1. THE CONSTRUCTION OF A REPRESENTATIVE HUMAN CRYOPRESERVED HEPATOCYTE POOL FOR METABOLISM STUDY Zhihong Zhang OBrien*, Troy Bremer, Kevin Holme and Yong Hee Lee* 1 In Vitro System, Lion Bioscience, San Diego, CA 92121 THE CONSTRUCTION OF A REPRESENTATIVE HUMAN CRYOPRESERVED HEPATOCYTE POOL FOR METABOLISM STUDY Zhihong Zhang OBrien*, Troy Bremer, Kevin Holme and Yong Hee Lee* 1 In Vitro System, Lion Bioscience, San Diego, CA 92121 Due to the extensive inter-individual difference and polymorphic distribution of liver enzymes, it is necessary to construct a representative human cryopreserved hepatocyte (HCH) pool for metabolism study. To obtain a representative HCH pool, enzyme activity database of total 51 donors (In Vitro Technology (IVT)) was analyzed using statistical tools. Due to the non-normal distribution of all isozymes from vendor database, a median value, its 99% confidence interval (CI) and confidence intervals of 67% typical population for each isozyme were defined. To select a representative pool and minimize the pool-to-pool variation, the following criteria will be applied: 1) at least 80% of individual isozyme activity values must be within their corresponding acceptable activity range; 2) no more than 30-35% of each enzyme activity value/X donors should be outside the acceptable activity range; and 3) mean activity for each enzyme /pool should fall into 99% CI of its corresponding median value. By doing so, it is expected to result in approximate one- to three-fold (except 4-fold for CYP1A2 and 6-fold for CYP2C19) variations of isozyme activities among pools. Thirty 6-donor pools were constructed based on the selection criteria described above, and four of them were tested for isozyme activities using 11 functional probe substrates. Our results showed that: 1) good correlation (0.8 slope 1.3 and R 2 0.8) is achieved between the mean of each isozyme activity per pool and median values of each enzyme in the database; 2) enzyme activity changing trends of the virtual HCH pools constructed through IVT database are compatible with our actual results; and 3) one- to three-fold isozyme activity differences are observed among pools. ABSTRACT INTRODUCTION METHODS RESULTS CONCLUSION Hepatocyte has been recognized as one of the most powerful in vitro tools in metabolism and toxicity studies. The advent of cryopreservation technologies opened a new field for using hepatocyte more conveniently and wisely, especially for human hepatocyte. Cryopreservation techniqe circumvents the huge waste of freshly isolated human hepatocytes due to the lack of appropriate storage, and provides a tremendous opportunity to minimize the inter- individual differences and polymorphic distribution of liver enzymes. To maintain the consistency of liver enzyme activities and reduce the inter-indivadual enzyme differences in hepatocyte system used in each study, it is better to construct a uniformed hepatocyte pool. However, it is impossible to have a same generic pool for each indivadual study. Then, what would be the reasonable way for the hepatocyte pool construction? Up to now, there hasnt been any strong interest shown in how to construct the best pool although almost everyone realizes the importance of doing so. Recently, our lab started with a total of 51 human cryopreserved hepatocyte donor database published by In Vitro Technology (IVT). To initiate the investigation, the enzyme activity database of 51 donors was analyzed by using statistical tools. Based on the statistical results, hepatocyte pool selection criteria was established. Thirty-four 6-donor pools were constructed according to the selection criteria and four of them were actually tested in our lab for isozyme activities using 11 functional marker compounds. This study provides a basis for obtaining a representative human cryopreserved hepatocyte (HCH) pool to reduce the chance of causing enzyme activity variation during each study. Table 1. IVT 51-donor enzyme activity database Conventional pool construction for liver fractions Human microsome and S9: 10 to 15 donors as an optimum pool Human cryopreserved hepatocyte: Not addressed yet Advantages for construction of a representative human cryopreserved hepatocyte pool Reduce the interindividual variations in human liver enzymes Make experimental results more comparible between studies or labs Provide experimental data more representation of a generic human Study Design: (1) Statistical analysis of IVT 51-donor database to establish the pool selection criteria: Determine type of distribution of 51-donors for each isozyme Define median value and its 99% confidence interval (CI) for each isozyme Define confidence intervals of 67% typical population for each isozyme (2) Construction of thirty-four HCH pools based on the pool selection criteria (3) Actual pool comparisons by measuring isozyme activities using 11 functional markers Table 2. Eleven functional marker compounds (1) Statistical analysis of IVT 51-donor database to establish the pool selection criteria: Table 3. Median confidence range and acceptable activity range of indivadual isozyme Donor selection criteria: At least 80% of individual isozyme activity values must be within their corresponding acceptable range. No more than 30-35% of each enzyme activity value/X donors should be outside the acceptable activity range. Mean activity for each isozyme/pool should fall into its corresponding median confidence range. The donor selection criteria based on the statistical analysis successfully provides a basis for obtaining a representative human cryopreserved hepatocyte (HCH) pool. (2) Construction of thirty-four HCH pools based on the pool selection criteria: (3) Actual pool comparisons by measuring isozyme activities using 11 functional markers: Good correlation (0.8 slope 1.3 and R 2 0.8) is achieved between the mean of each isozyme activity per pool and median values of each enzyme in the database. One- to three-fold isozyme activity differences are observed among pools. Enzyme activity changing trends of the virtual HCH pools constructed through IVT database are compatible with our actal results. Enzyme activity values are based on IVT database Enzyme activity values are based on Lion actual experimental data