Drug Metabolism in Drug Design and Development Basic Concepts and Practice

APPENDIX B: REACTION PHENOTYPING—MICROSOMAL

CHEMICAL INHIBITION

I. Objective

A. To specifically inhibit CYP450s in human liver microsomes to

determine which enzymes are contributing to metabolism by

observing the changes in the disappearance of parent compound.

II. Materials

A. Appropriate buffer pH 7.4 (i.e, 50-mM potassium phosphate buffer

(KP)).

B.b-NADPH (1 mM final concentration).

C. Human liver microsomes.

D. Stock Solutions

1. Compound
   a. Prepare a stock solution that can provide a final incubate
   drug concentration of 1mM or less ifKmknown (0.1–1.0 mM
   or 100–1000 final drug concentration).


2. Inhibitors
   a. Dissolve primary stocks in DMSO.
   b. Dilute primary stock with ACN so final stock is 75:25, %v/v
   (ACN:DMSO).


3. Organic solvent content added must be less than 1% of the total
   incubation volume and the content of DMSO is less than 0.1%
   of the total incubation volume.

III. Methods

A. Drug incubates

1. Drug incubations performed in triplicate (N= 3) for each
   inhibitor.


2. On ice, add all components of the incubation exceptb-NADPH.


3. Preincubate samples at 37C in shaking (80 rpm) water bath for
   at least 3 min.


4. Add prewarmedb-NADPH and swirl gently to start reaction,
   keep covered throughout incubation.


5. Sample quench
   a. Remove aliquots at predetermined times and transfer to
   Eppendorf tubes or 96-well plate containing quench solvent.


6. Samples may be stored at 80 C until analysis.

512 REACTION PHENOTYPING