Alfentanil N-Dealkylation: Monitoring the formation of N-phenylpropionamide (AMX) to determine CYP3A4 activity in vitroAlfentanil is a synthetic analgesic cleared exclusively by hepatic metabolism. Due to the highly linear correlation between alfentanil systemic clearance and CYP3A4 activity, as well as the direct pharmacologic effects on pupil size, it has been used as a non-invasive metabolic probe for CYP3A4 activity in vivo. AMX is a direct metabolite of alfentanil, evaluated in this study for its potential to measure CYP3A4 activity in vitro.
CYP4F Enzymes are the Major Enzymes in Human Liver Microsomes that Catalyze the O-Demethylation of the Antiparasitic Prodrug DB289DB289 is a prodrug that is converted by several steps to the active metabolite DB75. DB289 exhibits enhanced oral efficacy and reduced acute toxicity over DB75, an aromatic dicationic compound that is effective against a broad range of pathogens in vitro including African trypanosomiasis (African sleeping sickness). This reaction phenotyping study aimed to identify the enzymes responsible for oxidative O-demethylation; the first step in this conversion to DB75.
In vitro CYP inhibition studies are often performed with pooled human liver microsomes by an IC50 “shift” design in which IC50 values for inhibition of a given CYP enzyme by a drug candidate are measured with and without a preincubation with NADPH. Overall, these results show that studies employing long substrate incubation periods with what would ordinarily be considered a low concentration of microsomal protein (0.1 mg/mL) can potentially underestimate the metabolism-dependent inactivation of CYP enzymes, potentially leading to the incorrect conclusion that only direct inhibition occurs.
An Evaluation of the Selectivity of Commonly Used Mechanism-Based Inhibitors of CYP Enzymes
One of the approaches to in vitro reaction phenotyping involves the use of a panel of mechanism-based inhibitors that supposedly inactivates each of the major human cytochrome P450 (CYP) enzymes involved in drug metabolism. We evaluated the specificity of several metabolism-dependent inhibitors, demonstrating that the inhibitory effects of several of the commonly used mechanism-based inhibitors are not restricted exclusively to their targeted CYP enzyme. In most cases, however, the inhibition of non-target enzymes is the result of direct inhibition rather than metabolism-dependent inhibition, such that selectivity may be maintained through the use of a long pre-incubation time.
On the Problem of Underestimating Rates of Glucuronidation Based on In Vitro Studies with Human Liver Microsomes: Additive and Differential Effects of BSA and Alamethicin on UGT2B7 But Not UGT1A Enzymes
It is becoming widely appreciated that estimates of intrinsic clearance based on in vitro rates of drug glucuronidation by human liver microsomes can grossly underestimate in vivo clearance rates, as in the case of the anti-HIV drug zidovudine (AZT). We have examined a variety of in vitro conditions for their effects on the rate of glucuronidation of AZT and morphine, which is largely catalyzed by UGT2B7, and on four UGT1A enzymes. Although the combination of BSA and alamethicin greatly improved the measurement of in vitro intrinsic clearance by UGT2B7, this improvement did not extend to several UGT1A enzymes and cannot be recommended as a universal in vitro procedure to improve estimates of the clearance of drugs that are extensively glucuronidated.
Bergamottin and dihydroxybergamottin in rapid screens for CYP inhibition and transporter interactions
Evaluation of Ebastine Hydroxylation as a Specific Probe of CYP2J2 in Human Liver Microsomes
The hydroxylation of ebastine has been proposed as a specific marker of CYP2J2 in human liver microsomes, although it is possible that CYP3A4 also contributes to this reaction. The results from this study are consistent with previous proposals that ebastine hydroxylation is a useful probe of CYP2J2 activity in human liver microsomes.
A Comparison of p-Vanillin and Phthalazine as Substrates for Human Aldehyde Oxidase
In the present study, we evaluated the oxidation of p-vanillin to vanillic acid and phthalazine to phthalazinone as in vitro reactions to study the cytosolic molybdozyme aldehyde oxidase. The results of this study suggest that p-vanillin and phthalazine are equally selective substrates with which to measure aldehyde oxidase activity in human liver cytosol, and that aldehyde oxidase activity in human liver cytosol varies widely (approximately 20 fold) from one individual to the next.