As shown earlier,IfS Km;V=S¼Vmax=Km¼CL0
int; or
CL
0
int¼SðEi=ETÞVi=S;if multiple enzymes are involved: ð^13 :^13 Þ
V/Sis in essence the slope of the pseudolinear portion of the Michaelis–
Menten plot.
Again,Viwould preferably be detected, for each metabolic pathway, in
HLM or hepatocyte suspensions, if appropriate.
Scaling Factor CLintdeterminedin vitro(CL
0
int) must be multiplied by a SF to
be converted to CLintin units of L/min/kg from units of ml/min/mg protein
(determined using liver microsomal preparations), ml/min/million cells
(determined using hepatocyte suspensions), or ml/min/nmol enzymes (deter-
mined using enzyme preparations with known enzyme quantities). SFs based
on human physiologic parameters have been described previously, and are
listed in Table 13.6.
SFs could be derived using several different approaches (Naritomi et al.,
2003; Ito and Houston, 2005). Physiologically based SFs, although widely used
as indicated, do not necessarily lead to appropriatein vivopredictions, the
empirically derived SFs, sometime, might be considered as the alternative (Ito
and Houston, 2005). SFs, unlike typical constants, are potentially compound
dependent, due in part to their physicochemical properties (Ito and Houston,
2005; Naritomi et al., 2001). Moreover,CL
0
int, when calculated with nonspecific
protein binding taken into account, might correlate better with liver clearance
in some cases (Andersson et al., 2001; Ito and Houston, 2005; Obach, 2000;
Soars et al., 2002). Therefore, it has been suggested for serum or albumin to be
included in the reaction mixtures, to alleviate the discrepancy in protein
binding betweenin vitroassay systems and human liver, particularly for acidic
TABLE 13.6 Basic conversion factors for the scaling-up of CLintdetermined
in vitro.
Content References
CYPsa HLMb 0.32 nmol/mg protein Broly et al. (1990),
Richard et al. (1991)
Hepatocytes 0.14 nmol/mill cells Grant et al. (1987)
Liver 14.4 nmol/g liver Obach et al. (1997)
16.8 nmol/g liver Iwatsubo et al. (1997)
Microsomal proteins Liver 45 mg/g liver Obach et al. (1997)
52.5 mg/g liver Iwatsubo et al. (1997)
Hepatocytes Liver 120 mill cells/g liver Bayliss et al. (1990)
aCYP3A4, the abundant human hepatic CYP form, at the level of approximately 5 nmol/g liver, in
average. (Iwatsubo et al., 1997).
bHuman liver microsomal preparations.
436 DETERMINATION OF METABOLIC RATES AND ENZYME KINETICS