In Vitro to In Vivo Scaling Factors

It has previously been mentioned that the predictive accuracy of in vitro to in vivo extrapolation (IVIVE) approaches can depend greatly upon the in vitro systems of choice. It is also important to examine the extrapolative scaling factors used for sources of uncertainty and variability. The classical scaling approach (as described by Houston,50 eqn [6]) has been modified to reflect the challenges in scaling from both HLM (eqn [7]) and rhCYPs (eqn [8]), whilst taking into account established measures of interindividual variability. It is important to note that the units in both latter strategies account for the extreme variability of CYP expression in tissues by expressing the rates per unit CYP isoform. The strategy used for scaling total hepatic intrinsic clearance (CLH int) from in vitro data is outlined below. Central to these strategies are the amount of microsomal protein per gram of human liver (MPPGL) and the weight of the liver to be projected.

CLH,int = HLM CLint x MPPGL x liver weight


x CYPj abundance x MPPGL x liver weight

where hepatic intrinsic clearances are summed for each contributing CYP (/').


][]rhCYP CLint,j x ISEFj x CYPj abundance x MPPGL x liver weight

It has been demonstrated that human MPPGL has considerable interindividual variability and indeed can vary dramatically between investigating laboratories.51-58 The experimental technique of differential centrifugation used for preparing HLM can be highly variable and it is therefore essential to determine a recovery factor for each preparation in order to quantify reliably the true MPPGL rather than merely the experimental yield.58 The most common values in use of 52.5s4 and 4559 mgg-1 would appear to owe more to their assistance in overcoming underpredictions than any valid human physiological basis.60 A recent publication has attempted to define a value of MPPGL and also assess the true interindividual variability present.58 The authors have incorporated adjustments of both yield and assay variability by the means of recovery factor and cross-validated measurements. A further update61 to this work has established MPPGL in a total of 53 human liver samples and work is ongoing to examine relationships between MPPGL and age, disease state, or medication status of the donors. An overall geometric mean value of 28 mgg_ 1 (range 13-54 mgg_ 1) has been obtained which appears to encompass other, smaller, data sets. Such a large degree of interindividual variation can obviously be significant when extrapolating to a human population.

The abundance of CYP isozymes in the liver and other tissues is also an issue of great importance. In addition to helping contextualize the results of in vitro experiments from a particular liver (or pool of livers), these values are essential in the successful scaling of rhCYP data. A recent exhaustive metaanalysis of the literature,62 including the most commonly cited source of abundance data,63 has shown a large degree of interindividual variability for many of the CYPs most commonly involved in drug metabolism. For example, reported CYP3A (3A4) content varies from 3764 to 248 pmol mg_ 165 CYP3A4 is the major isoform of the CYP3A subfamily, both in terms of absolute amount and metabolic capacity.

A similar strategy has been pursued for the scaling of hepatocyte CLint (eqn [9]), with slightly greater success, although this system has particular disadvantages for predicting DDI liability, as previously discussed.

Hepatocyte CL¡n x HPGL x liver weight [9]

The approach to defining hepatocellularity (or HPGL, quantity of hepatocytes per gram of liver) is slightly more challenging due in part to the difficulties involved in sourcing, isolating, and handling primary human hepatocytes. The approach has been well described,7 although published literature has been unclear on the source of the most commonly accepted value of 120 x 106 cells g_ 1. Although widely used,54'59'66-69 the original methodologies used for determining this number have not been verified, although the original reference value70 is identical to that verified several years earlier in the rat.71 Initial estimates58 (n = 7) have given a geometric mean value of 107 x 106 cells g_ 1 (range 65-185 x 106cells g_ 1), although more recent data (n = 24) suggest that the true population will be somewhat lower than this.

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