Identification of cytochrome P450 isoforms involved in the metabolism of loperamide in human liver microsomes

Kyoung Ah Kim, Jaegul Chung, Dong Hae Jung, Ji-Young Park

Research output: Contribution to journalArticle

67 Citations (Scopus)

Abstract

Objective: The purpose of the present study was to elucidate the cytochrome P450 (P450) isoform(s) involved in the metabolism of loperamide (LOP) to N-demethylated LOP (DLOP) in human liver microsomes. Methods: Three established approaches were used to identify the P450 isoforms responsible for LOP N-demethylation using human liver microsomes and cDNA-expressed P450 isoforms: (1) correlation of LOP N-demethylation activity with marker P450 activities in a panel of human liver microsomes, (2) inhibition of enzyme activity by P450-selective inhibitors, and (3) measurement of DLOP formation by cDNA-expressed P450 isoforms. The relative contribution of P450 isoforms involved in LOP N-demethylation in human liver microsomes were estimated by applying relative activity factor (RAF) values. Results: The formation rate of DLOP showed biphasic kinetics, suggesting the involvement of multiple P450 isoforms. Apparent Km and Vmax values were 21.1 μM and 122.3 pmol/min per milligram of protein for the high-affinity component and 83.9 μM and 412.0 pmol/ min per milligram of protein for the low-affinity component, respectively. Of the cDNA-expressed P450 s tested, CYP2B6, CYP2C8, CYP2D6, and CYP3A4 catalyzed LOP N-demethylation. LOP N-demethylation was significantly inhibited when coincubated with quercetin (a CYP2C8 inhibitor) and ketoconazole (a CYP3A4 inhibitor) by 40 and 90%, respectively, but other chemical inhibitors tested showed weak or no significant inhibition. DLOP formation was highly correlated with CYP3A4-catalyzed midazolam 1-hydroxylation (rs = 0.829; P < 0.01), CYP2B6-catalzyed 7-ethoxy4- trifluoromethylcoumarin O-deethylation (rs = 0.691; P < 0.05), and CYP2C8-catalyzed paclitaxel 6α-hydroxylation (rs = 0.797; P < 0.05). Conclusion: CYP2B6, CYP2C8, CYP2D6, and CYP3A4 catalyze LOP N-demethylation in human liver microsomes, and among them, CYP2C8 and CYP3A4 may play a crucial role in LOP metabolism at the therapeutic concentrations of LOP. Coadministration of these P450 inhibitors may cause drug interactions with LOP. However, the clinical significance of potential interaction of LOP metabolism by CYP2C8 and CYP3A4 inhibitors should be studied further.

Original languageEnglish
Pages (from-to)575-581
Number of pages7
JournalEuropean Journal of Clinical Pharmacology
Volume60
Issue number8
DOIs
Publication statusPublished - 2004 Oct 1
Externally publishedYes

Fingerprint

Loperamide
Liver Microsomes
Cytochrome P-450 Enzyme System
Protein Isoforms
Cytochrome P-450 CYP3A
Cytochrome P-450 CYP2D6
Complementary DNA
Hydroxylation
Myeloma Proteins
Ketoconazole
Midazolam
Quercetin
Paclitaxel
Drug Interactions

Keywords

  • CYP2C8
  • CYP3A4
  • Cytochrome P450 (P450)
  • Human liver microsomes
  • Loperamide

ASJC Scopus subject areas

  • Pharmacology
  • Pharmacology (medical)

Cite this

Identification of cytochrome P450 isoforms involved in the metabolism of loperamide in human liver microsomes. / Kim, Kyoung Ah; Chung, Jaegul; Jung, Dong Hae; Park, Ji-Young.

In: European Journal of Clinical Pharmacology, Vol. 60, No. 8, 01.10.2004, p. 575-581.

Research output: Contribution to journalArticle

@article{1aaa4e9a258c4f0883f16e75875f7a7c,
title = "Identification of cytochrome P450 isoforms involved in the metabolism of loperamide in human liver microsomes",
abstract = "Objective: The purpose of the present study was to elucidate the cytochrome P450 (P450) isoform(s) involved in the metabolism of loperamide (LOP) to N-demethylated LOP (DLOP) in human liver microsomes. Methods: Three established approaches were used to identify the P450 isoforms responsible for LOP N-demethylation using human liver microsomes and cDNA-expressed P450 isoforms: (1) correlation of LOP N-demethylation activity with marker P450 activities in a panel of human liver microsomes, (2) inhibition of enzyme activity by P450-selective inhibitors, and (3) measurement of DLOP formation by cDNA-expressed P450 isoforms. The relative contribution of P450 isoforms involved in LOP N-demethylation in human liver microsomes were estimated by applying relative activity factor (RAF) values. Results: The formation rate of DLOP showed biphasic kinetics, suggesting the involvement of multiple P450 isoforms. Apparent Km and Vmax values were 21.1 μM and 122.3 pmol/min per milligram of protein for the high-affinity component and 83.9 μM and 412.0 pmol/ min per milligram of protein for the low-affinity component, respectively. Of the cDNA-expressed P450 s tested, CYP2B6, CYP2C8, CYP2D6, and CYP3A4 catalyzed LOP N-demethylation. LOP N-demethylation was significantly inhibited when coincubated with quercetin (a CYP2C8 inhibitor) and ketoconazole (a CYP3A4 inhibitor) by 40 and 90{\%}, respectively, but other chemical inhibitors tested showed weak or no significant inhibition. DLOP formation was highly correlated with CYP3A4-catalyzed midazolam 1-hydroxylation (rs = 0.829; P < 0.01), CYP2B6-catalzyed 7-ethoxy4- trifluoromethylcoumarin O-deethylation (rs = 0.691; P < 0.05), and CYP2C8-catalyzed paclitaxel 6α-hydroxylation (rs = 0.797; P < 0.05). Conclusion: CYP2B6, CYP2C8, CYP2D6, and CYP3A4 catalyze LOP N-demethylation in human liver microsomes, and among them, CYP2C8 and CYP3A4 may play a crucial role in LOP metabolism at the therapeutic concentrations of LOP. Coadministration of these P450 inhibitors may cause drug interactions with LOP. However, the clinical significance of potential interaction of LOP metabolism by CYP2C8 and CYP3A4 inhibitors should be studied further.",
keywords = "CYP2C8, CYP3A4, Cytochrome P450 (P450), Human liver microsomes, Loperamide",
author = "Kim, {Kyoung Ah} and Jaegul Chung and Jung, {Dong Hae} and Ji-Young Park",
year = "2004",
month = "10",
day = "1",
doi = "10.1007/s00228-004-0815-3",
language = "English",
volume = "60",
pages = "575--581",
journal = "European Journal of Clinical Pharmacology",
issn = "0031-6970",
publisher = "Springer Verlag",
number = "8",

}

TY - JOUR

T1 - Identification of cytochrome P450 isoforms involved in the metabolism of loperamide in human liver microsomes

AU - Kim, Kyoung Ah

AU - Chung, Jaegul

AU - Jung, Dong Hae

AU - Park, Ji-Young

PY - 2004/10/1

Y1 - 2004/10/1

N2 - Objective: The purpose of the present study was to elucidate the cytochrome P450 (P450) isoform(s) involved in the metabolism of loperamide (LOP) to N-demethylated LOP (DLOP) in human liver microsomes. Methods: Three established approaches were used to identify the P450 isoforms responsible for LOP N-demethylation using human liver microsomes and cDNA-expressed P450 isoforms: (1) correlation of LOP N-demethylation activity with marker P450 activities in a panel of human liver microsomes, (2) inhibition of enzyme activity by P450-selective inhibitors, and (3) measurement of DLOP formation by cDNA-expressed P450 isoforms. The relative contribution of P450 isoforms involved in LOP N-demethylation in human liver microsomes were estimated by applying relative activity factor (RAF) values. Results: The formation rate of DLOP showed biphasic kinetics, suggesting the involvement of multiple P450 isoforms. Apparent Km and Vmax values were 21.1 μM and 122.3 pmol/min per milligram of protein for the high-affinity component and 83.9 μM and 412.0 pmol/ min per milligram of protein for the low-affinity component, respectively. Of the cDNA-expressed P450 s tested, CYP2B6, CYP2C8, CYP2D6, and CYP3A4 catalyzed LOP N-demethylation. LOP N-demethylation was significantly inhibited when coincubated with quercetin (a CYP2C8 inhibitor) and ketoconazole (a CYP3A4 inhibitor) by 40 and 90%, respectively, but other chemical inhibitors tested showed weak or no significant inhibition. DLOP formation was highly correlated with CYP3A4-catalyzed midazolam 1-hydroxylation (rs = 0.829; P < 0.01), CYP2B6-catalzyed 7-ethoxy4- trifluoromethylcoumarin O-deethylation (rs = 0.691; P < 0.05), and CYP2C8-catalyzed paclitaxel 6α-hydroxylation (rs = 0.797; P < 0.05). Conclusion: CYP2B6, CYP2C8, CYP2D6, and CYP3A4 catalyze LOP N-demethylation in human liver microsomes, and among them, CYP2C8 and CYP3A4 may play a crucial role in LOP metabolism at the therapeutic concentrations of LOP. Coadministration of these P450 inhibitors may cause drug interactions with LOP. However, the clinical significance of potential interaction of LOP metabolism by CYP2C8 and CYP3A4 inhibitors should be studied further.

AB - Objective: The purpose of the present study was to elucidate the cytochrome P450 (P450) isoform(s) involved in the metabolism of loperamide (LOP) to N-demethylated LOP (DLOP) in human liver microsomes. Methods: Three established approaches were used to identify the P450 isoforms responsible for LOP N-demethylation using human liver microsomes and cDNA-expressed P450 isoforms: (1) correlation of LOP N-demethylation activity with marker P450 activities in a panel of human liver microsomes, (2) inhibition of enzyme activity by P450-selective inhibitors, and (3) measurement of DLOP formation by cDNA-expressed P450 isoforms. The relative contribution of P450 isoforms involved in LOP N-demethylation in human liver microsomes were estimated by applying relative activity factor (RAF) values. Results: The formation rate of DLOP showed biphasic kinetics, suggesting the involvement of multiple P450 isoforms. Apparent Km and Vmax values were 21.1 μM and 122.3 pmol/min per milligram of protein for the high-affinity component and 83.9 μM and 412.0 pmol/ min per milligram of protein for the low-affinity component, respectively. Of the cDNA-expressed P450 s tested, CYP2B6, CYP2C8, CYP2D6, and CYP3A4 catalyzed LOP N-demethylation. LOP N-demethylation was significantly inhibited when coincubated with quercetin (a CYP2C8 inhibitor) and ketoconazole (a CYP3A4 inhibitor) by 40 and 90%, respectively, but other chemical inhibitors tested showed weak or no significant inhibition. DLOP formation was highly correlated with CYP3A4-catalyzed midazolam 1-hydroxylation (rs = 0.829; P < 0.01), CYP2B6-catalzyed 7-ethoxy4- trifluoromethylcoumarin O-deethylation (rs = 0.691; P < 0.05), and CYP2C8-catalyzed paclitaxel 6α-hydroxylation (rs = 0.797; P < 0.05). Conclusion: CYP2B6, CYP2C8, CYP2D6, and CYP3A4 catalyze LOP N-demethylation in human liver microsomes, and among them, CYP2C8 and CYP3A4 may play a crucial role in LOP metabolism at the therapeutic concentrations of LOP. Coadministration of these P450 inhibitors may cause drug interactions with LOP. However, the clinical significance of potential interaction of LOP metabolism by CYP2C8 and CYP3A4 inhibitors should be studied further.

KW - CYP2C8

KW - CYP3A4

KW - Cytochrome P450 (P450)

KW - Human liver microsomes

KW - Loperamide

UR - http://www.scopus.com/inward/record.url?scp=8744278943&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=8744278943&partnerID=8YFLogxK

U2 - 10.1007/s00228-004-0815-3

DO - 10.1007/s00228-004-0815-3

M3 - Article

VL - 60

SP - 575

EP - 581

JO - European Journal of Clinical Pharmacology

JF - European Journal of Clinical Pharmacology

SN - 0031-6970

IS - 8

ER -