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

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

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

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

Tyrosine and aurora kinase inhibitors diminish transport function of multidrug resistance-associated protein (MRP) 4 and breast cancer resistance protein (BCRP)

Tyrosine and aurora kinases are important effectors in signal transduction pathways that are often involved in aberrant cancer cell growth. Tyrosine (TKI) and aurora (AKI) kinase inhibitors are anti-cancer agents specifically designed to target such signaling pathways through TKI/AKI binding to the ATP-binding pocket of kinases thereby leading to diminished kinase activity. Some TKIs have been identified as inhibitors of ATP-binding cassette (ABC) transporters such as P-glycoprotein and breast cancer resistance protein (BCRP), which are commonly upregulated in malignant cells. TKI/AKIs have been investigated as ABC transporter inhibitors in order to facilitate the accumulation of concomitantly administered chemo-therapeutics within cancer cells. However, ABC transporters are prominently expressed in the liver and other eliminating organs, and their inhibition has been linked to intracellular accumulation of drugs, altered disposition, and toxicity. The potential for TKIs/AKIs to inhibit other important hepatic efflux transporters, particularly multidrug resistance-associated proteins (MRPs), remains unknown. The aim of the current study was to compare the inhibitory potency of 20 selected TKI/AKIs against MRP4 and BCRP through the use of inverted membrane vesicle assays. Relative IC50 values were estimated by determining TKI/AKI inhibition of MRP4-mediated [3H]-dehydroepiandrosterone sulfate uptake and BCRP-mediated [3H]-estrone sulfate uptake. To provide insight to the clinical relevance of TKI/AKI inhibition of ABC efflux transporters, the ratio of the steady-state maximum total plasma concentration (Css) to the IC50 for each compound was calculated with Css/IC50 ratio >0.1 deemed potentially clinically relevant. Such analysis identified several potentially clinically relevant inhibitors of MRP4: alisertib, danusertib, erlotinib, lapatinib, neratinib, nilotinib, pazopanib, sorafenib, and tozasertib. The potentially clinically relevant inhibition of BCRP was much more extensive and included alisertib, barasertib, danusertib, enzastaurin, erlotinib, gefitinib, imatinib, neratinib, nilotinib, pazopanib, selumetinib, sorafenib, sunitinib, tozasertib, and vandetanib. These findings indicate the significant potential for TKI/AKIs to inhibit multiple ABC efflux transporters. The resulting inhibition data could provide insight regarding the clinical interpretation of pharmacokinetic/pharmacodynamic outcomes when TKI/AKIs are administered concomitantly with additional chemotherapeutic agents

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 (CL(int)(,basolateral)) of enalaprilat was 0.026 ± 0.012 µl/min; enalaprilat CL(int,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

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

Key Role for the 12-Hydroxy Group in the Negative Ion Fragmentation of Unconjugated C24 Bile Acids

Host-gut microbial interactions contribute to human health and disease states and an important manifestation resulting from this cometabolism is a vast diversity of bile acids (BAs). There is increasing interest in using BAs as biomarkers to assess the health status of individuals and, therefore, an increased need for their accurate separation and identification. In this study, the negative ion fragmentation behaviors of C24 BAs were investigated by UPLC-ESI-QTOF-MS. The step-by-step fragmentation analysis revealed a distinct fragmentation mechanism for the unconjugated BAs containing a 12-hydroxyl group. The unconjugated BAs lacking 12-hydroxylation fragmented via dehydration and dehydrogenation. In contrast, the 12-hydroxylated ones, such as deoxycholic acid (DCA) and cholic acid (CA), employed dissociation routes including dehydration, loss of carbon monoxide or carbon dioxide, and dehydrogenation. All fragmentations of the 12-hydroxylated unconjugated BAs, characterized by means of stable isotope labeled standards, were associated with the rotation of the carboxylate side chain and the subsequent rearrangements accompanied by proton transfer between 12-hydroxyl and 24-carboxyl groups. Compared to DCA, CA underwent further cleavages of the steroid skeleton. Accordingly, the effects of stereochemistry on the fragmentation pattern of CA were investigated using its stereoisomers. Based on the knowledge gained from the fragmentation analysis, a novel BA, 3β,7β,12α-trihydroxy-5β-cholanic acid, was identified in the postprandial urine samples of patients with nonalcoholic steatohepatitis. The analyses used in this study may contribute to a better understanding of the chemical diversity of BAs and the molecular basis of human liver diseases that involve BA synthesis, transport, and metabolism

Altered Bile Acid Metabolome in Patients with Nonalcoholic Steatohepatitis

BACKGROUND & AIMS: 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. METHODS: Liquid chromatography tandem mass spectroscopy was used to quantify 30 serum and 16 urinary bile acids from 15 healthy volunteers and 7 patients with biopsy-confirmed NASH. Bile acid concentrations were measured at two fasting and four post-prandial timepoints following a high-fat meal to induce gallbladder contraction and bile acid reabsorption from the intestine. RESULTS: Patients with NASH had significantly higher total serum bile acid concentrations than healthy subjects under fasting conditions (2.2- to 2.4-fold increase in NASH; NASH: 2595–3549 μM and healthy: 1171–1458 μM) and at all post-prandial time points (1.7- to 2.2-fold increase in NASH; NASH: 4444–5898 μM and healthy: 2634–2829 μM). These changes were driven by increased taurine- and glycine-conjugated primary and secondary bile acids. Patients with NASH exhibited greater variability in their fasting and post-prandial bile acid profile. CONCLUSIONS: Results indicate that patients with NASH have higher fasting and post-prandial exposure to bile acids, including the more hydrophobic and cytotoxic secondary species. Increased bile acid exposure may be involved in liver injury and the pathogenesis of NAFLD and NASH