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.
|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|
|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|
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.
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.
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