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

Abstract

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.

Figure 1

Measurement of the K_s for GW274150 and purified human iNOS in the oxyhaemoglobin assay.

Figure 2

Time-dependent inactivation of purified human iNOS by GW274150. (a) Progress plots of iNOS inhibition by GW27410 in the oxyhaemoglobin assay. (b) A plot of k_trans values against GW274150 concentration obtained from fitting the data for purified human iNOS inhibition by GW274150 in (a) to the integrated rate equation for slow binding inhibitors.

Figure 3

Increased potency of inhibition of iNOS in murine J774 cells by (a) GW274150 and (b) GW273629 over time. J774 cells were induced to express iNOS with LPS/IFNγ before addition of DAF-2DA and a range of inhibitor concentrations, and the fluorescence read at times indicated. Mean±s.d. from one experiment, representative of six.

Figure 4

Effect of GW274150 i.v. on LPS-induced elevation of mouse plasma NO_x. (a) The time course of mouse plasma NO_x inhibition by GW274150 over 24 h. LPS was dosed at zero time and GW274150 was dosed at 4 h. (b) Dose response to GW274150 on plasma NO_x. LPS was dosed at zero time and GW274150 was dosed at 4 h. Plasma was sampled at 18 h. Data represent means±s.e.m. for 4–6 animals. The 18 h LPS control NO_x (for convenience of presentation plotted at 0.01 mg kg⁻¹) was 1467±57 μM and the 4 h LPS control value (for convenience of presentation plotted at 1000 mg kg⁻¹) was 170±26 μM.

Figure 5

Effect of GW273629 i.v. on LPS-induced elevation of mouse plasma NO_x. (a) The time course of effects of GW273629 i.v. on LPS-induced elevation of mouse plasma NO_x over 12 h. LPS was dosed at zero time and GW273629 was dosed at 4 h. (b) Dose response to GW273629 on plasma NO_x. LPS was dosed at zero time and GW273629 was dosed at 4 h. Plasma was sampled at 6 h. The data shown are means±s.e.m. from four mice except 6 h LPS control which is an average and a range for two values. The 6 h LPS control NO_x (for convenience of presentation plotted at 0.001 mg kg⁻¹) was 335±20 μM and the 4 h LPS control value (for convenience of presentation plotted at 400 mg kg⁻¹) was 157±17 μM.

Figure 6

The effect of GW273629 on the blood pressure of endotoxin-shocked and normal mice. (a) GW273629 increases blood pressure in endotoxin-shocked mice. Left panel: the progressive fall in mean arterial blood pressure (MABP) over a 7 h period following endotoxin administration (E. coli, 026:B6, 12.5 mg kg⁻¹ i.v. bolus). Right panel: effect of GW273629 (1–1000 mg kg⁻¹ i.v. bolus) on MABP in the conscious mouse following endotoxin. The maximum increase in MABP was measured over the 20 min period between successive doses of GW273629. Each point is the mean±s.e.m. of six animals. (b) GW273629 has no effect on blood pressure in normal mice. Left panel: basal MABP over a 7 h period prior to GW273629 administration. Right panel: effect of GW273629 (1–1000 mg kg⁻¹ i.v. bolus) on MABP in the conscious mouse. The maximum increase in MABP was measured over a 20 min period between successive doses of GW273629. Each point is the mean±s.e.m. of six animals.