Importance of Hepatic Transporters, Including Basolateral Efflux Proteins, in Drug Disposition: Impact of Phospholipidosis and Non-Alcoholic Steatohepatitis

The objective of this dissertation project was to develop preclinical and clinical tools to assess the impact of liver pathology on transporter-mediated systemic and hepatic exposure to medications. A translational approach utilizing established pre-clinical hepatic transport systems, mathematical modeling, and a pivotal in vivo human study was employed. A novel application of rat sandwich-cultured hepatocytes (SCH) was developed to evaluate the impact of drug-induced phospholipidosis on the vectorial transport of probe substrates for hepatic basolateral and canalicular transport proteins. Results indicated that rat SCH treated with prototypical hepatic phospholipidosis inducers are a sensitive and selective model for drug-induced phospholipidosis; both organic anion transporting polypeptide-mediated uptake and bile salt export pump-mediated biliary excretion were reduced after the onset of phospholipidosis. Enalapril currently is being investigated for its anti-fibrotic effects in the treatment of patients with non-alcoholic steatohepatitis (NASH). Although the basolateral uptake transporters responsible for enalapril entry into the hepatocyte are well characterized, less is known about translocation of enalaprilat (the active metabolite) across the basolateral membrane into the systemic circulation. Studies were conducted using membrane vesicles prepared from transporter-expressing cells and a novel human SCH efflux protocol to assess the involvement of two hepatic basolateral efflux transporters known to be up-regulated in inflammatory liver disease [i.e., multidrug resistance-associated protein (MRP)3 and MRP4]; enalaprilat is transported across the basolateral membrane, in part, by MRP4. A novel clinical protocol was developed using morphine glucuronides as a probe for MRP3 function to determine the impact of altered hepatic transporter expression on drug disposition in patients with NASH. A physiologically-based mathematical model was constructed to inform study design. Systemic concentrations of morphine glucuronides were increased significantly in patients with NASH compared to healthy subjects, as predicted by the model. This work provides a mechanistic understanding of the impact that hepatic transporter function has on the disposition of drugs and endogenous compounds under normal conditions and in response to altered liver function due to drug-induced phospholipidosis or liver disease, specifically non-alcoholic steatohepatitis. This knowledge is fundamental to safe and efficacious dosing of medications that depend on these transporters for disposition and/or elimination.Doctor of Philosoph

Identification of Hepatic Phospholipidosis Inducers in Sandwich-Cultured Rat Hepatocytes, a Physiologically Relevant Model, Reveals Altered Basolateral Uptake and Biliary Excretion of Anionic Probe Substrates

Drug-induced phospholipidosis (PLD) is characterized by phospholipid accumulation within the lysosomes of affected tissues, resulting in lysosomal enlargement and laminar body inclusions. Numerous adverse effects and toxicities have been linked to PLD-inducing drugs, but it remains unknown whether drug-induced PLD represents a distinct toxicity or cellular adaptation. In silico and immortalized cellular models have been used to evaluate the PLD potential of new drugs, but these systems have some limitations. The aims of this study were to determine whether primary sandwich-cultured hepatocytes (SCH) can serve as a sensitive and selective model to evaluate hepatic drug-induced PLD, and to evaluate the impact of PLD on the uptake and biliary excretion of probe substrates, taurocholate (TC) and rosuvastatin (RSV). Rat SCH were cultured for 48 h with prototypic hepatic PLD-inducing drugs, amiodarone (AMD), chloroquine (CHQ), desipramine (DES), and azithromycin (AZI), as well as the renal PLD inducer gentamicin (GTM). LysoTracker Red localization and transmission electron microscopy indicated enlarged lysosomal compartments and laminar body inclusions in SCH treated with AMD, CHQ, DES, and AZI, but not GTM, relative to control. PLD resulted in a 51–92% decrease in the in vitro biliary clearance of both TC and RSV; the biliary excretion index significantly decreased for TC from 88 to 35–73%. These data suggested that PLD significantly reduced both organic anion transporting polypeptide-mediated uptake, and bile salt export pump-mediated biliary transport processes. The current study demonstrates that the rat SCH system is a promising model to study hepatic PLD in vitro. Altered hepatic transport of anionic substrates secondary to drug-induced PLD is a novel finding

Role of Multidrug Resistance-Associated Protein 4 in the Basolateral Efflux of Hepatically Derived Enalaprilat

Hepatic uptake and efflux transporters govern the systemic and hepatic exposure of many drugs and metabolites. Enalapril is a pharmacologically inactive prodrug of enalaprilat. Following oral administration, enalapril is converted to enalaprilat in hepatocytes and undergoes translocation into the systemic circulation to exert its pharmacologic effect by inhibiting angiotensin-converting enzyme. Although the transport proteins governing hepatic uptake of enalapril and the biliary excretion of enalapril and enalaprilat are well established, it remains unknown how hepatically derived enalaprilat translocates across the basolateral membrane into the systemic circulation. In this study, the role of ATP-binding cassette transporters in the hepatic basolateral efflux of enalaprilat was investigated using membrane vesicles. ATP-dependent uptake of enalaprilat into vesicles expressing multidrug resistance–associated protein (MRP) 4 was significantly greater (∼3.8-fold) than in control vesicles. In contrast, enalaprilat was not transported to a significant extent by MRP3, and enalapril was not transported by either MRP3 or MRP4. The functional importance of MRP4 in the basolateral excretion of derived enalaprilat was evaluated using a novel basolateral efflux protocol developed in human sandwich-cultured hepatocytes. Under normal culture conditions, the mean intrinsic basolateral efflux clearance (CLint,basolateral) of enalaprilat was 0.026 ± 0.012 µl/min; enalaprilat CLint,basolateral was significantly reduced to 0.009 ± 0.009 µl/min by pretreatment with the pan-MRP inhibitor MK-571. Results suggest that hepatically derived enalaprilat is excreted across the hepatic basolateral membrane by MRP4. Changes in MRP4-mediated basolateral efflux may alter the systemic concentrations of this active metabolite, and potentially the efficacy of enalapril

Characterization of the Cytochrome P450 epoxyeicosanoid pathway in non-alcoholic steatohepatitis

Non-alcoholic steatohepatitis (NASH) is an emerging public health problem without effective therapies. Cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid into bioactive epoxyeicosatrienoic acids (EETs), which have potent anti-inflammatory and protective effects. However, the functional relevance of the CYP epoxyeicosanoid metabolism pathway in the pathogenesis of NASH remains poorly understood. Our studies demonstrate that both mice with methionine-choline deficient (MCD) diet-induced NASH and humans with biopsy-confirmed NASH exhibited significantly higher free EET concentrations compared to healthy controls. Targeted disruption of Ephx2 (the gene encoding for soluble epoxide hydrolase) in mice further increased EET levels and significantly attenuated MCD diet-induced hepatic steatosis, inflammation and injury, as well as high fat diet-induced adipose tissue inflammation, systemic glucose intolerance and hepatic steatosis. Collectively, these findings suggest that dysregulation of the CYP epoxyeicosanoid pathway is a key pathological consequence of NASH in vivo, and promoting the anti-inflammatory and protective effects of EETs warrants further investigation as a novel therapeutic strategy for NASH

Risk Factors for Development of Cholestatic Drug-Induced Liver Injury: Inhibition of Hepatic Basolateral Bile Acid Transporters Multidrug Resistance-Associated Proteins 3 and 4

Impaired hepatic bile acid export may contribute to development of cholestatic drug-induced liver injury (DILI). The multidrug resistance-associated proteins (MRP) 3 and 4 are postulated to be compensatory hepatic basolateral bile acid efflux transporters when biliary excretion by the bile salt export pump (BSEP) is impaired. BSEP inhibition is a risk factor for cholestatic DILI. This study aimed to characterize the relationship between MRP3, MRP4, and BSEP inhibition and cholestatic potential of drugs. The inhibitory effect of 88 drugs (100 μM) on MRP3- and MRP4-mediated substrate transport was measured in membrane vesicles. Drugs selected for investigation included 50 BSEP non-inhibitors (24 non-cholestatic; 26 cholestatic) and 38 BSEP inhibitors (16 non-cholestatic; 22 cholestatic). MRP4 inhibition was associated with an increased risk of cholestatic potential among BSEP non-inhibitors. In this group, for each 1% increase in MRP4 inhibition, the odds of the drug being cholestatic increased by 3.1%. Using an inhibition cutoff of 21%, which predicted a 50% chance of cholestasis, 62% of cholestatic drugs inhibited MRP4 (P < 0.05); in contrast, only 17% of non-cholestatic drugs were MRP4 inhibitors. Among BSEP inhibitors, MRP4 inhibition did not provide additional predictive value of cholestatic potential; almost all BSEP inhibitors were also MRP4 inhibitors. Inclusion of pharmacokinetic predictor variables (e.g., maximal unbound concentration in plasma) in addition to percent MRP4 inhibition in logistic regression models did not improve cholestasis prediction. Association of cholestasis with percent MRP3 inhibition was not statistically significant, regardless of BSEP-inhibition status. Inhibition of MRP4, in addition to BSEP, may be a risk factor for the development of cholestatic DILI

Altered Bile Acid Metabolome in Patients with Nonalcoholic Steatohepatitis

The prevalence of non-alcoholic fatty liver disease (NAFLD) and steatohepatitis (NASH) is increasing at an alarming rate. The role of bile acids in the development and progression of NAFLD to NASH and cirrhosis is poorly understood. This study aimed to quantify the bile acid metabolome in healthy subjects and patients with non-cirrhotic NASH under fasting conditions and after a standardized meal

Altered morphine glucuronide and bile acid disposition in patients with nonalcoholic steatohepatitis

The functional impact of altered drug transport protein expression on the systemic pharmacokinetics of morphine, hepatically-derived morphine glucuronide (morphine-3- and morphine-6-glucuronide), and fasting bile acids was evaluated in patients with biopsy-confirmed non-alcoholic steatohepatitis (NASH) compared to healthy subjects. The maximum concentration (Cmax) and area under the concentration-time curve (AUC0-last) of morphine glucuronide in serum were increased in NASH patients (343 vs. 225nM and 58.8 vs. 37.2μM*min, respectively; P≤0.005); morphine pharmacokinetics did not differ between groups. Linear regression analyses detected an association of NASH severity with increased morphine glucuronide Cmax and AUC0-last (P<0.001). Fasting serum glycocholate, taurocholate and total bile acid concentrations were associated with NASH severity (P<0.006). Increased hepatic basolateral efflux of morphine glucuronide and bile acids is consistent with altered hepatic transport protein expression in patients with NASH and may partially explain differences in efficacy and/or toxicity of some highly transported anionic drugs/metabolites in this patient population

Dipivefrine

Dipivefrine is a member of a class of pharmaceutical agents known as prodrugs. Dipivefrine is not active itself but is biotransformed in the body to epinephrine. It is used in the treatment of open-angle and secondary glaucoma

Isoflurophate

Isoflurophate is a long acting cholinesterase inhibitor and potent miotic. It works as an indirect acting parasympathomimetic agent to reduce intraocular pressure. Also known as diisopropyl phosphofluoridate (Dyflos), it is an irreversible organophosphate cholinesterase inhibitor. It and its analogues were studied extensively during the Second World War as substances that could be employed as war gases because of their volatility and rapid absorption from the lungs. Due to its toxicity it is only used in the treatment of patients with open angle glaucoma or other chronic glaucomas which are not controlled with other less toxic short acting agents

Bioequivalence Study of Nabumetone: Tablet Versus Suspension

The orally administered tablet and suspension of the analgesic drug nabumetone (Relafen), a novel naphthylalkanone, were tested for bioequivalence. Nabumetone is rapidly metabolized to an active metabolite, 6-methoxy-2-naphthylacetic acid (BRL 10720). The pharmacokinetics of the metabolite were studied in 24 healthy adult male volunteers. Each received a 1-g dose of the nabumetone formulations in a balanced, randomized, two-way crossover investigation. Serum metabolite concentrations were determined over a 120-hour interval by high-performance liquid chromatography. The values of the pharmacokinetic parameters computed for tablet and suspension are presented in that order: area under the curve = 1,269:1,338 mg · hour/ml; absorption half-life = 1.04:0.83 hour; elimination half-life = 27.16:25.15 hours; lag time = 0.19:0.07 hour; peak concentration = 27.56:31.91 μg/ml, and time to peak concentration = 4.99:4.17 hours. The mean concentration of BRL 10720 was found to be higher during the first eight hours for the suspension than for the tablet. Using criteria for statistical significance, the values for peak concentration, time to peak concentration, elimination half-life, and lag time were found significant (p \u3c0.05). These results may well be reflecting the increased absorption characteristics of the suspension due to the pharmaceutical characteristics of the preparation. The formulations were found to be bioequivalent when compared on the premise that no significant difference was detected when area under the curve and all other parameters were compared, based upon the 75 75 rule analysis