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 [(3)H]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.

Variability in P-glycoprotein inhibitory potency (IC50) using various in vitro experimental systems: implications for universal digoxin drug-drug interaction risk assessment decision criteria.

A P-glycoprotein (P-gp) IC50 working group was established with 23 participating pharmaceutical and contract research laboratories and one academic institution to assess interlaboratory variability in P-gp IC50 determinations. Each laboratory followed its in-house protocol to determine in vitro IC50 values for 16 inhibitors using four different test systems: human colon adenocarcinoma cells (Caco-2; eleven laboratories), Madin-Darby canine kidney cells transfected with MDR1 cDNA (MDCKII-MDR1; six laboratories), and Lilly Laboratories Cells--Porcine Kidney Nr. 1 cells transfected with MDR1 cDNA (LLC-PK1-MDR1; four laboratories), and membrane vesicles containing human P-glycoprotein (P-gp; five laboratories). For cell models, various equations to calculate remaining transport activity (e.g., efflux ratio, unidirectional flux, net-secretory-flux) were also evaluated. The difference in IC50 values for each of the inhibitors across all test systems and equations ranged from a minimum of 20- and 24-fold between lowest and highest IC50 values for sertraline and isradipine, to a maximum of 407- and 796-fold for telmisartan and verapamil, respectively. For telmisartan and verapamil, variability was greatly influenced by data from one laboratory in each case. Excluding these two data sets brings the range in IC50 values for telmisartan and verapamil down to 69- and 159-fold. The efflux ratio-based equation generally resulted in severalfold lower IC50 values compared with unidirectional or net-secretory-flux equations. Statistical analysis indicated that variability in IC50 values was mainly due to interlaboratory variability, rather than an implicit systematic difference between test systems. Potential reasons for variability are discussed and the simplest, most robust experimental design for P-gp IC50 determination proposed. The impact of these findings on drug-drug interaction risk assessment is discussed in the companion article (Ellens et al., 2013) and recommendations are provided.

Case Study 4: Application of Basic Enzyme Kinetics to Metabolism Studies-Real-Life Examples.

An appreciation of enzyme kinetic principles can be applied in a number of drug metabolism applications. The concept for this chapter arose from a simple discussion on selecting appropriate time points to most efficiently assess metabolite profiles in a human Phase 1a clinical study (Subheading 4). By considering enzyme kinetics, a logical approach to the issue was derived. The dialog was an important learning opportunity for the participants in the discussion, and we have endeavored to capture this experience with other questions related to determination of Km and Vmax parameters, a consideration of the value of hepatocytes vs. liver microsomes, and enzyme inhibition parameters.

Case study 3. Application of basic enzyme kinetics to metabolism studies: real-life examples.

An appreciation of the principles of enzyme kinetics can be applied in a number of drug metabolism applications. The concept for this chapter arose from a simple discussion on selecting appropriate time points to most efficiently assess metabolite profiles in a human Phase 1a clinical study (Subheading 4). By considering enzyme kinetics, a logical approach to the issue was derived. The dialog was an important learning opportunity for the participants in the discussion, and we have endeavored to capture this experience with other questions related to determination of K m and V max parameters, a consideration of the value of hepatocytes versus liver microsomes and enzyme inhibition parameters.

Differential selectivity of efflux transporter inhibitors in Caco-2 and MDCK-MDR1 monolayers: a strategy to assess the interaction of a new chemical entity with P-gp, BCRP, and MRP2.

Determining the interaction of a molecule with membrane transporters is challenging because of overlapping substrate and inhibitor specificities and coexpression of multiple transporters. Caco-2 and MDCK-MDR1 cells were used to evaluate the selectivity of zosuquidar (LY335979), fumitremorgin C (FTC), and MK571 as inhibitors of P-glycoprotein (P-gp), breast cancer resistance protein (BCRP), and multidrug resistance-associated protein 2 (MRP2), respectively. Although these compounds are commonly used as transporter inhibitors, the concentrations at which they selectively inhibit P-gp, BCRP, and MRP2 have not been definitively assessed. In Caco-2 cells, which express P-gp, BCRP, and MRP2, FTC (1 µM) selectively inhibited the efflux of BCRP substrates estrone-3-sulfate and genistein; however, at 10 µM, FTC partially inhibited the efflux of P-gp substrates paclitaxel and digoxin. MK571 (50 µM), commonly used to inhibit MRP2, inhibited the efflux of P-gp and BCRP probe substrates in Caco-2 cells. In MDCK-MDR1 cells, which express human P-gp but not BCRP or MRP2, MK571 (50 µM) and FTC (10 µM) did not inhibit paclitaxel and digoxin efflux. Using Caco-2 cell monolayers, selected probe substrates, and optimized concentrations of LY335979 (3 µM) and FTC (1 µM), we propose a strategy to evaluate the interaction of a molecule with P-gp, BCRP, and MRP2.

Functional assessment of multiple P-glycoprotein (P-gp) probe substrates: influence of cell line and modulator concentration on P-gp activity.

Compounds known to modulate P-glycoprotein (P-gp) activity were evaluated in cell monolayers expressing P-gp for their effects on the secretory transport of P-gp substrates paclitaxel, vinblastine, and digoxin. Paclitaxel has been proposed to selectively interact with a binding site on P-gp that is distinct from the vinblastine and digoxin-binding site. Using Madin-Darby canine kidney (MDCK)-multidrug resistance-1 (MDR1), MDCK-wild-type (WT), and Caco-2 cell monolayers, the basal-to-apical (BL-AP) apparent permeability (Papp) of [3H]paclitaxel, [3H]vinblastine, and [3H]digoxin in the presence of various concentrations of a series of structurally diverse P-gp substrates and modulators of P-gp function were determined. MDCK-WT cell monolayers demonstrated active secretory transport of all P-gp substrate probes, although the sensitivity to inhibition by verapamil was lower than that demonstrated in MDCK-MDR1 cell monolayers. When evaluated as competitive inhibitors, several known P-gp substrates had no effect or only a slight modulatory effect on the BL-AP Papp of all probe substrates in MDCK-MDR1 cells. The secretory transport of P-gp substrates in MDCK-WT cells was more sensitive to inhibition by known P-gp modulators compared with MDCK-MDR1 cells. Low concentrations of ketoconazole (1-3 microM) activated the BL-AP Papp of [3H]vinblastine and [3H]digoxin in MDCK-MDR1 cells but not in MDCK-WT or Caco-2 cells. Determination of secretory transport in P-gp expressing cell monolayers, such as MDCK-MDR1 and Caco-2, may be complicated by substrate cooperativity and allosteric binding, which may result in the activation of P-gp. In addition, expression of other efflux transporters in these cell lines introduces additional complexity in distinguishing which transporter is responsible for substrate recognition and transport.