Stefano Orlandi

The third gas: H2S regulates perfusion pressure in both the isolated and perfused normal rat liver and in cirrhosis

The regulation of sinusoidal resistance is dependent on the contraction of hepatic stellate cells (HSC) around sinusoidal endothelial cell (SEC) through paracrine cross‐talk of vasoconstrictor and vasodilator agents. Hydrogen sulfide (H2S), a recently discovered gas neurotransmitter, is a putative vasodilator whose role in hepatic vascular regulation and portal hypertension is unexplored. Four‐week bile duct–ligated (BDL) rats with cirrhosis and control rats were treated daily with NaHS (56 μmol/kg) for 5 days. Isolated livers were perfused first with NaHS for 20 minutes and then with norepinephrine (NE) and the intrahepatic resistance studied. In normal rats and animals with cirrhosis, administration of NE resulted in a dose‐dependent increase of portal pressure. This effect was attenuated by H2S treatment (P < .05). The H2S‐induced relaxation of hepatic microcirculation was attenuated by glibenclamide, an adenosine triphosphate (ATP)‐sensitive K+ channel inhibitor. L‐Cysteine, a substrate of cystathionine‐gamma‐lyase (CSE), decreased vasoconstriction in normal rat livers (P < .05) but failed to do so in livers with cirrhosis. BDL resulted in a downregulation of CSE mRNA/protein levels and activity (P < .05). Our in vitro data demonstrate that CSE is expressed in hepatocytes, HSCs, but not in sinusoidal endothelial cells (SEC). HSC activation downregulates CSE mRNA expression, resulting in a defective production of H2S and abrogation of relaxation induced by L‐cysteine. In conclusion, CSE‐derived H2S is involved in the maintenance of portal venous pressure. The reduction of CSE expression in the liver with cirrhosis contributes to the development of increased intrahepatic resistance and portal hypertension. (HEPATOLOGY 2005.)

Enhanced activity of a hydrogen sulphide-releasing derivative of mesalamine (ATB-429) in a mouse model of colitis.

Mesalamine is the first‐line therapy for colitis, but it lacks potency and is only effective for mild‐to‐moderate forms of this disease. Hydrogen sulphide has been shown to be a potent, endogenous anti‐inflammatory substance, modulating leukocyte‐endothelial adhesion and leukocyte migration. The purpose of this study was to determine if an H2S‐releasing derivative of mesalamine (ATB‐429) would exhibit increased potency and effectiveness in a mouse model of colitis.

5-Amino-2-hydroxybenzoic Acid 4-(5-Thioxo-5H-[1,2]dithiol-3yl)-phenyl Ester (ATB-429), a Hydrogen Sulfide-Releasing Derivative of Mesalamine, Exerts Antinociceptive Effects in a Model of Postinflammatory Hypersensitivity

H2S functions as a neuromodulator and exerts anti-inflammatory activities. Recent data indicate that irritable bowel syndrome (IBS) is linked to inflammation of the gastrointestinal tract. In this study, we have investigated the role of a novel H2S-releasing derivative of mesalamine (5-amino-2-hydroxybenzoic acid 4-(5-thioxo-5H-[1,2]dithiol-3yl)-phenyl ester, ATB-429) in modulating nociception to colorectal distension (CRD), a model that mimics some features of IBS, in healthy and postcolitic rats. Four graded (0.4-1.6 ml of water) CRDs were produced in conscious rats, and colorectal sensitivity and pain were assessed by measuring the abdominal withdrawal response and spinal c-Fos expression. In healthy rats, ATB-429 dose dependently (25, 50, or 100 mg/kg) attenuated CRD-induced hypersensitivity and significantly inhibited CRD-induced overexpression of spinal c-FOS mRNA, whereas mesalamine had no effect. ATB-429-induced antinociception was reversed by glibenclamide, a ATP-sensitive K+ (KATP) channel inhibitor. The antinociceptive effect of ATB-429 was maintained in a rodent model of postinflammatory hypersensitivity (4 weeks after colitis induction). At a dose of 100 mg/kg, ATB-429 reversed the allodynic response caused by CRD in postcolitic rats. Colonic cyclooxygenase-2 and interkeukin-1β mRNA and spinal c-FOS mRNA expression were significantly down-regulated by ATB-429, but not by mesalamine. ATB-429, but not mesalamine, increased blood concentrations of H2S in both healthy and postcolitic rats. Taken together, these data suggest that ATB-429 inhibits hypersensitivity induced by CRD in both healthy and postcolitic, allodynic rats by a KATP channel-mediated mechanism. This study provides evidence that H2S-releasing drugs might have beneficial effects in the treatment of painful intestinal disorders.

Functional Characterization of the Semisynthetic Bile Acid Derivative INT-767, a Dual Farnesoid X Receptor and TGR5 Agonist

Two dedicated receptors for bile acids (BAs) have been identified, the nuclear hormone receptor farnesoid X receptor (FXR) and the G protein-coupled receptor TGR5, which represent attractive targets for the treatment of metabolic and chronic liver diseases. Previous work characterized 6α-ethyl-3α,7α-dihydroxy-5β-cholan-24-oic acid (INT-747), a potent and selective FXR agonist, as well as 6α-ethyl-23(S)-methyl-3α,7α,12α-trihydroxy-5β-cholan-24-oic acid (INT-777), a potent and selective TGR5 agonist. Here we characterize 6α-ethyl-3α,7α,23-trihydroxy-24-nor-5β-cholan-23-sulfate sodium salt (INT-767), a novel semisynthetic 23-sulfate derivative of INT-747. INT-767 is a potent agonist for both FXR (mean EC50, 30 nM by PerkinElmer AlphaScreen assay) and TGR5 (mean EC50, 630 nM by time resolved-fluorescence resonance energy transfer), the first compound described so far to potently and selectively activate both BA receptors. INT-767 does not show cytotoxic effects in HepG2 cells, does not inhibit cytochrome P450 enzymes, is highly stable to phase I and II enzymatic modifications, and does not inhibit the human ether-a-go-go-related gene potassium channel. In line with its dual activity, INT-767 induces FXR-dependent lipid uptake by adipocytes, with the beneficial effect of shuttling lipids from central hepatic to peripheral fat storage, and promotes TGR5-dependent glucagon-like peptide-1 secretion by enteroendocrine cells, a validated target in the treatment of type 2 diabetes. Moreover, INT-767 treatment markedly decreases cholesterol and triglyceride levels in diabetic db/db mice and in mice rendered diabetic by streptozotocin administration. Collectively, these preclinical results indicate that INT-767 is a safe and effective modulator of FXR and TGR5-dependent pathways, suggesting potential clinical applications in the treatment of liver and metabolic diseases.

NCX-1000, a nitric oxide-releasing derivative of ursodeoxycholic acid, ameliorates portal hypertension and lowers norepinephrine-induced intrahepatic resistance in the isolated and perfused rat liver

BACKGROUND/AIMS We studied whether acute administration of NCX-1000, a nitric oxide (NO)-releasing derivative of ursodeoxycholic acid (UDCA), to animals with established liver cirrhosis decreases intrahepatic resistance and modulates hepatic vascular hypereactivity to norepinephrine (NE). METHODS Four-week bile duct ligated (BDL) cirrhotic and control, sham-operated, rats were treated orally with 28 mg/kg per day NCX-1000 or 15 mg/kg per day UDCA for 5 days. Isolated normal and cirrhotic livers were perfused with NE, from 10 nM to 30 microM, in a recirculating system. RESULTS NCX-1000 administration to BDL cirrhotic rats decreased portal pressure (P<0.01) without affecting mean arterial pressure and heart rate. In the isolated perfused liver system, administration of NE resulted in a dose-dependent increase of intrahepatic resistance. Vasoconstriction caused by 30 microM NE was reduced by 60% in animals treated with NCX-1000 (P<0.001), while UDCA was uneffective. The same portal pressure lowering effect was documented in cirrhotic and sham operated rats. Administration of NCX-1000 to BDL and sham operated rats resulted in a similar increase of nitrite/nitrate and cGMP concentrations in the liver. CONCLUSIONS By selectively delivering NO to the liver, NCX-1000 increases cGMP concentrations and effectively counteracts the effect of endogenous vasoconstrictors on the hepatic vascular tone.