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Cytochrome P450 Inhibition (Time dependent IC50 shift)

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 time-dependent CYP450 inhibition assay (IC50 Shift) utilises drug-like probe substrates, HLM and the Phase I cofactor, NADPH and two pre-incubation times, 0 and 30 minutes.  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 following a pre-incubation period. 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
Pre-Incubation Conditions 30 and 0 minutes at 37°C
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 at 0 and 30 min pre-incubation. Compounds that exhibit a shift in their IC50 of >1.5 following a 30 min pre-incubation are considered time-dependent inhibitors

 

Results

 

Figure 1 In-house CYP2D6 time dependent Inhibition (IC50 Shift) Screening Output for Quinidine (reversible), Paroxetine (time-dependent) and 2 test compounds, A and B. Data shown are the mean of 2 replicates.

 

Figure 2  In-house CYP2C9 time dependent Inhibition (IC50 Shift) Screening Output for Sulfaphenazole (reversible) and  Tielinic acid  (time-dependent). Data shown are the mean of 2 replicates.

 

Figure 3  In-house CYP3A4 time dependent Inhibition (IC50 Shift) Screening Output for Ketoconazole (reversible) and  Azamulin (time-dependent). Data shown are the mean of 2 replicates.

 

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.