Metabolism

Drugs are metabolized by many sites in the body, including the gut wall, lungs, kidney and plasma. However, the majority of drug metabolism takes place in the liver which is the most metabolically active tissue per weight unit. We provide metabolic stability testing for small molecules including S9, microsomal and Hepatocytes stability assays.

S9 stability

S9 liver fraction is commonly used to support in vitro ADME studies, including phase I and phase II metabolism. S9 fraction is a mixture of microsomes and cytosol, which contains a wide variety of drug metabolizing enzymes and can be supplemented with cofactors such as NADPH and UDPGA. S9 fractions from different species are available to examine the differences in drug metabolism.

 

Standard S9 stability protocol:

Available species Human, rat, mouse, rabbit, dog, monkey
Test Compound Concentration 3 μM
S9 Concentration 1 mg/mL
Time Points 0, 5, 15, 30, 45 min
Temperature 37oC
Cofactors NADPH, UDPGA
Controls Blank sample
Sample without cofactor (45 min only)
Postive control (2 compounds)
Number of replicates

3 samples/time-point for test compound

1 sample/time-point for controls

Analysis Method LC-MS
Output data

Percent of the parent compound remaining after each incubation period

Intrinsic clearance
Half life

Microsomal stability

Liver microsomes are commonly used to support in vitro ADME studies. Liver microsomes contain a wide variety of drug metabolizing enzymes and are commonly used to examine the potential for first-pass metabolism of orally administered drugs. Microsomes are pooled form multiple donors to minimize lot-to-lot variation caused by interindividual variability. Microsomes from different species are available to examine the differences in drug metabolism.

 

Standard microsomal stability protocol:

Available species Human, rat, mouse, rabbit, dog, monkey
Test Compound Concentration 3 μM
Microsome Concentration 0.5 mg/mL
Time Points 0, 5, 15, 30, 45 min
Temperature 37oC
Cofactors NADPH
Controls Blank sample
Sample without cofactor (45 min only)
Postive control (2 compounds)
Number of replicates

3 samples/time-point for test compound

1 sample/time-point for controls

Analysis Method LC-MS
Output data

Percent of the parent compound remaining after each incubation period

Intrinsic clearance
Half life

Plasma stability

Stability of compounds in biological fluids is important parameter because substances which undergo rapid degradation exhibit low efficacy in vivo. Information about plasma instability is useful for other in vitro studies (e.g., plasma protein binding where data can be difficult to interpret), in vivo studies (e.g., storage and handling pre-clinical and clinical samples may be challenging) and for screening of drugs, where rapid conversion in plasma is desirable (e.g., prodrugs and antedrugs).

 

Standard plasma stability protocol:

Available species Human, rat, mouse, rabbit, dog, monkey, bovine
Test Compound Concentration 1 μM
Time-points 0, 15, 30, 60 and 120 min
Temperature 37oC
Controls

Blank sample

Positive control (1 compound)

Number of replicates

3 samples/time-point for test compound

1 sample/time-point for controls

Analysis Method LC-MS
Output data Percent of the parent compound remaining after each incubation period

Hepatocyte stability

Hepatocytes are commonly used to support in vitro ADME studies requiring intact cellular systems. Intact hepatocytes contain the major hepatic drug-metabolizing enzymes required to study the four categories of xenobiotic biotransformation: hydrolysis, reduction, oxidation and conjugation. The assay is performed on human hepatic cells Hep2G. Primary cryopreserved hepatocytes from different species are also available up on request to examine the differences in drug metabolism.

 

Standard hepatocyte stability assay protocol:

Test System Human Hep2G liver cells, Human, rat, mouse, dog, monkey, minipig (cryopreserved hepatocytes)
Test Compound Concentration 3 µM
Time points 0, 5, 10, 20, 40 and 60 min
Temperature 37oC
Controls Blank sample
Positive control (2 compounds)
Number of replicates

3 samples/time-point for test compound

1 sample/time-point for controls

Analysis Method LC-MS
Output data

Percent of the parent compound remaining after each incubation period

Intrinsic clearance
Half life