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ADME PK

Cytochrome P450 inhibition assay

Understand the potential drug-drug interaction liabilities of your compounds by using our cytochrome P450 (CYP450) reversible inhibition assay for a range of isoforms.

The cytochrome P450 inhibition assay is one of our portfolio of in vitro ADME screening services. Cyprotex deliver consistent, high quality data with cost-efficiency that comes from a highly automated approach.

Inhibition of cytochrome P450 (CYP450) enzymes

  • Cytochrome P450 are a family of enzymes which play a major role in the metabolism of drugs.
  • Assessment of the potential of a compound to 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.
  • In vitro cytochrome P450 inhibition data are useful in designing strategies for investigating clinical DDI Studies.
  • Cyprotex's Cytochrome P450 Inhibition assays use industry accepted probe substrates and human liver microsomes.
  • In Cyprotex's Cytochrome P450 Inhibition assay, a decrease in the formation of the metabolites compared to the vehicle control is used to calculate an IC50 value (test compound concentration which produces 50% inhibition).
The effects of new drugs on well characterized drug metabolism reactions known to be specific for various human drug-metabolizing enzymes are routinely examined using in vitro approaches.

1Obach RS, Walsky RL, Venkatakrishnan K, Gaman EA, Houston JB and Tremaine LM (2006) JPET 316; 336-348.

Protocol

Cytochrome P450 inhibition assay protocol

Test Article Concentration 0, 0.1, 0.25, 1, 2.5, 10, 25 μM
(different concentrations available)
CYP Isoforms CYP1A, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 (other isoforms are available)
Test Article Requirements Dependent on number of isoforms assessed
Controls Known isoform specific inhibitors
Analysis method LC-MS/MS (with the exception of ethoxyresorufin for CYP1A)
Data Delivery IC50
Standard error of IC50

Data

Data from Cyprotex's cytochrome P450 inhibition assay

Known cytochrome P450 inhibitors were screened in Cyprotex's Cytochrome P450 Inhibition assay in quadruplicate over 4 separate assays.

 
Cytochrome P450 Inhibition data for CYP3A4
Figure 1
Cyprotex's Cytochrome P450 Inhibition data for CYP3A4. The effect of 5 known CYP3A4 inhibitors (clotrimazole, ketoconazole, mibefradil, nicardipine and verapamil) on the 1-hydroxylation of midazolam was investigated on 4 separate occasions. Error bars represent the standard deviation of 4 replicates on each experiment. The data show good consistency for inhibitors with a range of inhibition potential.
Comparison of IC50 values
Figure 2
Comparison of Cyprotex's IC50 values (mean ± standard deviation) for the control inhibitors with literature(2,3,4,5,6,7,8) values.

Q&A

Questions and answers on cytochrome P450 inhibition

Please provide an overview of the Cyprotex's Cytochrome P450 Inhibition assay.

The seven main cytochrome P450 isoforms (CYP1A, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 - other isoforms are available on request) are investigated in Cyprotex's Cytochrome P450 Inhibition assay. Isoform-specific substrates are incubated individually with human liver microsomes and a range of test compound concentrations (typically 0.1 - 25 µM). 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).

Cytochrome p450 inhibition
Figure 3
Graph displaying a typical inhibition profile.

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.

Evaluating the cytochrome P450 inhibition of a new investigational drug is recommended in the draft FDA guidance for drug interaction studies (2012)9 and the EMA guideline on the investigation of drug interactions (adopted 2012)10.

How do I interpret the data from the cytochrome P450 inhibition assay?

Typically the compounds can be categorized into the following classification bands;

Potent inhibition IC50 < 1 µM
Moderate inhibition IC50 between 1 and 10 µM
No or weak inhibition IC50 > 10 µM

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 unfavorable 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.

IsoformSubstrate ReactionPositive 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
Table 1: Table showing the positive control inhibitors for each cytochrome P450 isoform reaction

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?

Metabolizing 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:

  1. The fluorescent probes which have been developed are not isoform-specific and can only be used with individually expressed enzymes11.
  2. Interference can occur from the inhibitor being tested and lead to false negatives11.
  3. These substrates are not suitable for testing in vivo 11.
  4. Fluorescent probes for in vitro studies are not recommended for regulatory submission11.
  5. There appears to be a poor correlation of inhibitory potential using different fluoroprobes12.

Can I further characterize 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.

References

1 Obach RS et al. (2006) JPET 316; 336-348
2 Bu HZ et al. (2001) Eur J Pharm Sci 12 (4); 447-52
3 Turpeinen M et al. (2004) Drug Metab Dispos 32 (6); 626-631
4 Back DJ et al. (1988) Br J Clin Pharmacol 26 (1); 23-29
5 Dierks EA et al. (2001) Drug Metab Dispos 29 (1); 23-9
6 Eagling VA et al. (1998) Br J Clin Pharmacol 45 (2); 107-114
7 Moody GC et al. (1999) Xenobiotica 29 (1); 53-75
8 Nomeir AA et al. (2001) Drug Metab Dispos 29 (5); 748-53
9 FDA Draft Guidance for Industry. Drug Interaction Studies – Study Design, Data Analysis, Implications for Dosing, and Labeling Recommendations (2012)
10 The European Medicines Agency (EMA) Guideline on the Investigation of Drug Interactions (Adopted in 2012)
11 Bjornsson TD et al. (2003) Drug Met Dispos 31; 815-832
12 Stresser DM et al. (2000) Drug Metab Dispos 28; 1440-1448

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