Nitric Oxide-independent Activation of Soluble Guanylate Cyclase by BAY 60-2770 in Experimental Liver Fibrosis

Summary

Liver cirrhosis is a chronic disease with high mortality rate and need for effective pharmacological intervention. The fibrotic remodelling of liver tissue is crucially dependent on hepatic stellate cell activation. Activation of hepatic stellate cells is reduced by an increase in cyclic guanosine monophosphate (cGMP). Stable cGMP analogues also reduce the contractile response of hepatic stellate cells. However, cGMP production is downregulated in the cirrhotic liver due to the reduced activity of the endothelial nitric oxide synthase.

Objective:

Here we report that the novel activator of soluble guanylate cyclase (sGC), BAY 60-2770 (4-({(4-carboxy-butyl)[2-(5-fluoro-2-{[4’-(trifluoromethyl) biphenyl-4-yl]methoxy}phenyl)ethyl]amino}methyl)benzoic acid), which increases the activity of sGC in a nitric oxide-independent manner, attenuates liver fibrosis in two rat models.

Methods:

The compound was studied in the pig serum model and the carbon tetrachloride model. Fibrosis was assessed by estimating the increase in fibrous collagen by micromorphometry of histological sections stained with Sirius Red/Fast Green and by measuring total hepatic collagen.

Results:

BAY 60-2770, on a recombinant sGC reporter cell line, stimulated the luminescence signals with an EC₅₀ value of 5.4 ±1.2 nmol/L. In the presence of [1] [2] [4] oxadiazolo [4,3-a] quinoxalin-1-one (ODQ; 10 µmol/L) the EC₅₀ was shifted to 0.39 ±0.11 nmol/L. In both fibrosis models, once daily oral administration of BAY 60-2770 concomittantly with the fibrotic stimulus prevented 60–75% of fibrosis, the lowest effective dose being 0.1 mg/kg in the pig serum model and 0.3 mg/kg in the carbon tetrachloride model. The treatment was well tolerated by all animals. The doses used were devoid of any significant influence on systemic blood pressure.

Conclusion:

Nitric oxide-independent activation of sGC might be an innovative therapeutic approach for the treatment of liver fibrosis of necro-inflammatory and immunological origin.

Key words

BAY 60-2770, activation of soluble guanylyl cyclase, antifibrotic effect - Guanylate cyclase, nitric oxide-independent activation, soluble - Liver fibrosis, carbon tetrachloride induced, pig serum induced - Liver therapeutics

• References

• 1 Friedman SL. Stellate cells: a moving target in hepatic fibrogenesis. Hepatology. 2004; 40: 1041-1003
• 2 Reynaert H, Thompson MG, Thomas T, Geerts A. Hepatic stellate cells: role in microcirculation and pathophysiology of portal hypertension. Gut. 2002; 50: 571-581
• 3 Gäbele E, Brenner D A, Rippe R A. Liver fibrosis: Signals leading to the amplification of the fibrinogenic hepatic stellate cell. Front Biosc. 2003; 8: d69-d77
• 4 Corpechot C, Barbu V, Wendum D, Kinnman N, Rey C, Poupon R et al. Hypoxia-induced VEGF and collagen I expressions are associated with angiogenesis and fibrogenesis in experimental cirrhosis. Hepatology. 2002; 35: 1010-1021
• 5 Kawada N, Kuroki T, Uoya M, Inue M, Kobayashi K. Smooth muscle alpha-actin expression in rat hepatic stellate cell is regulated by nitric oxide and cGMP production. Biochem. Biophys Res Commun. 1996; 229: 238-242
• 6 Tao J, Mallat A, Gallois C, Belmadani S, Mery PF, Nhieu JT et al. Biological effects of C-type natriuretic peptide in human myofibroblastic hepatic stellate cells. J Biol Chem. 1999; 274: 23761-23769
• 7 Rockey DC, Chung JJ, McKee CM, Noble PW. Stimulation of inducible nitric oxide synthase in rat liver by hyaluronan fragments. Hepatology. 1998; 27: 86-92
• 8 Van de Casteele M, Omasta A, Janssens S, Roskams T, Desmet V, Nevens F et al. In vivo gen transfer of endothelial nitric oxide synthase decreases portal pressure in anaesthetized CCl4 cirrhotic rats. Gut. 2002; 51: 440-445
• 9 Loureiro-Silva MR, Iwakiri Y, Abraldes JG, Haq O, Groszmann RJ. Increased phosphodiesterase-5 expression is involved in the decreased vasodilator response to nitric oxide in cirrhotic rat livers. J Hepatol. 2006; 44: 886-893
• 10 Stasch JP, Alonso-Alija C, Apeler H, Dembowsky K, Feurer A, Minuth T et al. Pharmacological actions of a novel NO-independent guanylyl cyclase stimulator, BAY 41– 8543 in vitro studies. Br J Pharmacol. 2002; a 135: 333-343
• 11 Stasch IP, Dembowsky K, Perzborn E, Stahl E, Schramm M. Cardiovascular actions of a novel NO independent guanylyl cyclase stimulator BAY 41– 8543 in vivo studies. Br J Pharmacol. 2002; b 135: 344-355
• 12 Stasch JP, Schmidt P, Alonso-Alija C, Apeler H, Dembowsky K, Haerter M, Heil M et al. NO– and haem-independent activation of soluble guanylyl cyclase: molecular basis and cardiovascular implications of a new pharmacological principle. Br J Pharmacol. 2002; c 136: 773-783
• 13 Alonso-Alija C, Heil M, Flubacher D, Naab P, Stasch JP, Wunder F et al. Novel derivatives of dicarboxylic acid having pharmaceuticals properties. Patent WO-119780-A-2001.03.22.2001
• 14 Wunder F, Stasch JP, Hütter J, Alonso-Alija C, Hüser J, Lohrmann E. A cell-based cGMP assay useful for ultra-high-throughput screening and identification of modulators of the NO/cGMP pathway. Anal Biochem. 2005; 339: 104-112
• 15 McLean E, McLean A, Sutton P. Instant Cirrhosis. An improved method for producing cirrhosis of the liver in rats by simultaneous administration of CCl₄ and phenobarbitone. Br J Exp Pathol. 1969; 50: 502-506
• 16 Bhunchet E, Wake K. Role of mesenchymal cell populations in porcine serum-induced rat liver fibrosis. Hepatology. 1992; 16: 1452-1473
• 17 Hoenicka M, Becker EM, Apeler H, Sirichoke T, Schröder H, Gerzer R et al. Purified soluble guanylyl cyclase expressed in a baculovirus/Sf9 system: stimulation by YC-1, nitric oxide, and carbon monoxide. J Mol Med. 1999; 77: 14-23
• 18 Prockop DJ, Udenfriend S. A specific method for the analysis of hydroxyproline in tissues and urine. Anal Biochem. 1960; 1: 228-239
• 19 Bergmeyer HU, Hørder M, Rej R. Approved recommendation on IFCC methods for the measurement of catalytic concentration of enzymes. Part 3. IFCC method for alanine aminotransferase. J Clin Chem Clin Biochem. 1986; a 24: 481-495
• 20 Bergmeyer HU, Hørder M, Rej R. Approved recommendation on IFCC methods for the measurement of catalytic concentration of enzymes. Part 2. IFCC method for aspartate aminotransferase. J Clin Chem Clin Biochem. 1986; b 24: 497-510
• 21 Doumas BT, Watson WA, Biggs HG. Albumin standards and the measurement of serum albumin with bromcresol green. Clin Chim Acta. 1971; 31: 87-96
• 22 Wahlefeld AW, Herz G, Bernt E. Modification of the Malloy-Evelyn-method for a simple, reliable determination of total bilirubin in serum. Scand J Clin Lab Invest. 1972; 29 (Suppl. 126) Abstr. 11.12
• 23 Menon KV, Kamath PS. Managing the complications of cirrhosis. Mayo Clin Proc. 2000; 75: 501-509
• 24 Bosch J, Garcia-Pagan JC. Complications of Cirrhosis. I. Portal hypertension. J Hepatol. 2000; 32 (1 Suppl) 141-156
• 25 Zhang J, Pegoli jr W, Clemens M. Endothelin-1 induces direct constriction of hepatic sinusoids. Am J Physiol. 1994; 266: G624-G632
• 26 Rockey DC, Weisinger RA. Endothelin induced contractility of stellate cells from normal and cirrhotic rat liver: implications for regulation of portal pressure and resistance. Hepatology. 1996; 24: 233-240
• 27 Rockey D, Chung J. Reduced nitric oxide production by endothelial cells in cirrhotic rat liver: Endothelial dysfunction in portal hypertension. Gastroenterology. 1996; 114: 344-351
• 28 Bathal PS, Grossmann HJ. Reduction of the increased portal vascular resistance of the isolated perfused cirrhotic rat liver by vasodilators. J Hepatol. 1985; 1: 325-329
• 29 Grose RD, Plevris JN, Redhead DN, Bouchier IAD, Hayes P. The acute and chronic effects of isosorbide-5-mononitrate on portal hemodynamics in cirrhosis. J Hepatol. 1994; 20: 542-547
• 30 Mela M, Mancuso A, Burroughs A. Drug treatment for portal hypertension. Ann Hepatol. 2002; 3: 102-120
• 31 Wiest R, Groszmann RJ. The paradox of nitric oxide in cirrhosis and portal hypertension: too much, not enough. Hepatology. 2002; 35: 478-491
• 32 Gentilini P, La Villa G, Laffi G, Pinzani M. Hemodynamic derangement and cardiac dysfunction in cirrhosis. Ann Ital Med Int. 2004; 19: 90-97
• 33 Reeves HL, Friedman SL. Activation of hepatic stellate cells-a-key issue in liver fibrosis. Front Biosc. 2002; 7: d808-d826
• 34 Murphy F, Arthur M, Iredale J. Developing strategies for liver fibrosis treatment. Expert Opin Invest Drugs. 2002; 11: 1575-1585
• 35 Gupta TK, Toruner M, Chung MK, Groszmann RJ. Endothelial dysfunction and decreased production of nitric oxide in the intrahepatic microcirculation of cirrhotic rats. Hepatology. 1998; 28: 926-931
• 36 Hobbs AJ. Soluble guanylate cyclase: an old therapeutic target revisited. Br J Pharmacol. 2002; 136: 637-640
• 37 Tulis DA. Salutary properties of YC-1 in the cardiovascular and hematological systems. Curr Med Chem. 2004; 2: 343-359
• 38 Minin EA, Buchalov IB, Wellner M, Palmes D, Spiegel H-U, Neumann J et al. L-Arginine-NO-cGMP signaling following acute liver injury in the rat. Exp Tox Path. 2005; 57: 161-171
• 39 Fiorucci S, Antonelli E, Morelli O, Mencarelli A, Casini A, Mello T et al. NCX-1000, a NO-releasing derivative of urodesoxcholic acid, selectively delivers NO to the liver and protects against development of portal hypertension. Proc Natl Acad Sci. 2002; 98: 8897-8902
• 40 Fiorucci S, Antonelli E, Tocchetti P, Morelli A. Treatment of portal hypertension with NCX-1000, a liver-specific NO donor. A review of its current status. Cardiovasc Drug Rev. 2004; 22: 135-146
• 41 Fiorucci S, Antonelli E, Brancaleone V, Sanpaolo L, Orlandi S, Distrutti E. NCX-1000, a nitric oxide-releasing derivative of orsodeoxycholic acid, ameliorates portal hypertension and lowers norepinephrine-induced intrahepatic resistance in the isolated and perfused rat liver. J Hepatol. 2003; 39: 932-939
• 42 Loureiro-Silva M, Cadelina G, Iwakiri Y, Groszmann R. A liver-specific nitric oxide donor improves the intra-hepatic vascular response to both portal blood flow increase and methoxamine in cirrhotic rats. J Hepatol. 2003; 39: 940-946
• 43 Moal F, Veal N, Vuillemin E, Barrière E, Wang J, Fizanne L. Hemodynamic and antifibrotic effects of a selective liver nitric oxide donor V-PYRRO/NO in bile duct ligated rats. World J Gastroenterol. 2006; 12: 6639-6645
• 44 Bosch J. Decreasing hepatic vascular tone by liver-specific NO donors: wishful thinking or a promising reality?. J Hepatol. 2003; 39: 1072-1075
• 45 Garcia-Pagan JC, Navasa M, Bosch J, Bru C, Pizcueta P, Rodes J. Enhancement of portal pressure reduction by the association of isosorbide-5- mononitrate to propranolol admionistration in patients with cirrhosis. Hepatology. 1990; 11: 230-238
• 46 Bellis l, Berzigotti A, Abraldes JG, Moitinho E, Garcia-Pagan JC, Bosch J. Low doses of isosorbide mononitrate attenuate the postprandial increase in portal pressure in patients with cirrhosis. Hepatology. 2003; 37: 378-384
• 47 Garcia-Pagan JC, Morillas R, Banares R, Albillos A, Villanueva C, Vila C. Spanish Variceal Bleeding Study Group: Propranolol plus placebo versus propranolol plus isosorbide-5 mononitrate in the prevention of a first variceal blee: A double-blind RCT. Hepatology. 2003; 37: 1260-1266
• 48 Merkel C, Marin R, Sacerdoti D, Donada C, Cavallarin G, Torboli P. Long-term results of a clinical trial of nadolol with or without isosorbide mononitrate for primary prophylaxis of variceal bleeding in cirrhosis. Hepatology. 2000; 31: 324-329
• 49 Li J, Billiar TR. Nitric oxide. IV. Determinants of nitric oxide protection and toxicity in liver. Am J Physiol. 1999; 276: G1069-G1073
• 50 Hon WM, Lee KH, Khoo HE. Nitric oxide in liver diseases:friendfoe or just passerby?. Ann NY Acad Sci. 2002; 962: 275-295
• 51 Chen T, Zamora R, Zuckerbraun B, Billiar TR. Role of nitric oxide in liver injury. Curr Mol Med. 2003; 3: 519-526
• 52 Dudenhoefer AA, Loureiro-Silva MR, Cadelina GW, Gupta T, Groszman RJ. Bioactivation of nitroglycerin and vasomotor response to nitric oxide are impaired in cirrhotic rat livers. Hepatology. 2002; 36: 381-385