Validated Assays for Human Cytochrome P450 Activities

Abstract

ABSTRACT:The measurement of the effect of new chemical entities on human cytochrome P450 marker activities using in vitro experimentation represents an important experimental approach in drug development. In vitro drug interaction data can be used in guiding the design of clinical drug interaction studies, or, when no effect is observed in vitro, the data can be used in place of an in vivo study to claim that no interaction will occur in vivo. To make such a claim, it must be assured that the in vitro experiments are performed with absolute confidence in the methods used and data obtained. To meet this need, 12 semiautomated assays for human P450 marker substrate activities have been developed and validated using approaches described in the GLP (good laboratory practices) as per the code of U.S. Federal Regulations. The assays that were validated are: phenacetin O-deethylase (CYP1A2), coumarin 7-hydroxylase (CYP2A6), bupropion hydroxylase (CYP2B6), amodiaquine N-deethylase (CYP2C8), diclofenac 4-hydroxylase and tolbutamide methylhydroxylase (CYP2C9), (S)-mephenytoin 4-hydroxylase (CYP2C19), dextromethorphan O-demethylase (CYP2D6), chlorzoxazone 6-hydroxylase (CYP2E1), felodipine dehydrogenase, testosterone 6␤-hydroxylase, and midazolam 1-hydroxylase (CYP3A4 and CYP3A5). High-pressure liquid chromatography-tandem mass spectrometry, using stable isotope-labeled internal standards, has been applied as the analytical method. This analytical approach, through its high sensitivity and selectivity, has permitted the use of very low incubation concentrations of microsomal protein (0.01-0.2 mg/ml). Analytical assay accuracy and precision values were excellent. Enzyme kinetic and inhibition parameters obtained using these methods demonstrated high precision and were within the range of values previously reported in the scientific literature. These methods should prove useful in the routine assessments of the potential for new drug candidates to elicit pharmacokinetic drug interactions via inhibition of cytochrome P450 activities.Drug-drug interactions are of great interest to scientists involved in drug research, regulatory authorities who are responsible for public safety, physicians, and their patients. Since "polypharmacy," or the practice of simultaneous prescription of more than one drug to treat one or more conditions in a single patient, has become a more common practice, drug interactions have been cited as one of the major reasons for hospitalization and even death (Lazarou et al., 1998). Thus, a great deal of effort is expended by researchers engaged in new drug research in avoiding the development of compounds that will cause drug-drug interactions.The most common mechanism underlying drug-drug interactions is the inhibition of cytochrome P450 activities. Several drugs in common use cause large increases in exposure to other drugs. Examples include ketoconazole, itraconazole, erythromycin, clarithromycin, diltiazem, and nefazodone (CYP3A); quinidine, paroxetine, and terbinafine (CYP2D6); ticlopidine (CYP2C19); ...

Profiling Induction of Cytochrome P450 Enzyme Activity by Statins Using a New Liquid Chromatography-Tandem Mass Spectrometry Cocktail Assay in Human Hepatocytes

“…The P450 activity cocktail assay presented here is focused on the seven most important P450s as judged by their roles in the metabolism of clinically used drugs (Zanger et al, 2008). For CYP1A2 (phenacetin), CYP2B6 (bupropion), CYP2C8 (amodiaquine), CYP2C9 (tolbutamide), and CYP2C19 (S-mephenytoin), we used single established marker substrates (Richter et al, 2004;Walsky and Obach, 2004;Turpeinen et al, 2009) and combined them in a cocktail assay with substrates not used before for CYP2D6 (propafenone) and CYP3A4 (atorvastatin).…”

Metabolism of Ramelteon in Human Liver Microsomes and Correlation with the Effect of Fluvoxamine on Ramelteon Pharmacokinetics

“…These are additionally corrected for intersystem scaling factors (Proctor et al, 2004) determined for the lots of recombinant enzymes used in this investigation, which are based off of previously determined V max values using standard marker substrate activities (Walsky and Obach, 2004); these were 0.48, 0.40, and 0.41 for CYP1A2, CYP2C19, and CYP3A4, respectively. Use of these intersystem scaling factor-corrected sum of CLЈ int values for each of the metabolites generated for the three recombinant P450 enzymes yields a total in vitro CLЈ int value of 514 l/(min ⅐ mg), which scales to an in vivo value of 462 ml/(min ⅐ kg).…”

The Pharmacogenetics of the Antimalarial Amodiaquine

“…This biotransformation occurs mostly in the liver and is almost exclusively performed by the cytochrome P450 (CYP) 2C8 (Li et al, 2002). The high specificity of CYP2C8 for this reaction has even led to the proposal of AQ as a specific probe drug for this P450 isoform (Walsky et al, 2004). Besides this main step, other putative AQ metabolites have been proposed and at least partially confirmed (figure 1).…”