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Oral administration of mRNA-encapsulated lipid nanoparticles (mRNA-LNPs) is challenging due to various factors, including the low efficiency of mRNA-LNP uptake by small intestinal epithelial cells due to the low levels of apolipoprotein-E in gastrointestinal fluid. Therefore, in this study, we aimed to improve mRNA-LNP uptake by intestinal cells by modifying the surface of mRNA-LNPs with bile acids. Bile acids are recognized by bile acid transporters in the small intestine. We synthesized a polyethylene glycol (PEG)-lipid bound to cholic acid, a type of bile acid, and prepared cholic acid-modified mRNA-LNPs (Cholic-PEG-LNPs) using an ethanol dilution method with a microfluidic device. Uptake of Cholic-PEG-LNPs by differentiated Caco-2 cells was higher than that of unmodified PEG-LNPs. Moreover, protein expression induced by Cholic-PEG-LNPs was higher than that induced by unmodified PEG-LNPs in differentiated Caco-2 cells, and no difference was observed in bile acid transporter-negative MCF-7 cells. These results suggest that the cholic acid modification of mRNA-LNPs enhances bile acid transporter-mediated cellular uptake and protein expression. Our strategy can be used to enhance the functionality of oral mRNA-LNPs. [ABSTRACT FROM AUTHOR]

Objective: Oral administration of insulin is a potential candidate for managing diabetes. However, it is obstructed by the gastrointestinal tract barriers resulting in negligible oral bioavailability. Methods: This investigation presents a novel nanocarrier platform designed to address these challenges. In this regard, the process involved amination of sodium alginate by ethylene diamine, followed by its conjugation with deoxycholic acid. Results: The resulting DCA@Alg@INS nanocarrier revealed a significantly high insulin loading content of 63.6 ± 1.03% and encapsulation efficiency of 87.6 ± 3.84%, with a particle size of 206 nm and zeta potentials of -3 mV. In vitro studies showed sustained and pH-dependent release profiles of insulin from nanoparticles. In vitro cellular studies, confocal laser scanning microscopy and flow cytometry analysis confirmed the successful attachment and internalization of DCA@Alg@INS nanoparticles in Caco-2 cells. Furthermore, the DCA@Alg@INS demonstrated a superior capacity for cellular uptake and permeability coefficient relative to the insulin solution, exhibiting sixfold and 4.94-fold enhancement, respectively. According to the uptake mechanism studies, the results indicated that DCA@Alg@INS was mostly transported through an energy-dependent active pathway since the uptake of DCA@Alg@INS by cells was significantly reduced in the presence of NaN3 by ~ 92% and at a low temperature of 4°C by ~ 94%. Conclusions: Given the significance of administering insulin through oral route, deoxycholic acid-modified alginate nanoparticles present a viable option to surmount various obstacles presented by the gastrointestinal. [ABSTRACT FROM AUTHOR]

Background & Aims: OATP1B3/SLCO1B3 is a human liver-specific transporter for the clearance of endogenous compounds (eg, bile acid [BA]) and xenobiotics. The functional role of OATP1B3 in humans has not been characterized, as SLCO1B3 is poorly conserved among species without mouse orthologs. Methods: Slc10a1-knockout (Slc10a1-/-), Slc10a1hSLCO1B3 (endogenous mouse Slc10a1 promoter-driven human-SLCO1B3 expression in Slc10a1-/- mice), and human SLCO1B3 liver-specific transgenic (hSLCO1B3-LTG) mice were generated and challenged with 0.1% ursodeoxycholic-acid (UDCA), 1% cholic-acid (CA) diet, or bile duct ligation (BDL) for functional studies. Primary hepatocytes and hepatoma-PLC/RPF/5 cells were used for mechanistic studies. Results: Serum BA levels in Slc10a1-/- mice were substantially increased with or without 0.1% UDCA feeding compared with wild-type (WT) mice. This increase was attenuated in Slc10a1hSLCO1B3-mice, indicating that OATP1B3 functions as a significant hepatic BA uptake transporter. In vitro assay using primary hepatocytes from WT, Slc10a1-/-, and Slc10a1hSLCO1B3-mice indicated that OATP1B3 has a similar capacity in taking up taurocholate/TCA as Ntcp. Furthermore, TCA-induced bile flow was significantly impaired in Slc10a1-/- mice but partially recovered in Slc10a1hSLC01B3-mice, indicating that OATP1B3 can partially compensate the NTCP function in vivo. Liver-specific overexpression of OATP1B3 markedly increased the level of hepatic conjugated BA and cholestatic liver injury in 1% CA-fed and BDL mice. Mechanistic studies revealed that conjugated BAs stimulated Ccl2 and Cxcl2 in hepatocytes to increase hepatic neutrophil infiltration and proinflammatory cytokine production (eg, IL-6), which activated STAT3 to repress OATP1B3 expression by binding to its promoter. Conclusions: Human OATP1B3 is a significant BA uptake transporter and can partially compensate Ntcp for conjugated BA uptake in mice. Its downregulation in cholestasis is an adaptive protective response.

Aim: To compare patient characteristics and outcomes between the overall and Japanese populations of GLIMMER. Methods: GLIMMER was a multicenter, double‐blind, randomized, placebo‐controlled, Phase IIb study evaluating linerixibat for the treatment of pruritus in patients with primary biliary cholangitis. Results: In total, 147 patients were randomized in the GLIMMER overall population with 38 patients comprising the Japanese population. Demographics and baseline clinical characteristics were similar across treatment groups and between both populations. A reduction in mean worst daily itch score from baseline to week 16 (primary endpoint) was seen in all groups, with the largest reduction observed with linerixibat 40 mg twice daily (BID; −2.92 [95% confidence interval: −5.07, −0.76] and −2.86 [95% confidence interval: −3.76, −1.95] for Japanese and overall populations, respectively). The highest proportion of responders was generally in the 40 mg BID group in both populations regardless of the responder definition applied. Improvements in health‐related quality of life were generally consistent in both populations. In the Japanese and overall populations, on‐treatment drug‐related adverse events were reported in 25% and 19% of patients in the placebo group and 0%–86% and 31%–78% of patients in the linerixibat groups, respectively. Consistent with the mechanism of action, the most common events were gastrointestinal in nature. The effects of linerixibat on pharmacodynamic biomarkers favored BID dosing. Conclusions: Therapeutic responses and safety of linerixibat were consistent between the Japanese and overall populations of GLIMMER. Linerixibat may provide an effective treatment option for cholestatic pruritus in patients with primary biliary cholangitis. Clinical Trial Registration: NCT02966834. [ABSTRACT FROM AUTHOR]

The prevalence of NAFLD and NASH is increasing worldwide, and there is no approved medical treatment until now. Evidence has emerged that interfering with bile acid metabolism may lead to improvement in NASH. In this study, 28 patients with elevated cholestatic liver function tests (especially GGT) were screened for bile acid gene polymorphisms and treated with UDCA. All patients had a bile acid gene polymorphism in ABCB4 or ABCB11. Treatment with UDCA for 12 months significantly reduced GGT in all patients and ALT in homozygous patients. No difference in fibrosis was observed using FIb-4, NFS, and transient elastography (TE). PNPLA3 and TM6SF2 were the most common NASH-associated polymorphisms, and patients with TM6SF2 showed a significant reduction in GGT and ALT with the administration of UDCA. In conclusion, NASH patients with elevated GGT should be screened for bile acid gene polymorphisms, as UDCA therapy may improve liver function tests. However, no difference in clinical outcomes, such as progression to cirrhosis, has been observed using non-invasive tests (NITs). [ABSTRACT FROM AUTHOR]

Owing to the lack of genetic animal models that adequately recreate key clinical characteristics of cirrhosis, the molecular pathogenesis of cirrhosis has been poorly characterized, and treatments remain limited. Hence, we aimed to better elucidate the pathological mechanisms of cirrhosis using a novel murine model. We report on the first murine genetic model mimicking human cirrhosis induced by hepatocyte-specific elimination of microspherule protein 1 (MCRS1), a member of non-specific lethal (NSL) and INO80 chromatin-modifier complexes. Using this genetic tool with other mouse models, cell culture and human samples, combined with quantitative proteomics, single nuclei/cell RNA sequencing and chromatin immunoprecipitation assays, we investigated mechanisms of cirrhosis. MCRS1 loss in mouse hepatocytes modulates the expression of bile acid (BA) transporters – with a pronounced downregulation of Na+-taurocholate cotransporting polypeptide (NTCP) – concentrating BAs in sinusoids and thereby activating hepatic stellate cells (HSCs) via the farnesoid X receptor (FXR), which is predominantly expressed in human and mouse HSCs. Consistently, re-expression of NTCP in mice reduces cirrhosis, and genetic ablation of FXR in HSCs suppresses fibrotic marks in mice and in vitro cell culture. Mechanistically, deletion of a putative SANT domain from MCRS1 evicts histone deacetylase 1 from its histone H3 anchoring sites, increasing histone acetylation of BA transporter genes, modulating their expression and perturbing BA flow. Accordingly, human cirrhosis displays decreased nuclear MCRS1 and NTCP expression. Our data reveal a previously unrecognized function of MCRS1 as a critical histone acetylation regulator, maintaining gene expression and liver homeostasis. MCRS1 loss induces acetylation of BA transporter genes, perturbation of BA flow, and consequently, FXR activation in HSCs. This axis represents a central and universal signaling event in cirrhosis, which has significant implications for cirrhosis treatment. By genetic ablation of MCRS1 in mouse hepatocytes, we generate the first genetic mouse model of cirrhosis that recapitulates human features. Herein, we demonstrate that the activation of the bile acid/FXR axis in liver fibroblasts is key in cirrhosis development. [Display omitted] • MCRS1 loss in hepatocytes deregulates the expression of BA transporters, causing perturbation of BA flow and cirrhosis. • Re-expression of the BA transporter NTCP in MCRS1-depleted hepatocytes restores the BA flow and prevents cirrhosis. • Accumulation of BAs in liver sinusoids activates liver fibroblasts via FXR leading to fibrosis. • MCRS1 loss increases histone acetylation of BA transporter genes, altering their expression levels. • MCRS1 and NTCP expression correlates with fibrotic marks in human cirrhosis samples. [ABSTRACT FROM AUTHOR]

Despite many ongoing and innovative approaches, there are still formidable challenges in the clinical translation of oral peptide drugs into marketable products due to their low absorption and poor bioavailability. Herein, a novel nanocarrier platform was developed that employs a hydrophobic ion-pairing (HIP) of model peptide (insulin) and the anionic bile salt (sodium glycodeoxycholate, SGDC), and markedly improves intestinal absorption via the bile acid pathway. The developed HIP-nanocomplexes (C1 and C2) were optimized, characterized, and in vitro and in vivo evaluation were performed to assess oral efficacy of these system. The optimal molar ratios of C1 and C2-nanocomplexes were 30:1 and 6:1 (SGDC:insulin), respectively. Compared to the insulin solution, the C1 and C2 nanocomplexes significantly enhanced the permeation of insulin across the Caco-2 cell monolayers, with 6.36- and 4.05-fold increases in apparent permeability, respectively. Uptake mechanism studies were conducted using different endocytosis inhibitors and apical sodium-dependent bile acid transporter (ASBT)-transfected MDCK cells, which demonstrated the involvement of the energy-dependent ASBT-mediated active transport. Furthermore, the intrajejunal administration of C1 and C2 resulted in their pharmacological availabilities (PA) being 6.44% and 0.10%, respectively, indicating increased potential for C1, when compared to C2. Similarly, the PA and the relative bioavailability with intrajejunal administration of the C1 were 17.89-fold and 16.82-fold greater than those with intracolonic administration, respectively, confirming better jejunal absorption of C1. Overall, these findings indicate that the HIP-nanocomplexes could be a prominent platform for the effective delivery of peptides with improved intestinal absorption. [Display omitted] [ABSTRACT FROM AUTHOR]

Background/AimsCholangiocytes are capable of reabsorbing bile salts from bile, but the pathophysiological significance of this process is unclear. To this end, we detected the expression and distribution of bile acid transport proteins in cholangiocytes from normal rat liver and analyzed the possible pathophysiological significance.Methods : Bile duct tissues of Sprague-Dawley rats were isolated by enzymatic digestion and mechanical isolation, and then divided into large and small bile duct tissues. Immunohistochemistry, real-time polymerase chain reaction and Western blotting were used to determine the expression of the apical sodium-dependent bile acid transporter (ASBT), ileal bile acid binding protein (IBABP), and basolateral organic solute transporter α (Ostα) in the biliary tract system of rats. Differences in the expression and distribution of these proteins were analyzed.Results : In cholangiocytes, ASBT and IBABP were mainly expressed in cholangiocytes of the large bile ducts, in which the expression of both was significantly higher than that in the small ducts (p0.05).Conclusion : sBile acid transporters are expressed and heterogeneously distributed in rat bile ducts, indicating that bile acid reabsorption by cholangiocytes might mainly occur in the large bile ducts. These findings may help explore the physiology of bile ducts and the pathogenesis of various cholangiopathies.

Apical sodium‐dependent bile acid transporter (ASBT) retrieves bile acids from the small intestine and plays a pivotal role in enterohepatic circulation. Currently, high‐resolution structures are available for two bacterial ASBT homologs (ASBTNM from Neisseria meningitides and ASBTYf from Yersinia frederiksenii), from which an elevator‐style alternating‐access mechanism has been proposed for substrate transport. A key concept in this model is that the substrate binds to the central cavity of the transporter so that the elevator‐like motion can expose the bound substrate alternatingly to either side of the membrane during a transport cycle. However, no structure of an ASBT has been solved with a substrate bound in its central cavity, so how a substrate binds to ASBT remains to be defined. In this study, molecular docking, structure determination and functional analysis were combined to define and validate the details of substrate binding in ASBTYf. The findings provide coherent evidence to provide a clearer picture of how the substrate binds in the central cavity of ASBTYf that fits the alternating‐access model. [ABSTRACT FROM AUTHOR]

Apical sodium‐dependent bile acid transporter (ASBT) mediates the uptake of bile acids from the ileum lumen into enterocytes and presents a potential target for the treatment of several metabolic diseases, including type 2 diabetes. It has been proposed that the underlying mechanism for transport by ASBT is an elevator‐style alternating‐access model, which was deduced mainly by comparing high‐resolution structures of two bacterial ASBT homologs (ASBTNM from Neisseria meningitides and ASBTYf from Yersinia frederiksenii) in different conformations. However, one important issue is that the only outward‐facing structure (PDB entry 4n7x) was obtained with an Na+‐binding site mutant of ASBTYf, which severely cripples its transport function, and therefore the physiological relevance of the conformation in PDB entry 4n7x requires further careful evaluation. Here, another crystal structure is reported of ASBTYf that was captured in a state closely resembling the conformation in PDB entry 4n7x using an engineered disulfide bridge. The introduced cysteine mutations avoided any proposed Na+‐ or substrate‐binding residues, and the resulting mutant retained both structural and functional integrity and behaved similarly to wild‐type ASBTYf. These data support the hypothesis that the PDB entry 4n7x‐like structure represents a functional outward‐facing conformation of ASBTYf in its transport cycle. [ABSTRACT FROM AUTHOR]

Background: Bile acids (BAs) are important in the metabolic effects of bariatric surgery. Most BAs are reabsorbed in the ileum and recycled back to the liver. We have reported that this enterohepatic circulation was shortened by duodenal-jejunal bypass (DJB), and the biliopancreatic (BP)-limb plays an important role in reabsorption of BAs. However, the mechanism of BA reabsorption in BP-limb remains uncertain. We aimed to investigate the mechanisms of BA reabsorption after DJB, especially focusing on carrier-mediated transport of BAs and the impact of the presence or absence of lipids on BA reabsorption. Methods: Otsuka-Long-Evans-Tokushima fatty rats or Sprague-Dawley rats were assigned to a control group and DJB group. BA levels in the divided small intestine were quantified with liquid chromatography-mass spectrometry. Labeled BA was injected and perfused with BA transporter inhibitors or mixture of lipids in the isolated BP-limb, and bile was sampled and analyzed. Results: Conjugated BA levels in the BP-limb were significantly higher than that of the control group. BA absorption tended to decrease by the apical sodium-dependent BA transporter inhibitor and was significantly decreased by the organic anion-transporting peptide (OATP) inhibitor. BA absorption tended to increase in the absence of lipid solutions compared with that in the presence of lipid solutions. Conclusion: We attributed the increased BA reabsorption in the BP-limb to lack of food in the BP-limb, which contains concentrated BAs and no lipids. OATP played an important role in BA reabsorption in the BP-limb. Therefore, BAs would be reabsorbed in different manners after DJB. [ABSTRACT FROM AUTHOR]

In vitro-in vivo extrapolation (IVIVE) integrated in physiologically-based pharmacokinetic (PBPK) models have been increasingly used during drug discovery and development processes to predict human pharmacokinetic (PK) parameters. Drug transporters can influence drug pharmacokinetics and are key aspects contributing to the development of a successful drug. This review provides a snapshot of challenges or shortcomings of in vitro and in vivo techniques for understanding the contribution of drug transporters to a drug’s pharmacokinetics. The paper also describes the potential of IVIVE-PBPK models as prospective approaches to predict the role of drug transporters in drug discovery and development.

Cholestasis resulting from hepatic bile acid efflux transporter inhibition may contribute to drug-induced liver injury (DILI). This condition is a common safety-related reason for drug attrition and withdrawal. To screen for safety risks associated with efflux transport inhibition, we developed a high-throughput cellular assay for different drug discovery phases. Hepatocytes isolated from chimeric mice with humanized livers presented gene expression resembling that of the human liver and demonstrated apical membrane polarity when sandwiched between Matrigel and collagen. The fluorescent bile acid-derivative cholyl- l -lysyl-fluorescein (CLF) was used to quantify drug-induced efflux transport inhibition in hepatocytes. Cyclosporine inhibited CLF accumulation in the apical bile canalicular lumen in a concentration-dependent manner. The assay had equivalent predictive power to a primary human hepatocyte-based assay and greater predictive power than an assay performed with rat hepatocytes. Predictive power was tested using 45 pharmaceutical compounds, and 91.3% of the compounds with cholestatic potential (21/23) had margins (IC50/ C max) < 20. In contrast, 90.9% (20/22) of compounds without cholestatic potential had IC50/ C max>20. Assay sensitivity and specificity were 91.3% and 90.9%, respectively. We suggest that this improved assay performance could result from higher expression of efflux transporters, metabolic pathways, and/or species differences. Given the long-term supply of cells from the same donor, the humanized mouse-derived hepatocyte-based CLF efflux assay could be a valuable tool for predicting cholestatic DILI. [ABSTRACT FROM AUTHOR]

We previously reported a cell-based toxicity assay using sandwich-cultured hepatocytes in combination with a titrated amount of human bile acid (BA) species. In this assay, test compound-induced inhibition of BA efflux from sandwich-cultured hepatocytes leads to BA-dependent cell toxicity (BA tox , i.e., cell death due to the accumulation of BAs). Using this assay, we investigated whether 1-aminobenzotriazole (1-ABT; a nonselective cytochrome P450 inhibitor) enhanced or suppressed test compound-induced BA tox . There was a tendency that BA tox of many compounds was enhanced by 1-ABT in human hepatocytes; in contrast, such a tendency was not observed in rat hepatocytes. In particular, 1-ABT tended to enhance BA tox of several compounds (clopidogrel, ticlopidine, everolimus, etc.) in human, whereas 1-ABT tended to enhance BA tox of only ticlopidine in rat. These results indicate that this system can be used to evaluate BA tox while taking into account drug metabolism and the existence of an interspecies difference in the effect of 1-ABT treatment on BA tox . [ABSTRACT FROM AUTHOR]

Background & Aims Hepatobiliary secretion of bile acids is an important liver function. Here, we quantified the hepatic transport kinetics of conjugated bile acids using the bile acid tracer [ N -methyl- 11 C]cholylsarcosine ( 11 C-CSar) and positron emission tomography (PET). Methods Nine healthy participants and eight patients with varying degrees of cholestasis were examined with 11 C-CSar PET and measurement of arterial and hepatic venous blood concentrations of 11 C-CSar. Results Results are presented as median (range). The hepatic intrinsic clearance was 1.50 (1.20–1.76) ml blood/min/ml liver tissue in healthy participants and 0.46 (0.13–0.91) in patients. In healthy participants, the rate constant for secretion of 11 C-CSar from hepatocytes to bile was 0.36 (0.30–0.62) min −1 , 20 times higher than the rate constant for backflux from hepatocytes to blood (0.02, 0.005–0.07 min −1 ). In the patients, rate constant for transport from hepatocyte to bile was reduced to 0.12 (0.006–0.27) min −1 , 2.3 times higher than the rate constant for backflux to blood (0.05, 0.04–0.09). The increased backflux did not fully normalize exposure of the hepatocyte to bile acids as mean hepatocyte residence time of 11 C-CSar was 2.5 (1.6–3.1) min in healthy participants and 6.4 (3.1–23.7) min in patients. The rate constant for transport of 11 C-CSar from intrahepatic to extrahepatic bile was 0.057 (0.023–0.11) min −1 in healthy participants and only slightly reduced in patients 0.039 (0.017–0.066). Conclusions This first in vivo quantification of individual steps involved in the hepatobiliary secretion of a conjugated bile acid in humans provided new insight into cholestatic disease. Lay summary Positron emission tomography (PET) using the radiolabelled bile acid ( 11 C-CSar) enabled quantification of the individual steps of the hepatic transport of bile acids from blood to bile in man. Cholestasis reduced uptake and secretion and increased backflux to blood. These findings improve our understanding of cholestatic liver diseases and may support therapeutic decisions. Clinical trial registration number: The trial is registered at ClinicalTrials.gov (NCT01879735). [ABSTRACT FROM AUTHOR]

The supply of inorganic carbon (Ci) at the site of fixation by Rubisco is a key parameter for efficient CO2 fixation in aquatic organisms including the green alga, Chlamydomonas reinhardtii. Chlamydomonas reinhardtii cells, when grown on limiting CO2, have a CO2-concentrating mechanism (CCM) that functions to concentrate CO2 at the site of Rubisco. Proteins thought to be involved in inorganic carbon uptake have been identified and localized to the plasma membrane or chloroplast envelope. However, current CCM models suggest that additional molecular components are involved in Ci uptake. In this study, the gene Cia8 was identified in an insertional mutagenesis screen and characterized. The protein encoded by Cia8 belongs to the sodium bile acid symporter subfamily. Transcript levels for this gene were significantly up-regulated when the cells were grown on low CO2. The cia8 mutant exhibited reduced growth and reduced affinity for Ci when grown in limiting CO2 conditions. Prediction programs localize this protein to the chloroplast. Ci uptake and the photosynthetic rate, particularly at high external pH, were reduced in the mutant. The results are consistent with the model that CIA8 is involved in Ci uptake in C. reinhardtii. [ABSTRACT FROM AUTHOR]

Expression of bile salt export pump (BSEP) is regulated by the bile acid/farnesoid X receptor (FXR) signaling pathway. Two FXR isoforms, FXRα1 and FXRα2, are predominantly expressed in human liver. We previously showed that human BSEP was isoform-dependently regulated by FXR and diminished with altered expression of FXRα1 and FXRα2 in patients with hepatocellular carcinoma. In this study, we demonstrate that FXRα1 and FXRα2 regulate human BSEP through two distinct FXR responsive elements (FXRE): IR1a and IR1b. As the predominant regulator, FXRα2 potently transactivated human BSEP through IR1a, while FXRα1 weakly transactivated human BSEP through a newly identified IR1b. Relative expression of FXRα1 and FXRα2 affected human BSEP expression in vitro and in vivo. Electrophoretic mobility shift and chromatin immunoprecipitation assays confirmed the binding and recruitment of FXRα1 and FXRα2 to IR1b and IR1a. Sequence analysis concluded that IR1b was completely conserved among species, whereas IR1a exhibited apparent differences across species. Sequence variations in IR1a were responsible for the observed species difference in BSEP transactivation by FXRα1 and FXRα2. In conclusion, FXR regulates BSEP in an isoform-dependent and species-specific manner through two distinct FXREs, and alteration of relative FXR isoform expression may be a potential mechanism for FXR to precisely regulate human BSEP in response to various physiological and pathological conditions.

Proprotein convertase subtilisin-kexin-9 (PCSK9) inhibition markedly augments the LDL lowering action of statins. The combination is being evaluated for long-term effects on atherosclerotic disease outcomes. However, effects of combined treatment on hepatic cholesterol and bile acid metabolism have not yet been reported. To study this, PCSK9-Y119X mutant (knockout) and wild-type mice were treated with or without atorvastatin for 12 weeks. Atorvastatin progressively lowered plasma LDL in each group, but no differences in liver cholesterol, cholesterol ester, or total bile acid concentrations, or in plasma total bile acid levels were seen. In contrast, atorvastatin increased fecal total bile acids (∼2-fold, P < 0.01) and cholesterol concentrations (∼3-fold, P < 0.01) versus controls for both PCSK9-Y119X and wild-type mice. All 14 individual bile acids resolved by LC-MS, including primary, secondary, and conjugated species, reflected similar increases. Expression of key liver bile acid synthesis genes CYP7A1 and CYP8B1 were ∼2.5-fold higher with atorvastatin in both strains, but mRNA for liver bile acid export and reuptake transporters and conjugating enzymes were not unaffected. The data suggest that hepatocyte cholesterol and bile acid homeostasis is maintained with combined PCSK9 and HMG-CoA reductase inhibition through efficient liver enzymatic conversion of LDL-derived cholesterol into bile acids and excretion of both, with undisturbed enterohepatic recycling.

The Bile Acid Sodium Symporter (BASS) family transports a wide array of molecules across membranes, including bile acids in humans, and small metabolites in plants. These transporters, many of which are sodium-coupled, have been shown to use an elevator mechanism of transport, but exactly how substrate binding is coupled to sodium ion binding and transport is not clear. Here we solve the crystal structure at 2.3 Å of a transporter from Neisseria Meningitidis (ASBT NM ) in complex with pantoate, a potential substrate of ASBT NM . The BASS family is characterised by two helices that cross-over in the centre of the protein in an arrangement that is intricately held together by two sodium ions. We observe that the pantoate binds, specifically, between the N-termini of two of the opposing helices in this cross-over region. During molecular dynamics simulations the pantoate remains in this position when sodium ions are present but is more mobile in their absence. Comparison of structures in the presence and absence of pantoate demonstrates that pantoate elicits a conformational change in one of the cross-over helices. This modifies the interface between the two domains that move relative to one another to elicit the elevator mechanism. These results have implications, not only for ASBT NM but for the BASS family as a whole and indeed other transporters that work through the elevator mechanism., Competing Interests: Competing interests: Authors declare no competing interests.

Chronic idiopathic constipation is highly prevalent among adults. Bile acids (BAs) and the enterohepatic BA circulation modulate colonic secretion and motility that affect transit. BAs in the colon have a dual action as osmotic and stimulant agents. Newer agents, such as elobixibat (A3309), an inhibitor of the ileal BA transporter, have the potential to improve significantly the management of chronic constipation, with minimal adverse effects. Elobixibat modulates the enterohepatic BA circulation, enhancing the delivery of BAs to the colon where they induce secretory and motor effects. Secondary effects of the inhibition of BA absorption are reduced activation of the farnesoid X receptor, decreased secretion of fibroblast growth factor-19 into the portal circulation, and increased BA synthesis. This review focuses on the role of BAs, the enterohepatic BA circulation, and an ileal BA transporter inhibitor (elobixibat) in chronic constipation. [ABSTRACT FROM PUBLISHER]

Abstract: Intestinal transporters are limited to the transport of small molecular substrates. Here, we describe the development of apical sodium-dependent bile acid transporter (ASBT)-targeted high-affinity oligomeric bile acid substrates that mediate the transmembrane transport of low molecular weight heparin (LMWH). Several oligomers of deoxycholic acid (oligoDOCA) were synthesized to investigate the substrate specificity of ASBT. To see the binding of oligoDOCA on the substrate-binding pocket of ASBT, molecular docking was used and the dissociation rate constants (KD) were measured using surface plasmon resonance. The KD for tetrameric DOCA (tetraDOCA) was 50-fold lower than that for monomeric DOCA, because tetraDOCA interacted with several hydrophobic grooves in the substrate-binding pocket of ASBT. The synthesized oligoDOCA compounds were subsequently chemically conjugated to macromolecular LMWH. In vitro, tetraDOCA-conjugated LMWH (LHe-tetraD) had highest selectivity for ASBT during its transport. Orally administered LHe-tetraD showed remarkable systemic anticoagulation activity and high oral bioavailability of 33.5±3.2% and 19.9±2.5% in rats and monkeys, respectively. Notably, LHe-tetraD successfully prevented thrombosis in a rat model of deep vein thrombosis. These results represent a major advancement in ASBT-mediated LMWH delivery and may facilitate administration of many important therapeutic macromolecules through a non-invasive oral route. [Copyright &y& Elsevier]

Effect of chronic renal failure on the hepatic, intestinal, and renal expression of bile acid transporters. Am J Physiol Renal Physiol 306: F130-F137, 2014. First published November 6, 2013; doi:10.1152/ajprenal.00114.2013.--Although the kidney is believed to play a minor role in bile acid (BA) excretion, chronic renal failure (CRF) has been reported to be associated with increased serum bile acid levels and alterations in BA homeostasis. The mechanisms for elevated BA levels are poorly understood in both clinical and experimental studies. This study was designed to examine the effects of naturally progressing CRF of longer duration on the hepatic and renal mRNA and protein levels of the BA-synthesizing enzyme Cyp7a1 and the BA transporters Ntcp, Bsep, Mrp3, Ost-α, and Ost-β. Sprague-Dawley rats were randomized to the CRF group (5/6 nephrectomy) or to the sham-operated control group and were analyzed 8 wk after surgery. Results obtained in the CRF rats were compared with those obtained in rats that had undergone uninephrectomy (UNX). The CRF group exhibited significantly increased plasma cholesterol and BA concentrations. Hepatic Cyp7a1 mRNA and protein levels were almost identical in the two groups. Hepatic Mrp3, Ost-α, and Ost-β expression was increased, suggesting increased basolateral efflux of bile acids into the blood. However, no such changes in BA transporter expression were observed in the remnant kidney. In UNX rats, similar changes in plasma BA levels and in the expression of BA transporters were found. We hypothesize that the increase in plasma BA is an early event in the progression of CRF and is caused by increased efflux across the basolateral hepatocyte membrane. [ABSTRACT FROM AUTHOR]

Abstract: Deoxycholic acid (DCA), a secondary bile acid (BA), and ursodeoxycholic acid (UDCA), a tertiary BA, cause opposing effects in vivo and in cell suspensions. Fluorescent analogues of DCA and UDCA could help investigate important questions about their cellular interactions and distribution. We have prepared a set of isomeric 3α- and 3β-amino analogues of UDCA and DCA and derivatised these with the discrete fluorophore, 4-nitrobenzo-2-oxa-1,3-diazol (NBD), forming the corresponding four fluorescent adducts. These absorb in the range 465–470nm and fluoresce at approx. 535nm. In order to determine the ability of the new fluorescent bile acids to mimic the parents, their uptake was studied using monolayers of Caco-2 cells, which are known to express multiple proteins of the organic anion-transporting peptide (OATP) subfamily of transporters. Cellular uptake was monitored over time at 4 and 37°C to distinguish between passive and active transport. All four BA analogues were taken up but in a strikingly stereo- and structure-specific manner, suggesting highly discriminatory interactions with transporter protein(s). The α-analogues of DCA and to a lesser extent UDCA were actively transported, whereas the β-analogues were not. The active transport process was saturable, with Michaelis–Menten constants for 3α-NBD DCA (5) being K m =42.27±12.98μM and V max =2.8±0.4nmol/(mg protein∗min) and for 3α-NBD UDCA (3) K m =28.20±7.45μM and V max =1.8±0.2nmol/(mg protein∗min). These fluorescent bile acids are promising agents for investigating questions of bile acid biology and for detection of bile acids and related organic anion transport processes. [Copyright &y& Elsevier]

Abstract: The objective was to synthesize prodrugs of niacin and ketoprofen that target the human apical sodium-dependent bile acid transporter (ASBT) and potentially allow for prolonged drug release. Each drug was conjugated to the naturally occurring bile acid chenodeoxycholic acid (CDCA) using lysine as a linker. Their inhibitory binding and transport properties were evaluated in stably transfected ASBT-MDCK monolayers, and the kinetic parameters K i, K t, normJ max, and P p were characterized. Enzymatic stability of the conjugates was evaluated in Caco-2 and liver homogenate. Both conjugates were potent inhibitors of ASBT. For the niacin prodrug, substrate kinetic parameter K t was 8.22μM and normJ max was 0.0917. In 4h, 69.4% and 26.9% of niacin was released from 1μM and 5μM of the conjugate in Caco-2 homogenate, respectively. For the ketoprofen prodrug, K t was 50.8μM and normJ max was 1.58. In 4h, 5.94% and 3.73% of ketoprofen was released from 1μM and 5μM of the conjugate in Caco-2 homogenate, and 24.5% and 12.2% of ketoprofen was released in liver homogenate, respectively. In vitro results showed that these bile acid conjugates are potential prolonged release prodrugs with binding affinity for ASBT. [Copyright &y& Elsevier]

The article focuses on a study about the role of Peroxisome proliferator-activated receptor &#x03B (PPAR &#x03B); in hepatobiliary injuries in mice induced by ammonium perfluorooctanoic acid (PFOA). Major hepatocellular damage and minor cholangiopathy cause by PFOA in wild type mice compared to treated PPAR&#x03B-null mice in which fat was accumulated, severe cholangiopathy and hepatocellular damage was observed. Results reported that PPAR&#x03B is significant against PFOA and biliary injury.

Organic anion transporting polypeptide 1B1 (OATP1B1, encoded by SLCO1B1) is a sinusoidal influx transporter of human hepatocytes. Our aim was to characterize the role of OATP1B1 in the hepatic uptake of bile acids in vivo.Fasting blood samples were drawn from 24 healthy volunteers with SLCO1B1 c.388AA-c.521TT (∗1A/∗1A) genotype, eight with c.388GG-c.521TT (∗1B/∗1B) genotype, 24 with c.521TC genotype, and nine with c.521CC genotype. Plasma concentrations of 15 endogenous bile acids, their synthesis marker, and cholesterol were determined by liquid chromatography-tandem mass spectrometry.The concentrations of ursodeoxycholic acid, glycoursodeoxycholic acid, chenodeoxycholic acid, and glycochenodeoxycholic acid were approximately 50-240% higher in individuals with the SLCO1B1 c.521CC, c.521TC, or c.388AA-c.521TT genotype than in those with the c.388GG-c.521TT genotype (P<0.05), with the largest differences seen between the c.521CC and c.388GG-c.521TT individuals. The concentration of tauroursodeoxycholic acid was approximately 120% higher in individuals with the c.521TC genotype and that of taurochenodeoxycholic acid 110% higher in individuals with the c.521CC or c.521TC genotype than in those with the c.388GG-c.521TT genotype (P<0.05). The cholic acid concentration was approximately 30% higher in individuals with the c.521CC or c.388AA-c.521TT genotype than in those with the c.388GG-c.521TT genotype (P<0.05), but its conjugates remained unaffected by the genotype. The bile acid synthesis marker 7α-hydroxy-4-cholesten-3-one/cholesterol concentration ratio was 62 or 45% higher in the c.388AA-c.521TT participants than in the c.388GG-c.521TT or c.521TC participants, respectively (P<0.05).SLCO1B1 polymorphism considerably affects the disposition of several endogenous bile acids and bile acid synthesis marker, indicating that OATP1B1 plays an important role in the hepatic uptake of bile acids in vivo in humans. [ABSTRACT FROM AUTHOR]

Hepatic uptake of bile salts is mediated by sodium-dependent and sodium-independent transport systems. During extrahepatic cholestasis, both the function and the expression of the Na+/taurocholate cotransporting polypeptide (Ntcp) are downregulated. To test whether sodium-independent organic anion-transporting polypeptides are also affected by extrahepatic cholestasis, the function and expression of all three Oatps have been determined in common bile duct-ligated (CBDL) rats. Oatp1/Oatp1a1 protein mass remained unchanged after CBDL for 1 day, but then declined by 75±7% and 90±17%, respectively, after 3 and 7 days. In contrast, Oatp2/Oatp1a4 and Oatp4/Oatp1b2 protein expression was not affected by CBDL as compared with controls. After CBDL, Oatp1 mRNA was rapidly downregulated by 68±21% of untreated controls ( P<0.05) within 24 h, and remained at similar levels at 3 and 7 days. Cytokine-inactivation studies with etanercept pretreatment demonstrated that TNF-α-dependent signals mediated the down-regulation of this transporter gene at both protein and mRNA levels during obstructive cholestasis. Sodium-independent uptake of taurocholate and cholate into freshly isolated hepatocyte suspensions showed neither significant differences in Km nor Vmax values. These results indicate that sodium-independent transport of bile salts may be mediated by Oatp2 and 4 during biliary obstruction, because its expression remains unaffected and may compensate for loss of Oatp1 expression and function in cholestatic hepatocytes. [ABSTRACT FROM AUTHOR]

Two proteins that mediate bile acid export from the ileal enterocyte, organic solute transporter (OST)-α and -β, have recently been identified. It is unclear whether these two proteins associate directly and how they interact to mediate transport function and membrane localization. In this study, the protein-protein interactions, transport functions, and membrane localization of human (h)OST-α and -β proteins were examined. The results demonstrated that coexpression of hOST-α and -β in transfected cells resulted in a three- to fivefold increase of the initial rate of taurocholate influx or efflux compared with cells expressing each protein individually and nontransfected cells. Confocal microscopy demonstrated plasma membrane colocalization of hOST-α and -β proteins in cells cotransfected with hOST-α and -β cDNAs. Protein-protein interactions between hOST-α and -β were demonstrated by mammalian two-hybrid and coimmunoprecipitation analyses. Truncation of the amino-terminal 50 amino acid extracellular residues of hOST-α abolished its interaction with hOST-β and led to an intracellular accumulation of the two proteins and to only background levels of taurocholate transport. In contrast, carboxyl-terminal 28 amino acid truncated hOST-α still interacted with hOST-β, and majority of this cytoplasmic tail-truncated protein was expressed on the basolateral membrane when it was stably cotransfected with hOST-β protein in Madin-Darby canine kidney cells. In summary, hOST-α and -β proteins are physically associated. The intracellular carboxyl-terminal domain of hOST-α is not essential for this interaction with hOST-β. The extracellular amino-terminal fragment of hOST-α may contain important information for the assembly of the heterodimer and trafficking to the plasma membrane. [ABSTRACT FROM AUTHOR]

Treatment of β-lactamase positive bacterial infections with a combination of amoxicillin (AMOX) and clavulanic acid (CLAV) causes idiosyncratic drug-induced liver injury (iDILI) in a relevant number of patients, often with features of intrahepatic cholestasis. This study aims to determine serum bile acid (BA) levels in amoxicillin/clavulanate (A+C)-iDILI patients and to investigate the mechanism of cholestasis by A+C in human in vitro hepatic models. In six A+C-iDILI patients, significant elevations of serum primary conjugated BA definitely demonstrated A+C-induced cholestasis. In cultured human Upcyte hepatocytes and HepG2 cells, CLAV was more cytotoxic than AMOX, and, at subcytotoxic concentrations, it altered the expression of more than 1,300 genes. CLAV, but not AMOX, downregulated the expression of key genes for BA transport (BSEP, NTCP, OSTα and MDR2) and synthesis (CYP7A1 and CYP8B1). CLAV also caused early oxidative stress, with reduced GSH/GSSG ratio, along with induction of antioxidant nuclear factor erythroid 2-related factor 2 (NRF2) target genes. Activation of NRF2 by sulforaphane also resulted in downregulation of NTCP, OSTα, ABCG5, CYP7A1 and CYP8B1. CLAV also inhibited the BA-sensor farnesoid X receptor (FXR), in agreement with the downregulation of FXR targets BSEP, OSTα and ABCG5. We conclude that CLAV, the culprit molecule in A+C, downregulates several key biliary transporters by modulating NRF2 and FXR signaling, thus likely promoting intrahepatic cholestasis. On top of that, increased ROS production and GSH depletion may aggravate the cholestatic injury by A+C. Mechanisms involved in CLAV-induced cholestasis. Schematic representation of a human hepatocyte with basolateral and canalicular biliary transporters. Blue lines with arrows represent the genes of biliary transporters. Green-pointed and red-blocked arrows represent activating or repressing events. An "X" through a point arrow represents a loss of activation. Green and red ovals represent proteins that result activated/upregulated or inhibited/downregulated by CLAV. ROS. Reactive oxygen species. BA: bile acids, PL: phospholipids, Chol: cholesterol, GS-conj: glutathione conjugates, Bil: bilirubin, GSSG, oxidized glutathione. [Display omitted] • Amoxicillin-clavulanate raises serum primary conjugated bile acids in DILI patients. • Clavulanate is the culprit of cholestasis by amoxicillin-clavulanate in hepatocytes. • Clavulanate downregulates expression of bile acid transporter and synthesis genes. • Clavulanate causes early oxidative stress, decreases GSH and induces NRF2 signalling. • Clavulanate inhibits FXR activation by agonists, in agreement with BSEP repression. [ABSTRACT FROM AUTHOR]

[Display omitted] Most anticancer drugs are not orally bioavailable due to their undesirable physicochemical properties and inherent physiological barriers. In this study, a polymeric prodrug strategy was presented to enhance the oral bioavailability of BCS class IV drugs using paclitaxel (PTX) as the model drug. PTX was covalently conjugated with cholic acid-functionalized PEG by a redox-sensitive disulfide bond. Cholic acid-functionalized PEGylated PTX (CPP) achieved remarkably improved PTX solubility (>30,000-fold), as well as favorable stability under the physiological environment and controlled drug release in the tumor. Meanwhile, CPP could self-assemble into nanoparticles with an average size of 56.18 ± 2.06 nm and drug loading up to 17.6% (w/w). Then, permeability study on Caco-2 cell monolayers demonstrated that CPP obtained an approximately 4-fold increase by apical sodium-dependent bile acid transporter (ASBT) mediated transport, compared with Taxol®. Pharmacokinetic studies carried out in rats confirmed that the oral bioavailability of CPP was 10-fold higher than that of Taxol®. Finally, significant improvement in the antitumor efficacy of CPP against breast cancer was confirmed on MDA-MB-231 cells. In summary, this prodrug-based cascade strategy offers new ways for chemotherapeutic drugs whose oral delivery is limited by solubility and permeability, also endows drugs with the capacity of tumor-specific release. [ABSTRACT FROM AUTHOR]

• The mechanism of conformational isomerization of ASBT NM remains undefined. • Na+ binding to ASBT NM does not favor an outward-facing conformation. • DEER distance measurements are in agreement with SDAF profiles. • ASBT NM exists as a monomer in detergent micelles and membranes. • The dynamics studies provide a new viewpoint of elevator-type transporters. Apical sodium-dependent bile acid transporter (ASBT) catalyses uphill transport of bile acids using the electrochemical gradient of Na+ as the driving force. The crystal structures of two bacterial homologues ASBT NM and ASBT Yf have previously been determined, with the former showing an inward-facing conformation, and the latter adopting an outward-facing conformation accomplished by the substitution of the critical Na+-binding residue glutamate-254 with an alanine residue. While the two crystal structures suggested an elevator-like movement to afford alternating access to the substrate binding site, the mechanistic role of Na+ and substrate in the conformational isomerization remains unclear. In this study, we utilized site-directed alkylation monitored by in-gel fluorescence (SDAF) to probe the solvent accessibility of the residues lining the substrate permeation pathway of ASBT NM under different Na+ and substrate conditions, and interpreted the conformational states inferred from the crystal structures. Unexpectedly, the crosslinking experiments demonstrated that ASBT NM is a monomer protein, unlike the other elevator-type transporters, usually forming a homodimer or a homotrimer. The conformational dynamics observed by the biochemical experiments were further validated using DEER measuring the distance between the spin-labelled pairs. Our results revealed that Na+ ions shift the conformational equilibrium of ASBT NM toward the inward-facing state thereby facilitating cytoplasmic uptake of substrate. The current findings provide a novel perspective on the conformational equilibrium of secondary active transporters. [ABSTRACT FROM AUTHOR]

Veronica ciliata Fisch. is a traditional medical herb that present in more than 100 types of Tibetan medicine prescriptions, most of which are used for liver disease therapy. Iridoid glycosides have been identified as the major active components of V.ciliata with a variety of biological activities. Aims of the study : The aim of this study is to explore the protective effect and potential mechanism of n-Butanol extract (BE) and iridoid glycosides (IG) from V.ciliata against ɑ-naphthyl isothiocyanate (ANIT)-induced hepatotoxicity and cholestasis in mice. Mice were intragastrically (i.g.) given BE and IG at different dose or positive control ursodeoxycholic acid (UCDA) once a day for 14 consecutive days, and were treated with ANIT to cause liver injury on day 12th. Serum levels of hepatic injury markers and cholestasis indicators, liver index and liver histopathology were measured to evaluate the effect of BE and IG on liver injury caused by ANIT. The protein levels of tumor necrosis factor-α (TNF-α), nuclear factor kappa B(NF-κB), interleukin-6 (IL-6), Na+/taurocholate cotransporting polypeptide (NTCP), bile salt export pump (BSEP), multidrug resistance-associated protein 2 (MRP2), and the levels of oxidative stress indicators in liver tissue were investigated to reveal the underlying protective mechanisms of BE and IG against ANIT-induced hepatotoxicity and cholestasis. The n-Butanol extract (BE) and iridoid glycosides (IG) isolated from V.ciliata significantly decreased serum level of cholestatic liver injury markers aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), γ-glutamyl transferase (GGT), total bile acid (TBA), total bilirubin (TBIL), and direct bilirubin (DBIL) in ANIT-treated mice. Histopathology of the liver tissue showed that pathological damages were relieved upon BE and IG treatment. Meanwhile, the results indicated BE and IG notably restored relative liver weights, inhibited oxidative stress induced by ANIT through increasing hepatic level of superoxide dismutase (SOD), reduced glutathione (GSH), catalase (CAT) and decreasing hepatic content of malondialdehyde (MDA). Western blot revealed that BE and IG inhibited the expression of pro-inflammatory factors TGF-α, IL-6 and NF-κB. Furthermore, the decreased protein expression of bile acid transporters NTCP, BSEP, MRP2 were upregulated by BE and IG in a dose-dependent manner. The results have demonstrated that BE and IG exhibited a dose-dependently protective effect against ANIT-induced liver injury with acute intrahepatic cholestasis in mice, which might be related to the regulation of oxidative stress, inflammatory response and bile acid transport. In addition, these findings pointed out that iridoid glycosides as main active components of V.ciliata play a critical role in hepatoprotective effect of V.ciliata. Schematic representation of possible mechanism of V.ciliata n-Butanol extract (BE) and iridoid glycosides (IG) on protective effects against ANIT-induced cholestatic liver injury. Image 1 [ABSTRACT FROM AUTHOR]

Introduction: Constipation-predominant irritable bowel syndrome (IBS-C) is a functional gastrointestinal (GI) disorder with an unknown etiology. A number of the drugs tested for IBS-C have also been applied to chronic constipation and chronic idiopathic constipation. Unfortunately, due to severe adverse effects, many drugs envisioned for IBS-C had been withdrawn from the market. Nevertheless, a number of potential new agents for this indication are now under development., Areas Covered: The following review describes the most recently developed agents in preclinical as well as Phase 1 and Phase 2 clinical studies. Information was obtained from published literature, abstracts and the latest results found in Clinicaltrial.gov database. The authors put a special interest on glucagon-like peptide 1 analogue, bile acid modulators, serotonergic agents, guanylate cyclase C and cannabinoid antagonists., Expert Opinion: To enter the market, a newly-developed drug has to meet several criteria, such as good bioavailability or the absence of drug-related adverse events. Taking into account constipation and abdominal pain as the main symptoms in IBS-C, a novel successful drug is usually able to improve both at the same time. Four out of fifteen investigational drugs described in this paper belong to the serotonergic family and have a good prognosis to reach the market; still, more long-term clinical studies are warranted.

Introduction: Chronic constipation (CC) is a common gastrointestinal (GI) motility disorder that significantly impairs the quality of life in affected subjects. As almost half of the patients suffering from CC are not satisfied with currently available medicines, there is a need to develop new molecules with better effectiveness and tolerability., Areas Covered: The authors include all experimental and clinical trials (up to Phase II) about new investigational drugs for the treatment of CC. The article identifies nine new agents: mitemcinal, TD-8954, YKP10811, itopride, RM-131, KWA-0711, elobixibat, velusetrag, and naronapride. All nine agents have shown prokinetic effects in different stages of the development. The mechanisms of new developing drugs include: the activation of 5-hydroxytryptamine type-4 (5-HT4), ghrelin and motilin receptors, antagonizing dopamine type-2 (D2) receptors, inhibition of ileal bile acid reabsorption and acetylcholine esterase, as well as water absorption from the GI tract., Expert Opinion: At this current point in time, new generations of 5-HT4 receptor agonists (velusetrag, noranopride and YKP10811) are hoped to progress, further in the future, due to better efficiency and safety. However, it is not possible to make a concise conclusion at this current time due to a lack of evidence. Further clinical trials with a longer duration and a larger sample size are warranted.