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Cytochrome P450 Inhibition (Reversible)

The cytochrome P450 (CYP450) enzymes play a significant role in the metabolism of both endogenous and exogenous compounds. Within this family, CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 are predominantly involved in the metabolism of drugs.

The measurement of the inhibition of each of these CYP450 enzymes in human liver microsomes (HLM) helps in predicting the potential for drug-drug interactions with co-administered drugs and in understanding the subsequent clinical consequences.

Sygnature’s standard reversible CYP450 inhibition assay utilises drug-like probe substrates, HLM and the Phase I cofactor, NADPH.  The assay monitors for the formation of the metabolites of the drug-like probe substrates in the absence and presence of a compound by LC-MS/MS. All assays have two replicates per compound and include a positive control inhibitor. Data output consists of the generation of an inhibition constant or IC50 value.

Protocol

Compound requirements 10mM in DMSO, 10µL
CYP Isoforms CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4/5 and others on request
Test Article Concentration 0.003, 0.009, 0.03, 0.08, 0.25, 0.74, 2.2, 6.7 and 20 µM
Analysis Method LC-MS/MS
Incubation Conditions CYP dependent; at 37°C
Cofactor NADPH (1 mM)
Controls Minus cofactor (0% control), no compound control (100% control)

Ion Suppression Control (Optional)

1 Positive control inhibitor (CYP450 isoform dependent)

Data Delivery IC50 value

Results

Figure 1 In-house CYP3A4 Inhibition Screening Output. Data shown are the mean of 2 replicates.

Figure 2 In-house CYP2D6 Inhibition Screening Output. Data shown are the mean of 2 replicates.

Figure 3 In-house CYP2C19 Inhibition Screening Output. Data shown are the mean of 2 replicates.

Figure 4 In-house CYP2C9 Inhibition Screening Output. Data shown are the mean of 2 replicates.

Figure 5 In-house CYP1A2 Inhibition Screening Output. Data shown are the mean of 2 replicates.

Figure 6 Sygnature Discovery’s IC50 values (mean ± standard deviation) for five CYP Isoforms control inhibitors.

About Us

The DMPK & Physical Sciences department at Sygnature Discovery is dedicated to understanding and optimising the absorption, distribution, metabolism and excretion of drug candidates by working in close partnership with clients and other departments within Sygnature to provide successful optimisation strategies.

We have extensive know-how and expertise to provide well validated, state-of-the-art assays and a comprehensive applied consultancy service for interpretation of the in vitro ADME and in vivo PK data.

Our corporate vision is to accelerate the discovery of new medicines, from the laboratory into development to treat patients.

Our DMPK mission is to deliver tailored DMPK expertise through innovation, quality and commitment.