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.

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 longer-term 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.

Preclinical metabolism and pharmacokinetics of NVS-CRF38, a potent and orally bioavailable corticotropin-releasing factor receptor 1 antagonist.

1. The pharmacokinetic properties and metabolism of NVS-CRF38 [7-(3,5-dimethyl-1H-1,2,4-triazol-1-yl)-3-(4-methoxy-2-methylphenyl)-2,6-dimethylpyrazolo[5,1-b]oxazole], a novel corticotropin-releasing factor receptor 1 (CRF1) antagonist, were determined in vitro and in animals. 2. NVS-CRF38 undergoes near complete absorption in rats and dogs. In both species the compound has low hepatic extraction and is extensively distributed to tissues. 3. In rat and human hepatic microsomes and cryopreserved hepatocytes from rat, dog, monkey and human, NVS-CRF38 was metabolised to form O-desmethyl NVS-CRF38 (M7) and several oxygen adducts (M1, M3, M4, M5 and M6). In hepatocytes further metabolites were observed, specifically the carboxylic acid (M2) and conjugates (sulphate and glucuronide) of M7. 4. Formation of primary metabolites in hepatocytes was blocked by the cytochrome P450 enzyme (P450) suicide inhibitor 1-aminobenzotriazole, implicating P450 enzymes in the primary metabolism of this compound. 5. NVS-CRF38 is weakly bound to plasma proteins from rat (fub = 0.19), dog (fub = 0.25), monkey (fub = 0.20) and humans (fub = 0.23). Blood-to-plasma partition for NVS-CRF38 approaches unity in rat and human blood. 6. The hepatic clearance of NVS-CRF38 in humans is predicted to be low (extraction ratio ∼ 0.2) based on scaling from drug depletion profiles in hepatic microsomes.

1-Aminobenzotriazole modulates oral drug pharmacokinetics through cytochrome P450 inhibition and delay of gastric emptying in rats.

The simultaneous effects of the cytochrome P450 inhibitor 1-aminobenzotriazole (ABT) on inhibition of in vivo metabolism and gastric emptying were evaluated with the test compound 7-(3,5-dimethyl-1H-1,2,4-triazol-1-yl)-3-(4-methoxy-2-methylphenyl)-2,6-dimethylpyrazolo[5,1-b]oxazole(NVS-CRF38), a novel corticotropin releasing factor receptor 1 (CRF1) antagonist with low water solubility, and the reference compound midazolam with high water solubility in rats. Pretreatment of rats with 100 mg/kg oral ABT administered 2 hours before a semisolid caloric test meal markedly delayed gastric emptying. ABT increased stomach weights by 2-fold; this is likely attributable to a prosecretory effect because stomach concentrations of bilirubin were comparable in ABT and control groups. ABT administration decreased the initial systemic exposure of orally administered NVS-CRF38 and increased Tmax 40-fold, suggesting gastric retention and delayed oral absorption. ABT increased the initial systemic exposure of midazolam, however for orally (but not subcutaneously) administered midazolam, extensive variability in plasma-concentration time profiles was apparent. Careful selection of administration routes is recommended for ABT use in vivo, variable oral absorption of coadministered compounds can be expected due to a disturbance of gastrointestinal transit.

Application of a deuterium replacement strategy to modulate the pharmacokinetics of 7-(3,5-dimethyl-1H-1,2,4-triazol-1-yl)-3-(4-methoxy-2-methylphenyl)-2,6-dimethylpyrazolo[5,1-b]oxazole, a novel CRF1 antagonist.

Deuterium isotope effects were evaluated as a strategy to optimize the pharmacokinetics of 7-(3,5-dimethyl-1H-1,2,4-triazol-1-yl)-3-(4-methoxy-2-methylphenyl)-2,6-dimethylpyrazolo[5,1-b]oxazole (NVS-CRF38), a novel corticotropin-releasing factor receptor 1 (CRF1) antagonist. In an attempt to suppress O-demethylation of NVS-CRF38 without losing activity against the CRF1 receptor, the protons at the site of metabolism were replaced with deuterium. For in vitro and in vivo studies, intrinsic primary isotope effects (KH/KD) were determined by the ratio of intrinsic clearance (CLint) obtained for NVS-CRF38 and deuterated NVS-CRF38. In vitro kinetic isotope effects (KH/KD) were more pronounced when CLint values were calculated based on the rate of formation of the O-desmethyl metabolite (KH/KD ∼7) compared with the substrate depletion method (KH/KD ∼2). In vivo isotope effects were measured in rats after intravenous (1 mg/kg) and oral (10 mg/kg) administration. For both administration routes, isotope effects calculated from in vivo CLint corresponding to all biotransformation pathways were lower (KH/KD ∼2) compared with CLint values calculated from the O-demethylation reaction alone (KH/KD ∼7). Comparative metabolite identification studies were undertaken using rat and human microsomes to explore the potential for metabolic switching. As expected, a marked reduction of the O-demethylated metabolite was observed for NVS-CRF38; however, levels of NVS-CRF38's other metabolites increased, compensating to some extent for the isotope effect.

Evaluation of recombinant cytochrome P450 enzymes as an in vitro system for metabolic clearance predictions.

The aim of this study was to explore the potential of recombinant cytochrome P450 (P450) enzymes for human metabolic clearance prediction. The relative abundance and relative activity approaches were compared as methods to bridge the gap between catalytic activities in recombinant P450 enzymes and human liver microsomes (HLMs). Relative activity factors were measured by determining the intrinsic clearance (CL(int)) of probe substrates (bufuralol-CYP2D6, diclofenac-CYP2C9, midazolam-CYP3A4, and phenacetin-CYP1A2) in recombinant P450s and 16 HLM donors. Simultaneous determination of drug depletion and metabolite formation profiles has enabled a direct comparison of these methods for CL(int) determination. Of the 110 drugs tested, 66% were metabolized by one or more P450 enzymes; of these 44% of were metabolized by CYP3A4 (0.3-21 microl/min/pmol of P450), 41% by CYP2D6 (0.6-60 microl/min/pmol of P450), 26% by CYP2C19 (0.4-8.1 microl/min/pmol of P450), 9% by CYP1A2 (0.4-2.5 microl/min/pmol of P450), and 4% by CYP2C9 (0.9-6.4 microl/min/pmol of P450). Recombinant enzymes demonstrated improved prediction reliability relative to HLMs and hepatocytes. The most reliable correlations in terms of lowest bias and highest precision were observed by comparing in vivo CL(int), calculated using the parallel-tube model and incorporating fraction unbound in blood, with in vitro CL(int) determined using relative activity factors and adjusted for nonspecific binding. Predictions were less reliable using the relative abundance approach. For these drugs, recombinant P450 enzymes offer improved assay sensitivity compared with HLMs and cryopreserved hepatocytes for CL(int) determination using the drug depletion method.