Characterization of human cytochrome P450 mediated bioactivation of amodiaquine and its major metabolite N-desethylamodiaquine

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Abstract

Aims: Oxidative bioactivation of amodiaquine (AQ) by cytochrome P450s to a reactive quinoneimine is considered as an important mechanism underlying its idiosyncratic hepatotoxicity. However, because internal exposure to its major metabolite N-desethylamodiaquine (DEAQ) is up to 240-fold higher than AQ, bioactivation of DEAQ might significantly contribute to covalent binding. The aim of the present study was to compare the kinetics of bioactivation of AQ and DEAQ by human liver microsomes (HLM) and to characterize the CYPs involved in bioactivation of AQ and DEAQ. Methods: Glutathione was used to trap reactive metabolites formed in incubations of AQ and DEAQ with HLM and recombinant human cytochrome P450s (hCYPs). Kinetics of bioactivation of AQ and DEAQ in HLM and involvement of hCYPs were characterized by measuring corresponding glutathione conjugates (AQ-SG and DEAQ-SG) using a high-performance liquid chromatography method. Results: Bioactivation of AQ and DEAQ in HLM both exhibited Michaelis–Menten kinetics. For AQ bioactivation, enzyme kinetical parameters were Km, 11.5 ± 2.0 μmol l–1, Vmax, 59.2 ± 3.2 pmol min−1 mg−1 and CLint, 5.15 μl min−1 mg−1. For DEAQ, parameters for bioactivation were Km, 6.1 ± 1.3 μmol l–1, Vmax, 5.5 ± 0.4 pmol min−1 mg−1 and CLint 0.90 μl min−1 mg−1. Recombinant hCYPs and inhibition studies with HLM showed involvement of CYP3A4, CYP2C8, CYP2C9 and CYP2D6 in bioactivation. Conclusions: The major metabolite DEAQ is likely to be quantitatively more important than AQ with respect to hepatic exposure to reactive metabolites in vivo. High expression of CYP3A4, CYP2C8, CYP2C9, and CYP2D6 may be risk factors for hepatotoxicity caused by AQ-therapy.

LanguageEnglish
Pages572-583
Number of pages12
JournalBritish Journal of Clinical Pharmacology
Volume83
Issue number3
DOIs
StatePublished - 1 Mar 2017

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Amodiaquine
Cytochrome P-450 Enzyme System
Liver Microsomes
Cytochromes
Cytochrome P-450 CYP3A
Cytochrome P-450 CYP2D6
Glutathione
desethylamodiaquine

Keywords

  • amodiaquine
  • bioactivation
  • glutathione conjugate
  • human cytochrome P450s
  • N-desethylamodiaquine

Cite this

@article{da5c73e2baee41ecb2ecb2da8f09b9f0,
title = "Characterization of human cytochrome P450 mediated bioactivation of amodiaquine and its major metabolite N-desethylamodiaquine",
abstract = "Aims: Oxidative bioactivation of amodiaquine (AQ) by cytochrome P450s to a reactive quinoneimine is considered as an important mechanism underlying its idiosyncratic hepatotoxicity. However, because internal exposure to its major metabolite N-desethylamodiaquine (DEAQ) is up to 240-fold higher than AQ, bioactivation of DEAQ might significantly contribute to covalent binding. The aim of the present study was to compare the kinetics of bioactivation of AQ and DEAQ by human liver microsomes (HLM) and to characterize the CYPs involved in bioactivation of AQ and DEAQ. Methods: Glutathione was used to trap reactive metabolites formed in incubations of AQ and DEAQ with HLM and recombinant human cytochrome P450s (hCYPs). Kinetics of bioactivation of AQ and DEAQ in HLM and involvement of hCYPs were characterized by measuring corresponding glutathione conjugates (AQ-SG and DEAQ-SG) using a high-performance liquid chromatography method. Results: Bioactivation of AQ and DEAQ in HLM both exhibited Michaelis–Menten kinetics. For AQ bioactivation, enzyme kinetical parameters were Km, 11.5 ± 2.0 μmol l–1, Vmax, 59.2 ± 3.2 pmol min−1 mg−1 and CLint, 5.15 μl min−1 mg−1. For DEAQ, parameters for bioactivation were Km, 6.1 ± 1.3 μmol l–1, Vmax, 5.5 ± 0.4 pmol min−1 mg−1 and CLint 0.90 μl min−1 mg−1. Recombinant hCYPs and inhibition studies with HLM showed involvement of CYP3A4, CYP2C8, CYP2C9 and CYP2D6 in bioactivation. Conclusions: The major metabolite DEAQ is likely to be quantitatively more important than AQ with respect to hepatic exposure to reactive metabolites in vivo. High expression of CYP3A4, CYP2C8, CYP2C9, and CYP2D6 may be risk factors for hepatotoxicity caused by AQ-therapy.",
keywords = "amodiaquine, bioactivation, glutathione conjugate, human cytochrome P450s, N-desethylamodiaquine",
author = "Y. Zhang and N.P.E. Vermeulen and J.N.M. Commandeur",
year = "2017",
month = "3",
day = "1",
doi = "10.111/bcp.13148",
language = "English",
volume = "83",
pages = "572--583",
journal = "British Journal of Clinical Pharmacology",
issn = "0306-5251",
publisher = "Wiley-Blackwell",
number = "3",

}

TY - JOUR

T1 - Characterization of human cytochrome P450 mediated bioactivation of amodiaquine and its major metabolite N-desethylamodiaquine

AU - Zhang,Y.

AU - Vermeulen,N.P.E.

AU - Commandeur,J.N.M.

PY - 2017/3/1

Y1 - 2017/3/1

N2 - Aims: Oxidative bioactivation of amodiaquine (AQ) by cytochrome P450s to a reactive quinoneimine is considered as an important mechanism underlying its idiosyncratic hepatotoxicity. However, because internal exposure to its major metabolite N-desethylamodiaquine (DEAQ) is up to 240-fold higher than AQ, bioactivation of DEAQ might significantly contribute to covalent binding. The aim of the present study was to compare the kinetics of bioactivation of AQ and DEAQ by human liver microsomes (HLM) and to characterize the CYPs involved in bioactivation of AQ and DEAQ. Methods: Glutathione was used to trap reactive metabolites formed in incubations of AQ and DEAQ with HLM and recombinant human cytochrome P450s (hCYPs). Kinetics of bioactivation of AQ and DEAQ in HLM and involvement of hCYPs were characterized by measuring corresponding glutathione conjugates (AQ-SG and DEAQ-SG) using a high-performance liquid chromatography method. Results: Bioactivation of AQ and DEAQ in HLM both exhibited Michaelis–Menten kinetics. For AQ bioactivation, enzyme kinetical parameters were Km, 11.5 ± 2.0 μmol l–1, Vmax, 59.2 ± 3.2 pmol min−1 mg−1 and CLint, 5.15 μl min−1 mg−1. For DEAQ, parameters for bioactivation were Km, 6.1 ± 1.3 μmol l–1, Vmax, 5.5 ± 0.4 pmol min−1 mg−1 and CLint 0.90 μl min−1 mg−1. Recombinant hCYPs and inhibition studies with HLM showed involvement of CYP3A4, CYP2C8, CYP2C9 and CYP2D6 in bioactivation. Conclusions: The major metabolite DEAQ is likely to be quantitatively more important than AQ with respect to hepatic exposure to reactive metabolites in vivo. High expression of CYP3A4, CYP2C8, CYP2C9, and CYP2D6 may be risk factors for hepatotoxicity caused by AQ-therapy.

AB - Aims: Oxidative bioactivation of amodiaquine (AQ) by cytochrome P450s to a reactive quinoneimine is considered as an important mechanism underlying its idiosyncratic hepatotoxicity. However, because internal exposure to its major metabolite N-desethylamodiaquine (DEAQ) is up to 240-fold higher than AQ, bioactivation of DEAQ might significantly contribute to covalent binding. The aim of the present study was to compare the kinetics of bioactivation of AQ and DEAQ by human liver microsomes (HLM) and to characterize the CYPs involved in bioactivation of AQ and DEAQ. Methods: Glutathione was used to trap reactive metabolites formed in incubations of AQ and DEAQ with HLM and recombinant human cytochrome P450s (hCYPs). Kinetics of bioactivation of AQ and DEAQ in HLM and involvement of hCYPs were characterized by measuring corresponding glutathione conjugates (AQ-SG and DEAQ-SG) using a high-performance liquid chromatography method. Results: Bioactivation of AQ and DEAQ in HLM both exhibited Michaelis–Menten kinetics. For AQ bioactivation, enzyme kinetical parameters were Km, 11.5 ± 2.0 μmol l–1, Vmax, 59.2 ± 3.2 pmol min−1 mg−1 and CLint, 5.15 μl min−1 mg−1. For DEAQ, parameters for bioactivation were Km, 6.1 ± 1.3 μmol l–1, Vmax, 5.5 ± 0.4 pmol min−1 mg−1 and CLint 0.90 μl min−1 mg−1. Recombinant hCYPs and inhibition studies with HLM showed involvement of CYP3A4, CYP2C8, CYP2C9 and CYP2D6 in bioactivation. Conclusions: The major metabolite DEAQ is likely to be quantitatively more important than AQ with respect to hepatic exposure to reactive metabolites in vivo. High expression of CYP3A4, CYP2C8, CYP2C9, and CYP2D6 may be risk factors for hepatotoxicity caused by AQ-therapy.

KW - amodiaquine

KW - bioactivation

KW - glutathione conjugate

KW - human cytochrome P450s

KW - N-desethylamodiaquine

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U2 - 10.111/bcp.13148

DO - 10.111/bcp.13148

M3 - Article

VL - 83

SP - 572

EP - 583

JO - British Journal of Clinical Pharmacology

T2 - British Journal of Clinical Pharmacology

JF - British Journal of Clinical Pharmacology

SN - 0306-5251

IS - 3

ER -