Background: Cytochrome P450 (CYP) enzymes represent a heme containing protein superfamily metabolizing a broad variety of xenobiotics, including drugs and toxic chemicals. 11 CYP families are expressed in a human liver and gastrointestinal tract (CYP1A2, CYP2A6, CYP2B6, CYP2C8/9/18/19, CYP2D6, CYP2E1, and CYP3A4/5). Five major isoforms (CYPs 1A2, 3A4, 2C9, 2C19, and 2D6) are involved in about 95% of the known drug metabolism. Recent studies showed the increasing role of CYP2C8 and CYP2B6 in metabolism of numerous drugs and important endogenous compounds. Cytochrome P450s are of critical importance due to the two of the most significant problems in clinical pharmacology: metabolism-mediated drug-drug interactions (DDI) and individual variability in drug metabolism. It is important to evaluate the potential inhibition of a new drug candidate for the most clinically relevant CYP450 enzymes. CYP450 inhibition may potentially lead to elevated in vivo plasma levels of a co-administered drug metabolized by the inhibited enzyme, and, consequently, to adverse drug reactions and toxicity. Assessment of the following CYP450 inhibition by a new drug candidate is recommended by FDA and EMA: CYP3A4, CYP2C9, CYP2C19, CYP2D6, CYP1A2, CYP2C8 and CYP2B6.

Service Details: The potential inhibition of 7 major cytochromes CYP1A2, CYP3A4, CYP2C9, CYP2C8, CYP2C19, CYP2D6, and CYP2B6 is assessed using LC-MS/MS based assay, in which biotransformations of the CYP450 specific substrates are used as markers to quantify the enzymatic activity. The FDA recommended CYP-selective substrates are used in the assay, which represent well characterized currently or previously marketed drugs. Quantification of a decrease in the formation of a metabolite in the presence of an inhibitor compared to the vehicle control is used to determine the CYP450 inhibition. Due to high specificity of the substrates to CYP450 isoforms not only individually expressed human cytochromes can be used in the assay but also human liver microsomes comprising a full set of CYP450 enzymes. The human liver microsomes assay is more comparable to the in vivo processes occurred in the liver and it is considered as ‘‘gold standard’’ for in vitro drug-drug interaction evaluation. This testing system is accepted for regulatory in vitro DDI studies.

Isoform specific CYP450 substrates, metabolites and inhibitors

CYP450 Substrate Metabolite Reference Inhibitor
1A2 Phenacetin Acetaminophen Furafylline
3A4 Testosterone 6β-Hydroxytestosterone Ketoconazole
2C9 Diclofenac 4’-Hydroxydiclofenac Sulphaphenazole
2C19 S-Mephenytoin S-4-Hydroxymephenytoin Tranylcypromine
2D6 Dextromethorphan Dextrorphan Quinidine
2B6 Efavirenz 8-Hydroxyefavirenz Ticlopidine
2C8 Amodiaquine Desethylamodiaquine Montelukast

 

For the rough estimate, single point assays are typically performed for each compound at 10 µM concentration or another concentration stipulated by the customer. If a noticeable inhibition is detected, the IC50 values for the tested compounds can be determined upon request. For this purpose, dose-response inhibition curves (8 points, 3-fold serial dilution) of the test compound and reference inhibitor are built starting at 100µM concentration. All test points are performed in duplicates. The IC50 values are calculated using Microsoft Excel and GraphPad Prism software. Reference inhibitors specific for each CYP enzyme are used to assess CYP inhibition in the control experiments for every batch of tested compounds. Test concentrations of the reference compounds correspond to approximately 4x fold of IC50 values for the corresponding cytochromes P450, which is expected to produce 80-100% inhibition in the properly performing assay.

LC-MS/MS based CYP450 inhibition assay conditions:

  • substrate conversion during the incubation is no more than 20% accordingly to FDA recommendations
  • substrate concentration is around Km value
  • good assay sensitivity that allows for quantification of the enzymatic activity even for 95% inhibited enzyme
  • final organic solvent concentration is low (<0.5%) to prevent strong enzyme inhibition
  • the extensively-characterized lots of pooled human liver microsomes (50 or 200 donors) are used to provide consistent and reproducible results

Advantages of LC-MS/MS method over fluorimetric assay:

  • LC-MS/MS analysis method is sensitive and specific, therefore human liver microsomes can be used, the fluorescent probes are not isoform-specific and should be used only with individual recombinant cytochromes
  • interference can occur from the test compound exhibiting fluorescence or fluorescence quenching and lead to false results

The service is available both in panel of 5, comprising the most clinically important CYPs (1A2, 3A4, 2C9, 2C19, and 2D6), and more expanded panel of 7 (1A2, 3A4, 2C9, 2C19, 2D6, 2B6, and 2C8) formats. Both individual recombinant cytochromes and human liver microsomes can be used. For the rough estimate, single point assays are typically performed for each compound at 10 µM concentration or another concentration stipulated by the customer. All test points are performed in duplicates. If a noticeable inhibition is detected, the IC50 values (test compound concentration which produces 50% inhibition) can be determined upon request. For this purpose, dose-response inhibition curves (8 points, 3-fold serial dilution) of the test compound and reference inhibitor are built starting at 100µM concentration. The IC50 values are calculated using Microsoft Excel and GraphPad Prism software. Reference inhibitors specific for each CYP enzyme are used to assess inhibition in the control experiments for every batch of tested compounds. Test concentrations of the reference compounds correspond to approximately 5x fold of IC50 values for the corresponding cytochromes P450, which is expected to produce 80-100% inhibition in the properly performing assay.

Figure 1. Test compounds CYP450 inhibition profiles.

Figure 2. IC50 of Sulphaphenazole

Deliverable: either single point assay data for each compound at 10 µM concentration or IC50 values for the tested compounds based on 8-point, 3-fold serial dilution dose-response inhibition curves (upon request). Full study report is provided.

Sample Submission: A minimal accurately weighable quantity of dry compound (~1 mg or 2 µmol) or 100 µL of 10-20 mM stock DMSO solution is required for this assay. For multiple assays, lesser amount of compound per assay may be sufficient, which should be discussed for each particular project.