In Vitro Inhibitory Effect of 1-Aminobenzotriazole on Drug Oxidations in Human Liver Microsomes: a Comparison with SKF-525A

Emoto, Chie; Murase, Shigeo; Sawada, Yasufusa; Iwasaki, Katsunori

doi:10.2133/dmpk.20.351

Cited by 36 publications

(33 citation statements)

References 27 publications

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“…In particular, for P450 2C9, tienilic acid as well as the competitive inhibitor sulfaphenazole were significantly more effective, with 11 and 27% remaining activity, respectively, compared with 58% with 1-ABT (Table 2). This notably weak inactivation of P450 2C9 was particularly interesting and is consistent with previous reports (Emoto et al, 2003(Emoto et al, , 2005. Prototypical substrates of P450 2C9 are carboxylic acids such as flurbiprofen, naproxen, ibuprofen, and diclofenac (Tracy et al, 1996(Tracy et al, , 1997Klose et al, 1998), which are anionic at pH 7.4, and proposed to be involved in a key binding interaction with Arg108, a et al, 2004).…”

Section: Resultssupporting

confidence: 86%

“…Thus, it has become a common in vitro practice to preincubate either human liver microsomes (HLMs) or hepatocytes with high concentrations of 1-ABT (ϳ1 mM) before the introduction of test compounds (substrate-depletion approach) to decipher P450 from non-P450 mediated-metabolism (Dalmadi et al, 2003;Williams et al, 2003;Kostrubsky et al, 2006). Although it is acknowledged that this approach is useful for determining P450-mediated metabolism, the characterization of the specific effects of 1-ABT on the major human P450 enzymes in vitro has been limited (Emoto et al, 2005). The purpose of the current research was to evaluate the use of 1-ABT as a nonselective inactivator of P450 by determining the remaining activity of the major human P450 enzymes (CYP1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4) in HLMs after pretreatment with 1 mM 1-ABT.…”

mentioning

confidence: 99%

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Is 1-Aminobenzotriazole an Appropriate in Vitro Tool as a Nonspecific Cytochrome P450 Inactivator?

Linder¹,

Renaud²,

Hutzler³

2008

Drug Metab Dispos

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ABSTRACT:1-Aminobenzotriazole (1-ABT) is generally considered to be a nonselective mechanism-based inactivator of both human and nonhuman cytochrome P450 (P450) enzymes. Thus, 1-ABT is routinely used when conducting in vitro reaction phenotyping studies with new chemical entities in drug discovery to decipher P450 from non-P450-mediated metabolism. Experiments with pooled human liver microsomes (HLMs) demonstrated that carbon monoxide binding, although substantially reduced after a 30-min preincubation with 1-ABT, was still measurable. Thus, remaining activity of nine major human P450s (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4) in HLMs was determined using established selective probe substrates after 30-min preincubation with either 1-ABT (1 mM), a positive control time-dependent inhibitor, or a competitive inhibitor. Whereas P450 2A6 and 3A4 activity was essentially eliminated upon 30-min pretreatment with 1-ABT, the other human P450s were less affected, with at least 20% activity remaining after pretreatment. In contrast, most of the known P450 selective timedependent inhibitors were more effective inactivators than 1-ABT at lower concentrations. A particularly interesting finding was that 1-ABT was quite ineffective at inactivating P450 2C9, with roughly 60% activity remaining after pretreatment, which suggests that 1-ABT is much less selective for certain human P450s. This collection of data clearly demonstrates that assuming 1-ABT is a nonselective P450 inhibitor in vitro is risky, and false conclusions regarding remaining metabolic activity being non-P450 mediated after 1-ABT pretreatment may be made.In vitro reaction phenotyping, where enzymes contributing to the biotransformation of new chemical entities are identified, is a routine practice in drug discovery (Williams et al., 2003;Zhang et al., 2007). This information is essential for understanding the relative contributions of metabolic pathways to overall clearance and, thus, the risk of pharmacokinetic drug-drug interactions and/or interpatient variability in drug exposure. One critical issue in conducting these in vitro studies is having the appropriate tool substrates and inhibitors of the various human cytochrome P450 (P450) enzymes. A particularly useful tool inhibitor historically has been 1-aminobenzotriazole (1-ABT), which is thought to inactivate P450 enzymes nonselectively by covalent modification of the heme prosthetic group following bioactivation (Ortiz de Montellano and Mathews, 1981). Thus, it has become a common in vitro practice to preincubate either human liver microsomes (HLMs) or hepatocytes with high concentrations of 1-ABT (ϳ1 mM) before the introduction of test compounds (substrate-depletion approach) to decipher P450 from non-P450 mediated-metabolism (Dalmadi et al., 2003;Williams et al., 2003;Kostrubsky et al., 2006). Although it is acknowledged that this approach is useful for determining P450-mediated metabolism, the characterization of the specific effects of 1-ABT on the major human P450 enzymes in vitro has been l...

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Section: Resultssupporting

confidence: 86%

mentioning

confidence: 99%

Is 1-Aminobenzotriazole an Appropriate in Vitro Tool as a Nonspecific Cytochrome P450 Inactivator?

Linder¹,

Renaud²,

Hutzler³

2008

Drug Metab Dispos

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“…Taken together, these findings underscore that the inhibition of human P450s by SKF525A and related compounds is complex and involves several mechanisms, and P450-MI complex formation only plays a role with CYP3A. The inhibition of multiple human P450s by the parent compound (SKF525A) has been observed previously (Ono et al, 1996;Emoto et al, 2005). In human liver microsomes, SKF525A was most inhibitory toward CYP2D6-, CYP2C19-, and CYP3A4/5-catalyzed reactions (Ono et al, 1996).…”

Section: P450 Isoformsupporting

confidence: 59%

“…In human liver microsomes, SKF525A was most inhibitory toward CYP2D6-, CYP2C19-, and CYP3A-catalyzed reactions (Ono et al, 1996). In studies with baculovirus-expressed enzymes (Emoto et al, 2003) and human liver microsomes (Emoto et al, 2005), a similar rank order was seen, although the degree of inhibition of CYP3A-catalyzed reactions showed considerable variation among the substrates/reactions used. Preincubation of SKF525A with human liver microsomes and NADPH decreased the inhibitory activity toward CYP2D6 and CYP2C19, variously either decreased or increased inhibitory activity toward CYP3A (depending on the substrate examined), and increased the inhibitory activity toward CYP1A2.…”

mentioning

confidence: 73%

2-Diethylaminoethyl-2,2-diphenylvalerate-HCl (SKF525A) Revisited: Comparative Cytochrome P450 Inhibition in Human Liver Microsomes by SKF525A, Its Metabolites, and SKF-Acid and SKF-Alcohol

Franklin¹,

Hathaway²

2008

Drug Metab Dispos

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ABSTRACT:When incubated with human liver microsomes, 2-diethylaminoethyl-2,2-diphenylvalerate-HCl (SKF525A) undergoes cytochrome P450 (P450)-dependent oxidative N-deethylation to the secondary amine metabolite 2-ethylaminoethyl-2,2-diphenylvalerate (SKF8742). P450-selective inhibitors indicated CYP3As catalyzed this reaction, and the deethylation rate correlated best with the CYP3A activity across a range of human liver microsomes. SKF525A and its metabolite and primary amine analog all inhibited CYP2B6-, CYP2C9-, CYP2C19-, CYP2D6-, and CYP3A-selective reactions to varying degrees but had little effect on CYP1A2, CYP2A6, and CYP2E1 reactions. Only the inhibition of CYP3A showed major enhancement when the inhibitors were preincubated with NADPH-fortified microsomes, and the extent of metabolic intermediate (MI) complex formation approximated typical CYP3A content. Two "lost with time" SKF525A derivatives devoid of the ethylamine moiety, 2,2-diphenylpropylethanol (SKF-Alcohol) and 2,2-diphenylpropylacetic acid (SKF-Acid) did not form an MI complex and were identified as selective inhibitors of CYP2C9. Although without detectable metabolism, their CYP2C9 inhibition fitted best with a competitive mechanism. Thus, not all the human P450s are inhibited by SKF525A and related compounds, and the mechanisms contributing to those that are inhibited vary with the isoform. P450 MI-complex formation only seems to play a role with CYP3As.

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“…APCs were preincubated for 1 h with 1-aminobenzotriazole (a nonselective suicide inhibitor; 1 mM) (32) or methimazole (an inhibitor of peroxidases and flavin-monooxygenase; 1 mM) (33).…”

Section: Enzyme-inhibition Experimentsmentioning

confidence: 99%

Drug Antigenicity, Immunogenicity, and Costimulatory Signaling: Evidence for Formation of a Functional Antigen through Immune Cell Metabolism

Elsheikh

Lavergne

Castrejón-Flores

et al. 2010

The Journal of Immunology

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Recognition of drugs by immune cells is usually explained by the hapten model, which states that endogenous metabolites bind irreversibly to protein to stimulate immune cells. Synthetic metabolites interact directly with protein generating antigenic determinants for T-cells; however, experimental evidence relating intracellular metabolism in immune cells and the generation of physiologically relevant antigens to functional immune responses is lacking. The aim of this study was to develop an integrated approach using both animal and human experimental systems to characterize sulfamethoxazole (SMX) metabolism-derived antigenic protein adduct formation in immune cells and define the relationship between adduct formation, cell death, co-stimulatory signalling and stimulation of a T-cell response. Formation of SMX-derived adducts in antigen presenting cells was dose- and time-dependent, detectable at non-toxic concentrations and dependent on drug metabolizing enzyme activity. Adduct formation above a threshold induced necrotic cell death, dendritic cell co-stimulatory molecule expression and cytokine secretion. Antigen presenting cells cultured with SMX for 16h, the time needed for drug metabolism, stimulated T-cells from sensitized mice and lymphocytes and T-cell clones from allergic patients. Enzyme inhibition decreased SMX-derived protein adduct formation and the T-cell response. Dendritic cells cultured with SMX and adoptively transferred to recipient mice initiated an immune response; however, T-cells were stimulated with adducts derived from SMX metabolism in antigen presenting cells, not the parent drug. This study shows that antigen presenting cells metabolize SMX; subsequent protein binding generates a functional T-cell antigen. Adduct formation above a threshold stimulates cell death, which provides a maturation signal for dendritic cells.

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In Vitro Inhibitory Effect of 1-Aminobenzotriazole on Drug Oxidations in Human Liver Microsomes: a Comparison with SKF-525A

Cited by 36 publications

References 27 publications

Is 1-Aminobenzotriazole an Appropriate in Vitro Tool as a Nonspecific Cytochrome P450 Inactivator?

Is 1-Aminobenzotriazole an Appropriate in Vitro Tool as a Nonspecific Cytochrome P450 Inactivator?

2-Diethylaminoethyl-2,2-diphenylvalerate-HCl (SKF525A) Revisited: Comparative Cytochrome P450 Inhibition in Human Liver Microsomes by SKF525A, Its Metabolites, and SKF-Acid and SKF-Alcohol

Drug Antigenicity, Immunogenicity, and Costimulatory Signaling: Evidence for Formation of a Functional Antigen through Immune Cell Metabolism

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