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

doi:10.2133/dmpk.20.351

Drug Metabolism and Pharmacokinetics

Volume 20, Issue 5, 2005, Pages 351-357

https://doi.org/10.2133/dmpk.20.351 Get rights and content

Summary:

1-Aminobenzotriazole (ABT) is extensively used as a non-specific cytochrome P450 (CYP) inhibitor. In this study, the inhibitory effect of ABT on CYP-dependent drug oxidations was investigated in human liver microsomes (HLM) and compared with that of SKF-525A, another non-specific inhibitor. The following probe activities for human CYP isoforms were determined using pooled HLM: phenacetin O-deethylation (CYP1A2); diclofenac 4'-hydroxylation (CYP2C9); S-mephenytoin 4'-hydroxylation, (CYP2C19); bufuralol 1'-hydroxylation (CYP2D6); chlorzoxazone 6-hydroxylation (CYP2E1); midazolam 1'-hydroxylation, nifedipine oxidation, and testosterone 6β-hydroxylation (CYP3A). ABT had the strongest inhibitory effect on the CYP3A-dependent drug oxidations and the weakest effect on the diclofenac 4'-hydroxylation. SKF-525A potently inhibited the bufuralol 1'-hydroxylation, but weakly inhibited chlorzoxazone 6-hydroxylation. The inhibitory effects of ABT and SKF-525A were increased by preincubation in some probe reactions, and this preincubation effect was greater in ABT than in SKF-525A. The remarkable IC₅₀ shift (> 10 times) by preincubation with ABT was observed on the phenacetin O-deethylation, chlorzoxazone 6-hydroxylation, and midazolam 1'-hydroxylation. In conclusion, ABT and SKF-525A had a wide range of IC₅₀ values in inhibiting the drug oxidations by HLM with and without preincubation.

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For drugs metabolized by oxidation, an initial experiment is needed to define whether these reactions are catalyzed by CYP enzymes or other enzymes that catalyze oxidations. This is most frequently done using the pan-CYP inhibitor, 1-aminobenzotriazole (ABT), which is a mechanism-based inactivator of several major CYP enzymes [30]. However, this needs to be supplemented with an inactivator of CYP2C9 which is more refractory to inactivation by ABT [31].
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As a mechanism-based inhibitor, without preincubation ABT almost didn't show inhibition toward major CYPs tested as expected (IC50 values >100 μM). That is consistent with what it is reported by the literature (Emoto et al., 2005). During CYP inhibition assessment in human and animal liver microsomes, midazolam 1′-hydroxylation in rat liver microsomes was the least inhibited by atipamezole among the seven CYPs tested (IC50 of 7.93 μM), whereas human CYP2B6 mediated bupropion hydroxylation was the most inhibited among the seven CYPs tested in human and animal liver microsomes (0.02–0.08 μM).
1-aminobenzotriazole (ABT) is a known P450 enzyme non-selective inactivator that serves as a tool for assessing P450-mediated metabolism. However, many findings demonstrated that ABT was ineffective with human CYP2C9. A profound pan-CYP450 inhibitor is desired avoid the risk of incomplete inhibition of P450, especially CYP2C9. Atipamezole is commonly used to recover animals from sedation-anesthesia induced by α₂₋adrenoceptor agonists. The purpose of this study is to evaluate atipamezole as a non-selective inhibitor of P450 enzymes and compare it with ABT. Inhibition toward seven major human CYP450 isoform was determined for atipamezole and ABT in human, rat, and dog liver microsomes for the direct and time-dependent inhibition potentials. IC₅₀ values toward human and animal CYPs without preincubation are 0.02–7.93 μM and 20.9–1798 μM for atipamezole and ABT, respectively. The IC₅₀ values of ABT after preincubation shift to 4.06–460 μM. Atipamezole has more effective inhibition to CYP2C9 mediated diclofenac hydroxylation in human and animal liver microsomes with IC₅₀ values of 1.50–5.20 μM than that of ABT at 74.7-460 μM. No IC₅₀ shift was observed for atipamezole to CYP isoforms. In vivo utility of atipamezole was assessed by co-dosing with diclofenac in rats. At 30 mg/kg via oral, atipamezole enhanced the AUC of diclofenac by 13.1-fold and the C_max by 5.6-fold. Similar enhancement also achieved for ABT (100 mg/kg) with AUC and C_max increased 9.5 and 4.8-fold. As a reversible pan-CYP inhibitor, atipamezole showed less species difference than ABT. It provides a better and easier to use alternative to ABT for ADME optimization and elucidating mechanistic drug metabolism or toxicity studies.
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The results demonstrated that inhibition of CYP1A2, 2B6, 2C9, 2C19, 2D6, and 3A4 was concentration-dependent, with less than 20% activity remaining for all the P450s examined.7 The inhibitory effect of ABT on CYP-dependent oxidative metabolism was also reported in human liver microsomes.9 By preincubation with ABT for 30 min, the IC50 for ABT-mediated inhibition of CYP1A2, 2C9, 2C19, 2D6, 2E1, and 3A ranked from 0.7 to 400 μM, with the weakest inhibitory effect of ABT on the CYP2C9-mediated diclofenac 4’-hydroxylation.9
1-Aminobenzotriazole (ABT) has been widely used as a nonspecific mechanism-based inhibitor of cytochrome P450 (P450) enzymes. It is extensively used in preclinical studies to determine the relative contribution of oxidative metabolism mediated by P450 in vitro and in vivo. The aim of present study was to understand the translation of fraction metabolized by P450 in dog hepatocytes to in vivo using ABT, for canagliflozin, known to be cleared by P450-mediated oxidation and UDP-glucuronosyltransferases–mediated glucuronidation, and 3 drug discovery project compounds mainly cleared by hepatic metabolism. In a dog hepatocyte, intrinsic clearance assay with and without preincubation of ABT, 3 Lilly compounds exhibited a wide range of fraction metabolized by P450. Subsequent metabolite profiling in dog hepatocytes demonstrated a combination of metabolism by P450 and UDP-glucuronosyltransferases. In vivo, dogs were pretreated with 50 mg/kg ABT or vehicle at 2 h before intravenous administration of canagliflozin and Lilly compounds. The areas under the concentration-time curve (AUC) were compared for the ABT-pretreated and vehicle-pretreated groups. The measured AUC_ABT/AUC_veh ratios were correlated to fraction of metabolism by P450 in dog hepatocytes, suggesting that in vitro ABT inhibition in hepatocytes is useful to rank order compounds for in vivo fraction of metabolism assessment.
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Millions of people are treated with anthelmintics to control soil-transmitted helminth infections; yet, drug distribution in the plasma and gastrointestinal tract compartments and the pathway of drug uptake into gastrointestinal nematodes responsible for the pharmacological effect are unknown. We assessed the distribution and uptake of albendazole, albendazole sulfoxide, albendazole sulfone in the hookworm Heligmosomoides polygyrus in vitro and in vivo as well as the distribution and uptake of albendazole, mebendazole, and oxantel pamoate in the whipworm Trichuris muris in vitro and in vivo. Oral and intraperitoneal treatments (100 mg/kg) were studied. Drug quantities in helminths and host compartments (stomach, the contents and mucosa of the small and large intestine, and the plasma) were determined using HPLC-UV/vis and anthelmintic activities were recorded using phenotypic readout. The influence of 1-aminobenzotriazole (ABT), an irreversible and unspecific cytochrome P450 inhibitor, on albendazole disposition in mice harboring H. polygyrus was evaluated. In vivo, albendazole was found in quantities up to 10 nmol per ten H. polygyrus and up to 31 nmol per ten T. muris. ABT did not change the levels of albendazole or its metabolites in the plasma of mice harboring H. polygyrus or in H. polygyrus, whereas drug levels in the gastrointestinal tract of host mice doubled. Mebendazole and oxantel pamoate quantities per ten T. muris were as high as 21 nmol and 34 nmol, respectively. Albendazole revealed a very dynamic distribution and high rate of metabolism, hence, H. polygyrus and T. muris are exposed to albendazole and both metabolites via multiple pathways. Diffusion through the cuticle seems to be the crucial pathway of oxantel pamoate uptake into T. muris, and likely also for mebendazole. No relationship between concentrations measured in helminths and concentrations in plasma, intestinal content and mucosa of mice, or drug efficacy was noted for any of the drugs studied.
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Regular ArticleIn Vitro Inhibitory Effect of 1-Aminobenzotriazole on Drug Oxidations in Human Liver Microsomes: a Comparison with SKF-525A

Summary:

Fundam. Appl. Toxicol.

Drug Metabol. Pharmacokinet.

Tetrahedron

Biochem. Pharmacol.

Biochem. Pharmacol.

J. Pharm. Sci

Arch. Biochem. Biophys.

P450 superfamily: update on new sequences, gene mapping, accession numbers and nomenclature

Pharmacogenetics

Extrahepatic metabolism of drugs in humans

Clin. Pharmacokinet.

Clinical importance of hepatic cytochrome P450 in drug metabolism

Drug Metab. Rev.

Autocatalytic alkylation of the cytochrome P-450 prosthetic haem group by 1-aminobenzotriazole

Biochem. J.

Effective dosing regimen of 1-aminobenzotriazole for inhibition of antipyrine clearance in rats, dogs, and monkeys

Drug Metab. Dispos.

Effective dosing regimen of 1-aminobenzotriazole for inhibition of antipyrine clearance in Guinea pigs and mice using serial sampling

Drug Metab. Dispos.

Inactivation of rabbit pulmonary cytochrome P-450 in microsomes and isolated perfused lungs by the suicide substrate 1-aminobenzotriazole

J. Pharmacol. Exp. Ther.

N-aralkylated derivatives of 1-aminobenzotriazole as isozyme-selective, mechanism-based inhibitors of guinea pig hepatic cytochrome P-450 dependent monooxygenase activity

Can. J. Physiol. Pharmacol.

Oxidation of cyclophosphamide to 4-hydroxycyclophosphamide and deschloroethylcyclophosphamide in human liver microsomes

Cancer Res.

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