| Why is it important to investigate cytochrome P450 inhibition? Cytochromes P450 are a family of enzymes which play a major role in the metabolism of drugs. Assessment of the potential of a compound to reversibly inhibit a specific cytochrome P450 enzyme is important as co-administration of compounds may result in one or both inhibiting the other’s metabolism. This may affect plasma levels in vivo and potentially lead to adverse drug reactions or toxicity. Please provide an overview of the Cloe Screen Cytochrome P450 Inhibition assay. The seven main cytochrome P450 isoforms (CYP1A, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4) are investigated in the Cloe Screen Cytochrome P450 Inhibition assay. Isoform-specific substrates are incubated individually with human liver microsomes and a range of test compound concentrations (typically 0.1 - 25µM). At the end of the incubation, the formation of metabolite is monitored by LC-MS/MS (or fluorescence in the case of CYP1A using ethoxyresorufin as substrate) at each of the test compound concentrations. An example IC50 profile is shown in Figure 3. A decrease in the formation of the metabolites compared to vehicle control is used to calculate an IC50 value (test compound concentration which produces 50% inhibition). Figure 3.
Graph displaying a typical inhibition profile. How do I interpret the data from the cytochrome P450 inhibition assay? Typically the compounds can be categorised into the following classification bands; | Potent inhibition | IC50 < 1µM | | Moderate inhibition | IC50 between 1 and 10µM | | No or weak inhibition | IC50 > 10µM | Inhibition potency must always be considered in the context of expected in vivo concentrations of the test compound. Although the criteria for acceptance are project and isoform-specific, potent inhibition is considered unfavourable and may preclude the development of a compound. What substrates and positive control inhibitors are used in the cytochrome P450 inhibition assay? We run a known specific positive control inhibitors for each of the isoform assays. Table 1: Table showing the positive control inhibitors for each cytochrome P450 isoform reaction | Isoform | Substrate Reaction | Positive Control Inhibitor | | CYP1A | Ethoxyresorufin O-deethylation | α-Naphthoflavone | | CYP2B6 | Bupropion hydroxylation | Ticlopidine | | CYP2C8 | Paclitaxel 6α-hydroxylation | Montelukast | | CYP2C9 | Tolbutamide 4-hydroxylation | Sulphaphenazole | | CYP2C19 | S-mephenytoin 4-hydroxylation | Tranylcypromine | | CYP2D6 | Dextromethorphan O-demethylation | Quinidine | | CYP3A4 | Midazolam 1-hydroxylation | Ketoconazole | | CYP3A4 | Testosterone 6ß-hydroxylation | Ketoconazole | What is the concentration of the probe substrates relative to Km? The substrate concentration is equivalent to the Km. Do you perform the assays for each isoform as a cocktail, or as separate incubations? We do not perform the incubations as a cocktail. All of our cytochrome P450 inhibition reactions are incubated seperately for each isoform. Why do you use human liver microsomes rather than cDNA expressed enzyme to study P450 inhibition? Metabolising systems which use recombinant enzymes are artificial as the enzyme is not present in its native environment and is often over-expressed. With these systems, there is an absence of competing enzymes and reactions. Human liver microsomes contain the full complement of P450 enzymes and so are more comparable to the in vivo situation. Why do you use LC-MS/MS rather than fluorescent probes? Conversion of ethoxyresorufin to resorufin is the only fluorescent reaction which we have chosen to use. Ethoxyresorufin is selective for CYP1A and so is appropriate to use with liver microsomes. However, for the remaining isoforms, traditional probe substrates are used with LC-MS/MS detection. There are several reasons for not using fluorescent probes for these isoforms, as listed below: - The fluorescent probes which have been developed are not isoform-specific and can only be used with individually expressed enzymes9.
- Interference can occur from the inhibitor being tested and lead to false negatives9.
- These substrates are not suitable for testing in vivo 9.
- Fluorescent probes for in vitro studies are not recommended for regulatory submission9.
- There appears to be a poor correlation of inhibitory potential using different fluoroprobes10.
Can I further characterise the type of inhibition determined? Following IC50 determination we can then determine the Ki for the test compound against the appropriate isoform. This will give information as to the potency of the inhibition and the type of inhibition (competitive, non-competitive, uncompetitive or mixed) and can be used to estimate the impact of any potential in vivo interactions.
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