Renal Organic Anion Transporters 1 and 3 In Vitro: Gone but Not Forgotten.

Organic anion transporters 1 and 3 (OAT1 and OAT3) play a crucial role in kidney function by regulating the secretion of multiple renally cleared small molecules and toxic metabolic by-products. Assessing the activity of these transporters is essential for drug development purposes as they can significantly impact drug disposition and safety. OAT1 and OAT3 are amongst the most abundant drug transporters expressed in human renal proximal tubules. However, their expression is lost when cells are isolated and cultured in vitro, which is a persistent issue across all human and animal renal proximal tubule cell models, including primary cells and cell lines. Although it is well known that the overall expression of drug transporters is affected in vitro, the underlying reasons for the loss of OAT1 and OAT3 are still not fully understood. Nonetheless, research into the regulatory mechanisms of these transporters has provided insights into the molecular pathways underlying their expression and activity. In this review, we explore the regulatory mechanisms that govern the expression and activity of OAT1 and OAT3 and investigate the physiological changes that proximal tubule cells undergo and that potentially result in the loss of these transporters. A better understanding of the regulation of these transporters could aid in the development of strategies, such as introducing microfluidic conditions or epigenetic modification inhibitors, to improve their expression and activity in vitro and to create more physiologically relevant models. Consequently, this will enable more accurate assessment for drug development and safety applications.

Molecular Mechanisms for Species Differences in Organic Anion Transporter 1, OAT1: Implications for Renal Drug Toxicity.

Species differences in renal drug transporters continue to plague drug development with animal models failing to adequately predict renal drug toxicity. For example, adefovir, a renally excreted antiviral drug, failed clinical studies for human immunodeficiency virus due to pronounced nephrotoxicity in humans. In this study, we demonstrated that there are large species differences in the kinetics of interactions of a key class of antiviral drugs, acyclic nucleoside phosphonates (ANPs), with organic anion transporter 1 [(OAT1) SLC22A6] and identified a key amino acid residue responsible for these differences. In OAT1 stably transfected human embryonic kidney 293 cells, the Km value of tenofovir for human OAT1 (hOAT1) was significantly lower than for OAT1 orthologs from common preclinical animals, including cynomolgus monkey, mouse, rat, and dog. Chimeric and site-directed mutagenesis studies along with comparative structure modeling identified serine at position 203 (S203) in hOAT1 as a determinant of its lower Km value. Furthermore, S203 is conserved in apes, and in contrast alanine at the equivalent position is conserved in preclinical animals and Old World monkeys, the most related primates to apes. Intriguingly, transport efficiencies are significantly higher for OAT1 orthologs from apes with high serum uric acid (SUA) levels than for the orthologs from species with low serum uric acid levels. In conclusion, our data provide a molecular mechanism underlying species differences in renal accumulation of nephrotoxic ANPs and a novel insight into OAT1 transport function in primate evolution.

Perspective on the Application of Microphysiological Systems to Drug Transporter Studies.

Transmembrane flux of a drug within a tissue or organ frequently involves a complex system of transporters from multiple families that have redundant and overlapping specificities. Current in vitro systems poorly represent physiology, with reduced expression and activity of drug transporter proteins; therefore, novel models that recapitulate the complexity and interplay among various transporters are needed. The development of microphysiological systems that bring simulated physiologic conditions to in vitro cell culture models has enormous potential to better reproduce the morphology and transport activity across several organ models, especially in tissues such as the liver, kidney, intestine, or the blood-brain barrier, in which drug transporters play a key role. The prospect of improving the in vitro function of organ models highly prolific in drug transporters holds the promise of implementing novel tools to study these mechanisms with far more representative biology than before. In this short review, we exemplify recent developments in the characterization of perfused microphysiological systems involving the activity of drug transporters. Furthermore, we analyze the challenges and opportunities for the implementation of such systems in the study of transporter-mediated drug disposition and the generation of clinically relevant physiology-based in silico models incorporating relevant drug transport activity.

A novel multi-parametric high content screening assay in ciPTEC-OAT1 to predict drug-induced nephrotoxicity during drug discovery.

Drug-induced nephrotoxicity is a major concern in the clinic and hampers the use of available treatments as well as the development of innovative medicines. It is typically discovered late during drug development, which reflects a lack of in vitro nephrotoxicity assays available that can be employed readily in early drug discovery, to identify and hence steer away from the risk. Here, we report the development of a high content screening assay in ciPTEC-OAT1, a proximal tubular cell line that expresses several relevant renal transporters, using five fluorescent dyes to quantify cell health parameters. We used a validation set of 62 drugs, tested across a relevant concentration range compared to their exposure in humans, to develop a model that integrates multi-parametric data and drug exposure information, which identified most proximal tubular toxic drugs tested (sensitivity 75%) without any false positives (specificity 100%). Due to the relatively high throughput (straight-forward assay protocol, 96-well format, cost-effective) the assay is compatible with the needs in the early drug discovery setting to enable identification, quantification and subsequent mitigation of the risk for nephrotoxicity.

A multicenter assessment of single-cell models aligned to standard measures of cell health for prediction of acute hepatotoxicity.

Assessing the potential of a new drug to cause drug-induced liver injury (DILI) is a challenge for the pharmaceutical industry. We therefore determined whether cell models currently used in safety assessment (HepG2, HepaRG, Upcyte and primary human hepatocytes in conjunction with basic but commonly used endpoints) are actually able to distinguish between novel chemical entities (NCEs) with respect to their potential to cause DILI. A panel of thirteen compounds (nine DILI implicated and four non-DILI implicated in man) were selected for our study, which was conducted, for the first time, across multiple laboratories. None of the cell models could distinguish faithfully between DILI and non-DILI compounds. Only when nominal in vitro concentrations were adjusted for in vivo exposure levels were primary human hepatocytes (PHH) found to be the most accurate cell model, closely followed by HepG2. From a practical perspective, this study revealed significant inter-laboratory variation in the response of PHH, HepG2 and Upcyte cells, but not HepaRG cells. This variation was also observed to be compound dependent. Interestingly, differences between donors (hepatocytes), clones (HepG2) and the effect of cryopreservation (HepaRG and hepatocytes) were less important than differences between the cell models per se. In summary, these results demonstrate that basic cell health endpoints will not predict hepatotoxic risk in simple hepatic cells in the absence of pharmacokinetic data and that a multicenter assessment of more sophisticated signals of molecular initiating events is required to determine whether these cells can be incorporated in early safety assessment.

Multiple compound-related adverse properties contribute to liver injury caused by endothelin receptor antagonists.

Drug-induced liver injury has been observed in patients treated with the endothelin receptor antagonists sitaxentan and bosentan, but not following treatment with ambrisentan. The aim of our studies was to assess the possible role of multiple contributory mechanisms in this clinically relevant toxicity. Inhibition of the bile salt export pump (BSEP) and multidrug resistance-associated protein 2 was quantified using membrane vesicle assays. Inhibition of mitochondrial respiration in human liver-derived HuH-7 cells was determined using a Seahorse XF(e96) analyzer. Cytochrome P450 (P450)-independent and P450-mediated cell toxicity was assessed using transfected SV40-T-antigen-immortalized human liver epithelial (THLE) cell lines. Exposure-adjusted assay ratios were calculated by dividing the maximum human drug plasma concentrations by the IC50 or EC50 values obtained in vitro. Covalent binding (CVB) of radiolabeled drugs to human hepatocytes was quantified, and CVB body burdens were calculated by adjusting CVB values for fractional drug turnover in vitro and daily therapeutic dose. Sitaxentan exhibited positive exposure-adjusted signals in all five in vitro assays and a high CVB body burden. Bosentan exhibited a positive exposure-adjusted signal in one assay (BSEP inhibition) and a moderate CVB body burden. Ambrisentan exhibited no positive exposure-adjusted assay signals and a low CVB body burden. These data indicate that multiple mechanisms contribute to the rare, but potentially severe liver injury caused by sitaxentan in humans; provide a plausible rationale for the markedly lower propensity of bosentan to cause liver injury; and highlight the relative safety of ambrisentan.

Systems toxicology: modelling biomarkers of glutathione homeostasis and paracetamol metabolism.

One aim of systems toxicology is to deliver mechanistic, mathematically rigorous, models integrating biochemical and pharmacological processes that result in toxicity to enhance the assessment of the risk posed to humans by drugs and other xenobiotics. The benefits of such 'in silico' models would be in enabling the rapid and robust prediction of the effects of compounds over a range of exposures, improving in vitro-in vivo correlations and the translation from preclinical species to humans. Systems toxicology models of organ toxicities that result in high attrition rates during drug discovery and development, or post-marketing withdrawals (e.g., drug-induced liver injury (DILI)) should facilitate the discovery of safe new drugs. Here, systems toxicology as applied to the effects of paracetamol (acetaminophen, N-acetyl-para-aminophenol (APAP)) is used to exemplify the potential of the approach.

Amplifying the impact of kidney microphysiological systems: predicting renal drug clearance using mechanistic modelling based on reconstructed drug secretion.

Accurate prediction of pharmacokinetic parameters, such as renal clearance, is fundamental to the development of effective and safe new treatments for patients. However, conventional renal models have a limited ability to predict renal drug secretion, a process that is dependent on transporters in the proximal tubule. Improvements in microphysiological systems (MPS) have extended our in vitro capabilities to predict pharmacokinetic parameters. In this study a kidney-MPS model was developed that successfully recreated renal drug secretion. Human proximal tubule cells grown in the kidney-MPS, resem­bling an in vivo phenotype, actively secreted the organic cation drug metformin and organic anion drug cidofovir, in contrast to cells cultured in conventional culture formats. Metformin and cidofovir renal secretory clearance were predicted from kid­ney-MPS data within 3.3- and 1.3-fold, respectively, of clinically reported values by employing a semi-mechanistic drug distribution model using kidney-MPS drug transport parameters together with in vitro to in vivo extrapolation. This approach introduces an effective application of a kidney-MPS model coupled with pharmacokinetic modelling tools to evaluate and predict renal drug clearance in humans. Kidney-MPS renal clearance predictions can potentially complement pharma-cokinetic animal studies and contribute to the reduction of pre-clinical species use during drug development.

Utilization of OATP1B Biomarker Coproporphyrin-I to Guide Drug-Drug Interaction Risk Assessment: Evaluation by the Pharmaceutical Industry.

Drug-drug interactions (DDIs) involving hepatic organic anion transporting polypeptides 1B1/1B3 (OATP1B) can be substantial, however, challenges remain for predicting interaction risk. Emerging evidence suggests that endogenous biomarkers, particularly coproporphyrin-I (CP-I), can be used to assess in vivo OATP1B activity. The present work under the International Consortium for Innovation and Quality in Pharmaceutical Development was aimed primarily at assessing CP-I as a biomarker for informing OATP1B DDI risk. Literature and unpublished CP-I data along with pertinent in vitro and clinical DDI information were collected to identify DDIs primarily involving OATP1B inhibition and assess the relationship between OATP1B substrate drug and CP-I exposure changes. Static models to predict changes in exposure of CP-I, as a selective OATP1B substrate, were also evaluated. Significant correlations were observed between CP-I area under the curve ratio (AUCR) or maximum concentration ratio (Cmax R) and AUCR of substrate drugs. In general, the CP-I Cmax R was equal to or greater than the CP-I AUCR. CP-I Cmax R < 1.25 was associated with absence of OATP1B-mediated DDIs (AUCR < 1.25) with no false negative predictions. CP-I Cmax R < 2 was associated with weak OATP1B-mediated DDIs (AUCR < 2). A correlation was identified between CP-I exposure changes and OATP1B1 static DDI predictions. Recommendations for collecting and interpreting CP-I data are discussed, including a decision tree for guiding DDI risk assessment. In conclusion, measurement of CP-I is recommended to inform OATP1B inhibition potential. The current analysis identified changes in CP-I exposure that may be used to prioritize, delay, or replace clinical DDI studies.

Challenges and Opportunities for Improved Drug-Drug Interaction Predictions for Renal OCT2 and MATE1/2-K Transporters.

Transporters contribute to renal elimination of drugs; therefore drug disposition can be impacted if transporters are inhibited by comedicant drugs. Regulatory agencies have provided guidelines to assess potential drug-drug interaction (DDI) risk for renal organic cation transporter 2 (OCT2) and multidrug and toxin extrusion 1 and 2-K (MATE1/2-K) transporters. Despite this, there are challenges with translating in vitro data using currently available tools to obtain a quantitative assessment of DDI risk in the clinic. Given the high number of drugs and new molecular entities showing in vitro inhibition toward OCT2 and/or MATE1/2-K and the lack of translation to clinically significant effects, it is reasonable to question whether the current in vitro assay design and modeling practice has led to unnecessary clinical evaluation. The aim of this review is to assess and discuss available in vitro and clinical data along with prediction models intended to provide clinical context of risk, including static models proposed by regulatory agencies and physiologically-based pharmacokinetic models, in order to identify best practices and areas of future opportunity. This analysis highlights that different in vitro assay designs, including substrate and cell systems used, strongly influence the derived concentration of drug producing 50% inhibition values and contribute to high variability observed across laboratories. Furthermore, the lack of sensitive index substrates coupled with specific inhibitors for individual transporters necessitates the use of complex models to evaluate clinical DDI risk.

Leveraging microphysiological systems to address challenges encountered during development of oligonucleotide therapeutics.

Oligonucleotide therapeutics (ONTs) encompass classes of medicines that selectively target and potentially ameliorate previously untreatable and often rare diseases. Several unique classes of ONTs provide versatility, enabling direct modu­lation of gene expression by virtue of Watson-Crick base pairing or modulation of cell signaling through structural mimicry or interference with protein-receptor interactions. Due to a lack of suitable in vitro models capable of recapitulating or predicting in vivo effects of ONTs, their discovery and optimization has relied heavily on animal studies for predicting efficacy and safety in humans. Since ONTs often lack cross-species activity, animal models with genetic humanization and/or species-specific surrogate ONTs are often required. Human microphysiological systems (MPS) offer an oppor­tunity to reduce the use of animals and may enable evaluation of drug mechanisms, optimization of cell and tissue targeting ligands or delivery vehicles, and characterization of pharmacokinetics (PK), pharmacodynamics (PD), and safety of candidate ONTs. The lack of published examples for MPS applications with ONT demonstrates the need for a focused effort to characterize and build confidence in their utility. The goals of this review are to summarize the current landscape of ONTs and highlight potential opportunities and challenges for application of MPS during ONT discovery and development. In addition, this review aims to raise awareness with ONT drug developers and regulatory authorities on the potential impact of MPS with respect to characterizing pharmacology, ADME, and toxicity and to educate MPS platform developers on unique design attributes needed to fully appreciate MPS advantages in ONT development.

Regulation of Drug Transport Proteins-From Mechanisms to Clinical Impact: A White Paper on Behalf of the International Transporter Consortium.

Membrane transport proteins are involved in the absorption, disposition, efficacy, and/or toxicity of many drugs. Numerous mechanisms (e.g., nuclear receptors, epigenetic gene regulation, microRNAs, alternative splicing, post-translational modifications, and trafficking) regulate transport protein levels, localization, and function. Various factors associated with disease, medications, and dietary constituents, for example, may alter the regulation and activity of transport proteins in the intestine, liver, kidneys, brain, lungs, placenta, and other important sites, such as tumor tissue. This white paper reviews key mechanisms and regulatory factors that alter the function of clinically relevant transport proteins involved in drug disposition. Current considerations with in vitro and in vivo models that are used to investigate transporter regulation are discussed, including strengths, limitations, and the inherent challenges in predicting the impact of changes due to regulation of one transporter on compensatory pathways and overall drug disposition. In addition, translation and scaling of in vitro observations to in vivo outcomes are considered. The importance of incorporating altered transporter regulation in modeling and simulation approaches to predict the clinical impact on drug disposition is also discussed. Regulation of transporters is highly complex and, therefore, identification of knowledge gaps will aid in directing future research to expand our understanding of clinically relevant molecular mechanisms of transporter regulation. This information is critical to the development of tools and approaches to improve therapeutic outcomes by predicting more accurately the impact of regulation-mediated changes in transporter function on drug disposition and response.

The analysis of acetaminophen (paracetamol) and seven metabolites in rat, pig and human plasma by U(H)PLC-MS.

A U(H)PLC-MS/MS method is described for the analysis of acetaminophen and its sulphate, glucuronide, glutathione, cysteinyl and N-acetylcysteinyl metabolites in plasma using stable isotope-labeled internal standards. P-Aminophenol glucuronide and 3-methoxyacetaminophen were monitored and semi-quantified using external standards. The assay takes 7.5 min/sample, requires only 5 µl of plasma and involves minimal sample preparation. The method was validated for rat plasma and cross validated for human and pig plasma and mouse serum. LOQ in plasma for these analytes were 0.44 µg/ml (APAP-C), 0.58 µg/ml (APAP-SG), 0.84 µg/ml (APAP-NAC), 2.75 µg/ml (APAP-S), 3.00 µg/ml (APAP-G) and 16 µg/ml (APAP). Application of the method is illustrated by the analysis of plasma following oral administration of APAP to male Han Wistar rats.

Using Quantitative Systems Toxicology to Investigate Observed Species Differences in CKA-Mediated Hepatotoxicity.

CKA, a chemokine receptor antagonist intended for treating inflammatory conditions, produced dose-dependent hepatotoxicity in rats but advanced into the clinic where single doses of CKA up to 600 mg appeared safe in humans. Because existing toxicological platforms used during drug development are not perfectly predictive, a quantitative systems toxicology model investigated the hepatotoxic potential of CKA in humans and rats through in vitro assessments of CKA on mitochondrial respiration, oxidative stress, and bile acid transporters. DILIsym predicted that single doses of CKA caused serum ALT >3xULN in a subset of the simulated rat population, while single doses in a simulated human population did not produce serum ALT elevations. Species differences were largely attributed to differences in liver exposure, but increased sensitivity to inhibition of mitochondrial respiration in the rat also contributed. We conclude that mechanistic modeling can elucidate species differences in the hepatotoxic potential of drug candidates.

Quantification of Drug-Induced Inhibition of Canalicular Cholyl-l-Lysyl-Fluorescein Excretion From Hepatocytes by High Content Cell Imaging.

We describe the use of a commercially available high content cell imaging algorithm (Cellomics Arrayscan Spot Detector) to quantify biliary excretion of the fluorescent probe substrate cholyl-l-lysyl-fluorescein (CLF) from rat hepatocytes cultured in collagen/matrigel sandwich configuration and to explore inhibition of this process by a variety of test compounds. The method provided robust, reproducible data. Twenty-nine pharmaceuticals inhibited biliary CLF efflux from hepatocytes and a broad range of potencies of inhibition were observed (IC50 values ranged between <1 and 794 µM). Thirteen drugs that inhibited CLF efflux also inhibited hepatocellular uptake of the probe substrate [(3)H]-taurocholate. Although no clear correlation between the potencies of inhibition of the 2 processes was evident, these data highlight the need to consider possible uptake transporter inhibition when interpreting hepatocyte CLF inhibition data. It has been reported that CLF is transported by MRP2. The CLF efflux inhibition data correlated closely with published data on inhibition by the drugs of the bile salt export pump (Bsep), which suggests that the tested drugs inhibit both Bsep and Mrp2. Calculation of the ratios between the maximum human plasma concentrations of the drugs and their CLF efflux inhibition IC50 values raised the possibility that for many, but not all, of them the in vitro effects may be functionally significant in vivo and that Mrp2 inhibition might be a drug-induced liver injury (DILI) risk factor. These data indicate that imaging hepatocyte CLF inhibition is a promising new method for quantification of biliary efflux inhibition by drugs, which could aid assessment of compound-related DILI risk.