Drug Metabolism in Drug Design and Development Basic Concepts and Practice

APPENDIX A: REACTION PHENOTYPINGEXPRESSED cDNA
ENZYME INCUBATION METHOD SHEET

I. Objective

A. To determine whether individually expressed human cDNA P450

enzymes are capable of contributing to metabolism by measuring

the disappearance of parent compound.

II. Materials

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

(KP)).

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

C. P450 expressed enzyme

1. Recombinant human P450 and NADPH-P450 reductase.


2. P450 control for expression system
   D. Drug stock solutions


3. 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).


4. 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 water bath for at least
   3 min.


3. Add prewarmedb-NADPH and swirl gently to start reaction,
   keep covered throughout incubation (for minus b-NADPH
   controls buffer is substituted forb-NADPH).


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


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

APPENDIX A: REACTION PHENOTYPINGEXPRESSED cDNA ENZYME 511