Hepatic Transporter Function in Liver Disease: Impact on Hepatobiliary Disposition and Pharmacokinetics

The objective of this doctoral dissertation research was to understand how hepatic transporter function in liver disease affects hepatobiliary disposition and pharmacokinetics of endogenous and exogenous compounds. In particular, this dissertation focused on two forms of liver disease: non-alcoholic steatohepatitis (NASH) and autosomal dominant polycystic kidney disease (ADPKD). A multi-experimental, translational approach including transporter over-expressing membrane vesicles, sandwich-cultured human hepatocytes, isolated perfused rat livers, metabolomic profiling, imaging, and an in vivo study in humans was employed. Based on reported changes in in vitro expression of organic anion transporting polypeptide (OATP), multidrug resistance-associated protein (MRP) 2 and MRP3 in liver tissue from patients with NASH, we tested the hypothesis that the disposition of 99mTechnetium-mebrofenin (MEB), and OATP, MRP2, and MRP3 probe, was altered in this patient population. Systemic and hepatic concentrations of MEB were increased in patients with NASH compared to age- and sex-matched healthy volunteers, consistent with impaired uptake and biliary excretion, and enhanced basolateral efflux of MEB. Tolvaptan and two metabolites, DM-4103 and DM-4107, were shown to be inhibitors of the major human hepatic bile acid transporters Na+-taurocholate cotransporting polypeptide (NTCP), bile salt export pump (BSEP), and MRP2, MRP3, and MRP4, which may contribute to tolvaptan-associated liver injury in patients with ADPKD. Although impaired bile acid transport by tolvaptan and/or its metabolites may negatively impact bile acid homeostasis, the fact that liver injury has been observed only in patients with ADPKD suggests that other mechanism(s) and/or patient susceptibility factors may contribute to drug-induced liver injury (DILI) in this patient population. Metabolomic profiling of bile acids in polycystic kidney (PCK) rats, a rodent model of human ADPKD, revealed significantly increased concentrations of total bile acids and bile acids associated with hepatotoxicity in the serum and liver when compared to wild-type rats. Total serum bile acids positively correlated with measures of liver impairment (i.e. liver weight, total liver bile acids, total liver bile acids associated with hepatotoxic, and cystic volume) suggesting that serum bile acids may be useful biomarkers for liver impairment in ADPKD. Increased bile acids concentrations in the liver due to ADPKD, independent of tolvaptan therapy, suggests that it is plausible that this patient population may be susceptible bile acid-mediated hepatotoxicity. Further mechanistic studies revealed, for the first time, decreased biliary excretion of 5(6)-carboxy-2’,7’-dichlorofluorescein (CDF), a fluorescent Oatp, Mrp2, and Mrp3 probe, in PCK rats using the isolated perfused rat liver model. In total, these data suggest that drug- and disease-specific factors may play a role in the potential for bile acid-mediated hepatotoxicity associated with tolvaptan use in patients with ADPKD. This research has resulted in novel contributions that provide important insight into the impact of liver disease on hepatic transporter function. This information will significantly enhance our ability to understand and predict altered hepatic transporter function, which will improve the safety and effectiveness of drugs.Doctor of Philosoph

Inhibition of Human Hepatic Bile Acid Transporters by Tolvaptan and Metabolites: Contributing Factors to Drug-Induced Liver Injury?

Tolvaptan is a vasopressin

Hepatocellular Disposition and Transporter Interactions with Tolvaptan and Metabolites in Sandwich-Cultured Human Hepatocytes

Tolvaptan is a selective V2-receptor antagonist primarily metabolized by CYP 3A. The present study investigated the hepatocellular disposition of tolvaptan and the generated tolvaptan metabolites, DM-4103 and DM-4107, as well as the potential for drug-drug interactions (DDIs) with metabolic and transport proteins in sandwich-cultured human hepatocytes (SCHH). Tolvaptan was incubated with SCHH and quantified by liquid chromatography–tandem mass spectrometry. Pioglitazone, verapamil, MK-571, and elacridar were used as inhibitors to investigate mechanisms of transport and metabolism of tolvaptan and metabolites. Taurocholate (TCA), pravastatin, digoxin, and metformin were used as transporter probes to investigate which transport proteins were inhibited by tolvaptan and metabolites. Cellular accumulation of tolvaptan (0.15–50 μM), DM-4103, and DM-4107 in SCHH was concentration-dependent. Tolvaptan accumulation (15 μM) in SCHH was not altered markedly by 50 μM pioglitazone, verapamil, MK-571, or 10 μM elacridar. Coincubation of tolvaptan with pioglitazone, verapamil, MK-571, and elacridar reduced DM-4107 accumulation by 45.6, 79.8, 94.5, and 23.0%, respectively, relative to control. Coincubation with increasing tolvaptan concentrations (0.15–50 μM) decreased TCA (2.5 μM) cell+bile accumulation and the TCA biliary excretion index (BEI; from 76% to 51%), consistent with inhibition of the bile salt export pump (BSEP). Tolvaptan (15 μM) had no effect on the cellular accumulation of 2.5 μM pravastatin or metformin. Digoxin cellular accumulation increased, and the BEI of digoxin decreased from 23.9 to 8.1% in the presence of 15 μM tolvaptan, consistent with inhibition of P-glycoprotein. In summary, SCHH studies revealed potential metabolic- and transporter-mediated DDIs involving tolvaptan and metabolites

Mu, delta, and kappa opioid receptor mRNA expression in the rat CNS: An in situ hybridization study

The Μ, Δ, and Κ opioid receptors are the three main types of opioid receptors round in the central nervous system (CNS) and periphery. These receptors and the peptides with which they interact are important in a number of physiological functions, including analgesia, respiration, and hormonal regulation. This study examines the expression of Μ, Δ, and Κ receptor mRNAs in the rat brain and spinal cord using in situ hybridization techniques. Tissue sections were hybridized with 35 S-labeled cRNA probes to the rat Μ (744–1, 064 b), Δ (304–1,287 b), and Κ (1,351–2,124 b) receptors. Each mRNA demonstrates a distinct anatomical distribution that corresponds well to known receptor binding distributions. Cells expressing Μ receptor mRNA are localized in such regions as the olfactory bulb, caudate-putamen, nucleus accumbens, lateral and medial septum, diagonal band of Broca, bed nucleus of the stria terminalis, most thalamic nuclei, hippocampus, amygdala, medial preoptic area, superior and inferior colliculi, central gray, dorsal and median raphe, raphe magnus, locus coeruleus, parabrachial nucleus, pontine and medullary reticular nuclei, nucleus ambiguus, nucleus of the solitary tract, nucleus gracilis and cuneatus, dorsal motor nucleus of vagus, spinal cord, and dorsal root ganglia. Cellular localization of Δ receptor mRNA varied from Μ or Κ, with expression in such regions as the olfactory bulb, allo- and neocortex, caudate-putamen, nucleus accumbens, olfactory tubercle, ventromedial hypothalamus, hippocampus, amygdala, red nucleus, pontine nuclei, reticulotegmental nucleus, motor and spinal trigeminal, linear nucleus of the medulla, lateral reticular nucleus, spinal cord, and dorsal root ganglia. Cells expressing, Κ receptor mRNA demonstrate a third pattern of expression, with cells localized in regions such as the claustrum, endopiriform nucleus, nucleus accumbens, olfactory tubercle, medial preoptic area, bed nucleus of the stria terminalis, amygdala, most hypothalamic nuclei, median eminence, infundibulum, substantia nigra, ventral tegmental area, raphe nuclei, paratrigeminal and spinal trigeminal, nucleus of the solitary tract, spinal cord, and dorsal root ganglia. These findings are discussed in relation to the physiologica functions associated with the opioid receptors.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/50064/1/903500307_ftp.pd

Studies on the nature and mechanism of the diuretic activity of the opioid analgesic ethylketocyclazocine

ABSTRACT Slizgi