Pubblicazioni 2019
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Biagioli M, Carino A, Fiorucci C, Annunziato G, Marchianò S, Bordoni M, Roselli R, Giorgio CD, Castiglione F, Ricci P, Bruno A, Faccini A, Distrutti E, Baldoni M, Costantino G, Fiorucci S. The Aryl Hydrocarbon Receptor (AhR) Mediates the Counter-Regulatory Effects of Pelargonidins in Models of Inflammation and Metabolic Dysfunctions. Nutrients. 2019 Aug 7;11(8):1820.
Abstract
Pelargonidins are anthocyanidins thought to be beneficial for the human health, although controversies exist over the doses needed and the unclear mechanism of action, along with poor systemic bioavailability. One putative target of pelargonidins is the aryl hydrocarbon receptor (AhR). A synthetic pelargonidin (Mt-P) was synthesized by the methylation of the pelargonidin (the natural compound indicated as P). Mt-P transactivated the AhR with an EC50 of 1.97 µM and was ~2-fold more potent than the natural compound. In vitro Mt-P attenuated pro-inflammatory activities of Raw264.7 macrophage cells in an AhR-dependent manner. In vivo, administration of the Mt-P in Balb/c mice resulted in a dose-dependent attenuation of signs and symptoms of colitis induced by TNBS. A dose of 5 mg/kg Mt-P, but not the natural compound P, reversed intestinal inflammation and increased expression of Tnf-α, Ifn-ƴ, and Il-6, while promoted the expansion of regulatory T cells and M2 macrophages. In C57BL/6J mice fed a high fat diet (HFD), Mt-P attenuated body weight gain, intestinal and liver inflammation, and ameliorated insulin sensitivity, while worsened liver steatosis by up-regulating the liver expression of Cd36 and Apo100b. These effects were abrogated by AhR gene ablation. Mt-P is a synthetic pelargonidin endowed with robust AhR agonist activity that exerts beneficial effects in murine models of inflammation and metabolic dysfunction.
Carino A, Biagioli M, Marchianò S, Fiorucci C, Zampella A, Monti MC, Scarpelli P, Ricci P, Distrutti E, Fiorucci S. Ursodeoxycholic acid is a GPBAR1 agonist and resets liver/intestinal FXR signaling in a model of diet-induced dysbiosis and NASH. Biochim Biophys Acta Mol Cell Biol Lipids. 2019 Oct;1864(10):1422-1437.
Abstract
Obeticholic acid (OCA) is a farnesoid-X-receptor (FXR) ligand, shown effective in reducing steatosis and fibrosis in NASH patients. However, OCA causes major side effects including pruritus, while increases the risk for liver decompensation in cirrhotic patients. Ursodeoxycholic acid (UDCA), is a safe and unexpensive bile acid used in the treatment of liver disorders whose mechanism of action is poorly defined. Here we have compared the effects of OCA and UDCA in a mouse model of NASH. In mice exposed to a diet rich in fat/cholesterol and fructose (HFD-F), treatment with OCA or UDCA effectively prevented body weight gain, insulin resistance, as demonstrated by OGTT, and AST plasma levels. After 12 weeks HFD-F mice developed liver microvesicular steatosis, inflammation and mild fibrosis, increased expression of inflammatory (TNFα, IL6, F4/80) and fibrosis (αSma, Col1α1, Tgfβ) markers, reduced liver expression of FXR, dysregulated liver FXR signaling and elevated levels of Tauro-α and β-muricholic acid (T-α and βMCA), two FXR antagonists in mice. Both compounds prevented these changes and improved liver histopathology. OCA reduced primary bile acid synthesis worsening the T-CA/T-βMCA ratio. UDCA effectively transactivated GPBAR1 in vitro. By RNAseq analysis we found that among over 2400 genes modulated by the HFD-F, only 32 and 60 genes were modulated by OCA and UDCA, with only 3 genes (Dbp, Adh7, Osgin1) being modulated by both agents. Both agents partially prevented the intestinal dysbiosis. CONCLUSIONS: UDCA is a GPBAR1 ligand and exerts beneficial effects in a rodent model of NASH by activating non-overlapping pathway with OCA.
Fiorucci S, Distrutti E. Chenodeoxycholic Acid: An Update on Its Therapeutic Applications. Handb Exp Pharmacol. 2019;256:265-282.
Abstract
Chenodeoxycholic acid (CDCA), 3α,7α-dihydroxy-5β-cholan-24-oic acid, is a primary bile acid generated in the liver from cholesterol. In liver cells CDCA is conjugated with glycine or taurine to form two bile salts, Glyco-CDCA and Tauro-CDCA, before being released into the bile ducts. In the intestine, CDCA is further metabolized to generate a 7β epimer, i.e., the ursodeoxycholic acid (UDCA), or dehydroxylate to generate lithocolic acid (LCA). In humans, CDCA is the physiological ligand for the bile acid sensor farnesoid X receptor (FXR), while LCA is a potent agonist for a G protein-coupled receptor, known as GPBAR1 (TGR5). Along with UDCA, CDCA has been clinically used for the dissolution of gallbladder stones at doses ranging from 375 to 750 mg/day, with a success rate of 8 to 18%. Because the efficacy of CDCA was significantly lower than that of UDCA and 18-30% of patients developed significant side effects, the most frequent being diarrhea and a reversible increase in aminotransferases plasma levels, this application has lost its therapeutic relevance. Additionally, the combination of CDCA with UDCA, generally at doses of 5-10 mg/kg each, has failed to provide significant advantages over UDCA alone. In 2017, CDCA has been approved as an orphan indication for the treatment of patients with cerebrotendinous xanthomatosis (CTX), a rare autosomal recessive disorder caused by mutations of sterol 27-hydroxylase (CYP27A1) gene. Since CYP27A1 is essential for cholesterol breakdown, CTX patients develop abnormal lipid storage with increased plasma and tissue levels of cholestanol and very low/absent production of CDCA. CDCA is a potent inhibitor of CYP27A1, and early initiation of CDCA therapy, at doses up to 750 mg/day, is considered the standard medical therapy for CTX resulting in decreased plasma levels of cholestanol and stabilization of neurologic symptoms. Studies in CTX patients have also shown that CDCA might suppress the activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase in the liver. Furthermore, CDCA promotes the release of glucagon-like peptide-1 (GLP-1) in diabetic patients, likely by activating GPBAR1.
Biagioli M, Carino A, Fiorucci C, Marchianò S, Di Giorgio C, Roselli R, Magro M, Distrutti E, Bereshchenko O, Scarpelli P, Zampella A, Fiorucci S. GPBAR1 Functions as Gatekeeper for Liver NKT Cells and provides Counterregulatory Signals in Mouse Models of Immune-Mediated Hepatitis. Cell Mol Gastroenterol Hepatol. 2019;8(3):447-473.
Abstract
Background & aims: GPBAR1, also known as TGR5, is a G protein-coupled receptor activated by bile acids. Hepatic innate immune cells are involved in the immunopathogenesis of human liver diseases and in several murine hepatitis models. Here, by using genetic and pharmacological approaches, we provide evidence that GPBAR1 ligation attenuates the inflammation in rodent models of hepatitis.
Material and methods: Hepatitis was induced by concanavalin A (Con A) or α-galactosyl-ceramide (α-GalCer). 6b-Ethyl-3a,7b-dihydroxy-5b-cholan-24-ol (BAR501), a selective agonist of GPBAR1, was administrated by o.s.
Results: In the mouse models of hepatitis, the genetic ablation of Gpabar1 worsened the severity of liver injury and resulted in a type I NKT cells phenotype that was biased toward a NKT1, a proinflammatory, IFN-γ producing, NKT cells subtype. Further on, NKT cells from GPBAR1-/- mice were sufficient to cause a severe hepatitis when transferred to naïve mice. In contrast, GPBAR1 agonism rescued wild-type mice from acute liver damage and redirects the NKT cells polarization toward a NKT10, a regulatory, IL-10 secreting, type I NKT cell subset. In addition, GPBAR1 agonism significantly expanded the subset of IL-10 secreting type II NKT cells. RNAseq analysis of both NKT cells type confirmed that IL-10 is a major target for GPABR1. Accordingly, IL-10 gene ablation abrogated protection afforded by GPBAR1 agonism in the Con A model.
Conclusion: Present results illustrate a role for GPBAR1 in regulating liver NKT ecology. Because NKT cells are an essential component of liver immune system, our data provide a compelling evidence for a GPBAR1-IL-10 axis in regulating of liver immunity.
Fiorucci S, Distrutti E. The Pharmacology of Bile Acids and Their Receptors. Handb Exp Pharmacol. 2019;256:3-18.
Abstract
This review provides a historical perspective of bile acids and their receptors as therapeutic targets. Bile acids are atypical steroids generated by the liver from cholesterol and have been used for almost half a century for treating liver and biliary disorders. Since the early 1970s of the last century, chenodeoxycholic acid (CDCA), a primary bile acid, and ursodeoxycholic acid (UDCA), a secondary bile acid and the 7βepimer of CDCA, have been shown effective in promoting the dissolution of cholesterol gallstones. However, lack of activity and side effects associated with the use of CDCA, along with the advent of laparoscopic cholecystectomy, have greatly reduced the clinical relevance of this application. At the turn of the century, however, the discovery that bile acids activate specific receptors, along with the discovery that those receptors are placed at the interface of the host and intestinal microbiota regulating physiologically relevant enterohepatic and entero-pancreatic axes, has led to a “bile acid renaissance.” Similarly to other steroids, bile acids bind and activate both cell surface and nuclear receptors, including the bile acid sensor farnesoid X receptor (FXR) and a G-protein-coupled bile acid receptor, known as GPBAR1 (TGR5). Both receptors have been proved druggable, and several highly potent, selective, and nonselective ligands for the two receptors have been discovered in the last two decades. Currently, in addition to obeticholic acid, a semisynthetic derivative of CDCA and the first in class of FXR ligands approved for clinical use, either selective or dual FXR and GPBAR1 ligands, have been developed, and some of them are undergoing pre-approval trials. The effects of FXR and GPBAR1 ligands in different therapeutic area are reviewed.
Fiorucci S, Di Giorgio C, Distrutti E. Obeticholic Acid: An Update of Its Pharmacological Activities in Liver Disorders. Handb Exp Pharmacol. 2019;256:283-295.
Abstract
Obeticholic acid (OCA), 6α-ethyl-3α,7α-dihydroxy-5-cholan-24-oic acid, is a semisynthetic derivative of the chenodeoxycholic acid (CDCA, 3α,7α-dihydroxy-5-cholan-24-oic acid), a relatively hydrophobic primary bile acid synthesized in the liver from cholesterol. OCA, also known as 6-ethyl-CDCA or INT-747, was originally described by investigators at the Perugia University in 2002 as a selective ligand for the bile acid sensor, farnesoid-X-receptor (FXR). In addition to FXR and similarly to CDCA, OCA also activates GPBAR1/TGR5, a cell membrane G protein-coupled receptor for secondary bile acids. In 2016, based on the results of phase II studies showing efficacy in reducing the plasma levels of alkaline phosphatase, a surrogate biomarker for disease progression in primary biliary cholangitis (PBC), OCA has gained approval as a second-line treatment for PBC patients nonresponsive to UDCA. The use of OCA in PBC patients associates with several side effects, the most common of which is pruritus, whose incidence is dose-dependent and is extremely high when this agent is used as a monotherapy. Additionally, the use of OCA associates with the increased risk for the development of liver failure in cirrhotic PBC patients. Currently, OCA is investigated for its potential in the treatment of nonalcoholic steatohepatitis (NASH). Phase II and III trials have shown that OCA might attenuate the severity of liver fibrosis in patients with NASH, but it has no efficacy in reversing the steatotic component of the disease, while reduces the circulating levels of HDL-C and increases LDL-C. In summary, OCA has been the first-in-class of FXR ligands advanced to a clinical stage and is now entering its third decade of life, highlighting the potential benefits and risk linked to FXR-targeted therapies.
Carino A, Marchianò S, Biagioli M, Fiorucci C, Zampella A, Monti MC, Morretta E, Bordoni M, Di Giorgio C, Roselli R, Ricci P, Distrutti E, Fiorucci S. Transcriptome Analysis of Dual FXR and GPBAR1 Agonism in Rodent Model of NASH Reveals Modulation of Lipid Droplets Formation. Nutrients. 2019 May 21;11(5):1132.
Abstract
Non-alcoholic steatohepatitis (NASH) is a progressive, chronic, liver disease whose prevalence is growing worldwide. Despite several agents being under development for treating NASH, there are no drugs currently approved. The Farnesoid-x-receptor (FXR) and the G-protein coupled bile acid receptor 1 (GPBAR1), two bile acid activated receptors, have been investigated for their potential in treating NASH. Here we report that BAR502, a steroidal dual ligand for FXR/GPBAR1, attenuates development of clinical and liver histopathology features of NASH in mice fed a high fat diet (HFD) and fructose (F). By RNAseq analysis of liver transcriptome we found that BAR502 restores FXR signaling in the liver of mice feed HFD-F, and negatively regulates a cluster of genes including Srebf1 (Srepb1c) and its target genes-fatty acid synthase (Fasn) and Cell death-inducing DFF45-like effector (CIDE) genes, Cidea and Cidec-involved in lipid droplets formation and triglycerides storage in hepatocytes. Additionally, BAR502 increased the intestinal expression of Fgf15 and Glp1 and energy expenditure by white adipose tissues. Finally, exposure to BAR502 reshaped the intestinal microbiota by increasing the amount of Bacteroidaceae. In conclusion, we have shown that dual FXR/GPBAR1 agonism might have utility in treatment of NASH.
De Vuono S, Ricci MA, Nulli Migliola E, Monti MC, Morretta E, Boni M, Ministrini S, Carino A, Fiorucci S, Distrutti E, Lupattelli G. Serum Bile Acid Levels Before and After Sleeve Gastrectomy and Their Correlation with Obesity-Related Comorbidities. Obes Surg. 2019 Aug;29(8):2517-2526.
Abstract
Background and aims: The rising prevalence of morbid obesity is increasing the demand for bariatric surgery. The benefits observed after bariatric surgery seems to be not fully explained by surgery-induced weight loss or traditional cardiovascular risk factors regression or improvement. Some evidences suggest that bile acid (BA) levels change after bariatric surgery, thus suggesting that BA concentrations could influence some of the metabolic improvement induced by bariatric surgery. In this report, we have characterized circulating BA patterns and compared them to metabolic and vascular parameters before and after sleeve gastrectomy (SG).
Patients and methods: Seventy-nine subjects (27 males, 52 females, aged 45 ± 12 years, mean BMI 45 ± 7 kg/m2) SG candidates were included in the study. Before and about 12 months after SG, all subjects underwent a clinical examination, blood tests (including lipid profile, plasma glucose and insulin, both used for calculating HOMA-IR, and glycated hemoglobin), ultrasound visceral fat area estimation, ultrasound flow-mediated dilation evaluation, and determination of plasma BA concentrations.
Results: Before SG, both primary and secondary BA levels were higher in insulin-resistant obese subjects than in non-insulin resistant obese, and BA were positively associated with the markers of insulin-resistance. After SG, total (conjugated and unconjugated) cholic acids significantly decreased (p 0.007), and total lithocholic acids significantly increased (p 0.017). SG-induced total cholic and chenodeoxycholic acid changes were directly associated with surgery-induced glycemia (p 0.011 and 0.033 respectively) and HOMA-IR (p 0.016 and 0.012 respectively) changes.
Conclusions: Serum BA are associated with glucose metabolism and particularly with markers of insulin-resistance. SG modifies circulating BA pool size and composition. SG-induced BA changes are associated with insulin-resistance amelioration. In conclusion, an interplay between glucose metabolism and circulating BA exists but further studies are needed.
Festa C, Finamore C, Marchianò S, Di Leva FS, Carino A, Monti MC, Del Gaudio F, Ceccacci S, Limongelli V, Zampella A, Fiorucci S, De Marino S. Investigation around the Oxadiazole Core in the Discovery of a New Chemotype of Potent and Selective FXR Antagonists. ACS Med Chem Lett. 2019 Jan 10;10(4):504-510.
Abstract
Recent findings have shown that Farnesoid X Receptor (FXR) antagonists might be useful in the treatment of cholestasis and related metabolic disorders. In this paper, we report the discovery of a new chemotype of FXR antagonists featured by a 3,5-disubstituted oxadiazole core. In total, 35 new derivatives were designed and synthesized, and notably, compounds 3f and 13, containing a piperidine ring, displayed the best antagonistic activity against FXR with promising cellular potency (IC50 = 0.58 ± 0.27 and 0.127 ± 0.02 μM, respectively). The excellent pharmacokinetic properties make compound 3f the most promising lead identified in this study.
Finamore C, Baronissi G, Marchianò S, Di Leva FS, Carino A, Monti MC, Limongelli V, Zampella A, Fiorucci S, Sepe V. Introduction of Nonacidic Side Chains on 6-Ethylcholane Scaffolds in the Identification of Potent Bile Acid Receptor Agonists with Improved Pharmacokinetic Properties. Molecules. 2019 Mar 16;24(6):1043.
Abstract
As a cellular bile acid sensor, farnesoid X receptor (FXR) and the membrane G-coupled receptor (GPBAR1) participate in maintaining bile acid, lipid, and glucose homeostasis. To date, several selective and dual agonists have been developed as promising pharmacological approach to metabolic disorders, with most of them possessing an acidic conjugable function that might compromise their pharmacokinetic distribution. Here, guided by docking calculations, nonacidic 6-ethyl cholane derivatives have been prepared. In vitro pharmacological characterization resulted in the identification of bile acid receptor modulators with improved pharmacokinetic properties.
Di Leva FS, Festa C, Carino A, De Marino S, Marchianò S, Di Marino D, Finamore C, Monti MC, Zampella A, Fiorucci S, Limongelli V. Discovery of ((1,2,4-oxadiazol-5-yl)pyrrolidin-3-yl)ureidyl derivatives as selective non-steroidal agonists of the G-protein coupled bile acid receptor-1. Sci Rep. 2019 Feb 21;9(1):2504.
Abstract
The G-protein bile acid receptor 1 (GPBAR1) has emerged in the last decade as prominent target for the treatment of metabolic and inflammatory diseases including type 2 diabetes, obesity, and non-alcoholic steatohepatitis. To date numerous bile acid derivatives have been identified as GPBAR1 agonists, however their clinical application is hampered by the lack of selectivity toward the other bile acid receptors. Therefore, non-steroidal GPBAR1 ligands able to selectively activate the receptor are urgently needed. With this aim, we here designed, synthesized and biologically evaluated ((1,2,4-oxadiazol-5-yl)pyrrolidin-3-yl) urea derivatives as novel potent GPBAR1 agonists. Particularly, compounds 9 and 10 induce the mRNA expression of the GPBAR1 target gene pro-glucagon and show high selectivity over the other bile acid receptors FXR, LXRα, LXRβ and PXR, and the related receptors PPARα and PPARγ. Computational studies elucidated the binding mode of 10 to GPBAR1, providing important structural insights for the design of non-steroidal GPBAR1 agonists. The pharmacokinetic properties of 9 and 10 suggest that the ((1,2,4-oxadiazol-5-yl)pyrrolidin-3-yl)ureydil scaffold might be exploited to achieve effective drug candidates to treat GPBAR1 related disorders.
Biagioli M, Capobianco D, Carino A, Marchianò S, Fiorucci C, Ricci P, Distrutti E, Fiorucci S. Divergent Effectiveness of Multispecies Probiotic Preparations on Intestinal Microbiota Structure Depends on Metabolic Properties. Nutrients. 2019 Feb 2;11(2):325.
Abstract
A growing body of evidence suggests that probiotic functionality is not accurately predicted by their taxonomy. Here, we have set up a study to investigate the effectiveness of two probiotic formulations containing a blend of seven bacterial species in modulating intestinal inflammation in two rodent models of colitis, induced by treating mice with 2,4,6-Trinitrobenzenesulfonic acid (TNBS) or dextran sodium sulfate (DSS). Despite the taxonomy of the bacterial species in the two probiotic formulations being similar, only one preparation (Blend 2-Vivomixx) effectively attenuated the development of colitis in both models. In the TNBS model of colitis, Blend 2 reduced the expression of pro-inflammatory genes while increasing the production of anti-inflammatory cytokines, promoting the expansion M2 macrophages and the formation of IL-10-producing Treg cells in the colon’s lamina propria. In the DSS model of colitis, disease attenuation and Treg formation was observed only in mice administered with Blend 2, and this effect was associated with intestinal microbiota remodeling and increased formation of lactate, butyrate, and propionate. None of these effects were observed in mice administered with Blend 1 (VSL#3). In summary, we have shown that two probiotic mixtures obtained by combining taxonomically similar species produced with different manufacturing methods exert divergent effects in mouse models of colitis.
Skledar DG, Carino A, Trontelj J, Troberg J, Distrutti E, Marchianò S, Tomašič T, Zega A, Finel M, Fiorucci S, Mašič LP. Endocrine activities and adipogenic effects of bisphenol AF and its main metabolite. Chemosphere. 2019 Jan;215:870-880.
Abstract
Bisphenol AF (BPAF) is a fluorinated analog of bisphenol A (BPA), and it is a more potent estrogen receptor (ER) agonist. BPAF is mainly metabolized to BPAF-glucuronide (BPAF-G), which has been reported to lack ER agonist activity and is believed to be biologically inactive. The main goal of the current study was to examine the influence of the metabolism of BPAF via glucuronidation on its ER activity and adipogenesis. Also, as metabolites can have different biological activities, the effects of BPAF-G on other nuclear receptors were evaluated. First, in-vitro BPAF glucuronidation was investigated using recombinant human enzymes. Specific reporter-gene assays were used to determine BPAF and BPAF-G effects on estrogen, androgen, glucocorticoid, and thyroid receptor pathways, and on PXR, FXR, and PPARγ pathways. Their effects on lipid accumulation and differentiation were determined in murine 3T3L1 preadipocytes using Nile Red, with mRNA expression analysis of the adipogenic markers adiponectin, Fabp4, Cebpα, and PPARγ. BPAF showed strong agonistic activity for hERα and moderate antagonistic activities for androgen and thyroid receptors, and for PXR. BPAF-G was antagonistic for PXR and PPARγ. BPAF (0.1 μM) and BPAF-G (1.0 μM) induced lipid accumulation and increased expression of key adipogenic markers in murine preadipocytes. BPAF-G is therefore not an inactive metabolite of BPAF. Further toxicological and epidemiological investigations of BPAF effects on human health are warranted, to provide better understanding of the metabolic end-elimination of BPAF.