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Microsomal Metabolic Stability

The liver is the major site of drug metabolism in the body, with well over 50% of marketed drugs being eliminated via hepatic mediated metabolism.  Hence, measurement of the rate of clearance and the identity of the metabolites of a drug is important. In addition, in support of projects, the rate of clearance values can be used to rank compounds and to predict in vivo hepatic clearance values.  Microsomes from a variety of species and tissues can be used to understand inter-species differences and non-hepatic metabolism.

Sygnature’s standard microsomal metabolic stability assay utilises the Phase I cofactor, NADPH.  We can also offer a combined Phase I/Phase II assay, utilising NADPH and UDPGA to determine both Phase 1 and Phase 2 (UDP-glucuronosyltransferase (UGT)) metabolism.  For the latter, these are accessed by pre-incubating the microsomes with the pore-forming agent, alamethicin.

Sygnature’s microsomal stability assay monitors the disappearance of a compound in the presence and absence of cofactor. All assays have two replicates per compound and are validated by the inclusion of up to 3 species-specific control compounds.  Data output consists of mean intrinsic clearance and half-life measurements.

Protocol

Compound requirements 10mM in DMSO, 10µL
Microsomal Concentration 0.5 mg/mL
Test Article Concentration 1 μM
Time Points 0, 5, 10, 15, 30 minutes
Analysis Method LC-MS/MS
Incubation Conditions Up to 30 minutes at 37°C
Cofactor NADPH (1 mM) or

NADPH (1 mM) + UDPGA (2 mM)

Controls Minus cofactor at all time points

3 Positive control compounds (species dependant)

Data Delivery Intrinsic clearance (µL/min/mg protein)

Half-life (minutes)

Results

Figure 1 In-house intrinsic clearance values compared with published data. Data shown are the mean ± standard deviation of 3 or more 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.