Characterization of the novel nitric oxide synthase inhibitor 7-nitro indazole and related indazoles: antinociceptive and cardiovascular effects.

1. 7-Nitro indazole (7-NI, 10-50 mg kg-1), 6-nitro indazole and indazole (25-100 mg kg-1) administered i.p. in the mouse produce dose-related antinociception in the late phase of the formalin-induced hindpaw licking and acetic acid-induced abdominal constriction assays. The ED50 values (mg kg-1) were as follows: 7-NI (27.5 and 22.5), 6-nitro indazole (62.5 and 44.0) and indazole (41.0 and 48.5) in the two assays respectively. 3-Indazolinone, 6 amino indazole and 6-sulphanilimido indazole (all 50 mg kg-1) were without effect. With the exception of 5-nitro indazole (50 mg kg-1) which produced sedation, none of the other indazole derivates examined caused overt behavioural changes. 2. The antinociceptive effect of 7-NI (25 mg kg-1, i.p.) in the late phase of the formalin-induced hindpaw licking assay was partially (46.7 +/- 16.2%, n = 18) reversed by pretreatment with L- but not D-arginine (both 50 mg kg-1, i.p.). 3. The time course of 7-NI induced antinociception in the mouse was correlated with inhibition of brain (cerebellum) nitric oxide synthase (NOS) activity. Maximum antinociceptive activity and NOS inhibition was detected 18-30 min following i.p. administration. In contrast, no antinociceptive effect or inhibition of cerebellar NOS was detected 75 min post-injection. 4. 7-NI, 6-nitro indazole, indazole, 3-indazolinone and 6-amino indazole (all 50 mg kg-1) failed to influence mean arterial pressure (MAP) over the 45 min after i.p. administration in the anaesthetized mouse.(ABSTRACT TRUNCATED AT 250 WORDS)     

Inhibition of rat cerebellar nitric oxide synthase by 7-nitro indazole and related substituted indazoles.

1. 7-Nitro indazole (7-NI) produces potent inhibition of rat cerebellar nitric oxide synthase (NOS) with an IC50 of 0.9 +/- 0.1 microM (n = 6). NOS activity is dependent on the presence of both exogenous CaCl2 and NADPH. The inhibitory potency of 7-NI remained unaltered in the presence of different concentrations of either CaCl2 (0.75-7.5 mM) or NADPH (0.05-5.0 mM). 2. Kinetic (Lineweaver-Burke) analysis of the effect of 7-NI on rat cerebellar NOS revealed that inhibition was of a competitive nature with a Ki value of 5.6 microM. The Km of of cerebellar NOS with respect to L-arginine was 2.5 microM. 3. The following indazole derivatives (IC50 values shown in parentheses, all n = 6) caused concentration-related inhibition of rat cerebellar NOS in vitro: 6-nitro indazole (31.6 +/- 3.4 microM), 5-nitro indazole (47.3 +/- 2.3 microM), 3-chloro indazole (100.0 +/- 5.5 microM), 3-chloro 5-nitro indazole (158.4 +/- 2.1 microM) and indazole (177.8 +/- 2.1 microM). The IC50 values for 5-amino indazole, 6-amino indazole and 6-sulphanilimido indazole were in excess of 1 mM; 3-indazolinone was inactive. 4. 7-NI (10 mg kg-1) administered i.p. to rats produced 60 min thereafter a significant inhibition of NOS activity in cerebellum (31.1 +/- 3.2%, n = 6), cerebral cortex (38.2 +/- 5.6%, n = 6), hippocampus (37.0 +/- 2.8%, n = 6) and adrenal gland (23.7 +/- 3.0%, n = 6). NOS activity in olfactory bulb and stomach fundus were unchanged.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synergistic anti-nociceptive effect of L-NG-nitro arginine methyl ester (L-NAME) and flurbiprofen in the mouse.

1. L-NG-nitro arginine methyl ester (L-NAME) administered i.p. produces anti-nociception in the mouse assessed by the formalin-induced paw licking and acetic acid-induced abdominal constriction models. The non-steroidal anti-inflammatory drug (NSAID), flurbiprofen, was similarly anti-nociceptive in both models. 2. Combination of a sub-threshold dose of L-NAME (10 mg kg-1) with increasing doses of flurbiprofen (25- 75 mg kg-1) or a sub-threshold dose of flurbiprofen (50 mg kg-1) with increasing doses of L-NAME (10- 100 mg kg-1) resulted in potentiated anti-nociception in the formalin model. Combined therapy with sub-threshold doses of L-NAME (10 mg kg-1) and indomethacin (10 mg kg-1) also resulted in significant anti-nociception. In addition, combining sub-threshold doses of L-NAME (12.5 mg kg-1) and flurbiprofen (2 mg kg-1) significantly reduced acetic acid-induced abdominal constriction. 3. L-NAME (10 mg kg-1) administered i.p. caused a significant (approximately 35%) increase in MAP in the urethane-anaesthetized mouse. Flurbiprofen (50 mg kg-1) was inactive. Combination treatment with L-NAME (10 mg kg-1) and flurbiprofen (50 mg kg-1) failed to elevate MAP above that observed with L-NAME alone. Neither L-NAME (10 mg kg-1) nor flurbiprofen (50 mg kg-1) either alone or in combination significantly altered mouse locomotor activity. 4. These results suggest that L-NAME and flurbiprofen/indomethacin act synergistically in their anti-nociceptive action in the mouse. Combination therapy with L-NAME and flurbiprofen and a similar NSAID may provide an alternative to the clinical control of pain in man.     

7-Nitro indazole, an inhibitor of nitric oxide synthase, exhibits anti-nociceptive activity in the mouse without increasing blood pressure.

7-Nitro indazole (7-NI) inhibits mouse cerebellar nitric oxide synthase (NOS) in vitro with an IC50 of 0.47 microM. Following i.p. administration in mice, 7-NI (10-50 mg kg-1) produces dose-related anti-nociception as evidenced by an inhibition of late phase (15-30 min) but not early phase (0-5 min) hindpaw licking time following subplantar injection of formalin (10 microliters, 5% v/v). The ED50 for this effect was 26 mg kg-1 (equivalent to 159.5 mumol kg-1). Similar i.p. administration of 7-NI (20 and 80 mg kg-1) in urethane-anaesthetized mice failed to increase MAP. Thus, 7-NI is a novel inhibitor of NOS which exhibits selectivity for the brain enzyme. Accordingly, 7-NI may be a useful starting point for the development of selective, centrally acting NOS inhibitors devoid of cardiovascular side effects and as a tool to study the central pharmacological effects of nitric oxide (NO).     

An investigation of some S-nitrosothiols, and of hydroxy-arginine, on the mouse anococcygeus.

1. The effect of five S-nitrosothiols, and of the stereoisomers of NG-hydroxy-arginine (HOARG), were investigated on the mouse anococcygeus. 2. All five S-nitrosothiols produced concentration-related (0.1-100 microM) relaxations of carbachol (50 microM)-induced tone; the order of potency was S-nitroso-L-cysteine (CYSNO) > S-nitroso-N-acetyl-D,L-penicillamine (SNAP) > S-nitrosoglutathione (GSNO) > S-nitrosocoenzyme A (CoASNO) > S-nitroso-N-acetyl-L-cysteine (NACNO). The relaxations were unaffected by the nitric oxide synthase (NOS) inhibitor, L-NG-nitro-arginine (10 microM) (L-NOARG). 3. Cold-storage of the tissue for 72 h resulted in loss of sympathetic and non-adrenergic, non-cholinergic (NANC) nerve function. NOS activity in the tissue was reduced by 97%. Despite this, relaxations induced by the S-nitrosothiols were unaffected. 4. Haemoglobin (50 microM) attenuated relaxations induced by NO and the S-nitrosothiols, although responses to 3-isobutyl-1-methyl-xanthine were unaffected. N-methyl-hydroxylamine (2 mM) which has been shown previously to produce selective inhibition of NANC and nitrovasodilator responses in this tissue, also reduced responses to all S-nitrosothiols. 5. Hydroquinone (100 microM) greatly reduced relaxations to CYSNO (by 88%) but had no effect on those to SNAP, GSNO, CoASNO or NACNO. Since hydroquinone does not reduce responses to NANC stimulation, CYSNO is unlikely to be the NANC transmitter. 6. L-HOARG by itself (up to 100 microM) had no significant effect on carbachol-induced tone or on NANC (10 Hz; 10 strain every 100 s) relaxations.(ABSTRACT TRUNCATED AT 250 WORDS)     

Anti-nociceptive activity of nitric oxide synthase inhibitors in the mouse: dissociation between the effect of l-NAME and l-NMMA

Abstract— The anti-nociceptive effect of selective inhibitors of nitric oxide synthase has been assessed in a formalin-induced paw-licking model in mice. l -N^G-Nitro arginine methyl ester (l -NAME) but not l -N^G-monomethyl arginine (l -NMMA) exhibited anti-nociceptive activity in both the early and late phases of paw licking following intraperitoneal administration. The effect on the late phase response was more pronounced. l -NAME (0·1–100 μg) and l -N^G-nitro arginine base (l -NOARG; 10 μg) but not d -NAME (10 μg) were also anti-nociceptive following intracerebroventricular administration. l -NAME (10 μg) administered by this route did not influence locomotor activity. l -NMMA was inactive at doses up to 40 μg by this route. At higher doses (75–200 μg) l -NMMA produced a similar and non-dose related reduction in early/late phase paw-licking time. d -NMMA (100 μg) was inactive. The greater anti-nociceptive effect of l -NAME in this model accords with recently published biochemical data indicating that l -NAME is several orders of magnitude more potent than l -NMMA as an inhibitor of brain nitric oxide synthase. These data support the use of l -NAME as a selective tool to investigate the central pharmacological effects of nitric oxide.     

L-NG-nitro arginine methyl ester exhibits antinociceptive activity in the mouse.

1. L-NG-nitro arginine methyl ester (L-NAME, 1-75 mg kg-1) administered intraperitoneally (i.p.) elicits dose-related antinociception in the mouse assessed by the formalin-induced paw licking procedure. Antinociceptive activity is still present 24 h after injection. L-NAME (75 mg kg-1, i.p.) is also antinociceptive in the acetic acid-induced abdominal constriction and hot plate procedures. 2. L-NAME additionally produces a dose-related inhibition of formalin-induced paw licking following intracerebroventricular (i.c.v., 0.1-100 microgram per mouse) and oral (p.o., 75-150 mg kg-1) administration. 3. L-Arginine (600 mg kg-1, i.p.) but not D-arginine (600 mg kg-1) or naloxone (5 mg kg-1) reverses the antinociceptive effect of L-NAME in the formalin test. 4. High doses of L-NAME (37.5-600 mg kg-1) but not D-NAME (75 mg kg-1) administered i.p. produce dose-related increases in blood pressure of the urethane-anaesthetized mouse whilst i.c.v. injected L-NAME (0.1 and 100 microgram per mouse) in inactive. 5. L-NAME (75 mg kg-1, i.p.) did not inhibit oedema formation in the formalin-injected mouse hindpaw. 6. L-NAME (75 mg kg-1) did not produce any overt behavioural changes in treated mice and failed to influence locomotor activity or the incidence of dipping, crossing, rearing or circling behaviour assessed by a modified 'head-dipping' board procedure. A high dose of L-NAME (600 mg kg-1) reduced dipping behaviour and locomotor activity suggesting a possible sedative effect.(ABSTRACT TRUNCATED AT 250 WORDS)