Impaired Itching Perception in Murine Models of Cholestasis Is Supported by Dysregulation of GPBAR1 Signaling.

In cholestatic syndromes, body accumulation of bile acids is thought to cause itching. However, the mechanisms supporting this effect remain elusive. Recently, GPBAR1 (TGR5) a G-protein coupled receptor has been shown to mediate itching caused by intradermal administration of DCA and LCA. 6α-ethyl-3α, 7α-dihydroxy-24-nor-5β-cholan-23-ol (BAR502) is a non-bile acid dual ligand for FXR and GPBAR1.Cholestasis was induced in wild type and GPBAR1-/- mice by administration of α-naphthyl-isothiocyanate (ANIT) or 17α-ethynylestradiol.In naïve mice skin application of DCA, TLCA, 6-ECDCA, oleanolic and betulinic acid induces a GPBAR1 dependent pruritogenic response that could be desensitized by re-challenging the mice with the same GPBAR1 agonist. In wild type and GPBAR1-/- mice cholestasis induced by ANIT fails to induce spontaneous itching and abrogates scratching behavior caused by intradermal administration of DCA. In this model, co-treatment with BAR502 increases survival, attenuates serum alkaline phosphatase levels and robustly modulates the liver expression of canonical FXR target genes including OSTα, BSEP, SHP and MDR1, without inducing pruritus. Betulinic acid, a selective GPBAR1 ligand, failed to rescue wild type and GPBAR1-/- mice from ANIT cholestasis but did not induced itching. In the 17α-ethynylestradiol model BAR502 attenuates cholestasis and reshapes bile acid pool without inducing itching.The itching response to intradermal injection of GPBAR1 agonists desensitizes rapidly and is deactivated in models of cholestasis, explain the lack of correlation between bile acids levels and itching severity in cholestatic syndromes. In models of non-obstructive cholestasis, BAR502 attenuates liver injury without causing itching

In vitro pharmacological characterization of BAR502.

<p>(A) Chemical structure of BAR502. (B-D) Serum starved HepG2 cells were stimulated 18 h with 10 M CDCA, 6-ECDCA or BAR501 and the relative mRNA expression of OSTα (B), BSEP (C) and SHP (D) was assayed by RT-PCR. Results are the mean ± SE of three experiments. *p<0.05 versus not treated cells (NT). (E) Serum starved THP-1 cells were stimulated with BAR501 or forskolin as indicated in materials and methods. At the end of stimulation intracellular cAMP levels were measured. (F) Glutag cells were stimulated 18 h with 10 M TLCA or BAR501 and the relative mRNA expression of pro-glucagon was assayed by RT-PCR. Results are the mean ± SE of three experiments. *p<0.05 versus not treated cells (NT). (G-L) Effect of FXR silencing on activity of BAR502 on FXR target genes. HepG2 cells were left untransfected or transfected with a cocktail of four siRNA directed against FXR. 48 hours post transfection cells were stimulated 18 with BAR502 (10 M) and the relative mRNA expression of FXR (G), OSTα (H), SHP (I) and BSEP (L) were assayed by RT-PCR. (M-N) Effect of FXR antagonism by suvanine. Serum starved HepG2 cells were stimulated 18 hours with BAR502 (10 M) or with the combination of BAR 502 plus suvanine (50 M). At the end of stimulation the relative mRNA expression of OSTα (M) and SHP (N) were assayed by RT-PCR.</p

Effects of administration of BAR502 on cholestasis induced by 17αE₂ for 8 days on relative mRNA expression of (A) OSTα, (B) NTCP, (C) BSEP, (D) MDR1, (E) SHP, (F) Cyp7α1 and (G) FXR.

<p>Values are normalized relative to GAPDH mRNA and are expressed relative to those of control animals, which were arbitrarily set to 1. Results are the mean ± SE of 4–8 mice per group. *p<0.05 versus control group (CTRL); #p<0.05 versus 17αE_<b>2</b> treated mice.</p

Quantitative evaluation of serum and gallbladder composition of bile acids after administration of 17αE₂ alone or 17αE₂ plus BAR502.

<p>(A) Gallbladder weight. (B-C) Quantitative evaluation of serum (B) and gallbladder (C) non conjugated bile acids from control animals (CTRL), animals treated with 17αE_<b>2</b> or animals co-administered with 17αE_<b>2</b> plus BAR502. (D-E) Quantitative evaluation of serum (D) and gallbladder (E) conjugated bile acids from either control animals (CTRL), animals treated with 17αE_<b>2</b> or animals co-administered with 17αE_<b>2</b> plus BAR502. Results are the mean ± SE of 4–8 animals per group. *p<0.05 versus control group (CTRL); #p<0.05 versus 17αE_<b>2</b> treated mice.</p

Dual FXR/TGR5 agonist BAR502 improves liver injury in both GPBAR1^+/+ and GPBAR1^-/- mice.

<p>GPBAR1^<b>+/+</b> and GPBAR1^<b>-/-</b> mice were treated for 10 days with ANIT or with the combination of ANIT plus BAR502. Serum levels of (A) bilirubin, (B) Alkaline phosphatase and (C) AST. Results are the mean ± SE of 4–8 mice per group. *p<0.05 versus GPBAR^<b>+/+</b> mice. °p<0.05 versus GPBAR1^<b>+/+</b> mice treated with ANIT. **p<0.05 versus GPBAR1^<b>-/-</b> mice. #p<0.05 versus GP-BAR^<b>-/-</b> mice administered with ANIT. (D) Representative histological pictures of H&E-stained livers. (E) Analysis of the number of lesions per field. Results are the mean ± SE of 4–8 mice per group. *p<0.05 versus GPBAR1^<b>+/+</b> mice. °p<0.05 versus GPBAR1^<b>+/+</b> mice treated with ANIT. **p<0.05 versus GPBAR1^<b>-/-</b> mice. #p<0.05 versus GPBAR1^<b>-/-</b> mice administered with ANIT.</p

Effects of BAR502 on scratching behavior in GPBAR1^+/+ and-^/- mice administered with ANIT.

<p>GPBAR1^<b>+/+</b> and-^<b>/-</b> mice were treated for 10 days with ANIT or with the combination of ANIT plus BAR502. At the end of the treatments mice were subjected to intradermal injection of DCA. (A-B) Survival of GPBAR1^<b>+/+</b> and-^<b>/-</b> mice in response to ANIT administration. (C) Effect of ANIT on spontaneous scratching and scratching induced by DCA. Scratching tests were performed at day 5. Results are expressed as the number of scratching events during 60 minutes of observation. Results are the mean ± SE of 4–8 mice per group. *p<0.05 versus GPBAR1^<b>+/+</b> mice subjected to intradermal injection of DCA. #p<0.05 versus GPBAR1^<b>-/-</b> mice subjected to intradermal injection of DCA.</p

Effect of betulinic acid on ANIT induced cholestasis in GPBAR1^+/+ and-^/- mice.

<p>GPBAR1^<b>+/+</b> and GPBAR1^<b>-/-</b> mice were administered with ANIT or with the combination of ANIT plus betulinic acid for 5 days. (A) Body weight; (B) Scratching test was performed at day 5. Results are expressed as the number of scratching events during 60 minutes of observation. Results are the mean ± SE of 4–8 mice per group. (C) Serum levels of AST, (D) Bilirubin and (E) Alkaline phosphatase. Results are the mean ± SE of 4–8 mice per group. *p<0.05 versus GP-BAR^<b>+/+</b> mice. **p<0.05 versus GPBAR1^<b>-/-</b> mice. (F) Representative histological pictures of H&E-stained livers.</p

Effects of BAR502 on scratching behavior in GPBAR1^+/+ and GPBAR1^-/- mice.

<p>(A) GPBAR1^<b>+/+</b> and GPBAR1^<b>-/-</b> mice were subjected to intradermal injection of DCA, TLCA, betulinic acid, oleanolic acid, PAR-2 agonist and BAR502. Each agent was tested at the dose of 25 μg. Results are expressed as the number of scratching events during 60 minutes of observation. Results are the mean ± SE of 4–8 mice per group. °p<0.05 versus control group (CTRL); *p<0.05 versus GPBAR1^<b>+/+</b> mice. B-D. Desensitization of the itching pathway by a second administration of a GPBAR1 ligand. Animals were administered DCA, TLCA or betulinic acid at the dose of 50 μg. After counting scratching for 60 minutes animals were rechallenged with a second dose of the given agonist and the scratching response was recorded for a further 60 minutes of observation. Data are mean ± SE of 6–7 animals per group.] p>0.01 versus first dose.</p

BAR502 is dual FXR and GPBAR1 ligand.

<p>Data are mean ± SE of 3 experiments in duplicate</p><p>* Activity of BAR502 toward GPBAR1 in a reporter assay was assessed in HEK293T cells transfected with a cAMP responsive element (CRE) cloned upstream to the luciferase gene (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129866#sec006" target="_blank">Materials and Methods</a>). For calculation of efficacy data, maximal transactivation of CRE caused by BAR502 (10 μM) was compared to maximal transactivation caused by TLCA (10 μM).</p><p>** Activity of BAR502 toward FXR in a reporter assay was assessed in HepG2 cells transfected with a FXR responsive element (FRE) cloned upstream to the luciferase gene (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0129866#sec006" target="_blank">Materials and Methods</a>). For calculation of efficacy data, maximal transactivation of FRE caused by BAR502 (10 μM) was compared to maximal transactivation caused by 6-ECDCA (10 μM).</p><p>BAR502 is dual FXR and GPBAR1 ligand.</p

BAR502 reduces inflammatory cytokines and regulates canonical FXR target genes during ANIT induced cholestasis.

<p>GPBAR1^<b>+/+</b> and GPBAR1^<b>-/-</b> mice were treated for 10 days with ANIT or with the combination of ANIT plus BAR502. Quantitative Real-Time PCR of (A) IL1β, (B) IL6, (C) TNFα, (D) SHP, (E) OSTα, (F) BSEP, (G) MDR1, (H) Cyp7α1, (I) FXR and (L) NTCP. Results are the mean ± SE of 4–8 mice per group. *p<0.05 versus GPBAR1^<b>+/+</b> mice. #p<0.05 versus GPBAR1^<b>+/+</b> mice treated with ANIT. **p<0.05 versus GPBAR1^<b>-/-</b> mice. °p<0.05 versus GPBAR1^<b>-/-</b> mice administered ANIT.</p