Hepatic intracellular free drug concentration and unbound partition coefficient: development and validation of an in vitro methodology to predict in vivo liver drug exposure

The Free drug hypothesis states that only the unbound drug molecules are able, in the site of action, to interact with the target and exert the pharmacodynamic effect. Therefore, in drug discovery it is of great interest knowing the unbound tissue concentration, especially for drugs that have their target placed intracellularly, because it can be significantly useful to better predict the pharmacokinetic and pharmacodynamic profiles, but also the toxicity of any potential drug. In vivo assays to measure the unbound partition coefficient Kp,uu (that is the drug’s ratio of the unbound tissue concentration to the unbound plasma concentration) are routinely applied, but only few compounds might be tested per experiment due to the preclinical species being inevitably sacrificed in the process. Meanwhile for human Kp,uu more complex techniques are required (e.g. PET imaging) that make even more desirable finding an alternative to determine Kp,uu. This thesis was focused on establishing and validating a methodology to measure in vitro Kp,uu with the aim to accurately predict the respective in vivo Kp,uu value in liver for the tested compounds. To achieve this goal the compounds were tested in suspended hepatocytes to measure the partition coefficient (Kp), indicator of the total (bound + unbound) drug accumulation. In a separate experiment the compounds were tested for the cell drug binding and medium drug binding. These parameters allowed to calculate the hepatic in vitro Kp,uu, representative of the unbound drug accumulation in the hepatocytes. The in vitro Kp,uu was determined for eighteen commercial drugs using both Male Sprague-Dawley Rat and Mixed Gender Human cryopreserved hepatocytes. The results were compared with the data already available in the scientific literature for the tested compounds. The rat and human experimental in vitro Kp,uu was in accordance with the literature counterpart with most of the values having less than a 2-fold difference, if the same method to determined in vitro Kp,uu was applied. The greatest discrepancies were reported if in vitro Kp,uu_Experimental and in vitro Kp,uu_Literature were determined with a different in vitro methodology instead. The rat in vitro Kp,uu results showed also good correlation with in vivo Kp,uu, with most of the data staying within the 3-fold difference. Although in a limited number for the comparison, also the human Kp,uu showed a trend of good in vitro-in vivo correlation. Finally, the methodology was applied to determine the in vitro Kp,uu of NCEs, of which the in vivo Kp,uu was already available from internal projects of Evotec SE, with a positive overall result for the IVIVC even in this case. In conclusion the proposed in vitro assay to measure hepatic Kp,uu had shown to have the potential to be a useful tool to study the unbound drug accumulation in the hepatocytes with the great advantage of giving a good estimation of hepatic drug exposure in vivo. This approach could be also applied in other in vitro assays for a better prediction of drug potency and clearance.