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.
Background and Aims
The prevalence of nonalcoholic 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 time points 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.
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.
Background
New treatment options with improved safety and novel mechanisms of actions are needed for patients with peanut allergy.
Objectives
To evaluate the safety, tolerability, and immunogenicity ...of ASP0892, a peanut DNA vaccine, after intradermal (id) or intramuscular (im) administration in adult or adolescent patients with peanut allergy in two phase 1 studies.
Methods
ASP0892 or placebo was administered every 2 weeks for a total of 4 doses. The doses were 1 mg or 4 mg id or 4 mg im for adults, and 1 mg or 4 mg id for adolescents. Immunologic parameters were assessed longitudinally.
Results
Thirty‐one adults (mean age 24.3 years, 17 males) received ASP0892 (9, 8, 8 patients for 1 mg id, 4 mg id or 4 mg im, respectively) or placebo (2 patients/group). Twenty adolescents (mean age 14.2 years, 11 males) received ASP0892 (8 patients/group) or placebo (2 patients/group). In both studies, the most common treatment‐emergent adverse event (TEAE) was injection site pruritus. No deaths or treatment withdrawal were related to TEAEs. No serious TEAEs related to treatment were observed in adult or adolescent patients. ASP0892 treatment led to modest increases in allergen‐specific IgG and/or IgG4 in adults (1 mg id, 4 mg im) and adolescents (1 mg id, 4 mg id). No improvements in clinical outcomes, including double‐blind placebo‐controlled food challenge, were found after ASP0892 treatment.
Conclusions
In two phase 1 studies, ASP0892 was well tolerated with modest but not clinically relevant changes in immune responses.
ClinicalTrials.gov Identifiers
NCT02851277, NCT03755713.
This study evaluates the safety, tolerability, and immunogenicity of a peanut vaccine, ASP0892, after intradermal or intramuscular injection in adults and adolescents with peanut allergy in two phase 1 clinical studies. Both studies of ASP0892 (ARA‐LAMP‐Vax) vaccine showed no significant safety issues and modest but non‐relevant modifications in clinical or immune responses in adults or adolescents with peanut allergy. The safety and immunogenicity potential of ASP0892 may warrant future explorations of LAMP‐Vax vaccines for immunotherapeutic treatment of other diseases. Ara h, Arachis hypogaea; id, intradermal; Ig, immunoglobulin; im, intramuscular; LAMP, lysosomal‐associated membrane protein.Abbreviations: ARA, arachis; id, intradermal; Ig, immunoglobulin; im, intramuscular; LAMP, lysosomal‐associated membrane protein.
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.
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Despite similar clinical symptoms, peanut-allergic (PA) individuals may respond quite differently to the same therapeutic interventions.
This study aimed to determine whether inherent ...qualities of cell response at baseline could influence response to peanut oral immunotherapy (PnOIT).
We first performed ex vivo T-cell profiling on peanut-reactive CD154+CD137+ T (pTeff) cells from 90 challenge-confirmed PA individuals. We developed a gating strategy for unbiased assessment of the phenotypic distribution of rare pTeff cells across different memory CD4+ T-cell subsets to define patient immunotype. In longitudinal samples of 29 PA participants enrolled onto the IMPACT trial of PnOIT, we determined whether patient immunotype at baseline could influence response to PnOIT.
Our data emphasize the heterogeneity of pTeff cell responses in PA participants with 2 mutually exclusive phenotypic entities (CCR6−CRTH2+ and CCR6+CRTH2−). Our findings lead us to propose that peanut allergy can be classified broadly into at least 2 discrete subtypes, termed immunotypes, with distinct immunologic and clinical characteristics that are based on the proportion of TH2A pTeff cells. PnOIT induced elimination of TH2A pTeff cells in the context of the IMPACT clinical trial. Only 1 PA patient with a low level of TH2A pTeff cells at baseline experienced long-lasting benefit of remission after PnOIT discontinuation.
Dividing PA patients according to their individual peanut-specific T-cell profile may facilitate patient stratification in clinical settings by identifying which immunotypes might respond best to different therapies.
Basolateral efflux clearance (CLBL) contributes significantly to rosuvastatin (RSV) elimination in sandwich-cultured hepatocytes (SCH). The contribution of CLBL to RSV hepatic elimination was ...determined in single-pass isolated perfused livers (IPLs) from wild-type (WT) and multidrug resistance-associated protein 2 (Mrp2)-deficient (TR(-)) rats in the absence and presence of the P-glycoprotein and breast cancer resistance protein (Bcrp) inhibitor, elacridar (GF120918); clearance values were compared with SCH. RSV biliary clearance (CLBile) was ablated almost completely by GF120918 in TR(-) IPLs, confirming that Mrp2 and Bcrp primarily are responsible for RSV CLBile. RSV appearance in outflow perfusate was attributed primarily to CLBL, which was impaired in TR(-) IPLs. CLBL was ≈ 6-fold greater than CLBile in the linear range in WT IPLs in the absence of GF120918. Recovery of unchanged RSV in liver tissue increased in TR(-) compared with WT (≈ 25 versus 6% of the administered dose) due to impaired CLBL and CLBile. RSV pentanoic acid, identified by high-resolution liquid chromatography-tandem mass spectroscopy, comprised ≈ 40% of total liver content and ≈ 16% of the administered dose in TR(-) livers at the end of perfusion, compared with ≈ 30 and 3% in WT livers, consistent with impaired RSV excretion and "shunting" to the metabolic pathway. In vitro-ex vivo extrapolation between WT SCH and IPLs (without GF120918) revealed that uptake clearance and CLBL were 4.2- and 6.4-fold lower, respectively, in rat SCH compared with IPLs; CLBile translated almost directly (1.1-fold). The present IPL data confirmed the significant role of CLBL in RSV hepatic elimination, and demonstrated that both CLBL and CLBile influence RSV hepatic and systemic exposure.
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.
•The functional relevance of the CYP epoxyeicosanoid pathway in NASH was evaluated.•Experimental induction of NASH increased hepatic and circulating EET levels in mice.•Humans with biopsy-confirmed ...NASH had higher EET levels versus healthy volunteers.•Disruption of sEH-mediated EET hydrolysis attenuated the progression of NASH in mice.•These data suggest the therapeutic effects of EETs in NASH warrant further study.
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.
Objectives. Homicidal poisoning is an intriguing but poorly described phenomenon. This study describes homicidal poisoning deaths in the United States during 1999-2005. Methods. A trend analysis of ...homicidal poisoning death was performed using Vital Statistics data. The National Mortality Statistics database was queried using "homicide" as injury intent and "poisoning" as mechanism for the years 1999-2005. Counts and rates were obtained for subgrouping by using demographic data and ICD-10 codes. Chi-square analysis calculated subgroup odds ratios (OR) and 95% confidence intervals (CI). Results. A total of 523 homicidal poisoning deaths were identified. The overall rate for the period was 0.26 million year. Males were significantly (OR 1.34, CI 1.13-1.59) more likely to victims than females. Death was more likely at extremes of age. Rates were 2.05 million (OR 8.72, CI 6.63-11.49) in children <1 year and 0.56 million (OR 2.20, CI 1.38-3.52) at ages ≥85 years. Rates also varied by race and were more common among Blacks (0.43 million; OR 1.83, CI 1.47-2.26). The greatest rate was observed in Black infants (5.3 million; OR 21.34, CI 14.17-32.15). ICD-10 codes indicated that medications were the most common poison. Rates were significantly higher in the West and lower in the Northeast. Conclusion. The overall homicidal poisoning death rate was low between 1999 and 2005. The most common type of poison used was medications. Substantially higher rates were observed in vulnerable populations at extremes of age, particularly infants, and among Blacks. This challenges common notions of the victims of homicidal poisoning and the underlying motivation of their poisoners.
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