GW274150

GW274150, a potent and highly selective inhibitor of iNOS, reduces experimental renal ischemia/reperfusion injury

Background: Generation of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) may contribute to renal ischemia/reperfusion (I/R) injury. The aim of this study was to investigate the effects of GW274150, a novel, highly selective, potent and long-acting inhibitor of iNOS activity in rat and mouse models of renal I/R. Methods: Rats were administered GW274150 (5 mg/kg intravenous bolus administered 30 minutes prior to I/R) and subjected to bilateral renal ischemia (45 minutes) followed by reperfusion (6 hours). Serum and urinary indicators of renal dysfunction, tubular and reperfusion injury were measured, specifically, serum urea, creatinine, aspartate aminotransferase (AST) and N-acetyl-beta-d-glucosaminidase (NAG) enzymuria. In addition, renal sections were used for histologic scoring of renal injury and for immunologic evidence of nitrotyrosine formation and poly [adenosine diphosphate (ADP)-ribose] (PAR). Nitrate levels were measured in rat plasma using the Griess assay. Mice (wild-type, administered 5 mg/kg GW274150, and iNOS-/-) were subjected to bilateral renal ischemia (30 minutes) followed by reperfusion (24 hours) after which renal dysfunction (serum urea, creatinine), renal myeloperoxidase (MPO) activity and malondialdehyde (MDA) levels were measured. Results: GW274150, administered prior to I/R, significantly reduced serum urea, serum creatinine, AST, and NAG indicating reduction of renal dysfunction and injury caused by I/R. GW274150 reduced histologic evidence of tubular injury and markedly reduced immunohistochemical evidence of nitrotyrosine and PAR formation, indicating reduced peroxynitrite formation and poly (ADP-ribose) polymerase (PARP) activation, respectively. GW274150 abolished the rise in the plasma levels of nitrate (indicating reduced NO production). GW274150 also reduced the renal dysfunction in wild-type mice to levels similar to that observed in iNOS-/- mice subjected to I/R. Renal MPO activity and MDA levels were significantly reduced in wild-type mice administered GW274150 and iNOS-/- mice subjected to renal I/R, indicating reduced polymorphonuclear leukocyte (PMN) infiltration and lipid peroxidation. Conclusions: These results suggest that (1). an enhanced formation of NO by iNOS contributes to the pathophysiology of renal I/R injury and (2). GW274150 reduces I/R injury of the kidney. We propose that selective inhibitors of iNOS activity may be useful against renal dysfunction and injury associated with I/R of the kidney.

GW274150 and GW273629 are potent and highly selective inhibitors of inducible nitric oxide synthase in vitro and in vivo

1 GW274150 ([2-[(1-iminoethyl) amino]ethyl]-L-homocysteine) and GW273629 (3-[[2-[(1-iminoethyl)amino]ethyl]sulphonyl]-L-alanine) are potent, time-dependent, highly selective inhibitors of human inducible nitric oxide synthase (iNOS) vs endothelial NOS (eNOS) (>100-fold) or neuronal NOS (nNOS) (>80-fold). GW274150 and GW273629 are arginine competitive, NADPH-dependent inhibitors of human iNOS with steady state K(d) values of <40 and <90 nM, respectively. 2 GW274150 and GW273629 inhibited intracellular iNOS in J774 cells in a time-dependent manner, reaching IC(50) values of 0.2+/-0.04 and 1.3+/-0.16 microM, respectively. They were also acutely selective in intact rat tissues: GW274150 was >260-fold and 219-fold selective for iNOS against eNOS and nNOS, respectively, while GW273629 was >150-fold and 365-fold selective for iNOS against eNOS and nNOS, respectively. 3 The pharmacokinetic profile of GW274150 was biphasic in healthy rats and mice with a terminal half-life of approximately 6 h. That of GW273629 was also biphasic in rats, producing a terminal half-life of approximately 3 h. In mice however, elimination of GW273629 appeared monophasic and more rapid (approximately 10 min). Both compounds show a high oral bioavailability (>90%) in rats and mice. 4 GW274150 was effective in inhibiting LPS-induced plasma NO(x) levels in mice with an ED(50) of 3.2+/-0.7 mg kg(-1) after 14 h intraperitoneally (i.p.) and 3.8+/-1.5 mg kg(-1) after 14 h when administered orally. GW273629 showed shorter-lived effects on plasma NO(x) and an ED(50) of 9+/-2 mg kg(-1) after 2 h when administered i.p. 5 The effects of GW274150 and GW273629 in vivo were consistent with high selectivity for iNOS, as these inhibitors were of low potency against nNOS in the rat cerebellum and did not cause significant effects on blood pressure in instrumented mice.

GW274150 inhibits nitric oxide production by primary cultures of rat proximal tubular cells

Background: Production of nitric oxide (NO) subsequent to expression of inducible NO synthase (iNOS) contributes to the development of ischemic renal injury and inflammation. Here we investigate the effects of GW274150, a potent, long-acting and highly selective inhibitor of iNOS activity, on NO production by primary cultures of rat proximal tubular cells (PTC).
Material/methods: Pure populations of PTC were isolated from the cortex of kidneys obtained from male Wistar rats using a combination of collagenase digestion, sieving and Percoll centrifugation. Confluent PTC cultures were incubated for 1-24 h with MEM, interferon-gamma (IFN-gamma, 100 microg/ml), bacterial lipopolysaccharide (LPS, 10 microg/ml) in combination after which NO production was determined. PTC were also incubated with IFN-gamma (100 microg/ml) and LPS (10 microg/ml) and increasing concentrations of GW274150 or L-N6-(1-iminoethyl)lysine (L-NIL) (10 nM-1 mM) for 24 h after which nitrite levels in the incubation medium were measured.
Results: IFN-g and LPS in combination produced a significant, time-dependent increase in NO production. Both GW274150 and L-NIL produced a significant and concentration-dependent inhibition of NO production. However, GW274150 was markedly more potent (EC50 approximately 100 nM) than L-NIL (EC50 approximately 10 microM).
Conclusions: GW274150 inhibits NO production by primary cultures of PTCs and may therefore be useful in conditions associated with nitrosative stress of the kidney.

GW274150, a novel and highly selective inhibitor of the inducible isoform of nitric oxide synthase (iNOS), shows analgesic effects in rat models of inflammatory and neuropathic pain

Nitric oxide (NO), synthesised by different isoforms of nitric oxide synthase (NOS), has been linked with the development and maintenance of nociception. We studied the role of the inducible isoform, iNOS, in two different rat pain models with an inflammatory component. iNOS was immunohistochemically detected locally in the paw 6h after Freund's Complete Adjuvant (FCA) injection, showing a plateau at 24-72 h and falling slowly in the following weeks. This correlated with the late phase of the hypersensitivity to pain revealed in the behavioural tests. A highly selective iNOS inhibitor GW274150 (1-30 mg/kg orally, 24h after FCA) suppressed the accumulation of nitrite in the inflamed paw indicating substantial iNOS inhibition. At the same time it partially reversed FCA-induced hypersensitivity to pain and edema in a dose-dependent manner. After Chronic Constriction Injury (CCI) surgery to the sciatic nerve, iNOS presence was only detected locally in the region of the nerve (inflammatory cells). GW274150 (3-30 mg/kg orally, 21 days after surgery) also reversed significantly the CCI-associated hypersensitivity to pain. No iNOS was detectable in dorsal root ganglia, spinal cord or brain in either model. This study demonstrates a role for peripherally-expressed iNOS in pain conditions with an inflammatory component and the potential value of iNOS inhibitors in such conditions.

Lack of efficacy of the selective iNOS inhibitor GW274150 in prophylaxis of migraine headache

Introduction: This study investigated the efficacy and tolerability of the highly selective iNOS inhibitor GW274150 in prophylaxis of migraine headache.
Subjects and methods: The study was conducted in two parts, each comprising a 4-week baseline period, a 12-week, double-blind, parallel-group treatment period, and a 4-week follow-up period. The study had an adaptive design in that findings of Part 1 of the study were used to inform the conduct of Part 2. Following an interim analysis at the end of Part 1, the trial could be stopped for futility or continued in Part 2 to study the full-dose response or to increase sample size in case initial assumptions had been violated. The primary end-point in both parts of the study was the probability of the occurrence of a migraine headache day during the baseline period and the treatment period.
Results: In Part 1, adult male and female patients with migraine received GW274150 60 mg (n = 37), 120 mg (n = 37), or placebo (n = 38) once daily for 12 weeks. In Part 2, female patients with migraine received GW274150 60 mg (n= 160) or placebo (n = 154) once daily for 12 weeks. GW274150 was no more effective than placebo for the primary efficacy end-point or any secondary efficacy end-point in Part 1 or Part 2. GW274150 was generally well tolerated.
Conclusions: GW274150 at doses predicted to inhibit iNOS >80% did not differ from placebo in the prophylaxis of migraine. The results do not support a role of iNOS inhibition in migraine prevention.