Effective Dosing Regimen of 1-Aminobenzotriazole for Inhibition of Antipyrine Clearance in Guinea Pigs and Mice Using Serial Sampling

Abstract

ABSTRACT:Single-dose pharmacokinetics of 1-aminobenzotriazole (ABT), a potent nonspecific inhibitor of cytochromes P450 (P450s), were characterized after oral administration to mice and guinea pigs at doses of 50, 100, and 150 mg/kg using serial sampling in both species. Only 30-l blood samples were drawn from jugular veincannulated mice using Microvette capillary tubes containing lithium heparin. A comparison of the pharmacokinetics of antipyrine (AP) administered i.v. at 20 mg/kg to mice followed by serial and terminal sampling techniques yielded similar results. The ABT concentrations in plasma were sustained at high levels (5-100 M) for at least 12 h in both species. Pretreatment of animals with ABT 2 h prior to AP administration decreased the plasma AP clearance by about 95% in mice at all ABT doses studied and 84, 95, and 95% in guinea pigs at a dose of 50, 100, and 150 mg/kg ABT, respectively. In vitro, the dissociation constants (K I ) for ABT as the P450 mechanism-based inactivator were determined to be 45.6 and 193 M, and the maximal inactivation rate constants (k inact ) were determined to be 0.089 and 0.075 min ؊1 for the mouse and guinea pig liver microsomes, respectively. The projected P450 inactivations at the plasma C max of ABT agreed with the inhibitions of P450-mediated AP clearance observed in vivo. For mechanistic studies in vivo overall, a 2-h prior oral pretreatment with ABT at 50 mg/kg in mice and 100 mg/kg in guinea pigs would provide significant systemic concentrations of the inhibitor over 24 h and inhibition of P450-dependent clearance of test compounds.In our last communication (Balani et al., 2002), the dosing regimen of 1-aminobenzotriazole (ABT), a nonspecific inhibitor of cytochromes P450 (P450s) (Huijzer et al., 1989;Constan et al., 1999), was established in rats, dogs, and monkeys to effectively inhibit P450s and hence decrease the plasma clearance and increase the exposure of antipyrine (AP), a nonspecific probe substrate of P450s (Engel et al., 1996;Sharer and Wrighton, 1996;Matzke et al., 2000). All P450s were shown to be affected by ABT treatment (Balani et al., 2002). Due to wide distribution of ABT in rats, P450 inhibition is expected to be general in the body tissues (Town et al., 1993). The literature previously contained varied treatment of animals with ABT (e.g., dosing route, dose level, pretreatment time, and frequency of dosing). The current study extends our previous studies to mice and guinea pigs, which are routinely used for mechanistic PK, toxicity, and pharmacology studies; thus, it is intended to provide guidelines for the pretreatment of animals with ABT to significantly alter the oxidative metabolism of test compounds (e.g., to evaluate metabolite versus parent compound-based toxicities or boost compound concentration available for a target enzyme or receptor). Although the safety assessment of chronic dosing of ABT in mice and guinea pigs has not been reported, its safety in rats has clearly been demonstrated (Mico et al., 1988). This report also highligh...

Application of Osmotic Pumps for Sustained Release of 1-Aminobenzotriazole and Inhibition of Cytochrome P450 Enzymes in Mice: Model Comparison with the Hepatic P450 Reductase Null Mouse

“…A summary of antipyrine subcutaneous pharmacokinetics after 2-hour and 24-hour bolus ABT pretreatment is provided in Fig. 1A and Table 1 . Antipyrine was administered by subcutaneous, rather than intravenous, dosing in our experimental design (compared with the report by Balani et al, 2004). Results from a bioavailability (F) study (Supplemental Fig.…”

“…We considered the duration of ABT's inhibitory effect after a single dose. A report by Balani et al (2004), describing the interaction between ABT and antipyrine in mice, indicated clear inhibition of P450 activity to 8 hours postdose; however, low levels of antipyrine were observed at the 24-hour time point. Assuming a constant level of P450 inhibition over 24 hours (and monoexponential elimination of antipyrine), one would have expected much higher antipyrine levels at 24 hours.…”

“…This observation led us to speculate that after a single bolus dose of ABT, restoration of liver P450 activity occurs between 8 and 24 hours after inhibitor administration. We repeated the study by Balani et al (2004) and marked changes for antipyrine pharmacokinetic parameters were apparent for mice that received 2-hour prior subcutaneous treatment with ABT. The area under the concentration-time curve for antipyrine increased 8-fold and clearance was reduced by 88% compared with the control group.…”

“…The utility of ABT for in vivo P450 inhibition has been evaluated in a range of species, including mice, rats, guinea pigs, dogs, and monkeys (Balani et al, 2002(Balani et al, , 2004. For each species, efficient inhibition of metabolism was demonstrated with antipyrine, a nonspecific substrate of P450 enzymes (Engel et al, 1996;Sharer and Wrighton, 1996).…”

Role of Biotransformation Studies in Minimizing Metabolism-Related Liabilities in Drug Discovery

“…ABT undergoes bioactivation to a reactive intermediate that alkylates most CYPs, resulting in their inactivation (46). Thus, the pretreatment of animals with ABT during a toxicology study (a dose of 50 mg/kg administered 2 h before administration of the test article is a common practice (47,48)) may reduce the severity of adverse events, but only if such events are mediated by a toxic metabolite and not the parent compound itself. This approach has been used to implicate metabolites in the onset of hepatotoxicity in mice following administration of furosemide, (49) and nephrotoxicity in rats following treatment with [2,[3][4][5][6][7][8][9][10][11][12][13][14] C]-N-(3,5-dichlorophenyl)succinimide (50).…”