Drug Metabolism in Drug Design and Development Basic Concepts and Practice

If metabolic stability is determined in a suspension of hepatocytes,

SF¼ðGcell=WliverÞðWliver=WbodyÞ=Ccell

whereGcellis the average number of cells in the liver,Ccell, the cell

density (or concentration) in the reaction mixtures (e.g., 10^6 cells/mL).

Gcell/Wliveris approximately 120 106 cells/g for humans (Bayliss

et al., 1990); thus,

CLint¼ 24000 : 693 =ðt 1 = 2 CcellÞ¼ 1663 =ðt 1 = 2 CcellÞðmL=min=kgÞ;or

CLint¼ 1 : 663 =ðt 1 = 2 CcellÞwith units of L=min=kg: ð 13 : 5 Þ

It should be noted that the SFs used here are physiologically based.

However, despite their general acceptance, these SFs may not always

lead to reasonable in vivo predictions, and SFs other than the

physiologically-based should be also considered (Ito and Houston,

2005; Naritomi et al., 2001).

The previously described assays for detecting substrate disappearance

or half-life, however, are not suitable for the determination of metabolic

rates, which are required for the kinetic delineation of individual

metabolic pathways.

ii. CLintcan be estimated from rates of metabolism, usually gauged from

the formation of the metabolites, if authentic metabolite standards or

radioactively labeled parent compounds are available. However, the

metabolite standards or radioactively labeled compounds are often

unavailable, particularly in early discovery. The rates of product

formation should be determined as the initial step towards an under-

standing of the enzymatic basis of metabolism.

Rate calculation is straightforward when the amount of enzyme in the

reaction system is known:

V¼M=ðtEÞð 13 : 6 Þ

whereVis reaction rate,Mis the quantity of metabolite formed during

the incubation periodt, andEis the quantity of the enzymes or proteins

in the incubation mixture.Vis often in units of pmol metabolites/min/

mg protein or pmol metabolites/min/pmol enzyme.

The incubation timet, although theoretically as short as possible, is

typically between 15 and 45 min. Such a reaction period is based

largely on the following factors: the metabolite formation rates, the

linearity of metabolite formation rates, the responsible enzyme

thermostabilities, and substrate and enzyme concentrations. For

CYP-mediated reactions the rates of metabolite formation are often

moderate, and the CYP enzymes are often thermostable, resulting in

DETERMINATION OF METABOLIC STABILITY 423