and/or microsomal protein binding). Using this
well-stirred model, it has proved possible to predict
the hepatic clearance fromin vitrointrinsic clear-
ance rates in rat, dog and human (Table 8.2). The
hepaticclearancevaluefortherat(0.972 ml min^1
mg^1 protein) was approximately one-tenth the
actual clearance foundin vivo; well in agreement
with the observation thatin vivocompound X was
eliminated by the rat, largely unchanged, by the
kidneys (90%).
To predict hepatic clearance of compound X in
humans, humanin vitrointrinsic clearance could
then be scaled to hepatic clearance, using a tech-
nique that had been validated in rat (Ashforttet al.,
1995). Renal clearance is subject to an allometric
relationship and can generally be scaled across
species (see below). The predictedin vivorenal
Cl for rat (estimated by multiplying the predicted
hepatic Cl by 9) may be scaled allometrically to
obtain a prediction for the humanin vivorenal
clearance. Total or systemic Cl in humans can
then be estimated by adding the two clearance
parameters (hepatic and renal) together; in prac-
tice, for compound X, later first-in-human data
revealed an actual in vivo Cl nearly identical
to the predicted total Cl (2.15 vs. 1.87–2.45 ml
min^1 mg^1 , respectively; Table 8.2). Here, then,
is a real-world example of, first, how ratin vitroand
in vivopreclinical data were used to develop and
validate a scaling method for compound X in rat;
and second, how the scaling method success-
fully predictedin vivooverall drug clearance in
humans.
However, if the same methods are used for com-
pound X in dog, things initially appear to be dif-
ferent. Scaling thein vitrointrinsic clearance to
hepatic Cl using the rat-validated method, in con-
junction with allometric scaling of renal Cl,
resulted in a five-fold under-prediction of the
total or systemic clearance in vivo. However,
further metabolism studies in the dog in vivo
revealed that compound X undergoes significant
additional biotransformation, particularly N-
methylation, which is unique (as far as we are
aware) to this species, and invalidates some of
ourin vitroassumptions. This canine biotransfor-
mation pathway was not detected by our initial
microsomal studies because there are noN-methyl
transferases in microsomes. Thus, although we did
not successfully predict dog systemic clearance for
compound X, our scaling tactics did eventually
teach us about a new clearance mechanism, and
how important this was for the systemic clearance
of compound X in dog.
This is an example of howin vitrostudies can be
combined within vivopreclinical data, leading to
useful prediction of human systemic drug clear-
ance. Nonetheless, several caveats are encountered
in such scaling exercises, which warrant restating.
The first caveat is that all clearance pathways
(hepatic, renal, biliary or other) must be taken into
consideration. If a compound undergoes a high
level of hepatic clearance, thenin vitro–in vivo
scaling may be used to predict the fraction of
systemic clearance expected from this pathway.
If a compound undergoes a high level of renal
elimination, allometric scaling may be also used
to predict the clearance attributed to this pathway.
The second caveat is that, in order to accurately
predict hepatic clearance, the correctin vitrosys-
tem must be chosen. If the candidate drug is pri-
marily oxidatively metabolized, then liverTable 8.2 Comparison of the predictedin vivohepatic clearance and the actual clearance values for compound X
Predictedin vivohepatic Predictedin vivorenal Predictedin vivototal Actualin vivo
Cl (ml min^1 kg^1 ) Cl (ml min^1 kg^1 ) Cl (ml min^1 kg^1 )Cl(ml min^1 kg^1 )Rat 0.972 8.75 9.72 8.17–10.7
Human 0.223 1.93 2.15 1.87–2.45
Dog 0.463 3.74 4.20 21.2–22.5
Predicted values were scaled fromin vitrohalf-life data using liver microsomes and the well-stirred model of hepatic extraction.
HepaticClpredictions were corrected for plasma and microsomal protein binding. Predicted totalClwas obtained by adding in renal
Clestimates which were, in turn, scaled allometrically (Y¼aW^0 :^75 ).
8.1 THE IN VITRO/IN VIVO PREDICTION 83