Are there differences in the catalytic activity per unit enzyme of recombinantly expressed and human liver microsomal cytochrome P450 2C9? A systematic investigation into inter-system extrapolation factors.

The 'relative activity factor' (RAF) compares the activity per unit of microsomal protein in recombinantly expressed cytochrome P450 enzymes (rhCYP) and human liver without separating the potential sources of variation (i.e. abundance of enzyme per mg of protein or variation of activity per unit enzyme). The dimensionless 'inter-system extrapolation factor' (ISEF) dissects differences in activity from those in CYP abundance. Detailed protocols for the determination of this scalar, which is used in population in vitro-in vivo extrapolation (IVIVE), are currently lacking. The present study determined an ISEF for CYP2C9 and, for the first time, systematically evaluated the effects of probe substrate, cytochrome b5 and methods for assessing the intrinsic clearance (CL(int) ). Values of ISEF for S-warfarin, tolbutamide and diclofenac were 0.75 ± 0.18, 0.57 ± 0.07 and 0.37 ± 0.07, respectively, using CL(int) values derived from the kinetic values V(max) and K(m) of metabolite formation in rhCYP2C9 + reductase + b5 BD Supersomes™. The ISEF values obtained using rhCYP2C9 + reductase BD Supersomes™ were more variable, with values of 7.16 ± 1.25, 0.89 ± 0.52 and 0.50 ± 0.05 for S-warfarin, tolbutamide and diclofenac, respectively. Although the ISEF values obtained from rhCYP2C9 + reductase + b5 for the three probe substrates were statistically different (p < 0.001), the use of the mean value of 0.54 resulted in predicted oral clearance values for all three substrates within 1.4 fold of the observed literature values. For consistency in the relative activity across substrates, use of a b5 expressing recombinant system, with the intrinsic clearance calculated from full kinetic data is recommended for generation of the CYP2C9 ISEF. Furthermore, as ISEFs have been found to be sensitive to differences in accessory proteins, rhCYP system specific ISEFs are recommended.

Determination of a quantitative relationship between hepatic CYP3A5*1/*3 and CYP3A4 expression for use in the prediction of metabolic clearance in virtual populations.

The creation of virtual populations allows the estimation of pharmacokinetic parameters, such as metabolic clearance in extreme individuals rather than the 'average human'. Prediction of variability in metabolic clearance within genetically diverse populations relies on understanding the covariation in the expression of enzymes. A number of statistically significant positive correlations have been observed in the hepatic expression of cytochrome P450 drug metabolising enzymes. However, these rarely provided a quantitative description of the relationships which is required in creating virtual populations. Collation of data from 40 human liver microsomal samples in the current study indicated a significant positive relationship between hepatic microsomal CYP3A5*1/*3 and CYP3A4 content. Having developed a model describing the relationship between hepatic CYP3A4 and CYP3A5*1/*3, the Simcyp Population-based Simulator(®) was used to investigate the consequences of either incorporating or ignoring the relationship between the two enzymes on estimates of drug clearance. Simulations indicated that for a compound with greater metabolism by CYP3A5 than CYP3A4, such as tacrolimus, incorporation of the correlation between CYP3A4 and CYP3A5 does have an impact on the prediction of oral clearance. Failure to consider the relationship between CYP3A4 and CYP3A5 when creating the virtual population led to a 32% lower estimate of oral clearance in individuals possessing both the CYP3A5*1/*3 genotype and high basal concentrations of CYP3A4. Potential clinical implications may include an inadequate dose estimation during clinical study design, the consequences of which may include organ rejection in transplant recipients using immunosuppressants such as tacrolimus or toxicity due to elevated concentrations of circulating metabolites.

Covariation of human microsomal protein per gram of liver with age: absence of influence of operator and sample storage may justify interlaboratory data pooling.

Scaling of metabolic clearance values from liver microsomal data or recombinantly expressed cytochrome P450 enzymes to predict human hepatic clearance requires knowledge of the amount of microsomal protein per gram of liver (MPPGL). Identification of physiological covariates of MPPGL requires analysis of values from large diverse populations, which necessitates pooling of data from numerous sources. To ensure compatibility between results obtained within and between studies, the impact of interoperator differences and sample storage on values of MPPGL was investigated. With use of triplicate samples from one liver (HL86), no statistically significant difference was detected between values of MPPGL prepared from samples stored at -80 degrees C (23.5 +/- 1.2 mg g(-1)) and those determined using fresh tissue (21.9 +/- 0.3 mg g(-1)). Although there was a significant difference in the yield of microsomal protein obtained from another liver sample (HL43) by three different operators (17 +/- 1, 19 +/- 2, and 24 +/- 1 mg g(-1); p = 0.004, analysis of variance), no difference was observed in the estimated MPPGL after application of appropriate correction factors for each operator (28 +/- 1, 30 +/- 5, and 31 +/- 4 mg g(-1)). The result provided justification for pooling reported values of MPPGL for use in covariate analysis. Investigation of the relationship between age and MPPGL provided preliminary evidence that MPPGL values increase from birth to a maximum of 40 mg g(-1) [95% confidence interval for the geometric mean (95% CI mean(geo)): 37-43 mg g(-1) at approximately 28 years followed by a gradual decrease in older age (mean of 29 mg g(-1) at 65 years; 95% CI mean(geo): 27-32 mg g(-1)). Accordingly, appropriate age-adjusted scaling factors should be used in extrapolating in vitro clearance values to clinical studies.

Disparity in holoprotein/apoprotein ratios of different standards used for immunoquantification of hepatic cytochrome P450 enzymes.

An analysis of reported hepatic abundances of CYP3A4 and 3A5 indicated that values determined by immunoquantification using commercially available, unpurified recombinant enzymes as standards are significantly lower than those determined using purified enzymes or human liver microsomes characterized with lysosomal peptides (CYP3A4: mean 45 versus 121 pmol/mg protein, p < 0.01; CYP3A5: mean 28 versus 83 pmol/mg protein, p < 0.05). When immunoquantifying cytochromes P450 (P450s), it is assumed that the holoprotein (holo)/apoprotein ratio is the same in the samples and the standard. Estimates of holo/apoprotein ratios from data reported for a range of P450s purified from human liver and non-commercial recombinant systems indicated less than complete and variable heme coupling dependent on enzyme and system.