Drug Metabolism in Drug Design and Development Basic Concepts and Practice

16.2.1 Materials and Reagents

Chemicals available from the commercial sources below: phenacetin,
acetaminophen, diclofenac, cortisone, flufenamic acid, propranolol, 4^0 -hydroxy-
butyranilide, sulfaphenazole, quinidine, testosterone, ketoconazole, 6b-OH-
testosterone, taxol, quercetin, baccatin, 4^0 -hydroxydiclofenac, 4-hydroxytria-
zolam, (R)-(+)-propranolol, phenytoin, dextromethorphan, testosterone,
dextrorphan,a-naphthoflavone, and NADPH from Sigma (St Louis, MO);
fluvoxamine maleate from Tocris (Ballvin, MO); midazolam, 1-OH-midazo-
lam, (S)-mephenytoin, (+)-N-3-benzylnirvanol, bufuralol, 1^0 -OH-bufurarol, 4^0 -
OH-mephenytoin from BD Biosciences (Woburn, MA, USA); (R)-N-3-benzyl-
phenobarbital, and L-000706631 from Merck compound library; and 6b-OH-
progesterone from Steraloids, Inc. (Newport, RI, USA). Pooled HLM were
purchased from Tissue Transformation Technologies (Exton, PA) and from
Xenotech (Kansas, KS), respectively. Water was purified by a Mill-Q-System
from Millipore Corp. (Milford, MA). Formic acid (analytical grade) was from
J.T. Baker (Phillipsburg, NJ). All chemical inhibitors and substrates were
dissolved in 50% (v/v) acetonitrile/water or DMSO. The final volume of
acetonitrile or DMSO in the reaction mixture was less than 2% (v/v) or 0.2%,
respectively. Activity in the presence of the solvent alone was assigned as
‘‘control’’ (100%). Reaction 96-well plates (300mL), preparation 96-well plates
(1.5 mL), and 96-well plate receivers (1.5 mL) were purchased from VWR
(West Chester, PA). The reaction and preparation plates were treated with
acetonitrile, reconditioned with water and dried by centrifugation prior to use.
Hydrophobic and hydrophilic 96-well filtration plates (0.45mm polytetrafluor-
oethylene) were purchased from Millipore Corp (Billerica, MA).

16.2.2 Instrument

A TECAN Genesis RSP 200 liquid handling workstation equipped with eight
tips, shaking and temperature control (heating block) was used for sample

E+S

+

I

EI+S ESI

ES

+

I

E+ P

K

Ki αKi

k

αKS

EI+P

βkp

s p

SCHEME 16.1 General kinetics for competitive ([ESI] = 0), noncompetitive (a=1
andb= 0), uncompetitive (a=1,b= 0 and [EI] = 0), and mixed type inhibition
(a6¼1 and 0<b<1).KS= dissociation constant for substrate (S);Ki= dissociation
constant for inhibitor (I);kp= rate constant for the formation of product;aandbare
factors that changeKSorKiandkpwhen the second molecule (either I or S) is bound.
Rate equations for above inhibition kinetics are given in Table 16.4 (competitive,
noncompetitive, uncompetitive and mixed type).

516 ANALYSIS OFIN VITROCYTOCHROME P450 INHIBITION