Use of Phenotypically Poor Metabolizer Individual Donor Human Liver Microsomes To Identify Selective Substrates of UGT2B10.

UDP-glucuronosyltransferase (UGT)1A4 and UGT2B10 are the human UGT isoforms most frequently involved in N-glucuronidation of drugs. UGT2B10 exhibits higher affinity than UGT1A4 for numerous substrates, making it potentially the more important enzyme for metabolism of these compounds in vivo. Clinically relevant UGT2B10 polymorphisms, including a null activity splice site mutation common in African populations, can lead to large exposure differences for UGT2B10 substrates that may limit their developability as marketed drugs. UGT phenotyping approaches using recombinantly expressed UGTs are limited by low enzyme activity and lack of validation of scaling to in vivo. In this study, we describe the use of an efficient experimental protocol for identification of UGT2B10-selective substrates (i.e., those with high fraction metabolized by UGT2B10), which exploits the activity difference between pooled human liver microsomes (HLM) and HLM from a phenotypically UGT2B10 poor metabolizer donor. Following characterization of the approach with eight known UGT2B10 substrates, we used ligand-based virtual screening and literature precedents to select 24 potential UGT2B10 substrates of 140 UGT-metabolized drugs for testing. Of these, dothiepin, cidoxepin, cyclobenzaprine, azatadine, cyproheptadine, bifonazole, and asenapine were indicated to be selective UGT2B10 substrates that have not previously been described. UGT phenotyping experiments and tests comparing conjugative and oxidative clearance were then used to confirm these findings. These approaches provide rapid and sensitive ways to evaluate whether a potential drug candidate cleared via glucuronidation will be sensitive to UGT2B10 polymorphisms in vivo. SIGNIFICANCE STATEMENT: The role of highly polymorphic UDP-glucuronosyltransferase (UGT)2B10 is likely to be underestimated currently for many compounds cleared via N-glucuronidation due to high test concentrations often used in vitro and low activity of UGT2B10 preparations. The methodology described in this study can be combined with the assessment of UGT versus oxidative in vitro metabolism to rapidly identify compounds likely to be sensitive to UGT2B10 polymorphism (high fraction metabolized by UGT2B10), enabling either chemical modification or polymorphism risk assessment before candidate selection.

Simultaneous Assessment of Clearance, Metabolism, Induction, and Drug-Drug Interaction Potential Using a Long-Term In Vitro Liver Model for a Novel Hepatitis B Virus Inhibitor.

Long-term in vitro liver models are now widely explored for human hepatic metabolic clearance prediction, enzyme phenotyping, cross-species metabolism, comparison of low clearance drugs, and induction studies. Here, we present studies using a long-term liver model, which show how metabolism and active transport, drug-drug interactions, and enzyme induction in healthy and diseased states, such as hepatitis B virus (HBV) infection, may be assessed in a single test system to enable effective data integration for physiologically based pharmacokinetic (PBPK) modeling. The approach is exemplified in the case of (3S)-4-[[(4R)-4-(2-Chloro-4-fluorophenyl)-5-methoxycarbonyl-2-thiazol-2-yl-1,4-dihydropyrimidin-6-yl]methyl]morpholine-3-carboxylic acid RO6889678, a novel inhibitor of HBV with a complex absorption, distribution, metabolism, and excretion (ADME) profile. RO6889678 showed an intracellular enrichment of 78-fold in hepatocytes, with an apparent intrinsic clearance of 5.2 µl/min per mg protein and uptake and biliary clearances of 2.6 and 1.6 µl/min per mg protein, respectively. When apparent intrinsic clearance was incorporated into a PBPK model, the simulated oral human profiles were in good agreement with observed data at low doses but were underestimated at high doses due to unexpected overproportional increases in exposure with dose. In addition, the induction potential of RO6889678 on cytochrome P450 (P450) enzymes and transporters at steady state was assessed and cotreatment with ritonavir revealed a complex drug-drug interaction with concurrent P450 inhibition and moderate UDP-glucuronosyltransferase induction. Furthermore, we report on the first evaluation of in vitro pharmacokinetics studies using HBV-infected HepatoPac cocultures. Thus, long-term liver models have great potential as translational research tools exploring pharmacokinetics of novel drugs in vitro in health and disease.

Correction to: In Vitro to In Vivo Extrapolation of Metabolic Clearance for UGT Substrates Using Short-Term Suspension and Long-Term Co-cultured Human Hepatocytes.

During production, the figure captions for Fig. 1 and Fig. 2 were inadvertently switched in the proofing stage.

In Vitro to In Vivo Extrapolation of Metabolic Clearance for UGT Substrates Using Short-Term Suspension and Long-Term Co-cultured Human Hepatocytes.

The use of micro-patterned co-cultured hepatocytes for human hepatic clearance predictions has previously been demonstrated using drugs metabolized by cytochrome P450 enzymes. The present study evaluates the in vitro to in vivo extrapolation (IVIVE) performance for UDP-glucuronosyltransferase (UGT) substrates. In vitro intrinsic clearances for 13 drugs mainly cleared by UGTs were determined using HepatoPac and suspended hepatocytes. The in vivo intrinsic clearance was predicted from in vitro intrinsic clearance and compared with weighted mean in vivo intrinsic clearance estimated from several clinical studies. A conventional scaling methodology accounting for protein binding in plasma and incubation medium was used for the IVIVE assuming that only free drug is accessible at the site of metabolism. The in vivo hepatic intrinsic clearance was predicted within threefold error for six and nine out of thirteen drugs using suspended hepatocytes and HepatoPac, respectively. A reduced under-estimation of hepatic intrinsic clearance was observed in the average fold error (AFE) in HepatoPac (AFE, 0.69) compared with the suspended hepatocytes (AFE, 0.37). The current study shows reasonable performance of hepatic clearance prediction of drugs mainly metabolized by UGT enzymes using HepatoPac with a similar under-prediction bias as obtained in the reported IVIVEs for cytochrome P450 substrates. This study provides a validation of the approach for drugs cleared via UGT conjugation mechanisms and discusses potential causes for outlier behavior considering pharmacokinetic or physicochemical properties.

Impact of Intracellular Concentrations on Metabolic Drug-Drug Interaction Studies.

Accurate prediction of drug-drug interactions (DDI) is a challenging task in drug discovery and development. It requires determination of enzyme inhibition in vitro which is highly system-dependent for many compounds. The aim of this study was to investigate whether the determination of intracellular unbound concentrations in primary human hepatocytes can be used to bridge discrepancies between results obtained using human liver microsomes and hepatocytes. Specifically, we investigated if Kpuu could reconcile differences in CYP enzyme inhibition values (Ki or IC50). Firstly, our methodology for determination of Kpuu was optimized for human hepatocytes, using four well-studied reference compounds. Secondly, the methodology was applied to a series of structurally related CYP2C9 inhibitors from a Roche discovery project. Lastly, the Kpuu values of three commonly used CYP3A4 inhibitors-ketoconazole, itraconazole, and posaconazole-were determined and compared to compound-specific hepatic enrichment factors obtained from physiologically based modeling of clinical DDI studies with these three compounds. Kpuu obtained in suspended human hepatocytes gave good predictions of system-dependent differences in vitro. The Kpuu was also in fair agreement with the compound-specific hepatic enrichment factors in DDI models and can therefore be used to improve estimations of enrichment factors in physiologically based pharmacokinetic modeling.

Metabolic Profiling of Human Long-Term Liver Models and Hepatic Clearance Predictions from In Vitro Data Using Nonlinear Mixed-Effects Modeling.

Early prediction of human clearance is often challenging, in particular for the growing number of low-clearance compounds. Long-term in vitro models have been developed which enable sophisticated hepatic drug disposition studies and improved clearance predictions. Here, the cell line HepG2, iPSC-derived hepatocytes (iCell®), the hepatic stem cell line HepaRG™, and human hepatocyte co-cultures (HµREL™ and HepatoPac®) were compared to primary hepatocyte suspension cultures with respect to their key metabolic activities. Similar metabolic activities were found for the long-term models HepaRG™, HµREL™, and HepatoPac® and the short-term suspension cultures when averaged across all 11 enzyme markers, although differences were seen in the activities of CYP2D6 and non-CYP enzymes. For iCell® and HepG2, the metabolic activity was more than tenfold lower. The micropatterned HepatoPac® model was further evaluated with respect to clearance prediction. To assess the in vitro parameters, pharmacokinetic modeling was applied. The determination of intrinsic clearance by nonlinear mixed-effects modeling in a long-term model significantly increased the confidence in the parameter estimation and extended the sensitive range towards 3% of liver blood flow, i.e., >10-fold lower as compared to suspension cultures. For in vitro to in vivo extrapolation, the well-stirred model was used. The micropatterned model gave rise to clearance prediction in man within a twofold error for the majority of low-clearance compounds. Further research is needed to understand whether transporter activity and drug metabolism by non-CYP enzymes, such as UGTs, SULTs, AO, and FMO, is comparable to the in vivo situation in these long-term culture models.

Hydrophilic interaction chromatography of intact, soluble proteins.

The separation of intact proteins by means of Hydrophilic Interaction Chromatography (HILIC) was demonstrated with human apoA-I, recombinant human apoM, and equine cytochrome C. Five different commercially available HILIC columns were compared. Using one of these columns, different glycosylated isoforms of apoM were separated from each other and from the aglyco-form.