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

Abstract

The effectiveness of controlled release 1-aminobenzotriazole (ABT) administration to inhibit cytochrome P450 (P450) enzymes has been evaluated in mice. To maximize the duration of P450 inhibition in vivo, ABT was administered via an osmotic pump. The degree of P450 inhibition was compared with that achieved with a single bolus dose of ABT. Two-hour prior subcutaneous treatment of mice with ABT (50 mg/kg) inhibited antipyrine clearance by 88%. A less pronounced inhibitory effect (29% reduction in clearance) was observed when ABT was administered 24-hours before antipyrine administration, indicating partial restoration of P450 activity during this longer pretreatment time. The duration of ABT in mice was very short (mean residence time = 1.7 hours) after subcutaneous bolus administration. When the inhibitor was delivered by an osmotic pump, maximum blood concentrations of the inhibitor were observed 24 hours after device implantation and were maintained at steady state for 6 days. Inhibition of P450 activity, as measured by antipyrine clearance, was confirmed at 24 hours and 120 hours after pump implantation, highlighting the utility of this method as a longerterm model for P450 inhibition in mice. The magnitude of P450 inhibition in ABT-treated mice was compared with that in hepatic P450 reductase null mice and both models were comparable. In vivo ABT administration by an osmotic pump offers an effective approach for longer-term P450 inhibition in mice and avoids the necessity for multiple dosing of the inhibitor. Introduction1-Aminobenzotriazole (ABT) is a time-dependant inhibitor of cytochrome P450 (P450) enzymes (Ortiz de Montellano et al., 1984) and is often used as a tool compound for in vitro (Furnes and Schlenk, 2005) and in vivo (Stringer et al., 2014;Boily et al., 2015) drug metabolism studies. 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). To avoid the need for repeated ABT administration, we have explored the use of osmotic pumps as a technology to achieve sustained release of ABT and sustained inhibition of P450 enzymes in mice.To assess the effectiveness of the in vivo ABT approach, we compared the degree of P450 inhibition using this method with that in the hepatic reductase null (HRN) mouse. The HRN mouse only has residual hepatic P450 activity as a result of a liver-specific knockout of the P450 reductase enzyme during the postnatal period (Henderson et al., 2003), and extrahepatic P450 is unaffected. Marked differences in drug disposition have been observed in HRN mice compared with wild-type mice (Pickup et al., 2012;Boggs et al., 2014;Grimsley et al., 2014). After a single bolus dose of ABT to mice, the duration of inhibitory activity was relatively short: clearance of antipyrin...

Towards the Development of an In vivo Chemical Probe for Cyclin G Associated Kinase (GAK)

“…CYP2C9 was particularly resistant to inhibition by ABT, with 60% of the activity remaining. A separate study indicated that ABT caused a concentration-dependent inhibition of CYP1A2, -2B6, -2C9, -2C19, -2D6, and -3A4 [25]. Taken together, our results suggest that CYP2C9 or -2C8 may be responsible for the metabolism of the benzo[ d ][1,3]dioxol-4-yl group of 9 and 14 in MLM.…”

“…Long-term dosing of ABT is also possible. ABT has displayed a well-behaved pharmacokinetic profile over five days when dosing with an osmotic pump [25]. Minimal side effects were observed for chronic ABT dosing, the most prominent of which was delayed gastric emptying [33].…”

Development of SGC-GAK-1 as an orally active in vivo probe for cyclin G associated kinase through cytochrome P450 inhibition

“…For example, ABT displayed a well-behaved pharmacokinetic profile over 5 days dosing via osmotic pump. 24 Minimal side effects were observed on chronic ABT dosing, the most prominent of which is gastric emptying. 25 Second, there is potential for 1 and ABT to be utilized in species other than mice in in vivo studies.…”

1-Aminobenzotriazole: A Mechanism-Based Cytochrome P450 Inhibitor and Probe of Cytochrome P450 Biology

“…It has also been reported that 1-ABT delays gastric emptying in rats and can alter systemic exposure and T max of coadministered agents [ 30 ]. The observation that 50 mg/kg of 1-ABT inhibited antipyrine clearance by 88% when given shortly before the antipyrine, but only by 29% when given 24 h before, suggested that the duration of 1-ABT was relatively short [ 31 ]. However, The pharmacokinetic profiles of oral doses of 1-ABT ranging from 5 to 200 mg/kg, determined in rats, dogs, and monkeys, showed that a single dose of 50 mg/kg in rats and 20 mg/kg in dogs and monkeys produced high plasma concentrations that were sustained over 24 hours [ 32 ].…”

“…Administration of 1-ABT 2 hours before intravenous antipyrine decreased plasma antipyrine clearance by approximately 95% in mice and 84–95% in guinea pigs at all the doses of 1-ABT examined [ 33 ]. Osmotic pumps can be used to maintain maximum blood concentrations of 1-ABT for at least 6 days [ 31 ]. Indeed, ALZET osmotic pumps were able to maintain the 1-ABT plasma concentration above 4.1 mg/ml over 336 hours without overt toxicity [ 34 ].…”