Christoph Funk

The endothelin antagonist bosentan inhibits the canalicular bile salt export pump: A potential mechanism for hepatic adverse reactions

During clinical trials bosentan, the first orally active endothelin receptor antagonist, caused asymptomatic transaminase elevations in some patients. In this study we investigated whether inhibition of the hepatocanalicular bile salt export pump (rodents, Bsep; humans, BSEP ABCB11) could account for bosentan‐induced liver injury.

Cholestatic Potential of Troglitazone as a Possible Factor Contributing to Troglitazone-Induced Hepatotoxicity: In Vivo and in Vitro Interaction at the Canalicular Bile Salt Export Pump (Bsep) in the Rat

Troglitazone is a thiazolidinedione insulin sensitizer drug for the treatment of type 2 non-insulin-dependent diabetes mellitus (NIDDM). Based on an increasing number of reports on troglitazone-associated liver toxicity, the cholestatic potential of troglitazone has been investigated. Rapid and dose-dependent increases in the plasma bile acid concentrations were observed in rats after a single intravenous administration of troglitazone. A radiolabeled taurocholic acid tracer accumulated in liver tissue, indicating an interference with the hepatobiliary export of bile acids. In isolated canalicular rat liver plasma membrane preparations, troglitazone competitively inhibited the ATP-dependent taurocholate transport (apparent K(i) value, 1.3 microM), mediated by the canalicular bile salt export pump (Bsep). Troglitazone sulfate, the main troglitazone metabolite eliminated into bile, also showed competitive Bsep inhibition with an apparent K(i) value of 0.23 microM. A comparable inhibition was observed for both compounds in canalicular plasma membrane vesicles prepared from Mrp2-deficient (TR(-)) rats, suggesting a direct (cis-) inhibition of Bsep by troglitazone and troglitazone sulfate. A high accumulation potential was observed for troglitazone sulfate in rat liver tissue, indicating that the hepatobiliary export of this conjugated metabolite might represent a rate-limiting step in the overall elimination process of troglitazone. This accumulation in combination with the high Bsep inhibition potential suggested that mainly troglitazone sulfate was responsible for the interaction with the hepatobiliary export of bile acids at the level of the canalicular Bsep in rats. Such an interaction might lead to a troglitazone-induced intrahepatic cholestasis in humans as well, contributing to the formation of a troglitazone-induced liver toxicity.

Substrate-Dependent Drug-Drug Interactions between Gemfibrozil, Fluvastatin and Other Organic Anion-Transporting Peptide (OATP) Substrates on OATP1B1, OATP2B1, and OATP1B3

Hepatic uptake carriers of the organic anion-transporting peptide (OATP) family of solute carriers are more and more recognized as being involved in hepatic elimination of many drugs and potentially associated drug-drug interactions. The gemfibrozil-statin interaction was studied at the level of active hepatic uptake as a model for such drug-drug interactions. Active, temperature-dependent uptake of fluvastatin into primary human hepatocytes was shown. Multiple transporters are involved in this uptake as Chinese hamster ovary or HEK293 cells expressing either OATP1B1 (Km = 1.4–3.5 μM), OATP2B1 (Km = 0.7–0.8 μM), or OATP1B3 showed significant fluvastatin uptake relative to control cells. For OATP1B1 the inhibition by gemfibrozil was substrate-dependent as the transport of fluvastatin (IC50 of 63 μM), pravastatin, simvastatin, and taurocholate was inhibited by gemfibrozil, whereas the transport of estrone-3-sulfate and troglitazone sulfate (both used at 3 μM) was not affected. The OATP1B1- but not OATP2B1-mediated transport of estrone-3-sulfate displayed biphasic saturation kinetics, with two distinct affinity components for estrone-3-sulfate (0.23 and 45 μM). Only the high-affinity component was inhibited by gemfibrozil. Recombinant OATP1B1-, OATP2B1-, and OATP1B3-mediated fluvastatin transport was inhibited to 97, 70, and 62% by gemfibrozil (200 μM), respectively, whereas only a small inhibitory effect by gemfibrozil (200 μM) on fluvastatin uptake into primary human hepatocytes was observed (27% inhibition). The results indicate that the in vitro engineered systems can not always predict the behavior in more complex systems such as freshly isolated primary hepatocytes. Therefore, selection of substrate, substrate concentration, and in vitro transport system are critical for the conduct of in vitro interaction studies involving individual liver OATP carriers.

Design, Data Analysis, and Simulation of in Vitro Drug Transport Kinetic Experiments Using a Mechanistic in Vitro Model

The use of in vitro data for quantitative predictions of transporter-mediated elimination in vivo requires an accurate estimation of the transporter Michaelis-Menten parameters, Vmax and Km, as a first step. Therefore, the experimental conditions of in vitro studies used to assess hepatic uptake transport were optimized regarding active transport processes, nonspecific binding, and passive diffusion (Pdif). A mechanistic model was developed to analyze and accurately describe these active and passive processes. This two-compartmental model was parameterized to account for nonspecific binding, bidirectional passive diffusion, and active uptake processes based on the physiology of the cells. The model was used to estimate kinetic parameters of in vitro transport data from organic anion-transporting peptide model substrates (e.g., cholecystokinin octapeptide deltorphin II, fexofenadine, and pitavastatin). Data analysis by this mechanistic model significantly improved the accuracy and precision in all derived parameters [mean coefficient of variations (CVs) for Vmax and Km were 19 and 23%, respectively] compared with the conventional kinetic method of transport data analysis (mean CVs were 58 and 115%, respectively, using this method). Furthermore, permeability was found to be highly temperature-dependent in Chinese hamster ovary (CHO) control cells and artificial membranes (parallel artificial membrane permeability assay). Whereas for some compounds (taurocholate, estrone-3-sulfate, and propranolol) the effect was moderate (1.5–6-fold higher permeability at 37°C compared with that at 4°C), for fexofenadine a 16-fold higher passive permeability was seen at 37°C. Therefore, Pdif was better predicted if it was evaluated under the same experimental conditions as Vmax and Km, i.e., in a single incubation of CHO overexpressed cells or rat hepatocytes at 37°C, instead of a parallel control evaluation at 4°C.

The role of hepatic transporters in drug elimination

Background: Hepatic drug transporters of the solute carrier (OATPs, OAT2, OCT1, NTCP) and ABC transporter superfamilies (MDR1, MRPs, BCRP, BSEP) can significantly modulate drug ADME routes. Objective: The currently available literature was reviewed with focus on hepatic drug transporters, related drug–drug interactions and available tools for transporter assessment and extrapolation to in vivo. Conclusions: Hepatic drug transporters have gained additional importance in drug clearance by optimization for basic DMPK properties early on in drug research. However, the lack of selective substrates and inhibitors, proper assessment of the kinetic properties due to interfering passive permeability and multiple binding sites, as well as endogenous transporters present in many cellular assays, are some of the hurdles in studying active drug transport processes. Therefore, data from these in vitro assays have to be carefully evaluated to allow sound extrapolation to the in vivo situation.

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.

Metabolic Profiles of Minimally Absorbed Orlistat in Obese/Overweight Volunteers

To determine the metabolic profile of minimally absorbed orlistat in obese/overweight patients, an open‐label, single‐dose study was performed in eight obese/overweight volunteers between 23 and 68 years of age. Each subject received a single oral dose of 360 mg orlistat containing approximately 400 μCi of 14C‐labeled orlistat. Serial blood samples were collected at specified times over 10 hours after administration of orlistat for determination of total radioactivity, unchanged orlistat, and major metabolites in the plasma. Urine samples were collected over 24 hours and analyzed to evaluate the urinary recovery of total radioactivity and the profile of orlistat metabolites in the urine. In addition, all fecal samples were collected and analyzed for total radioactivity. Urinary and fecal recovery of the administered dose of total radioactivity were 1.13 ± 0.50% (24‐hour data only) and 96.4 ± 18.1% (n = 7), respectively. Maximum observed concentration (Cmax) and time to Cmax (tmax) values of plasma total radioactivity were 150 ± 51 ng·eq/mL and 6.8 ± 1.5 hrs, respectively. All these parameters obtained in obese/overweight subjects were similar to those reported previously in healthy subjects. On the basis of the area under the concentration‐time curve from 0 to 10 hours (AUC0–10), two major metabolites comprise a total of ∼42% of the total radioactivity in plasma. The primary metabolite (M1) has a short half‐life (∼2 hours), whereas the secondary metabolite (M3) disappeared at a slower rate. No strikingly apparent difference in the urinary metabolic profile was observed between two gender groups. It is concluded that the disposition of orlistat appears to be similar between normal and obese/overweight subjects. Of the minimal fraction of the dose that was absorbed systemically, the presence of two major metabolites accounts for ∼42%.

Mechanistic modeling of hepatic transport from cells to whole body: application to napsagatran and fexofenadine.

A mechanistic model was applied to quantitatively derive the kinetic parameters from in vitro hepatic uptake transport data. These parameters were used as input to simulate in vivo elimination using a fully mechanistic physiologically based pharmacokinetic (PBPK) model. Fexofenadine and napsagatran, both BDDCS class 3 drugs, were chosen as model compounds. In rat, both compounds are hardly metabolized and are eliminated unchanged mostly through biliary excretion. Uptake was estimated in this study based on plated rat hepatocytes, and a mechanistic model was used to derive the active and passive transport parameters, namely Michaelis-Menten uptake parameters (V(maxI) and K(mI,u)) together with passive diffusion (P(dif)) and nonspecific binding. Maximum transport velocity and passive diffusion were scaled to in vivo parameters (J(maxI) and PS(TC)) using hepatocellularity. Biliary excretion, through passive and active transport, was assessed from in vivo studies. These transport parameters were then used as input in a whole body physiologically based model in which the liver compartment was parametrized for the different passive and active transport processes. Each of the processes was linked to the free concentration in the relevant compartment. For napsagatran hepatic uptake, no passive diffusion and no binding were detected in vitro besides the active transport (K(mI,u) = 88.4 +/- 8.1 microM, V(maxI) = 384 +/- 19 pmol/mg/min). Fexofenadine was rapidly taken up into rat hepatocytes (K(mI,u) = 271 +/- 35 microM, V(maxI) = 3162 +/- 274 pmol/mg/min), and some contribution of passive diffusion to the uptake (P(dif) = 2.08 +/- 0.67 microL/mg/min) was observed. For fexofenadine, the biliary export rate was found to be slower than the uptake, leading to drug accumulation in liver. No accumulation was observed for napsagatran where excretion was faster than hepatic uptake. Observed plasma, liver and bile concentration time profiles were compared to PBPK simulations based on scaled in vitro transport kinetic parameters. An uncertainty analysis indicated that for both compounds the scaled in vitro uptake clearance had to be adjusted with an additional empirical scaling factor of 10 to match the plasma and liver concentrations and biliary excretion profiles. Applying this model, plasma clearance (CL(P)) and half-life (t(1/2)), maximum liver concentration (C(maxL)) and fraction excreted in bile (f(bile)) were predicted within 2-fold. In vitro uptake data had most impact on the simulated plasma and biliary excretion profiles, while accurate simulations of liver concentrations required also quantitative estimates of biliary excretion transport. This study indicated that the mechanistic model allowed for accurate evaluation of in vitro experiments; and the scaled kinetic parameters of hepatic uptake transport enabled the prediction of in vivo PK profiles and plasma clearances, using PBPK modeling.

Pharmacology of Basimglurant (RO4917523, RG7090), a Unique Metabotropic Glutamate Receptor 5 Negative Allosteric Modulator in Clinical Development for Depression

Major depressive disorder (MDD) is a serious public health burden and a leading cause of disability. Its pharmacotherapy is currently limited to modulators of monoamine neurotransmitters and second-generation antipsychotics. Recently, glutamatergic approaches for the treatment of MDD have increasingly received attention, and preclinical research suggests that metabotropic glutamate receptor 5 (mGlu5) inhibitors have antidepressant-like properties. Basimglurant (2-chloro-4-[1-(4-fluoro-phenyl)-2,5-dimethyl-1H-imidazol-4-ylethynyl]-pyridine) is a novel mGlu5 negative allosteric modulator currently in phase 2 clinical development for MDD and fragile X syndrome. Here, the comprehensive preclinical pharmacological profile of basimglurant is presented with a focus on its therapeutic potential for MDD and drug-like properties. Basimglurant is a potent, selective, and safe mGlu5 inhibitor with good oral bioavailability and long half-life supportive of once-daily administration, good brain penetration, and high in vivo potency. It has antidepressant properties that are corroborated by its functional magnetic imaging profile as well as anxiolytic-like and antinociceptive features. In electroencephalography recordings, basimglurant shows wake-promoting effects followed by increased delta power during subsequent non–rapid eye movement sleep. In microdialysis studies, basimglurant had no effect on monoamine transmitter levels in the frontal cortex or nucleus accumbens except for a moderate increase of accumbal dopamine, which is in line with its lack of pharmacological activity on monoamine reuptake transporters. These data taken together, basimglurant has favorable drug-like properties, a differentiated molecular mechanism of action, and antidepressant-like features that suggest the possibility of also addressing important comorbidities of MDD including anxiety and pain as well as daytime sleepiness and apathy or lethargy.

Metabotropic Glutamate Receptor 5 Negative Allosteric Modulators: Discovery of 2-Chloro-4-[1-(4-fluorophenyl)-2,5-dimethyl-1H-imidazol-4-ylethynyl]pyridine (Basimglurant, RO4917523), a Promising Novel Medicine for Psychiatric Diseases

Negative allosteric modulators (NAMs) of metabotropic glutamate receptor 5 (mGlu5) have potential for the treatment of psychiatric diseases including depression, fragile X syndrome (FXS), anxiety, obsessive-compulsive disorders, and levodopa induced dyskinesia in Parkinsons disease. Herein we report the optimization of a weakly active screening hit 1 to the potent and selective compounds chloro-4-[1-(4-fluorophenyl)-2,5-dimethyl-1H-imidazol-4-ylethynyl]pyridine (basimglurant, 2) and 2-chloro-4-((2,5-dimethyl-1-(4-(trifluoromethoxy)phenyl)-1H-imidazol-4-yl)ethynyl)pyridine (CTEP, 3). Compound 2 is active in a broad range of anxiety tests reaching the same efficacy but at a 10- to 100-fold lower dose compared to diazepam and is characterized by favorable DMPK properties in rat and monkey as well as an excellent preclinical safety profile and is currently in phase II clinical studies for the treatment of depression and fragile X syndrome. Analogue 3 is the first reported mGlu5 NAM with a long half-life in rodents and is therefore an ideal tool compound for chronic studies in mice and rats.