DONOR SELECTION CRITERIA

1. THE IMPACT OF HUMAN CRYOPRESERVED HEPATOCYTE (HCH) POOL DONOR SUBSTITUTIONS ON PREDICTIVE METABOLISM MODEL PERFORMANCE Ellen Okamoto, Zhihong O’Brien, Melanie Hann, Chelsea Delgado, Daniel A. Norris, Yong Hee Lee, Troy Bremer, Kevin Holme Lion Bioscience, San Diego, CA 92121 ABSTRACT DONOR SELECTION CRITERIA The Donor Selection Criteria uses the enzyme activity data from IVT’s website for HCH donors to evaluate the pools. Pools are tested against two ranges: a median confidence interval for the pool and an acceptable activity range for each donor. The median confidence interval was determined as approximately the 99% median confidence interval of a large population of donors and is used to test the mean enzyme activity values of the six donor pool. The acceptable activity range was determined as approximately the 67% median confidence interval of a large population of donors and is used to evaluate the individual enzyme activity values for each donor. The median confidence interval and acceptable activity ranges for IVT’s enzyme activity are listed in Table 2. The Donor Selection Criteria uses the two ranges in the three criteria that must be met for a pool to be acceptable (Table 3). Purpose: To evaluate the impact of changing donors within a HCH pool on predictions of bioavailability (FH) from a predictive simulation model of metabolism (iDEA, LION bioscience). Methods: HCH assay data at 6 initial concentrations and 4 time points were collected for 15 known drug compounds using 3 different pools of six HCH donors selected based on published characteristics available from the vendor. The pools were designated training, mirror, and modified. The training pool consisted of the six donors used to collect the majority of the data for use in building of the predictive metabolism simulation model. The mirror pool consisted of donors with characteristics similar to the donors in the training pool. The modified pool consisted of donors where at least one enzyme had an elevated rate of metabolism. The experimental data from each pool of donors was used to predict the FH for each compound using iDEA. Results: The mirror and modified pools were evaluated by comparing the predicted FH of the test pools to the training pool. Three and five compounds had FH differences greater than 15 when using the mirror pool and modified pool, respectively. A Student’s t-Test analysis indicated the FH predictions from the mirror pool were not significantly different than the training pool results (p-value = 0.35), while the modified pool results were significantly different from the training pool (p-value = 0.02). Conclusions: The substitution of donors within an HCH pool may have an effect on the outcome of bioavailability predictions using predictive models. These effects will not be significant if efforts are made to select substitute donors with enzyme characteristics similar to donors used in the training pool. Selection of donors with known enzyme characteristics different from training donor profiles will result in significant changes in FH predictions. INTRODUCTION The use of human cryopreserved hepatocytes (HCH) for metabolic in-vitro testing in early drug discovery is a relatively recent advancement. One concern surrounding the use of HCH in an assay is donor availability and substitution. As part of the validation of the robustness and reproducibility of the HCH assay, LION has determined the significance of donor pool changes on iDEA predictions of the fraction of a dose that reaches the hepatic vein (FH). The Metabolism Module of the iDEA Predictive ADME Simulation System predicts FH using LION’s in vitro HCH assay and fraction protein binding data along with results from the iDEA Absorption Module. The iDEA Adsorption Module uses a combination of chemical structure, Caco2 permeability and solubility data to predict the fraction of dose that reaches the portal vein (FDP). The iDEA Predictive ADME Simulation System is built with both physiological and structural models, and represents cutting-edge technology that combines the best of the computational and pharmacokinetic sciences. RESULTS In order to complete testing on all 15 compounds, two sets of HCH pools were needed for each of the training, mirror and modified pools. Each pool was tested against the Donor Selection Criteria and the results are listed in Table 4. The training and mirror pools passed all three criteria. The modified pools failed all three criteria. Table 4 shows results with number of enzymes that fell outside ranges in parentheses. METHODS In summary, significant differences in Metabolism Module performance (changes in FH predictions) were not observed when the HCH donor pool was chosen with the Donor Selection Criteria. In the other case when donors were selected that contain enzyme activities outside the Donor Selection Criteria, and thus were outside median population enzyme activities, significant effects on iDEA FH predictions were observed. STUDY OVERVIEW Three hepatocyte pools were selected to evaluate the effect of using different donors on FH predictions. Each pool consisted of six liver donors. Selection of the three different HCH pools (Table 1) was based on enzyme characterization using data published on IVT’s website and Lion’s Donor Selection Criteria. Kinetic data was collected for each of 15 compounds with the 3 donor pools. Metabolic turnover in the HCH assay was measured as loss of parent compound at 6 concentrations and 4 timepoints done in triplicate. Determinations of Vmax and Km and FH predictions were made with iDEA from the kinetic data. The FH results were compared to determine the significance of pool changes on FH predictions from iDEA. CONCLUSION Fifteen compounds were tested to evaluate the sensitivity of the FH prediction of the Metabolism Module to variations in three HCH donor pools, as determined by published enzyme activities. When comparing the mirror pool to the training pool, FH had a mean change of 3.76 units and p-value of 0.35, showing the difference between the enzyme activities of the pools is not significant. In contrast, when comparing the modified pool to the training pool, FH had a mean change of –10.85 units and a p-value of 0.02, showing the difference between the enzyme activities of the pools is significant.By following LION’s recommendations for selection of an HCH pool laboratories can help assure their data is comparable to that used to create and train the iDEA Metabolism Module. The LION HCH assay is demonstrated to give robust and reproducible results with different donor pools that fall within the Donor Selection Criteria. iDEA FH predictions with the training, mirror and modified pools are shown in Table 5. The Student’s t-Test was used to determine if the mirror and modified pools gave significantly different predictions from the training pool for the 15 compounds (Table 6). The mirror pool did not have significantly different predictions of FH from the training pool (p=0.35). In comparing the mirror pool to the training pool, 9 out of 14 compounds had higher FH predictions than the training pool and the mean difference in FH predictions was 3.76 FH units. Three compounds were predicted 15 FH units or more different from the training pool predictions (two were predicted with higher FH values and one was predicted with a lower FH value).  The modified pool had significantly different FH predictions from the training pool (p=0.02), with trend of slight underestimation of FH. In comparing the modified pool to the training pool, the mean difference in FH predictions was –10.85 FH units and 11 of 15 compounds had a lower FH than the training pool. Five compounds were predicted 15 FH units or more different from the training set (four were predicted with lower FH values and one was predicted with a higher FH value). iDEA HCH ASSAY Human cryopreserved hepatocytes were purchased from In-Vitro Technologies (IVT, Baltimore, MD) and a six-donor pool was used for the hepatocyte assay. After thawing and iso-Percoll centrifugation, viable cells were counted and viable cell yields were calculated. Cells were suspended with an appropriate volume of WME to obtain 0.5 million viable cells/ml suspension. One hundred l cell suspension was mixed with 100 l substrates at six different concentrations (0.4, 2, 10, 50, 125 and 250 M). After 5 min pre-incubation in a cell culture incubator at 37oC with 5% CO2 and 95% humidity, 200 l chilled methanol was added to stop the reaction at the appropriate time points (0, 30, 120 & 240 min). The mixtures were then vortexed and centrifuged at approximately 2000 g for 20 minutes to precipitate proteins. The supernatants were transferred to analytical plates for the quantitation of parents.