Drug Metabolism in Drug Design and Development Basic Concepts and Practice

where [E] is the active enzyme concentration at time t; [I] the inhibitor
concentration,kinactthe inactivation rate constant, and KIthe inactivator
concentration that produces half-maximal rate of inactivation. The potency of
an inhibitor can be described by the partitioning ratio of the rate for metabolite
formation to that for enzyme inactivation (Eq. 16.4). Partition ratio is a
measure of the efficiency of inhibitor (average number of cycles the enzyme can
traverse before it is inactivated). It can vary from 0 to several thousands. The
value of less than 10 is usually defined as a potent inhibitor. The ratio is
dependent on the reactivity of the reactive intermediate, rate of release of the
reactive intermediate from active site (k 3 ), rate of enzyme inactivation (k 4 ) and
the proximity of an appropriate target molecule(s) for the covalent bond
formation (Jushchyshyn et al., 2003; Walsh et al., 1978).

Partioning¼

kcat

kinact

¼

k 3

k 4

ð 16 : 4 Þ

16.3.2 Measurements of Kinetic Parameters

Evaluation of compounds as mechanism-based inhibitors of major CYP
isoforms (i.e., CYP3A4, CYP1A2, CYP2C8, CYP2C9, CYP2C19, and
CYP2D6) in a high throughput mode has been implemented with the marker
substrates at early stage of drug discovery in pharmaceutical industry. The
analytical assays are similar to those described in the reversible enzyme
inhibition. The MBI is commonly performed in preincubation of an inhibitor
at a single concentration and variable incubation times. Liver microsomes
(0.5–2 mg/mL) from different species can be used to serve as enzyme sources.
The inactivation (activity remaining) is monitored by a reaction with each
of individual isoform-specific substrates after preincubation time with
the inhibitor of test. At the discovery stage, screening for mechanism-
based inhibition potential is generally conducted at 10 or 50mM of test
compound, using a preincubation time period of 30 min. A decrease of enzyme

E+I EI EI

k 1 k 2

E+P

k 3

k- 1

′k^4 Einact

SCHEME 16.3 Proposed kinetic scheme for mechanism-based enzyme inhibition. E, I
and P stand for enzyme, inhibitor and product, respectively, and EI is the initial binding
of inhibitor to enzyme (the enzyme–inhibitor complex) and EI^0 is the active form of the
complex in which the inhibitor is catalyzed to intermediate (reactive metabolite).Einactis
the inactivated enzyme by the reactive metabolite formed. Inactivation of the enzyme is
an irreversible process over the time scale of the experiment. At the given concentrations
of inhibitor and enzyme, the reactions are governed by the first-order rate constantsk 1 ,
k 1 ,k 2 ,k 3 andk 4 , respectively.

IRREVERSIBLE INHIBITION 529