Exploration and application of a liver-on-a-chip device in combination with modelling and simulation for quantitative drug metabolism studies.

Microphysiological systems (MPS) are complex and more physiologically realistic cellular in vitro tools that aim to provide more relevant human in vitro data for quantitative prediction of clinical pharmacokinetics while also reducing the need for animal testing. The PhysioMimix liver-on-a-chip integrates medium flow with hepatocyte culture and has the potential to be adopted for in vitro studies investigating the hepatic disposition characteristics of drug candidates. The current study focusses on liver-on-a-chip system exploration for multiple drug metabolism applications. Characterization of cytochrome P450 (CYP), UDP-glucuronosyl transferase (UGT) and aldehyde oxidase (AO) activities was performed using 15 drugs and in vitro to in vivo extrapolation (IVIVE) was assessed for 12 of them. Next, the utility of the liver-on-a-chip for estimation of the fraction metabolized (fm) via specific biotransformation pathways of quinidine and diclofenac was established. Finally, the metabolite identification opportunities were also explored using efavirenz as an example drug with complex primary and secondary metabolism involving a combination of CYP, UGT and sulfotransferase enzymes. A key aspect of these investigations was the application of mathematical modelling for improved parameter calculation. Such approaches will be required for quantitative assessment of metabolism and/or transporter processes in systems where medium flow and system compartments result in non-homogeneous drug concentrations. In particular, modelling was used to explore the effect of evaporation from the medium and it was found that the intrinsic clearance (CLint) might be underestimated by up to 40% for low clearance compounds if evaporation is not accounted for. Modelling of liver-on-a-chip in vitro data also enhanced the approach to fm estimation allowing objective assessment of metabolism models of different complexity. The resultant diclofenac fm,UGT of 0.64 was highly comparable with values reported previously in the literature. The current study demonstrates the integration of mathematical modelling with experimental liver-on-a-chip studies and illustrates how this approach supports generation of high quality of data from complex in vitro cellular systems.

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.

Construction and Verification of Physiologically Based Pharmacokinetic Models for Four Drugs Majorly Cleared by Glucuronidation: Lorazepam, Oxazepam, Naloxone, and Zidovudine.

Physiologically based pharmacokinetic (PBPK) modeling is less well established for substrates of UDP-glucuronosyltransferases (UGT) than for cytochrome P450 (CYP) metabolized drugs and more verification of simulations is necessary to increase confidence. To address specific challenges of UGT substrates, we developed PBPK models for four drugs cleared majorly via glucuronidation (lorazepam, oxazepam, naloxone, and zidovudine). In vitro to in vivo scaling of intrinsic clearance generated with co-cultured human hepatocytes was applied for hepatic metabolism and extra-hepatic clearance was extrapolated based on relative expression of UGT isoforms in the liver, kidney, and intestine. Non-metabolic clearance and the contributions of individual UGT isoforms to glucuronidation were based on in vitro and in vivo studies taken from the literature and simulations were verified and evaluated with a broad set of clinical pharmacokinetic data. Model evaluation showed systemic clearance predictions within 1.5-fold for all drugs and all simulated parameters were within 2-fold of observed. However, during the verification step, top-down model fitting was necessary to adjust for under-prediction of zidovudine VSS and renal clearance and over estimation of intestinal first pass for lorazepam, oxazepam, and zidovudine. The impact of UGT2B15 polymorphisms on the pharmacokinetics of oxazepam and lorazepam was simulated and glucuronide metabolites were also simulated for all four drugs. To increase confidence in predicting extra-hepatic clearance, improvement of enzyme phenotyping for UGT substrates and more quantitative tissue expression levels of UGT enzymes are both needed. Prediction of glucuronide disposition is also challenging when active transport processes play a major role.

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.

Application of New Cellular and Microphysiological Systems to Drug Metabolism Optimization and Their Positioning Respective to In Silico Tools.

New cellular model systems for drug metabolism applications, such as advanced 2D liver co-cultures, spheroids, and microphysiological systems (MPSs), offer exciting opportunities to reproduce human biology more closely in vitro with the aim of improving predictions of pharmacokinetics, drug-drug interactions, and efficacy. These advanced cellular systems have quickly become established for human intrinsic clearance determination and have been validated for several other absorption, distribution, metabolism, and excretion (ADME) applications. Adoption will be driven through the demonstration of clear added value, for instance, by more accurate and precise clearance predictions and by more reliable extrapolation of drug interaction potential leading to faster progression to pivotal proof-of-concept studies. New experimental systems are attractive when they can (1) increase experimental capacity, removing optimization bottlenecks; (2) improve measurement quality of ADME properties that impact pharmacokinetics; and (3) enable measurements to be made that were not previously possible, reducing risk in ADME prediction and candidate selection. As new systems become established, they will find their place in the repository of tools used at different stages of the research and development process, depending on the balance of value, throughput, and cost. In this article, we give a perspective on the integration of these new methodologies into ADME optimization during drug discovery, and the likely applications and impacts on drug development.

Pharmacological profile of mephedrone analogs and related new psychoactive substances.

BACKGROUND: Mephedrone is a synthetic cathinone and one of the most popular recreationally used new psychoactive substances. The aim of the present study was to characterize the in vitro pharmacology of novel analogs of mephedrone and related newly emerged designer stimulants. METHODS: We determined norepinephrine, dopamine, and serotonin transporter inhibition potencies and monoamine release in transporter-transfected human embryonic kidney 293 cells. We also assessed monoamine receptor and transporter binding affinities. RESULTS: Mephedrone analogs potently inhibited the norepinephrine transporter and, with the exception of 3-methylmethcathinone (3-MMC), inhibited the serotonin transporter more potently than the dopamine transporter. Similar to classic amphetamines, mephedrone analogs were substrate-type monoamine releasers. 5-(2-Aminopropyl)indole (5-IT) was a highly potent monoamine transporter inhibitor and a releaser of dopamine and serotonin. 4-Methylamphetamine (4-MA) mediated efflux of all three monoamines and inhibited the serotonin transporter more potently than the dopamine transporter, unlike amphetamine. N-methyl-2-aminoindane (N-methyl-2-AI) was a selective norepinephrine transporter inhibitor and norepinephrine releaser, whereas 5-methoxy-6-methyl-2-aminoindane (MMAI) was a selective serotonin transporter inhibitor and serotonin releaser. All of the drugs interacted with monoamine receptors. CONCLUSION: The predominant actions on serotonin vs. dopamine transporters suggest that dimethylmethcathinones, 4-MA, and MMAI cause entactogenic effects similar to 3,4-methylenedioxymethamphetamine, whereas 3-MMC, 5-IT, and N-methyl-2-AI have more stimulant-type properties like amphetamine. Because of pharmacological and structural similarity to mephedrone, similar health risks can be expected for these analogs. This article is part of the Special Issue entitled 'Designer Drugs and Legal Highs.'