Microsomal stability assays use liver microsomes containing cytochrome P450 enzymes to assess the in vitro metabolic stability of compounds. Liver microsomes are subcellular fragments that contain drug-metabolizing enzymes. The percentage of the parent compound remaining over time is measured in the presence of microsomes and cofactors. This allows the calculation of intrinsic clearance and half-life to investigate Phase I metabolism. Controls are used to characterize specific cytochrome P450 isozymes. The amount of parent compound is quantified by HPLC-MS/MS to determine metabolic stability.

1. Microsomal Stability Assay
(mouse, rat, dog, monkey, human)

2. • Microsomes are vesicle-like artifacts re-formed from
pieces of the endoplasmic reticulum (ER) when
eukaryotic cells are broken-up in the laboratory;
microsomes are not present in healthy, living cells.
 Microsomes are necessary to analyze the
metabolic activity of CYPs.

3. Measurement of in vitro intrinsic
clearance using microsomes
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The liver is the most important site of drug
metabolism in the body. Approximately 60 % of
marketed compounds are cleared by hepatic CYP-
mediated metabolism.
Liver microsomes are subcellular fractions which
contain membrane bound drug metabolising
enzymes.
Microsomes can be used to determine the in vitro
intrinsic clearance of a compound.

4. •
•
Microsomes can be concentrated and separated from
other cellular debris by differential centrifugation.
Unbroken cells, nuclei, and mitochondria sediment out
whereas soluble enzymes and fragmented ER, which
contains cytochrome P450 (CYP), remain in solution.
Isolation of microsomes

5. ASSAY

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Metabolic stability, as defined as the percentage
of parent compound lost over time, is assessed
in the presence of liver microsomes.
The microsomal stability assay is primarily used
to investigate Phase I metabolism using
NADPH as an enzyme co-factor.

6. Determinations:
% remaining at time point, t 1/2 determination, and
intrinsic clearance estimation(cl int)
Controls:
NADPH;
Dextromethorphan (CYP2D6),
Midazolam (CYP3A4),
Phenacetin (CYP1A2),
Diclofenac (CYP2C9),
Omeprazole (CYP2C19).

7. Assay description
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Working solutions of each compound are prepared
from 10 mM stock solution in DMSO diluted to a
final concentration of 100 μM in 0.05 M phosphate
buffer (pH 7.4).
Aliquots of Liver Microsome working solution are
transferred into 1.1 mL tubes using a multichannel
pipette.
Positive control (5 mixed) and test compound
working solutions are transferred into the tubes.
using a multichannel pipette and vortexed gently.

8. Cont'd...
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At each time point of 0 min, 5 min, 15 min, 30 min and
60 min with NADPH or 0, 30 min and 60 min without
NADPH, an aliquot is removed from each tube.
Terfenadine/tolbutamide in ACN/MeOH (1:1, v/v) is
added to quench and precipitate the microsomal
incubations.
Samples are capped and vigorously vortexed and
then centrifuged at 4 °C.

9. 
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•
•
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An aliquot of each supernatant is transferred for
LC-MS/MSC analysis.
Each compound is analyzed by reversed phase
HPLC using mobile phases
Solvent A: water with 0.1% formic acid
Solvent B: ACN with 0.1% formic acid..
The amount of parent compound is determined
on the basis of the peak area ratio for each time
point.
Cont'd..

10. Data analysis
•
•
Calculations.
In the determination of the in vitro t1/2, the analyte/ISTD peak
height ratios were converted to percentage drug remaining,
using the T = 0 peak height ratio values as 100%.
The slope of the linear regression from log percentage
remaining versus incubation time relationships (−k) was used
in the conversion to in vitro T1/2, values by in vitro T1/2 =
−0.693/k.

11. Cont'd
′Conversion to in vitro CL′int (in units of ml/min/kg)
was done using the following formula
CL'Int
=0.693/in vitro T1/2 * ml incubation/mg microsomes *
45 mg microsomes/gm liver*20 gm liver/kg b.w.

12. Cont'd
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•
For those compounds in which renal excretion of unchanged
drug represents a significant component of total clearance,
clearance values were corrected to nonrenal clearance
values by:
CLnonrenal=CLtotal⋅(1−fraction of dose excreted unchanged
in urine)

13. Abbreviations
ACN Acetonitrile
DMSO Dimethylsulfoxide
HPLC High-performance liquid chromatography
LC Liquid chromatography
MS Mass spectrometry
NADPH Nicotinamide adenine dinucleotide
phosphate

14. Advantages of microsomal assays
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The use of species-specific microsomes can be used
to enable an understanding of interspecies
differences in drug metabolism.
Easy to prepare, use and store enabling cost
efficiencies over whole cell models.
Microsomes are pooled from multiple donors to
minimise the effect of interindividual variability.
Microsomes are fully characterised using probe
substrates to ensure activity is maintained between
batches.

15. Thank you...