Direct comparison of UDP-glucuronosyltransferase and cytochrome P450 activities in human liver microsomes, plated and suspended primary human hepatocytes from five liver donors

Shalenie P den Braver-Sewradj, Michiel W den Braver, Audrey Baze, Joachim Decorde, Massimiliano Fonsi, Philippe Bachellier, Nico P E Vermeulen, Jan N M Commandeur, Lysiane Richert, J Chris Vos

Research output: Scientific - peer-reviewArticle

Abstract

UDP-glucuronosyltransferases (UGTs) and cytochrome P450s (CYPs) are the major enzymes involved in hepatic metabolism of drugs. Hepatic drug metabolism is commonly investigated using human liver microsomes (HLM) or primary human hepatocytes (PHH). We describe the development of a sensitive assay to phenotype activities of six major hepatic UGT isoforms (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9 and UGT2B7) in intact PHH by analysis of glucuronidation of selective probe substrates. The non-selective, general substrate 7-hydroxycoumarin was included for comparison. For each liver donor preparation (five donors) UGT activities in cryopreserved suspended and plated PHH were compared to HLM prepared from the same donors. Standard CYP reaction phenotyping of seven major isoforms was performed in parallel. For all donors, CYP- and UGT-isoforms activity profiles were comparable in PHH and HLM, indicating that reaction phenotyping with selective probe substrates in intact cells primarily reflects respective CYP or UGT activity. System-dependent effects on UGT and CYP isoform activity were still found. While UGT activity of UGT1A1 was equivalent in plated and suspended PHH, UGT1A3, UGT1A6 and UGT2B7 activity was higher in suspended PHH and UGT1A9 and UGT1A4 activity was higher in plated PHH. The well-known decrease in activity of most CYP isoforms in plated compared to suspended PHH was confirmed. Importantly, we found a significant loss in CYP2C19 and CYP2B6 in HLM, activity being lower than in intact cells. Taken together, these findings implicate that, dependent on the UGT or CYP isoforms involved in the metabolism of a given compound, the outcome of metabolic assays is strongly dependent on the choice of the in vitro system. The currently described UGT- and CYP- activity profiling method can be used as a standard assay in intact cells and can especially aid in reaction phenotyping of in vitro systems for which a limited number of cells are available.

Original languageEnglish
Pages (from-to)96-110
Number of pages15
JournalEuropean Journal of Pharmaceutical Sciences
Volume109
DOIs
StateE-pub ahead of print - 1 Aug 2017

Cite this

den Braver-Sewradj, Shalenie P; den Braver, Michiel W; Baze, Audrey; Decorde, Joachim; Fonsi, Massimiliano; Bachellier, Philippe; Vermeulen, Nico P E; Commandeur, Jan N M; Richert, Lysiane; Vos, J Chris / Direct comparison of UDP-glucuronosyltransferase and cytochrome P450 activities in human liver microsomes, plated and suspended primary human hepatocytes from five liver donors.

In: European Journal of Pharmaceutical Sciences, Vol. 109, 01.08.2017, p. 96-110.

Research output: Scientific - peer-reviewArticle

@article{9b943383e4af478abf69e4851dc6f7fb,
title = "Direct comparison of UDP-glucuronosyltransferase and cytochrome P450 activities in human liver microsomes, plated and suspended primary human hepatocytes from five liver donors",
abstract = "UDP-glucuronosyltransferases (UGTs) and cytochrome P450s (CYPs) are the major enzymes involved in hepatic metabolism of drugs. Hepatic drug metabolism is commonly investigated using human liver microsomes (HLM) or primary human hepatocytes (PHH). We describe the development of a sensitive assay to phenotype activities of six major hepatic UGT isoforms (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9 and UGT2B7) in intact PHH by analysis of glucuronidation of selective probe substrates. The non-selective, general substrate 7-hydroxycoumarin was included for comparison. For each liver donor preparation (five donors) UGT activities in cryopreserved suspended and plated PHH were compared to HLM prepared from the same donors. Standard CYP reaction phenotyping of seven major isoforms was performed in parallel. For all donors, CYP- and UGT-isoforms activity profiles were comparable in PHH and HLM, indicating that reaction phenotyping with selective probe substrates in intact cells primarily reflects respective CYP or UGT activity. System-dependent effects on UGT and CYP isoform activity were still found. While UGT activity of UGT1A1 was equivalent in plated and suspended PHH, UGT1A3, UGT1A6 and UGT2B7 activity was higher in suspended PHH and UGT1A9 and UGT1A4 activity was higher in plated PHH. The well-known decrease in activity of most CYP isoforms in plated compared to suspended PHH was confirmed. Importantly, we found a significant loss in CYP2C19 and CYP2B6 in HLM, activity being lower than in intact cells. Taken together, these findings implicate that, dependent on the UGT or CYP isoforms involved in the metabolism of a given compound, the outcome of metabolic assays is strongly dependent on the choice of the in vitro system. The currently described UGT- and CYP- activity profiling method can be used as a standard assay in intact cells and can especially aid in reaction phenotyping of in vitro systems for which a limited number of cells are available.",
keywords = "Journal Article",
author = "{den Braver-Sewradj}, {Shalenie P} and {den Braver}, {Michiel W} and Audrey Baze and Joachim Decorde and Massimiliano Fonsi and Philippe Bachellier and Vermeulen, {Nico P E} and Commandeur, {Jan N M} and Lysiane Richert and Vos, {J Chris}",
note = "Copyright © 2017. Published by Elsevier B.V.",
year = "2017",
month = "8",
doi = "10.1016/j.ejps.2017.07.032",
volume = "109",
pages = "96--110",
journal = "European Journal of Pharmaceutical Sciences",
issn = "0928-0987",
publisher = "Elsevier",

}

Direct comparison of UDP-glucuronosyltransferase and cytochrome P450 activities in human liver microsomes, plated and suspended primary human hepatocytes from five liver donors. / den Braver-Sewradj, Shalenie P; den Braver, Michiel W; Baze, Audrey; Decorde, Joachim; Fonsi, Massimiliano; Bachellier, Philippe; Vermeulen, Nico P E; Commandeur, Jan N M; Richert, Lysiane; Vos, J Chris.

In: European Journal of Pharmaceutical Sciences, Vol. 109, 01.08.2017, p. 96-110.

Research output: Scientific - peer-reviewArticle

TY - JOUR

T1 - Direct comparison of UDP-glucuronosyltransferase and cytochrome P450 activities in human liver microsomes, plated and suspended primary human hepatocytes from five liver donors

AU - den Braver-Sewradj,Shalenie P

AU - den Braver,Michiel W

AU - Baze,Audrey

AU - Decorde,Joachim

AU - Fonsi,Massimiliano

AU - Bachellier,Philippe

AU - Vermeulen,Nico P E

AU - Commandeur,Jan N M

AU - Richert,Lysiane

AU - Vos,J Chris

N1 - Copyright © 2017. Published by Elsevier B.V.

PY - 2017/8/1

Y1 - 2017/8/1

N2 - UDP-glucuronosyltransferases (UGTs) and cytochrome P450s (CYPs) are the major enzymes involved in hepatic metabolism of drugs. Hepatic drug metabolism is commonly investigated using human liver microsomes (HLM) or primary human hepatocytes (PHH). We describe the development of a sensitive assay to phenotype activities of six major hepatic UGT isoforms (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9 and UGT2B7) in intact PHH by analysis of glucuronidation of selective probe substrates. The non-selective, general substrate 7-hydroxycoumarin was included for comparison. For each liver donor preparation (five donors) UGT activities in cryopreserved suspended and plated PHH were compared to HLM prepared from the same donors. Standard CYP reaction phenotyping of seven major isoforms was performed in parallel. For all donors, CYP- and UGT-isoforms activity profiles were comparable in PHH and HLM, indicating that reaction phenotyping with selective probe substrates in intact cells primarily reflects respective CYP or UGT activity. System-dependent effects on UGT and CYP isoform activity were still found. While UGT activity of UGT1A1 was equivalent in plated and suspended PHH, UGT1A3, UGT1A6 and UGT2B7 activity was higher in suspended PHH and UGT1A9 and UGT1A4 activity was higher in plated PHH. The well-known decrease in activity of most CYP isoforms in plated compared to suspended PHH was confirmed. Importantly, we found a significant loss in CYP2C19 and CYP2B6 in HLM, activity being lower than in intact cells. Taken together, these findings implicate that, dependent on the UGT or CYP isoforms involved in the metabolism of a given compound, the outcome of metabolic assays is strongly dependent on the choice of the in vitro system. The currently described UGT- and CYP- activity profiling method can be used as a standard assay in intact cells and can especially aid in reaction phenotyping of in vitro systems for which a limited number of cells are available.

AB - UDP-glucuronosyltransferases (UGTs) and cytochrome P450s (CYPs) are the major enzymes involved in hepatic metabolism of drugs. Hepatic drug metabolism is commonly investigated using human liver microsomes (HLM) or primary human hepatocytes (PHH). We describe the development of a sensitive assay to phenotype activities of six major hepatic UGT isoforms (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9 and UGT2B7) in intact PHH by analysis of glucuronidation of selective probe substrates. The non-selective, general substrate 7-hydroxycoumarin was included for comparison. For each liver donor preparation (five donors) UGT activities in cryopreserved suspended and plated PHH were compared to HLM prepared from the same donors. Standard CYP reaction phenotyping of seven major isoforms was performed in parallel. For all donors, CYP- and UGT-isoforms activity profiles were comparable in PHH and HLM, indicating that reaction phenotyping with selective probe substrates in intact cells primarily reflects respective CYP or UGT activity. System-dependent effects on UGT and CYP isoform activity were still found. While UGT activity of UGT1A1 was equivalent in plated and suspended PHH, UGT1A3, UGT1A6 and UGT2B7 activity was higher in suspended PHH and UGT1A9 and UGT1A4 activity was higher in plated PHH. The well-known decrease in activity of most CYP isoforms in plated compared to suspended PHH was confirmed. Importantly, we found a significant loss in CYP2C19 and CYP2B6 in HLM, activity being lower than in intact cells. Taken together, these findings implicate that, dependent on the UGT or CYP isoforms involved in the metabolism of a given compound, the outcome of metabolic assays is strongly dependent on the choice of the in vitro system. The currently described UGT- and CYP- activity profiling method can be used as a standard assay in intact cells and can especially aid in reaction phenotyping of in vitro systems for which a limited number of cells are available.

KW - Journal Article

U2 - 10.1016/j.ejps.2017.07.032

DO - 10.1016/j.ejps.2017.07.032

M3 - Article

VL - 109

SP - 96

EP - 110

JO - European Journal of Pharmaceutical Sciences

T2 - European Journal of Pharmaceutical Sciences

JF - European Journal of Pharmaceutical Sciences

SN - 0928-0987

ER -