CYTOCHROME P450 INHIBITION (REVERSIBLE)
Background
Protocol
The cytochrome P450 (CYP450) enzymes play a significant role in
Compound requirements
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.
10mM in DMSO, 10µL
CYP Isoforms
CYP1A2, CYP2B6, CYP2C8, CYP2C9,
CYP2C19, CYP2D6 and CYP3A4/5
Test Article Concentrations
0.001, 0.003, 0.01, 0.03, 0.1, 0.3, 1, 3, 10
and 30 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.
Results
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 Synagture 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.
Sygnature Discovery Limited
BioCity
Pennyfoot Street
Nottingham
NG1 1GF
United Kingdom
Telephone: +44 1159415401
Email: [email protected]