Cytochrome P450 Identification

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Unequivocal identification of one or more specific cytochrome P450 enzymes responsible for the metabolism of new therapeutic agents is the cornerstone of in vitro metabolism studies. This information is also critical for the follow-up cytochrome P450 inhibition and induction studies in the overall evaluation of in vitro drug-drug interactions. For all these studies, the experimental conditions should be that the measured initial reaction rates (in terms of product formation) are linear with respect to enzyme concentration and incubation time. It is preferable to use low enzyme concentration (e.g., below 0.5mg human liver microsomal protein per mL) and short incubation time (less than 20 min) to minimize protein binding and depletion of substrate and inhibitor (no more than 20% consumption, preferably less than 10%). If the analytical sensitivity is not an issue, lower enzyme concentration and shorter incubation time are highly desirable. In case of a slow substrate turnover, higher enzyme concentration and longer incubation time can be used as long as the initial metabolic rates are being measured.

If the cytochrome P450-mediated metabolism represents a significant clearance mechanism for the NME, cytochrome P450 reaction phenotyping should be carried out, generally, with human liver microsomes and recombinant cytochrome P450s using a combination of several basic approaches [22]. The NME concentrations used are generally at or below the Km values. Initial reaction rates are measured in the absence and the presence of antibodies or chemical inhibitors, or with a panel of human liver microsomes for correlation analysis with various cytochrome P450 probe substrates. If there is an indication for the involvement of more than one cytochrome P450 in the metabolism of the drug, several drug concentrations (e.g., low, medium, and high-spanning two to three orders of magnitude) should be used for inhibition studies.

Chemical Inhibitors and Inhibitory Antibodies. Specific and potent inhibitors are valuable for cytochrome P450 reaction phenotyping. In this respect, inhibitory antibodies (particularly monoclonal antibodies) with demonstrated specificity and potency can be useful [23], as illustrated in a recent paper by Granvil et al. [24]. These investigators described that the 4-hydroxylation of debrisoquine, a well-recognized probe reaction of CYP2D6, is mediated not only by CPY2D6 but also by human CYP1A1. Whereas quinidine, a recognized selective inhibitor of CYP2D6, inhibits the

4-hydroxylation of debrisoquine by both CYP2D6 and human CYP1A1, anti-CYP2D6 monoclonal anitbody inhibits specifically CYP2D6-medicated reaction, and not CYP1A1-dependent metabolism. To date, specific and potent monoclonal as well as polyclonal antibodies have not been widely used by the pharmaceutical industry possibly due to their high cost and limited availability from commercial sources.

A desirable antibody inhibition study can be conducted in two stages. Initially, metabolism of a drug by pooled human liver microsomes is examined in the presence of antibodies against all major human cytochrome P450s at a single high concentration (known to give greater than 80-95% inhibition with probe substrates) to determine which antibodies significantly inhibit the metabolism. This study establishes that one or more cytochrome P450 is involved in the metabolism of an NME. In subsequent studies, the effect of those inhibitory antibodies on the metabolism of the NME is studied in more detail using a series of antibody concentrations. A well-designed study should show that metabolism is inhibited strongly by the specific antibody in a concentration-dependent manner at low antibody concentrations and then reaches maximum inhibition at higher antibody concentrations [25] as illustrated in Fig. 1 (curves A and D). A steep inhibition slope indicates high potency of the antibody against specific cytochrome P450. The extent of the maximum inhibition indicates the extent (%) of the metabolism of the NME by this particular cytochrome P450 enzyme. No meaningful conclusion can be made regarding the role of a specific cytochrome P450 in the metabolism of an NME when an antibody inhibition study showed a shallow inhibition slope (an indication of low antibody potency) and failed to demonstrate maximum inhibition (Fig. 1, curve B). Thus, a good antibody inhibition study establishes not only the involvement but also the quantitative importance of a particular cytochrome P450 in the metabolism of the NME. When it is desirable to obtain information regarding the variability of cytochrome P450 involvement, particularly when more than one cytochrome P450 enzymes are involved, similar studies can be carried out with a panel of human liver microsomal preparations. Frequently, one can demonstrate a wide range of involvement of specific cytochrome P450 in the metabolism of a particular drug with microsomes from different donors [23].

Although specific chemical inhibitors for individual human cytochrome P450 are rare, isoform-selective inhibitors are generally available at most pharmaceutical laboratories and are valuable when properly used. Table 1 lists preferred probe substrates and inhibitors for individual cytochrome P450 enzyme [21]. Similar to antibody inhibition studies, chemical inhibition studies can be carried out first with a single inhibitor concentration (known to give strong inhibition with probe substrates) to

0 2 4 6 8 10 12

mg IgG/nmol P450

FIGURE 1 Inhibition of human liver microsomal drug metabolism by antibodies against cytochrome P450. Curve A depicts the strong inhibition of compound A metabolism by anti-CYP3A4 antibodies. The steep inhibition slope at low antibody concentrations indicates high potency of this antibody preparation. Maximum inhibition at higher antibody concentrations indicates that greater than 90% of the metabolism of compound A is mediated by CYP3A4 in this pooled human liver microsomal sample. Curve B shows the inhibition of compound A metabolism in human liver microsome by a different anti-CYP3A4 antibody preparation. The shallow inhibition slope indicates that either this antibody has a low potency against CYP3A4 or it cross-reacts with another cytochrome P450. No conclusion can be made regarding the role of CYP3A4 in the metabolism of compound A. Curve C is the control experiment showing lack of inhibition of compound A metabolism by pre-immune IgG. Curve D depicts the inhibition of the metabolism of compound B by anti-CYP3A4 antibodies. The steep inhibition slope is noted at low concentrations of this potent antibody. CYP3A4 is responsible for 50% of the determine which probe inhibitors significantly inhibit the metabolism of the NME, followed by a more detailed study involving a series of concentrations of the inhibitors. As shown in Fig. 2 (curves A and B), a good chemical inhibitor selective for a given cytochrome P450 isoform should give strong inhibition (a steep inhibition slope) in the metabolism of an NME at low inhibitor concentrations and reach maximum inhibition at higher inhibitor concentrations so that the quantitative involvement of this cytochrome P450 isoform in metabolism can be established. Gradual increase in inhibition with a wide range of inhibitor concentrations (i.e., a shallow inhibition slope, Fig. 2, curve C) would suggest that the inhibitor either has low potency toward the particular cytochrome P450 or it acts as a poor substrate of the enzyme. In this case inhibition results from the study have limited values. When studies are carried out using a panel of human

Substrates

Inhibitors

Substrates

Inhibitors

CYP

"Preferred"

"Acceptable"

"Preferred"

"Acceptable"

1A2

Ethoxyresorufin

Caffeine (low turnover)

Furafylline

a-naphthoflavone (but can also

Phenacetin

Theophylline (low turnover) Acetanilide (mostly applied in hepatocytes) Methoxyresorufin

activate and Inhibit CYP3A4)

2A6

Coumarin

Coumarin (but high turnover)

2B6

S-Mephenytoin (N-desmethyl metabolite)

Bupropion (availability of metabolite standards?)

Sertraline (but also inhibits CYP2D6)

2C8

Paclitaxel (availability of standards?)

("glitazones"—availability of standards?)

2C9

S-Warfarin Diclofenac

Tolbutamide (low turnover)

Sulphaphenazole

2C19

S-Mephenytoln (4-hydroxy metabolite) Omeprazole

Ticlopidine (but also Inhibits CYP2D6) Nootkatone (but also inhibits CYP2A6)

Dextromethorphan

Metoprolol Debrisoquine Codeine

(all with no problems, but less commonly used)

Quinidine

2E1

Chlorzoxazone

4-nitrophenol Laurie acid

4-methyl pyrazole

3A4

Midazolam

Nifedipine

Ketoconazole (but recent evidence

Cyclosporin

Testosterone

Felodipine

indicates that It is also a potent

(strongly recommended to use at

Cyclosporin

inhibitor of CYP2C8)

least two structurally unrelated

Terfenadine

Troleandomycin

substrates)

Erythromycin Simvastatin

ID 5

0 20 40 60 80 100 120

Chemical inhibitor "x" [uM]

FIGURE 2 Inhibition of human liver microsomal drug metabolism by a chemical inhibitor of CYP3A4. Curve A depicts the strong inhibition of compound A metabolism by this inhibitor. The steep inhibition slope at low inhibitor concentrations indicates that this inhibitor of CYP3A4 is very potent. CYP3A4 contributes to approximately 90% of the metabolism of compound A in this pooled microsomal preparation. Curve B shows that CYP3A4 contributes to 50% of the microsomal metabolism of compound B. Curve C depicts the shallow inhibition slope indicating poor inhibition of the metabolism of compound C even at high inhibitor concentrations. No conclusions can be made regarding the role of CYP3A4 in the metabolism of compound C.

liver microsomal preparations, different degrees of maximum inhibition in metabolism provide information regarding the variability of specific cytochrome P450 involvement in the metabolism of the NME among individual subjects.

Recombinant Human Cytochrome P450 Enzymes. Microsomes containing individually expressed human cytochrome P450s provide a different approach for cytochrome P450 reaction phenotyping. This approach establishes the intrinsic capability of the individual cytochrome P450 in the metabolism of an NME, in the absence of other cytochrome P450 species. If one or more cytochrome P450 species are involved in an NME's metabolism, it is important to examine the contribution of each cytochrome P450 to human liver microsomal metabolism using inhibitory antibodies or chemical inhibitors. Sometimes, a recombinant cytochrome P450 found to be involved in an NME's metabolism, based on a recombinant enzyme study, may later be shown to play little or no role in liver microsomal metabolism of the drug in the presence of other cytochrome P450s, based on an inhibition study. Furthermore, for these cytochrome enzymes for which activities are observed initially, a determination of the enzyme kinetics (Km and Vmax) may be warranted so that the intrinsic clearance and the relative importance of these different cytochrome P450 species contributing to the metabolism of the NME can be evaluated [26-28].

Correlation Analysis. Using this approach, the drug is incubated with a panel of human liver microsomes (preferably more than 10 preparations) and the reaction rates of an NME determined in each preparation are correlated with the reaction rates of a cytochrome P450 probe substrate measured in the same microsomal preparation. If a particular cytochrome P450 is responsible for the metabolism of the NME, a high correlation should be observed between the metabolic rates of the drug and the marker substrate. However, this type of correlation analysis appears to be less reliable in identifying specific cytochrome P450 enzymes responsible for the metabolism of an NME. For example, Weaver et al. [29] reported that 58C80 hydroxylation is catalyzed by CYP2C9 based on inhibition and recombinant cytochrome P450 studies; however, there is no correlation between 58C80 hydroxylation and CYP2C9 probe substrate activity (r=0.023). In another study, Heyn et al. [30] reported that although high correlations between S-mephenytoin N-demethylation and CYP2B6 (r=0.91), CYP2A6 (r=0.88), and CYP3A4 (r=0.74) were observed, other approaches showed CYP2B6 to be the major enzyme responsible for S-mephenytoin N-demethylation while CYP2A6 and CYP3A4 played no significant role in this reaction.

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Your Metabolism - What You Need To Know

Your Metabolism - What You Need To Know

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