Human Ileal Bile Acid Transporter Research Articles

Effect of ileal bile acid transporter inhibitor GSK2330672 on pruritus in primary biliary cholangitis: a double-blind, randomised, placebo-controlled, crossover, phase 2a study

Up to 70% of patients with primary biliary cholangitis develop pruritus (itch) during the course of their disease. Treatment of pruritus in primary biliary cholangitis is challenging and novel therapies are needed. Ursodeoxycholic acid, the standard first-line treatment for primary biliary cholangitis, is largely ineffective for pruritus. We investigated the efficacy and safety of GSK2330672, a selective inhibitor of human ileal bile acid transporter (IBAT), in patients with primary biliary cholangitis with pruritus. We conducted this phase 2a, double-blind, randomised, placebo-controlled, crossover trial in two UK medical centres. Following 2 weeks of open placebo run-in, patients were randomly assigned in a 1:1 ratio with a block size of 4 to receive GSK2330672 or placebo twice daily during two consecutive 14-day treatment periods in a crossover sequence. The treatment periods were followed by a 14-day single-blinded placebo follow-up period. The primary endpoints were safety of GSK2330672, assessed using clinical and laboratory parameters, and tolerability as rated by the Gastrointestinal Symptom Rating Scale. The secondary endpoints were changes in pruritus scores measured using the 0 to 10 numerical rating scale (NRS), primary biliary cholangitis-40 (PBC-40) itch domain score and 5-D itch scale, changes in serum total bile acids and 7 alpha hydroxy-4-cholesten-3-one (C4), and changes in the pharmacokinetic parameters of ursodeoxycholic acid and its conjugates. The trial was registered with ClinicalTrials.gov, number NCT01899703. Between March 10, 2014, and Oct 7, 2015, we enrolled 22 patients. 11 patients were assigned to receive intervention followed by placebo (sequence 1), and 11 patients were assigned to receive placebo followed by intervention (sequence 2). One patient assigned to sequence 2 withdrew consent prior to receiving randomised therapy. One patient did not attend the placebo follow-up period, but was included in the final analysis. GSK2330672 treatment for 14 days was safe with no serious adverse events reported. Diarrhoea was the most frequent adverse event during treatment with GSK2330672 (seven with GSK2330672 vs one with placebo) and headache was the most frequent adverse event during treatment with placebo (seven with placebo vs six with GSK2330672). After GSK2330672 treatment, the percentage changes from baseline itch scores were -57% (95% CI -73 to -42, p<0·0001) in the NRS, -31% (-42 to -20, p<0·0001) in the PBC-40 itch domain and -35% (-45 to -25, p<0·0001) in the 5-D itch scale. GSK2330672 produced significantly greater reduction from baseline than the double-blind placebo in the NRS (-23%, 95% CI -45 to -1; p=0·037), PBC-40 itch domain, (-14%, -26 to -1; p=0·034), and 5-D itch scale (-20%, -34 to -7; p=0·0045). After GSK2330672 treatment, serum total bile acid concentrations declined by 50% (95% CI -37 to -61, p<0·0001) from 30 to 15 μM, with a significant 3·1-times increase (95% CI 2·4 to 4·0, p<0·0001) in serum C4 concentrations from 7·9 to 24·7ng/mL. In patients with primary biliary cholangitis with pruritus, 14 days of ileal bile acid transporter inhibition by GSK2330672 was generally well tolerated without serious adverse events, and demonstrated efficacy in reducing pruritus severity. GSK2330672 has the potential to be a significant and novel advance for the treatment of pruritus in primary biliary cholangitis. Diarrhoea, the most common adverse event associated with GSK2330672 treatment, might limit the long-term use of this drug. GlaxoSmithKline and National Institute for Health Research.

S1302 Dose-Related Effects of Chenodeoxycholate on Gastrointestinal and Colonic Transit and Bowel Function in Female Patients With Constipation-Predominant Irritable Bowel Syndrome

Background: A3309 modulates the enterohepatic circulation of bile acids (BA) by inhibiting the intestinal bile acid transporter (IBAT). This will result in an increased concentration of BA in the colon. The synthesis of bile acids in the liver can be measured indirectly by plasma concentrations of the intermediate 7-α-hydroxy-4-cholesten-3-one (C4). Methods: In Vitro part: A3309 potency was tested in transfected HEK293 cells. The cells expressing the various BA transporters were grown in Cytostar 96 w Scintillation Proximity Assay plates and the effect of increasing concentrations of A3309 on accumulation of 30 μM of radiolabelled glycocholic acid was monitored. In Vivo part: In dogs, constipation was induced by shifting the standard diet to ground beef dosed once daily until the faeces weight was <30 g/day. Vehicle or test compounds (A3309 or the active comparator tegaserod) were administered once daily for 3 days, and faeces was collected and weighed on a daily basis. The pharmacodynamic effect of A3309 was investigated in a 1-month study in 24 dogs by measuring the plasma concentration of C4. Results: A3309 was found to be highly potent and selective for IBAT with IC50 =0.53±0.17, 0.13±0.03 and 5.8±1.6 nM for the human, mouse and canine transporters, respectively, whereas the corresponding value for the human liver (basolateral) sodium/bile acid co-transporter was found to be 0.24±0.02 μM (400-fold higher than for IBAT inhibition). Furthermore, inhibition of amino acid uptake via the neutral amino acid transporter(s) in the HEK293 cells was at least 1000 times lower than that of BA transport via the human ileal bile acid transporter. In the constipated dog, A3309 increased faeces weight from 9.4+12.4 g/day (mean±SEM n=36) after vehicle to 25.8 g/day (1.5 μmol/kg, n=2), 34.4 g/day (5 μmol/kg, n=2), 31.3±4,5 g/day (15 μmol/kg, n=6) and for tegaserod 14 g/day (3.3 μmol/kg, n=1), 18.1±10,2g/day (3.3 μmol /kgx2, n=3), 23.9±7,1 g/day (10 μmol /kg, n=3) and 68.3 g/day (33 μmol /kgx2, n=2). In the dog, a dose dependant increase of C4 in plasma was observed already after single doses. After 26 days treatment with A3309 the plasma concentration of C4 increased 3 to 7 fold (43.0±8.43,150±48.8 ,190±47.0 and 350±49.0 nmol/L, pre-values, 5, 25, 200 μmol/kg, respectivly). Conclusion: A3309 was found to be highly potent and selective for IBAT and ameliorate meat-induced constipation in dogs. The increased BA synthesis effect last for at least 1 month. IBAT inhibitors have a potential clinical utility in the treatment of constipation.

Synthesis and Biological Evaluation of Novel Steroidal Pyrazoles as Substrates for Bile Acid Transporters.

A series of novel steroidal pyrazoles was synthesized as substrates for bile acid transporters to explore their potential as drug carriers. The selected pyrazole fused bile acids were further conjugated with drugs and drug surrogates. Their in vitro transport activities were evaluated in human ileal bile acid transporter (hIBAT) and human liver bile acid transporter (hLBAT) expressing Chinese hamster ovary (CHO)-cells and Xenopus laevis oocytes. The results of synthetic efforts and transporter assays studies are described herein.

Synthesis and biological evaluation of novel steroidal pyrazoles as substrates for bile acid transporters

A series of novel steroidal pyrazoles was synthesized as substrates for bile acid transporters to explore their potential as drug carriers. The selected pyrazole fused bile acids were further conjugated with drugs and drug surrogates. Their in vitro transport activities were evaluated in human ileal bile acid transporter (hIBAT) and human liver bile acid transporter (hLBAT) expressing Chinese hamster ovary (CHO)-cells and Xenopus laevis oocytes. The results of synthetic efforts and transporter assays studies are described herein.

Bile acid-induced negative feedback regulation of the human ileal bile acid transporter.

Ileal expression of the apical sodium-dependent bile acid transporter (ASBT) in the rat is unaffected by bile salts, yet in the mouse it is under negative-feedback regulation. The bile acid responsiveness of human ASBT is unknown. The human ASBT promoter linked to a luciferase reporter was studied in Caco-2 cells treated with chenodeoxycholic acid (CDCA) and transfected with expression plasmids for farnesoid X-receptor (FXR), short heterodimer partner (SHP), and retinoic acid receptor/retinoid X receptor (RAR/RXR). CDCA treatment of Caco-2 cells led to a 75% reduction in steady-state ASBT messenger RNA levels and a 78% reduction in human ASBT promoter activity. A dominant negative FXR abrogated the response to CDCA. Site-directed mutagenesis of an RAR/RXR cis element in the human ASBT promoter reduced its activity by 50% and eliminated the bile acid response. Retinoic acid activated the human ASBT promoter fourfold. SHP repressed the activity of the ASBT promoter and reduced activation by retinoic acid. Antisense mediated knock-down of SHP in Caco-2 cells partially offset the bile acid mediated repression of ASBT promoter activity. In conclusion, the human ASBT is positively regulated by retinoic acid. Bile acids induce a negative feedback regulation of human ASBT via an FXR-mediated, SHP-dependent effect upon RAR/RXR activation of ASBT.

Human ileal bile acid transporter gene ASBT (SLC10A2) is transactivated by the glucocorticoid receptor

Background: Patients with Crohn’s disease suffer from intestinal bile acid malabsorption. Intestinal bile acid absorption is mediated by the apical sodium dependent bile acid transporter ASBT/IBAT ( SLC10A2 ). In rats, ASBT is induced by glucocorticoids. Aims: To study whether human ASBT is activated by glucocorticoids and to elucidate the mechanism of regulation. Patients and methods: ASBT expression in ileal biopsies from patients with Crohn’s disease and from healthy subjects was quantified by western blot. ASBT promoter function was studied in luciferase assays and by electrophoretic mobility shift assay. Results: In 16 patients with Crohn’s disease, ASBT expression was reduced to 69 (7.5)% compared with healthy controls (mean (SEM); p = 0.01). In 10 healthy male volunteers, ASBT protein expression was increased 1.34 (0.11)-fold (mean (SEM); p Conclusions: Human ASBT is induced by glucocorticoids in vitro and in vivo. Induction of ASBT by glucocorticoids could be beneficial in patients with Crohn’s disease who exhibit reduced ASBT expression. This study identifies ASBT as a novel target of glucocorticoid controlled gene regulation in the human intestine.

Bile acid induced negative feedback regulation of the human ileal bile acid transporter is mediated by the farnesoid X-receptor (FXR), the short heterodimer partner (SHP), and the retinoid X receptor (RAR/RXR)

Bile acid induced negative feedback regulation of the human ileal bile acid transporter is mediated by the farnesoid X-receptor (FXR), the short heterodimer partner (SHP), and the retinoid X receptor (RAR/RXR)

Introductory Lecture: In Vitro Translation Analysis of Integral Membrane Proteins

A method of in vitro translation scanning was applied to a variety of polytopic integral membrane proteins, a transition metal P type ATPase from Helicobacter pylori, the SERCA 2 ATPase, the gastric H+,K+ ATPase, the CCK-A receptor and the human ileal bile acid transporter. This method used vectors containing the N terminal region of the gastric H+,K+ ATPase or the N terminal region of the CCK-A receptor, coupled via a linker region to the last 177 amino acids of the β-subunit of the gastric H+,K+ ATPase. The latter contains 5 potential N-linked glycosylation sites. Translation of vectors containing the cDNA encoding one, two or more putative transmembrane domains in the absence or presence of microsomes allowed determination of signal anchor or stop transfer properties of the putative transmembrane domains by the molecular weight shift on SDS PAGE. The P type ATPase from Helicobacter pylori showed the presence of 8 transmembrane segments with this method. The SERCA 2 Ca2+ ATPase with this method had 9 transmembrane co-translational insertion domains and coupled with other evidence these data resulted in a 11 transmembrane segment model. Translation of segments of the gastric H+,K+ ATPase provided evidence for only 7 transmembrane segments but coupled with other data established a 10 membrane segment model. The G7 protein, the CCK-A receptor showed the presence of 6 of the 7 transmembrane segments postulated for this protein. Translation of segments of the human ileal bile acid transporter showed the presence of 8 membrane insertion domains. However, translation of the intact protein provided evidence for an odd number of transmembrane segments, resulting in a tentative model containing 7 or 9 transmembrane segments. Neither G7 type protein appeared to have an arrangement of sequential topogenic signals consistent with the final assembled protein. This method provides a useful addition to methods of determining membrane domains of integral membrane proteins but must in general be utilized with other methods to establish the number of transmembrane α-helices.

Identification of a dysfunctional mutation in the human ileal bile acid transporter gene: [formula omitted] Dept. of Internal Medicine, Bowman Gray School of Medicine, Winston-Salem, NC 27157

Identification of a dysfunctional mutation in the human ileal bile acid transporter gene: [formula omitted] Dept. of Internal Medicine, Bowman Gray School of Medicine, Winston-Salem, NC 27157