Donald J. Tweedie

Membrane transporters in drug development

Membrane transporters can be major determinants of the pharmacokinetic, safety and efficacy profiles of drugs. This presents several key questions for drug development, including which transporters are clinically important in drug absorption and disposition, and which in vitro methods are suitable for studying drug interactions with these transporters. In addition, what criteria should trigger follow-up clinical studies, and which clinical studies should be conducted if needed. In this article, we provide the recommendations of the International Transporter Consortium on these issues, and present decision trees that are intended to help guide clinical studies on the currently recognized most important drug transporter interactions. The recommendations are generally intended to support clinical development and filing of a new drug application. Overall, it is advised that the timing of transporter investigations should be driven by efficacy, safety and clinical trial enrolment questions (for example, exclusion and inclusion criteria), as well as a need for further understanding of the absorption, distribution, metabolism and excretion properties of the drug molecule, and information required for drug labelling.

Managing the challenge of chemically reactive metabolites in drug development

The normal metabolism of drugs can generate metabolites that have intrinsic chemical reactivity towards cellular molecules, and therefore have the potential to alter biological function and initiate serious adverse drug reactions. Here, we present an assessment of the current approaches used for the evaluation of chemically reactive metabolites. We also describe how these approaches are being used within the pharmaceutical industry to assess and minimize the potential of drug candidates to cause toxicity. At early stages of drug discovery, iteration between medicinal chemistry and drug metabolism can eliminate perceived reactive metabolite-mediated chemical liabilities without compromising pharmacological activity or the need for extensive safety evaluation beyond standard practices. In the future, reactive metabolite evaluation may also be useful during clinical development for improving clinical risk assessment and risk management. Currently, there remains a huge gap in our understanding of the basic mechanisms that underlie chemical stress-mediated adverse reactions in humans. This Review summarizes our views on this complex topic, and includes insights into practices considered by the pharmaceutical industry.

The Conduct of In Vitro and In Vivo Drug‐Drug Interaction Studies: A PhRMA Perspective

Current regulatory guidances do not address specific study designs for in vitro and in vivo drug‐drug interaction studies. There is a common desire by regulatory authorities and by industry sponsors to harmonize approaches to allow for a better assessment of the significance of findings across different studies and drugs. There is also a growing consensus for the standardization of cytochrome P450 (CYP) probe substrates, inhibitors, and inducers and for the development of classification systems to improve the communication of risk to health care providers and patients. While existing guidances cover mainly CYP‐mediated drug interactions, the importance of other mechanisms, such as transporters, has been recognized more recently and should also be addressed. This paper was prepared by the Pharmaceutical Research and Manufacturers of America (PhRMA) Drug Metabolism and Clinical Pharmacology Technical Working Groups and represents the current industry position. The intent is to define a minimal best practice for in vitro and in vivo pharmacokinetic drug‐drug interaction studies targeted to development (not discovery support) and to define a data package that can be expected by regulatory agencies in compound registration dossiers.

Transporter Studies in Drug Development: Experience to Date and Follow up on Decision Trees from the International Transporter Consortium.

The International Transporter Consortium (ITC) organized a second workshop in March 2012 to expand on the themes developed during the inaugural ITC workshop held in 2008. The final session of the workshop provided perspectives from regulatory and industry‐based scientists, with input from academic scientists, and focused primarily on the decision trees published from the first workshop. These decision trees have become a central part of subsequent regulatory drug–drug interaction (DDI) guidances issued over the past few years.

Meeting the Challenge of Predicting Hepatic Clearance of Compounds Slowly Metabolized by Cytochrome P450 Using a Novel Hepatocyte Model, HepatoPac

Generating accurate in vitro intrinsic clearance data is an important aspect of predicting in vivo human clearance. Primary hepatocytes in suspension are routinely used to predict in vivo clearance; however, incubation times have typically been limited to 4–6 hours, which is not long enough to accurately evaluate the metabolic stability of slowly metabolized compounds. HepatoPac is a micropatterened hepatocyte-fibroblast coculture system that can be used for continuous incubations of up to 7 days. This study evaluated the ability of human HepatoPac to predict the in vivo clearance (CL) of 17 commercially available compounds with low to intermediate clearance (<12 ml/min per kg). In vitro half-life for disappearance of each compound was converted to hepatic clearance using the well stirred model, with and without correction for plasma protein binding. Hepatic CL, using three individual donors, was accurately predicted for 10 of 17 compounds (59%; predicted clearance within 2-fold of observed human in vivo clearance values). The accuracy of prediction increased to 76% (13 of 17 compounds) with an acceptance criterion defined as within 3-fold. When considering only low clearance compounds (<5 ml/min per kg), which represented 10 of the 17 compounds, the accuracy of prediction was 60% within 2-fold and 90% within 3-fold. In addition, the turnover of three slowly metabolized compounds (alprazolam, meloxicam, and tolbutamide) in HepatoPac was directly compared with turnover in suspended hepatocytes. The turnover of alprazolam and tolbutamide was approximately 2-fold greater using HepatoPac compared with suspended hepatocytes, which was roughly in line with the extrapolated values (correcting for the longer incubation time and lower cell number with HepatoPac). HepatoPac, but not suspended hepatocytes, demonstrated significant turnover of meloxicam. These results demonstrate the utility of HepatoPac for prediction of in vivo hepatic clearance, particularly with low clearance compounds.

Highlights from the International Transporter Consortium second workshop.

The Second International Transporter Consortium (ITC) Workshop was held with the purpose of expanding on previous white‐paper recommendations, discussing recent regulatory draft guidance documents on transporter–drug interactions, and highlighting transporter‐related challenges in drug development. Specific goals were to discuss additional clinically relevant transporters (MATEs, MRP2, BSEP) and best‐practice methodologies and to re‐evaluate ITC decision trees based on actual case studies. The outcome of the workshop will be a series of white papers targeted for publication in 2013.

Bridging In Vitro and In Vivo Metabolism and Transport of Faldaprevir in Human Using a Novel Cocultured Human Hepatocyte System, HepatoPac

An increased appreciation of the importance of transporter and enzyme interplay in drug clearance and a desire to delineate these mechanisms necessitates the utilization of models that contain a full complement of enzymes and transporters at physiologically relevant activities. Additionally, the development of drugs with longer half-lives requires in vitro systems with extended incubation times that allow characterization of metabolic pathways for low-clearance drugs. A recently developed coculture hepatocyte model, HepatoPac, has been applied to meet these challenges. Faldaprevir is a drug in late-stage development for the treatment of hepatitis C. Faldaprevir is a low-clearance drug with the somewhat unique characteristic of being slowly metabolized, producing two abundant hydroxylated metabolites (M2a and M2b) in feces (∼40% of the dose) without exhibiting significant levels of circulating metabolites in humans. The human HepatoPac model was investigated to characterize the metabolism and transport of faldaprevir. In human HepatoPac cultures, M2a and M2b were the predominant metabolites formed, with extents of formation comparable to in vivo. Direct glucuronidation of faldaprevir was shown to be a minor metabolic pathway. HepatoPac studies also demonstrated that faldaprevir is concentrated in liver with active uptake by multiple transporters (including OATP1B1 and Na+-dependent transporters). Overall, human HepatoPac cultures provided valuable insights into the metabolism and disposition of faldaprevir in humans and demonstrated the importance of enzyme and transporter interplay in the clearance of the drug.

A Perspective on the Contribution of Metabolites to Drug-Drug Interaction Potential: The Need to Consider Both Circulating Levels and Inhibition Potency

The 2012 drug-drug interaction (DDI) guidance from the European Medicines Agency (EMA) and the draft DDI guidance from the Food and Drug Administration (FDA) have proposed that metabolites present at >25% of the parent area under the time-concentration curve (AUC) (EMA and FDA) and >10% of the total drug-related exposure (EMA) should be investigated in vitro for their DDI potential. This commentary attempts to rationalize the clinically relevant levels of metabolite(s) that contribute to DDI by considering not only the abundance but also inhibition potency, physicochemical properties, and structural alerts of the metabolite. A decision tree is proposed for levels of metabolites that could trigger in vitro DDI assessment. When the parent is an inhibitor of cytochrome P450s (P450s), clinical DDI studies will assess the in vivo DDI effect of the combination of parent and metabolite(s). When the parent is not a P450 inhibitor, it is important to assess the inhibition potential of abundant metabolites in vitro. The proposal is to apply a default cutoff value of metabolite level which is 100% of the parent AUC. It is important to note that exceptions can occur, and different metabolite levels may be considered depending on the physiochemical properties of metabolites (e.g., increased lipophilicity) and whether the metabolite contains structural alerts for DDI (e.g., mechanism-based inhibition). A key objective of this commentary is to stimulate discussions among the scientific community on this important topic, so that appropriate in vitro metabolism studies are conducted on metabolites, to ensure the safety of drugs in development balanced with the desire to avoid creating unnecessary studies that will add little to no value in ensuring patient safety.

Mechanisms Underlying Benign and Reversible Unconjugated Hyperbilirubinemia Observed with Faldaprevir Administration in Hepatitis C Virus Patients

Faldaprevir, an investigational agent for hepatitis C virus treatment, is well tolerated but associated with rapidly reversible, dose-dependent, clinically benign, unconjugated hyperbilirubinemia. Multidisciplinary preclinical and clinical studies were used to characterize mechanisms underlying this hyperbilirubinemia. In vitro, faldaprevir inhibited key processes involved in bilirubin clearance: UDP glucuronosyltransferase (UGT) 1A1 (UGT1A1) (IC50 0.45 µM), which conjugates bilirubin, and hepatic uptake and efflux transporters, organic anion–transporting polypeptide (OATP) 1B1 (IC50 0.57 µM), OATP1B3 (IC50 0.18 µM), and multidrug resistance–associated protein (MRP) 2 (IC50 6.2 µM), which transport bilirubin and its conjugates. In rat and human hepatocytes, uptake and biliary excretion of [3H]bilirubin and/or its glucuronides decreased on coincubation with faldaprevir. In monkeys, faldaprevir (≥20 mg/kg per day) caused reversible unconjugated hyperbilirubinemia, without hemolysis or hepatotoxicity. In clinical studies, faldaprevir-mediated hyperbilirubinemia was predominantly unconjugated, and levels of unconjugated bilirubin correlated with the UGT1A1*28 genotype. The reversible and dose-dependent nature of the clinical hyperbilirubinemia was consistent with competitive inhibition of bilirubin clearance by faldaprevir, and was not associated with liver toxicity or other adverse events. Overall, the reversible, unconjugated hyperbilirubinemia associated with faldaprevir may predominantly result from inhibition of bilirubin conjugation by UGT1A1, with inhibition of hepatic uptake of bilirubin also potentially playing a role. Since OATP1B1/1B3 are known to be involved in hepatic uptake of circulating bilirubin glucuronides, inhibition of OATP1B1/1B3 and MRP2 may underlie isolated increases in conjugated bilirubin. As such, faldaprevir-mediated hyperbilirubinemia is not associated with any liver injury or toxicity, and is considered to result from decreased bilirubin elimination due to a drug-bilirubin interaction.

The Role of Transporters in Toxicity and Disease

The significance of transporters in the disposition, metabolism, and elimination of drugs is well recognized. One gap in our knowledge is a comprehensive understanding of how drug transporters change functionality (their amount and activity) in response to disease and how disease and its inevitable pathology change transporter expression. In this issue of Drug Metabolism and Disposition a series of review and primary research articles are presented to highlight the importance of transporters in toxicity and disease. Because of the central role of the liver in drug metabolism, many of the articles in this theme issue focus on transporters in the liver and how pathology or alterations in physiology affects transporter expression. The contributing authors have also considered the role of transporters in drug interactions as well as drug-induced liver injury. Noninvasive approaches to assessing transporter function in vivo are also described. Several articles highlight important issues in oncology where toxicity must be balanced against efficacy. In total, this theme issue will provide a stepping-stone to future studies that will establish a more comprehensive understanding of transporters in disease.