CYP4F enzymes are the major enzymes in human liver microsomes that catalyze the O-demethylation of the antiparasitic prodrug DB289 [2,5-bis(4- amidinophenyl)furan-bis-O-methylamidoxime]

Michael Zhuo Wang, Janelle Y. Saulter, Etsuko Usuki, Yen Ling Cheung, Michael Hall, Arlene S. Bridges, Greg Loewen, Oliver T. Parkinson, Chad E. Stephens, James L. Allen, Darryl C. Zeldin, David W. Boykin, Richard R. Tidwell, Andrew Parkinson, Mary F. Paine, James Edwin Hall

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Abstract

DB289 [2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime] is biotransformed to the potent antiparasitic diamidine DB75 [2,5-bis(4- amidinophenyl) furan] by sequential oxidative O-demethylation and reductive N-dehydroxylation reactions. Previous work demonstrated that the N-dehydroxylation reactions are catalyzed by cytochrome b5/NADH- cytochrome b5 reductase. Enzymes responsible for catalyzing the DB289 O-demethylation pathway have not been identified. We report an in vitro metabolism study to characterize enzymes in human liver microsomes (HLMs) that catalyze the initial O-demethylation of DB289 (M1 formation). Potent inhibition by 1-aminobenzotriazole confirmed that M1 formation is catalyzed by P450 enzymes. M1 formation by HLMs was NADPH-dependent, with a Km and Vmax of 0.5 μM and 3.8 nmol/min/mg protein, respectively. Initial screening showed that recombinant CYP1A1, CYP1A2, and CYP1B1 were efficient catalysts of M1 formation. However, none of these three enzymes was responsible for M1 formation by HLMs. Further screening showed that recombinant CYP2J2, CYP4F2, and CYP4F3B could also catalyze M1 formation. An antibody against CYP4F2, which inhibited both CYP4F2 and CYP4F3B, inhibited 91% of M1 formation by HLMs. Two inhibitors of P450-mediated arachidonic acid metabolism, HET0016 (N-hydroxy-N′-(4-n-butyl-2-methylphenyl)formamidine) and 17-octadecynoic acid, effectively inhibited M1 formation by HLMs. Inhibition studies with ebastine and antibodies against CYP2J2 suggested that CYP2J2 was not involved in M1 formation by HLMs. Additionally, ketoconazole preferentially inhibited CYP4F2, but not CYP4F3B, and partially inhibited M1 formation by HLMs. We conclude that CYP4F enzymes (e.g., CYP4F2, CYP4F3B) are the major enzymes responsible for M1 formation by HLMs. These findings indicate that, in human liver, members of the CYP4F subfamily biotransform not only endogenous compounds but also xenobiotics.

Original languageEnglish (US)
Pages (from-to)1985-1994
Number of pages10
JournalDrug Metabolism and Disposition
Volume34
Issue number12
DOIs
StatePublished - Dec 1 2006

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Antiparasitic Agents
Prodrugs
Liver Microsomes
Enzymes
Cytochrome-B(5) Reductase
Pentamidine
Cytochromes b5
pafuramidine
Cytochrome P-450 CYP1A2
Cytochrome P-450 CYP1A1
Ketoconazole
Antibodies
Xenobiotics
NADP
Arachidonic Acid
Cytochrome P-450 Enzyme System

ASJC Scopus subject areas

  • Pharmacology
  • Pharmaceutical Science

Cite this

CYP4F enzymes are the major enzymes in human liver microsomes that catalyze the O-demethylation of the antiparasitic prodrug DB289 [2,5-bis(4- amidinophenyl)furan-bis-O-methylamidoxime]. / Wang, Michael Zhuo; Saulter, Janelle Y.; Usuki, Etsuko; Cheung, Yen Ling; Hall, Michael; Bridges, Arlene S.; Loewen, Greg; Parkinson, Oliver T.; Stephens, Chad E.; Allen, James L.; Zeldin, Darryl C.; Boykin, David W.; Tidwell, Richard R.; Parkinson, Andrew; Paine, Mary F.; Hall, James Edwin.

In: Drug Metabolism and Disposition, Vol. 34, No. 12, 01.12.2006, p. 1985-1994.

Research output: Contribution to journalArticle

Wang, MZ, Saulter, JY, Usuki, E, Cheung, YL, Hall, M, Bridges, AS, Loewen, G, Parkinson, OT, Stephens, CE, Allen, JL, Zeldin, DC, Boykin, DW, Tidwell, RR, Parkinson, A, Paine, MF & Hall, JE 2006, 'CYP4F enzymes are the major enzymes in human liver microsomes that catalyze the O-demethylation of the antiparasitic prodrug DB289 [2,5-bis(4- amidinophenyl)furan-bis-O-methylamidoxime]', Drug Metabolism and Disposition, vol. 34, no. 12, pp. 1985-1994. https://doi.org/10.1124/dmd.106.010587
Wang, Michael Zhuo ; Saulter, Janelle Y. ; Usuki, Etsuko ; Cheung, Yen Ling ; Hall, Michael ; Bridges, Arlene S. ; Loewen, Greg ; Parkinson, Oliver T. ; Stephens, Chad E. ; Allen, James L. ; Zeldin, Darryl C. ; Boykin, David W. ; Tidwell, Richard R. ; Parkinson, Andrew ; Paine, Mary F. ; Hall, James Edwin. / CYP4F enzymes are the major enzymes in human liver microsomes that catalyze the O-demethylation of the antiparasitic prodrug DB289 [2,5-bis(4- amidinophenyl)furan-bis-O-methylamidoxime]. In: Drug Metabolism and Disposition. 2006 ; Vol. 34, No. 12. pp. 1985-1994.
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title = "CYP4F enzymes are the major enzymes in human liver microsomes that catalyze the O-demethylation of the antiparasitic prodrug DB289 [2,5-bis(4- amidinophenyl)furan-bis-O-methylamidoxime]",
abstract = "DB289 [2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime] is biotransformed to the potent antiparasitic diamidine DB75 [2,5-bis(4- amidinophenyl) furan] by sequential oxidative O-demethylation and reductive N-dehydroxylation reactions. Previous work demonstrated that the N-dehydroxylation reactions are catalyzed by cytochrome b5/NADH- cytochrome b5 reductase. Enzymes responsible for catalyzing the DB289 O-demethylation pathway have not been identified. We report an in vitro metabolism study to characterize enzymes in human liver microsomes (HLMs) that catalyze the initial O-demethylation of DB289 (M1 formation). Potent inhibition by 1-aminobenzotriazole confirmed that M1 formation is catalyzed by P450 enzymes. M1 formation by HLMs was NADPH-dependent, with a Km and Vmax of 0.5 μM and 3.8 nmol/min/mg protein, respectively. Initial screening showed that recombinant CYP1A1, CYP1A2, and CYP1B1 were efficient catalysts of M1 formation. However, none of these three enzymes was responsible for M1 formation by HLMs. Further screening showed that recombinant CYP2J2, CYP4F2, and CYP4F3B could also catalyze M1 formation. An antibody against CYP4F2, which inhibited both CYP4F2 and CYP4F3B, inhibited 91{\%} of M1 formation by HLMs. Two inhibitors of P450-mediated arachidonic acid metabolism, HET0016 (N-hydroxy-N′-(4-n-butyl-2-methylphenyl)formamidine) and 17-octadecynoic acid, effectively inhibited M1 formation by HLMs. Inhibition studies with ebastine and antibodies against CYP2J2 suggested that CYP2J2 was not involved in M1 formation by HLMs. Additionally, ketoconazole preferentially inhibited CYP4F2, but not CYP4F3B, and partially inhibited M1 formation by HLMs. We conclude that CYP4F enzymes (e.g., CYP4F2, CYP4F3B) are the major enzymes responsible for M1 formation by HLMs. These findings indicate that, in human liver, members of the CYP4F subfamily biotransform not only endogenous compounds but also xenobiotics.",
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TY - JOUR

T1 - CYP4F enzymes are the major enzymes in human liver microsomes that catalyze the O-demethylation of the antiparasitic prodrug DB289 [2,5-bis(4- amidinophenyl)furan-bis-O-methylamidoxime]

AU - Wang, Michael Zhuo

AU - Saulter, Janelle Y.

AU - Usuki, Etsuko

AU - Cheung, Yen Ling

AU - Hall, Michael

AU - Bridges, Arlene S.

AU - Loewen, Greg

AU - Parkinson, Oliver T.

AU - Stephens, Chad E.

AU - Allen, James L.

AU - Zeldin, Darryl C.

AU - Boykin, David W.

AU - Tidwell, Richard R.

AU - Parkinson, Andrew

AU - Paine, Mary F.

AU - Hall, James Edwin

PY - 2006/12/1

Y1 - 2006/12/1

N2 - DB289 [2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime] is biotransformed to the potent antiparasitic diamidine DB75 [2,5-bis(4- amidinophenyl) furan] by sequential oxidative O-demethylation and reductive N-dehydroxylation reactions. Previous work demonstrated that the N-dehydroxylation reactions are catalyzed by cytochrome b5/NADH- cytochrome b5 reductase. Enzymes responsible for catalyzing the DB289 O-demethylation pathway have not been identified. We report an in vitro metabolism study to characterize enzymes in human liver microsomes (HLMs) that catalyze the initial O-demethylation of DB289 (M1 formation). Potent inhibition by 1-aminobenzotriazole confirmed that M1 formation is catalyzed by P450 enzymes. M1 formation by HLMs was NADPH-dependent, with a Km and Vmax of 0.5 μM and 3.8 nmol/min/mg protein, respectively. Initial screening showed that recombinant CYP1A1, CYP1A2, and CYP1B1 were efficient catalysts of M1 formation. However, none of these three enzymes was responsible for M1 formation by HLMs. Further screening showed that recombinant CYP2J2, CYP4F2, and CYP4F3B could also catalyze M1 formation. An antibody against CYP4F2, which inhibited both CYP4F2 and CYP4F3B, inhibited 91% of M1 formation by HLMs. Two inhibitors of P450-mediated arachidonic acid metabolism, HET0016 (N-hydroxy-N′-(4-n-butyl-2-methylphenyl)formamidine) and 17-octadecynoic acid, effectively inhibited M1 formation by HLMs. Inhibition studies with ebastine and antibodies against CYP2J2 suggested that CYP2J2 was not involved in M1 formation by HLMs. Additionally, ketoconazole preferentially inhibited CYP4F2, but not CYP4F3B, and partially inhibited M1 formation by HLMs. We conclude that CYP4F enzymes (e.g., CYP4F2, CYP4F3B) are the major enzymes responsible for M1 formation by HLMs. These findings indicate that, in human liver, members of the CYP4F subfamily biotransform not only endogenous compounds but also xenobiotics.

AB - DB289 [2,5-bis(4-amidinophenyl)furan-bis-O-methylamidoxime] is biotransformed to the potent antiparasitic diamidine DB75 [2,5-bis(4- amidinophenyl) furan] by sequential oxidative O-demethylation and reductive N-dehydroxylation reactions. Previous work demonstrated that the N-dehydroxylation reactions are catalyzed by cytochrome b5/NADH- cytochrome b5 reductase. Enzymes responsible for catalyzing the DB289 O-demethylation pathway have not been identified. We report an in vitro metabolism study to characterize enzymes in human liver microsomes (HLMs) that catalyze the initial O-demethylation of DB289 (M1 formation). Potent inhibition by 1-aminobenzotriazole confirmed that M1 formation is catalyzed by P450 enzymes. M1 formation by HLMs was NADPH-dependent, with a Km and Vmax of 0.5 μM and 3.8 nmol/min/mg protein, respectively. Initial screening showed that recombinant CYP1A1, CYP1A2, and CYP1B1 were efficient catalysts of M1 formation. However, none of these three enzymes was responsible for M1 formation by HLMs. Further screening showed that recombinant CYP2J2, CYP4F2, and CYP4F3B could also catalyze M1 formation. An antibody against CYP4F2, which inhibited both CYP4F2 and CYP4F3B, inhibited 91% of M1 formation by HLMs. Two inhibitors of P450-mediated arachidonic acid metabolism, HET0016 (N-hydroxy-N′-(4-n-butyl-2-methylphenyl)formamidine) and 17-octadecynoic acid, effectively inhibited M1 formation by HLMs. Inhibition studies with ebastine and antibodies against CYP2J2 suggested that CYP2J2 was not involved in M1 formation by HLMs. Additionally, ketoconazole preferentially inhibited CYP4F2, but not CYP4F3B, and partially inhibited M1 formation by HLMs. We conclude that CYP4F enzymes (e.g., CYP4F2, CYP4F3B) are the major enzymes responsible for M1 formation by HLMs. These findings indicate that, in human liver, members of the CYP4F subfamily biotransform not only endogenous compounds but also xenobiotics.

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U2 - 10.1124/dmd.106.010587

DO - 10.1124/dmd.106.010587

M3 - Article

VL - 34

SP - 1985

EP - 1994

JO - Drug Metabolism and Disposition

JF - Drug Metabolism and Disposition

SN - 0090-9556

IS - 12

ER -