.
Milani, N., Qiu, N., Molitor, B., Badée, J., Cruciani, G., & Fowler, S. (2020). Use of Phenotypically Poor Metabolizer Individual Donor Human Liver Microsomes To Identify Selective Substrates of UGT2B10. Drug Metabolism and Disposition: the Biological Fate of Chemicals, 48(3), 176-186. https://doi.org/10.1124/dmd.119.089482
Milani N, et al. Use of Phenotypically Poor Metabolizer Individual Donor Human Liver Microsomes to Identify Selective Substrates of UGT2B10. Drug Metab Dispos. 2020;48(3):176-186. PubMed PMID: 31839590.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Use of Phenotypically Poor Metabolizer Individual Donor Human Liver Microsomes To Identify Selective Substrates of UGT2B10. AU - Milani,Nicolo, AU - Qiu,NaHong, AU - Molitor,Birgit, AU - Badée,Justine, AU - Cruciani,Gabriele, AU - Fowler,Stephen, Y1 - 2019/12/15/ PY - 2019/09/23/received PY - 2019/12/02/accepted PY - 2019/12/17/pubmed PY - 2019/12/17/medline PY - 2019/12/17/entrez SP - 176 EP - 186 JF - Drug metabolism and disposition: the biological fate of chemicals JO - Drug Metab. Dispos. VL - 48 IS - 3 N2 - UDP-glucuronosyltransferase (UGT)1A4 and UGT2B10 are the human UGT isoforms most frequently involved in N-glucuronidation of drugs. UGT2B10 exhibits higher affinity than UGT1A4 for numerous substrates, making it potentially the more important enzyme for metabolism of these compounds in vivo. Clinically relevant UGT2B10 polymorphisms, including a null activity splice site mutation common in African populations, can lead to large exposure differences for UGT2B10 substrates that may limit their developability as marketed drugs. UGT phenotyping approaches using recombinantly expressed UGTs are limited by low enzyme activity and lack of validation of scaling to in vivo. In this study, we describe the use of an efficient experimental protocol for identification of UGT2B10-selective substrates (i.e., those with high fraction metabolized by UGT2B10), which exploits the activity difference between pooled human liver microsomes (HLM) and HLM from a phenotypically UGT2B10 poor metabolizer donor. Following characterization of the approach with eight known UGT2B10 substrates, we used ligand-based virtual screening and literature precedents to select 24 potential UGT2B10 substrates of 140 UGT-metabolized drugs for testing. Of these, dothiepin, cidoxepin, cyclobenzaprine, azatadine, cyproheptadine, bifonazole, and asenapine were indicated to be selective UGT2B10 substrates that have not previously been described. UGT phenotyping experiments and tests comparing conjugative and oxidative clearance were then used to confirm these findings. These approaches provide rapid and sensitive ways to evaluate whether a potential drug candidate cleared via glucuronidation will be sensitive to UGT2B10 polymorphisms in vivo. SIGNIFICANCE STATEMENT: The role of highly polymorphic UDP-glucuronosyltransferase (UGT)2B10 is likely to be underestimated currently for many compounds cleared via N-glucuronidation due to high test concentrations often used in vitro and low activity of UGT2B10 preparations. The methodology described in this study can be combined with the assessment of UGT versus oxidative in vitro metabolism to rapidly identify compounds likely to be sensitive to UGT2B10 polymorphism (high fraction metabolized by UGT2B10), enabling either chemical modification or polymorphism risk assessment before candidate selection. SN - 1521-009X UR - https://www.unboundmedicine.com/medline/citation/31839590/Use_of_Phenotypically_Poor_Metabolizer_Individual_Donor_Human_Liver_Microsomes_To_Identify_Selective_Substrates_of_UGT2B10 L2 - http://dmd.aspetjournals.org/cgi/pmidlookup?view=long&pmid=31839590 DB - PRIME DP - Unbound Medicine ER -
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