Antianginal effects of FK409, a new spontaneous NO releaser.

1. The aim of this study was to compare antianginal effects of (+/-)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide (FK409), a new spontaneous nitric oxide releaser, with those of isosorbide dinitrate (ISDN). We used two types of rat angina model; methacholine- and arginine vasopressin (AVP)-induced coronary vasospasm models. 2. In the in vitro study, FK409 showed 80 times more potent vasorelaxant effect in dog isolated coronary artery than ISDN (EC50 = 16.7 +/- 4.8 and 1340 +/- 320 nM, respectively). 3. In the rat methacholine-induced coronary vasospasm model, FK409 suppressed the elevation of ST segment dose-dependently and significantly at 0.1 mg kg-1, i.d. On the other hand, ISDN suppressed it significantly at 3.2 mg kg-1, i.d. In addition, the efficacy of 3.2 mg kg-1 ISDN in the model was almost the same as that of 0.1 mg kg-1 FK409. 4. In the above experiments, FK409 and ISDN decreased mean blood pressure significantly at the maximum dose tested (1.0 mg kg-1, i.d. and 3.2 mg kg-1, i.d., respectively) but did not change heart rate at these doses. Therefore, the hypotensive effect of FK409 was 10 times weaker than the antianginal effect of the compound, while those of ISDN were almost the same. 5. In the rat AVP-induced coronary vasospasm model, 32 mg kg-1 FK409 significantly inhibited the depression of ST segment 60 min after oral administration. On the other hand, 32 mg kg-1 ISDN did not inhibit it at 60 and 120 min after oral administration.(ABSTRACT TRUNCATED AT 250 WORDS)     

Close correlation of the cardioprotective effect of FK409, a spontaneous NO releaser, with an increase in plasma cyclic GMP level.

FK409 ((+/-)-(E)-ethyl-2-[(E)-hydroxyimino]-5-nitro-3-hexeneamide) , which has been reported by us to be a new spontaneous nitric oxide (NO) releaser, prevented myocardial infarction following occlusion and reperfusion in rat coronary artery and increased plasma cyclic GMP level in rats, dose-dependently and significantly at 32 mg kg-1. Isosorbide dinitrate (ISDN), which is the most popular orally active NO donor used in the treatment of ischaemic cardiovascular diseases, did not show significant effects at 32 mg kg-1 in either experiment. Therefore, it is suggested that FK409 can attenuate myocardial injury during ischaemia and reperfusion in contrast to ISDN and a change in plasma cyclic GMP level may serve as an indicator of the cardioprotective effect of NO-releasing drugs.     

Acceleration of Nitric Oxide (NO) Release from FK409, a Spontaneous NO Releaser,in the Presence of Sulfhydryl-bearing Compounds

Purpose. Recently, we have reported that FK409 spontaneously releases nitric oxide (NO) in solution. In the present study, the influence of L-cysteine (Cys) and glutathione (GSH), which are typical sulfhydryl group-bearing compounds, on NO release from FK409 and biological action of FK409 was examined. Methods. We evaluated the effects of Cys and GSH on NO release from FK409 by nitrite analysis or detection with a chemiiluminesence analyzer. In a biological study, the influence of Cys on inhibition of rat platelet aggregation of FK409 was investigated. In addition, the above mentioned characteristics of FK409 were compared with those of isosorbide dinitrate (ISDN). Results. FK409 decomposed spontaneously with generation of nitrite in solution. Both Cys and GSH accelerated decomposition of FK409 and nitrite generation from FK409 in a concentration-dependent manner. When the NO levels in the headspace of FK409 solutions (0.5 mM) reached equilibrium with and without 25 mM Cys, the constant rate for NO release from FK409 in the presence of Cys was 13 times larger than that in the absence of Cys. In biological study, FK409 (100 µM) showed 56 and 90% inhibition of rat platelet aggregation in the absence and presence of 10 mM Cys, respectively, whereas ISDN (100 µM) showed 10 and 23% inhibition, respectively. Conclusions. Decomposition of FK409 with generation of NO is spontaneous, and is accelerated in the presence of sulfhydryl group-bearing compounds, thereby potentiating the biological action of FK409.     

Pre-clinical pharmacology of ICI D2138, a potent orally-active non-redox inhibitor of 5-lipoxygenase.

1. This paper describes the pre-clinical pharmacology of ICI D2138, a potent orally-active non-redox inhibitor of 5-lipoxygenase which is undergoing clinical evaluation. 2. ICI D2138 potently inhibited leukotriene synthesis in murine peritoneal macrophages (IC50 = 3 nM) and human blood (IC50 = 20 nM). In human and dog blood, ICI D2138 did not inhibit thromboxane B2 synthesis at a concentration of 500 microM, thus the selectivity ratio (cyclo-oxygenase: 5-lipoxygenase) was greater than 20,000. In contrast, zileuton (a 5-lipoxygenase inhibitor also undergoing clinical evaluation) exhibited a selectivity ratio of 15-100. 3. ICI D2138 potently and dose-dependently inhibited ex vivo leukotriene B4 (LTB4) synthesis by rat blood with ED50 values of 0.9, 4.0 and 80.0 mg kg-1 p.o. at 3, 10 and 20 h respectively after dosing. Similar activity was observed for inhibition of LTB4 production in a zymosan-inflamed rat air pouch model. Zileuton produced ED50 values of 5 and 20 mg kg-1 at 3 and 10 h respectively. 4. Oral administration of 1, 3 or 10 mg kg-1 ICI D2138 to dogs produced maximal inhibition of ex vivo LTB4 synthesis by blood for 5, 9 and 31 h respectively. A dose of 5 mg kg-1 p.o. of zileuton caused maximal inhibition of LTB4 for 24 h. 5. Oral administration of 10 mg kg-1 ICI D2138 caused total inhibition of LTB4 production in zymosan-inflamed rabbit knee joint. 6. Topical administration of ICI D2138 to rabbit skin caused a dose-related inhibition of arachidonic acid-induced plasma extravasation with an ID30 of 1.08 nmol per site.(ABSTRACT TRUNCATED AT 250 WORDS)     

Evaluation of the bronchodilator properties of Ro 31-6930, a novel potassium channel opener, in the guinea-pig

1. Ro 31-6930 (0.001-0.3 microM), cromakalim (0.03-3.0 microM), salbutamol (0.001-0.3 microM) and theophylline (0.3-100 microM) evoked dose-related reductions in guinea-pig spontaneous tracheal tone with IC50 values of 0.044, 0.20, 0.021 and 21.0 microM respectively. All four agents also relaxed tone supported by betahistine, carbachol, 5-hydroxytryptamine (5-HT), leukotriene D4 (LTD4), U46619 and prostaglandin D2 (PGD2). The order of potency of tracheal relaxants was always salbutamol greater than Ro 31-6930 greater than cromakalim greater than theophylline. 2. All four agents evoked dose-related reductions in 5-HT- and histamine-induced bronchoconstriction in pithed vagotomised guinea-pigs. The dose of Ro 31-6930 producing 50% inhibition of a 5-HT bronchoconstriction was 11.6 micrograms kg-1 and the dose producing 50% inhibition of a histamine bronchoconstriction was 4.4 micrograms kg-1. Salbutamol was approximately 4-5 times more potent than Ro 31-6930 whilst cromakalim was approximately 10 times less potent than Ro 31-6930 as a bronchodilator. Theophylline was markedly less potent than any of the other agents. 3. Ro 31-6930, cromakalim, salbutamol and theophylline each protected conscious guinea-pigs from histamine-induced respiratory distress. Ro 31-6930 and salbutamol were each effective at oral doses of 1.0 and 3.0 mg kg-1 whilst cromakalim was effective at oral doses of 3.0 and 10.0 mg kg-1. Theophylline showed activity only at 300 mg kg-1 p.o. 4. Ro 31-6930 is a novel potassium channel opener which is a potent relaxant of guinea-pig tracheal smooth muscle in vitro and a bronchodilator in vivo.     

Investigation of the 5-hydroxytryptamine receptor mechanism mediating the short-circuit current response in rat colon.

1. The interactions between carbenoxolone and nitric oxide (NO) were examined by investigating their effects on human platelet aggregation, on rat aortic strips precontracted by phenylephrine and on protection of rat gastric mucosa against ethanol-induced injury. 2. Carbenoxolone (100-300 microM) caused a significant and concentration-dependent potentiation of rat peritoneal neutrophil (RPN)- 3-morpholino-syndnonimine (SIN-1)- or iloprost-induced inhibition of platelet aggregation. Higher concentrations (500 microM) of carbenoxolone alone markedly inhibited platelet aggregation. Pretreatment with carbenoxolone (100-300 microM) antagonized the reversal of the RPN- or SIN-1-induced antiaggregatory effect by oxyhaemoglobin (10 microM). 3. Rat aortic strips with intact endothelium precontracted by phenylephrine (0.1-0.3 microM) were relaxed by carbenoxolone (100-300 microM) in a concentration-dependent manner. Relaxations were abolished by mechanical removal of the endothelium or by incubation with methylene blue (10 microM) or NG-nitro-L-arginine (L-NNA, 100 microM). Sodium nitroprusside (10 nM)-induced relaxations of endothelium-denuded rat aortic strips were potentiated by carbenoxolone (100 microM). . The carbenoxolone (200 mg kg-1, p.o.)-induced gastroprotection against ethanol was antagonized by L-NNA (5-40 mg kg-1) in a dose-dependent manner. Pretreatment of rats with indomethacin (10 mg kg-1, s.c.) increased the effect of L-NNA. 5. The results suggest that the activity of carbenoxolone in the experimental systems tested is due to phosphodiesterase inhibition, although radical scavenging properties of the drug could contribute to some of the effects observed. In the rat gastric mucosa both increased prostaglandin levels and effects on the NO system could contribute to the protective action of carbenoxolone.     

Antithrombotic effect of TRK-100, a novel, stable PGI2 analogue

TRK-100, a stable PGI2 analogue structurally different from carbacyclines, was compared with other antiplatelet drugs for its effect on platelet functions using animal models. TRK-100 (10-300 nM) inhibited rat platelet aggregation induced by ADP (3 microM), collagen (12.5 micrograms/ml) and A23187 (10 microM), and its potency was about 1/3-1/7 that of PGI2. TRK-100 (0.3-3 mg/kg, p.o.) dose-dependently inhibited rabbit platelet adhesion (ED50: 2.2 mg/kg), and its effect lasted over at least 5 hr. In contrast, aspirin and ticlopidine (both at 300 mg/kg, p.o.) showed only a slight inhibition (4-7%). In the thrombocytopenia induced by collagen injection in rats, TRK-100 (3-300 micrograms/kg, i.v.; 0.1-3 mg/kg, p.o.) dose-dependently inhibited a decrease in platelet number, and its ED50 was 0.48-0.62 mg/kg orally and 13.7-16.4 micrograms/kg intravenously, while the inhibition by aspirin and ticlopidine (both at 1000 mg/kg, p.o.) was 40 and 37%, respectively. In the experimental thread thrombosis in rats. TRK-100 (0.03-3 mg/kg, p.o.) dose-dependently inhibited thrombus formation, and its ED50 was 0.46 mg/kg, being 21 and 87 times as potent as aspirin and ticlopidine, respectively. These results reveal that TRK-100 has a potent antiplatelet activity and is orally and intravenously effective for a variety of thrombosis models, suggesting that it may have a therapeutic value as an antithrombotic drug.     

Triazolodiazepines: dissociation of their Paf (platelet activating factor) antagonistic and CNS activity.

The relationship between the activity of thieno- or benzo-triazolodiazepines on platelet-activating factor (Paf)-induced effects and on the CNS (central nervous system) was studied in vitro and in vivo. Brotizolam and triazolam inhibited Paf-induced human platelet aggregation. The IC50 -values were 0.54 and 7.6 microM, respectively. This inhibitory effect was not blocked by the specific central-type benzodiazepine (BDZ) antagonist, Ro 15-1788, or the specific peripheral-type BDZ ligand, Ro 5-4846. These BDZ ligands also showed an inhibitory effect on Paf-induced platelet aggregation (IC50 = 200 and 560 microM, respectively). Ro 15-1788 or Ro 5-4846 in combination with brotizolam or triazolam enhanced the Paf inhibitory effect of these triazolodiazepines. In guinea-pigs, Ro 15-1788, 100 mg kg-1 p.o. and 10 mg kg-1 i.v. completely inhibited the hypnogenic effect of 10 mg kg-1 p.o. and 1 mg kg-1 i.v. of brotizolam, respectively. Similar results were obtained with triazolam but at higher doses. In anaesthetized guinea-pigs, a dose of 100 mg kg-1 p.o. of Ro 15-1788 did not inhibit bronchoconstriction and hypotension caused by Paf (30 ng kg-1 min-1 i.v.). The combination of brotizolam (10 mg kg-1 p.o.) or triazolam (200 mg kg-1 p.o.) with this BDZ antagonist (100 and 400 mg kg-1 p.o., respectively) did not affect the Paf inhibitory activity of these triazolodiazepines. These results show that the Paf antagonistic properties of the triazolodiazepine can be dissociated from their CNS activity. It is conceivable that compounds of this structural type could be the forerunners of a novel series of potent Paf antagonists.     

Antagonism of fenfluramine-induced hyperthermia in rats by some, but not all, selective inhibitors of 5-hydroxytryptamine uptake

The injection of fenfluramine (7.5 mg kg-1,i.p.) to rats housed at 27-28 degrees C was associated with an elevation of core body temperature which peaked at approximately 1 h post-injection. One h pretreatment with citalopram (20 mg kg-1, i.p.), chlorimipramine (10 mg kg-1, i.p.), femoxetine (10 mg kg-1, i.p.) and fluoxetine (20 mg kg-1, i.p.) resulted in an attenuated response to fenfluramine. In contrast, Org 6582 (20 mg kg-1) and zimelidine (20 mg kg-1) were devoid of an effect on fenfluramine-induced hyperthermia. The response to fenfluramine was was also blocked by i.p. injections of metergoline (0.2 mg kg-1), methysergide (5 mg kg-1) and mianserin (0.5 mg kg-1). Rectal temperature was unaltered by both the 5-hydroxytryptamine (5-HT) uptake inhibitors and the 5-HT receptor antagonists. The IC50 values (nM) for in vitro inhibition of [3H]-5-HT uptake into rat hypothalamic synaptosomes were for citalopram 2.4, chlorimipramine 8.8, femoxetine 14, fluoxetine 16, Org 6582 75 and zimelidine 250. The injection of all six compounds (20 mg kg-1, i.p.) 1 h before death was associated with an inhibition of [3H]-5-HT uptake into rat hypothalamic synaptosomes which ranged from 47.2% for chlorimipramine to 83.3% for citalopram. Rat hypothalamic 5-HT levels were decreased by approximately 50% 3 h after the injection of fenfluramine (15 mg kg-1, i.p.). This effect was blocked by a 1 h pretreatment with fluoxetine, Org 6582 and zimelidine (all 20 mg kg-1, i.p.). Ki values for displacement of specifically bound [3H]-5-HT (1 nM) to rat hypothalamic membranes were for metergoline 26 nM, methysergide 1.1 microM, mianserin 3.6 microM, chlorimipramine 9.2 microM and fluoxetine 32.7 microM. Values for citalopram, femoxetine, Org 6582 and zimelidine were in excess of 65.4 microM.(ABSTRACT TRUNCATED AT 250 WORDS)     

Comparison of the profiles of agonists as stimulants of the beta 3-adrenoceptor in vitro with their gastroprotective effects in the conscious rat.

1. This paper compares the activity of a range of agonists as stimulants of the beta 3-adrenoceptor in rat isolated oesophagus with their ability to afford protection against indomethacin-induced gastric damage in the conscious rat. 2. The beta 3-adrenoceptor agonists, CL 316243 and BRL 37344, the non-selective beta-adrenoceptor agonist, isoprenaline and the selective beta 2-adrenoceptor agonist, salmeterol, all evoked concentration-dependent relaxation of precontracted muscularis mucosa from rat oesophagus. The rank order of agonist potency was BRL 37344 > CL 316243 > isoprenaline >> salmeterol. The selective beta 1-adrenoceptor agonist, denopamine, did not relax the preparation. 3. The relaxant responses to all agonists were resistant to blockade by atenolol (10 microM), and ICI 118551 (1 microM) thus suggesting that they were not mediated by either beta 1- or beta 2-adrenoceptor stimulation. In contrast, cyanopindolol and propranolol did inhibit responses to BRL 37344, CL 316243 and isoprenaline, giving pA2 values or pKB estimates which were consistent with an interaction at beta 3-adrenoceptors (i.e. approximately 8.0 and 6.5 respectively). However, responses to salmeterol were resistant to blockade by all the antagonists tested, which suggests that the high (> 1 microM) concentrations of salmeterol used exerted non-specific relaxant effects. 4. The agonist effects of CL 316243 and BRL 37344 on beta 1- and beta 2-adrenoceptors were assessed on guinea-pig right atrium and precontracted trachea respectively. Both agonists had minimal activity as stimulants of heart rate, but did relax trachea, being 380 (CL 316243) and 21 (BRL 37344) fold less potent than isoprenaline. 5. CL 316243 and BRL 37344 were potent inhibitors of indomethacin-induced gastric antral ulceration in the conscious rat (ED50 values = 0.24 and 0.09 mumol kg-1, p.o.) Salmeterol was approximately 100 times less potent than BRL 37344 as a gastroprotective agent and denopamine was without effect. 6. The gastroprotective effects of CL 316243 and BRL 37344 were resistant to blockade by ICI 118551 (10 mg kg-1, p.o.) and propranolol (10 mg kg-1, p.o.). In contrast, both antagonists caused dose-related inhibition of the protective action of salmeterol (10 mg kg-1, p.o.). Cyanopindolol was not assessed as an antagonist in vivo because preliminary experiments revealed that it exacerbated indomethacin-induced gastric damage in its own right. 7. In conclusion, the beta 3-adrenoceptor agonists CL 316243 and BRL 37344 were potent inhibitors of indomethacin-induced gastric antral ulceration in the rat. These data suggest that an agonist which is potent and selective for the human beta 3-adrenoceptor may confer mucosal protection in man.     

Pharmacologic effects of Wy 27569: a combined calcium channel blocker and thromboxane synthetase inhibitor

Wy 27569 (1,4 dihydro-2-[imidazol-1-yl-methyl]-6-methyl-4- [3-nitrophenyl] pyridine-3,5, dicarboxylic acid 3-ethyl 5-methyl diester) is a combined calcium channel blocker and thromboxane synthetase inhibitor. This article reports the in vivo and in vitro pharmacological studies demonstrating these properties. Wy 27569 evoked rightward shifts and depressed the maximum of calcium dose response curves in potassium depolarised rat aortas [concentration required to inhibit the response by 50% (IC50) = 7.3 nM]. The calcium channel blocker nitrendipine exhibited a similar profile to, although more potent than, Wy 27569 (IC50 = 0.28 nM). Comparison of the data obtained in aortas with the effects of these compounds in electrically stimulated isolated ventricle strips (IC50 for Wy 27569 = 8.3 microM, IC50 for nitrendipine = 0.41 microM) suggests that Wy 27569, like nitrendipine, is a vascular selective calcium channel blocker. Wy 27569 and the thromboxane synthetase inhibitor dazoxiben inhibited the collagen-stimulated production of thromboxane B2 (TXB2, IC50 = 3.9 and 2.8 microM, respectively) and, over the same concentration range, enhanced the production of immunoreactive 6 keto prostaglandin F1 alpha (6 keto-PGF1 alpha) by human platelet rich plasma. Single doses of Wy 27569 (0.3-10 mg kg-1 p.o.) or dazoxiben (3-10 mg kg-1 p.o.) evoked a dose-related reduction of TXB2 and enhancement of immunoreactive 6 keto PGF1 alpha levels in rat plasma and serum. Nitrendipine (0.3-10 mg kg-1 p.o.) had no significant effect on either eicosanoid.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation by nitric oxide of spontaneous motility of the rat isolated duodenum: role of tachykinins.

1. Incubation of proximal segments of the rat isolated duodenum with NG-nitro-L-arginine (L-NOARG; 3-100 microM) produced a concentration-dependent increase in both resting tone and the amplitude of the spontaneous contractions. These effects were attenuated by concurrent incubation with L-arginine (1 mM) but not D-arginine (1 mM). 2. These changes in resting tone and motility induced by L-NOARG (30 microM) were substantially reduced by concurrent incubation with tetrodotoxin (1 microM) or hexamethonium (10 microM), implicating the involvement of a local neuronal response. 3. The L-NOARG-induced increase in duodenal motility was not, however, inhibited by atropine (1 microM), guanethidine (6.4 microM) phentolamine (1 microM), or indomethacin (10 microM), indicating a non-cholinergic, non-adrenergic and non-prostanoid-mediated contractile response. 4. The NK1/NK2 tachykinin receptor antagonist, (D-Pro2, D-Trp7.9 substance P, 1-10 microM), and the NK2-receptor antagonists, MEN 10,207 and MEN 10,376 (1-5 microM), concentration-dependently reduced the effect of L-NOARG (30 microM) on spontaneous duodenal motility. 5. The resting tone and amplitude of the spontaneous contractions was likewise increased by incubation with NG-monomethyl-L-arginine (L-NMMA; 100-1000 microM). However, incubation with L-NMMA (100 microM) attenuated the actions of more potent L-NOARG (30 microM) on resting motility. 6. Administration of E.coli endotoxin (3 mg kg-1, i.v.) to the rat 5 h prior to tissue removal, at a time of known induction of NO synthase, reduced the amplitude of spontaneous contractions of the isolated duodenum, an effect inhibited by pretreatment of the rats with dexamethasone (1 mg kg-1) 2 h prior to endotoxin challenge. 7. These findings indicate a role of endogenous NO in the modulation of spontaneous tone and motility in the rat duodenum. Induction of NO synthase may result in a reduction in spontaneous motility of the tissue. By contrast, inhibition of constitutive NO biosynthesis unmasks a contractile response that is neuronally mediated and involves tachykinin NK2 receptors.     

Nitric oxide donors inhibit 5-hydroxytryptamine (5-HT) uptake by the human 5-HT transporter (SERT)

1. The aim was to test the hypothesis that nitric oxide (NO) donor drugs can inhibit the 5-hydroxytryptamine (5-HT) transporter, SERT. 2. The NO donors, MAHMA/NO (a NONOate; (Z)-1-[N-methyl-N-[6-(N-methylammoniohexyl)-amino]]diazen-1-ium-1,2-diolate), SIN-1 (a sydnonimine; 5-amino-3-(4-morpholinyl)-1,2,3-oxadiazolium chloride), FK409 (an oxime; (+/-)-(4-ethyl-2E-(hydroxyimino)-5-nitro-3E-hexenamide)) and peroxynitrite, but not Angeli's salt (source of nitroxyl anion) or sodium nitrite, caused concentration-dependent inhibition of the specific uptake of [3H]-5-HT in COS-7 cells expressing human SERT. 3. Superoxide dismutase (150 U ml(-1)) plus catalase (1200 U ml(-1)), used to remove superoxide and hence prevent peroxynitrite formation, prevented the inhibitory effect of SIN-1 (which generates superoxide) but not of MAHMA/NO or FK409. 4 The inhibitory effects of the NO donors were not affected by the free radical scavenger, hydroxocobalamin (1 mM) or the guanylate cyclase inhibitor, ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one; 3 microM). 5. L-Cysteine (1 mM; source of excess thiol residues) abolished or markedly reduced the inhibitory effects of MAHMA/NO, SIN-1, FK409 and peroxynitrite. 6. It is concluded that inhibition of SERT by the NO donors cannot be attributed exclusively to NO free radical nor to nitroxyl anion. It does not involve guanosine-3',5'-cyclic monophosphate, but may involve nitrosation of cysteine residues on the SERT protein. Peroxynitrite mediates the effect of SIN-1, but not the other drugs. 7. Data in mice with hypoxic pulmonary hypertension suggest that SERT inhibitors may attenuate pulmonary vascular remodelling. Thus, NO donors may be useful in pulmonary hypertension, not only as vasodilators, but also because they inhibit SERT, provided they display this effect in vivo at appropriate doses.     

Modulation of central noradrenergic function by RS-15385-197.

1. RS-15385-197, a highly potent and selective alpha 2-adrenoceptor antagonist, was examined in a variety of in vitro and in vivo functional tests to assess the selectivity of its interaction with central noradrenergic neurones in the rat. 2. In hypothalamic slices, RS-15385-197 was potent in augmenting K(+)-evoked release of [3H]-noradrenaline, with an EC50 of 9 nM. Idazoxan and yohimbine showed 100 fold less activity. This was due to its antagonist action at presynaptic alpha 2-adrenoceptors, as RS-15385-197 (10 microM), did not directly release [3H]-noradrenaline from cortical slices unlike reserpine (10 microM), and did not inhibit noradrenaline re-uptake into cortical synaptosomes. 3. In vivo, RS-15385-197 (0.5 mg kg-1, p.o.) increased levels of 3-methoxy-4-hydroxy-phenylglycol (MHPG) in the cerebral cortex without modifying levels of 5-hydroxyindoleacetic acid (5-HIAA). This dose, but not a lower dose (0.1 mg kg-1, p.o.) caused beta-adrenoceptor down-regulation in the cortex when administered once daily for 14 days whereas 5-HT2 receptor number was unaltered, indicating a selective effect on noradrenergic transmission. 4. Selective depletion of cortical 5-HT by administration of p-chlorophenylalanine (PCPA; 100 mg kg-1, i.p. for 14 days) or 5,7-dihydroxytryptamine (5,7-DHT; 150 micrograms i.c.v.) prevented the beta-adrenoceptor down-regulation caused by RS-15385-197, indicating that a tonic 5-hydroxytryptaminergic input was required for it to elicit beta-adrenoceptor down-regulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

The antinociceptive effect of 1-(2-trifluoromethylphenyl) imidazole (TRIM), a potent inhibitor of neuronal nitric oxide synthase in vitro, in the mouse.

1-(2-trifluoromethylphenyl)imidazole (TRIM) is a potent inhibitor of neuronal (mouse cerebellar) and inducible (lung from endotoxin-pretreated rats) isoforms of nitric oxide synthase (NOS) with IC50 values of 28.2 microM and 27.0 microM, respectively. In contrast, TRIM is a poor inhibitor of bovine aortic endothelial NOS with an IC50 of 1057.5 microM. TRIM (10-50 mg kg-1) administered i.p. exhibits dose-related antinociceptive activity in the mouse (assessed as inhibition of late phase formalin-induced hindpaw licking behaviour) with an ED50 of 85.8 mumol kg-1. In contrast, TRIM (50 mg kg-1, i.p.) failed to influence mean arterial blood pressure in the urethane-anaesthetized mouse. Thus, TRIM may be of use as an experimental tool with which to investigate the biological roles of nitric oxide (NO) within the central nervous system.     

Characterization of the capsaicin-sensitive component of cyclophosphamide-induced inflammation in the rat urinary bladder.

1. Cyclophosphamide (CYP) (150 mg kg-1, i.p. 0.5-48 h before) caused a time-dependent plasma protein extravasation in the rat urinary bladder with the maximal extravasation occurring at between 2 and 4 h after administration of the drug. 2. Prior capsaicin desensitization of capsaicin-sensitive primary afferent neurones (CSPANs) (50 mg kg-1, s.c., 4 days before) resulted in approximately 50% inhibition of the magnitude of the extravasation response at the 2 h time-point. 3. Intraperitoneal (i.p.) pretreatment with the tachykinin NK1 receptor antagonist, RP 67,580 (0.44 mg kg-1) or the bradykinin B2 receptor antagonist, Hoe 140 (0.13 mg kg-1) had significant inhibitory effects, giving responses of 56 +/- 6% and 39 +/- 4% of the control extravasation response to CYP treatment after 2 h. Pretreatment with the tachykinin NK2 receptor antagonist, SR 48,968 (0.3 mg kg-1, i.p.), the histamine H1 receptor blocker, chlorpheniramine (10 mg kg-1, i.p.), the 5-HT receptor blocker, methysergide (6 mg kg-1, i.p.) or the cyclo-oxygenase inhibitor indomethacin (5 mg kg-1, i.p.) had no significant effect upon the development of the extravasation response at this same time-point. 4. In rat isolated urinary bladder strips, the active metabolite of CYP, acrolein (1-300 microM) produced a concentration-dependent contraction that was significantly reduced by in vitro capsaicin desensitization (10 microM for 15 min) indicating direct stimulation of CSPANs. CYP was without appreciable effect. 5. The effect of acrolein in vitro was significantly reduced by pretreatment of the bladder with a combination of tachykinin NK1 and NK2 receptor antagonists, RP 67,580 (3 microM) and SR 48,968 (1 microM). The dose-response curve to acrolein was also significantly inhibited by treatment with indomethacin (10 microM) and slightly affected by Hoe 140 (1 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

FR146801, a novel nitric oxide donating agent,prevents neointimal formation after balloon injury in rats

FR 146801 (±)‐N‐[(E)‐4‐ethyl‐3‐[(Z)‐hydroxyimino]‐6‐methyl‐5‐nitro‐3‐heptenyl]‐3‐pyridinecarboxamide is a novel nitric oxide (NO) donating agent. In the present study, we examined the effect of FR146801, a slow NO releaser, on intimal thickening of rat carotid arteries following balloon injury, in comparison with FK409, a rapid NO releaser. In this model, vehicle or each drug was administered orally, twice daily from 2 days before to 13 days after injury and the intimal thickening was assessed at 14 days after injury. FR146801 suppressed neointimal formation after balloon injury dose‐dependently and significantly. FR146801 (32 mg/kg) decreased neointima/media ratio by 36.4% (P<0.05) vs. vehicle‐treated group. FR146801 (1–100 μM) inhibited the growth of cultured rat vascular smooth muscle cells concentration‐dependently. In measurements of mean blood pressure with conscious rats, FR146801 showed no hypotensive effect at 32 mg/kg p.o., the dose required for significant suppression of intimal thickening. In contrast, FK409 showed hypotensive effect at 10 mg/kg p.o. at which it suppressed neointimal formation with almost the same efficacy as FR 146801. These data demonstrate that FR146801 could be a useful inhibitor of intimal hyperplasia after vascular injury and, moreover, that its inhibitory effect could be separated from its hypotensive effect. Drug Dev. Res. 61:37–43, 2004. © 2004 Wiley‐Liss, Inc.     

Specificity of different organic nitrates to elicit NO formation in rabbit vascular tissues and organs in vivo.

1. In the present study we assessed the formation of nitric oxide (NO) from classical and thiol-containing organic nitrates in vascular tissues and organs of anaesthetized rabbits, and established a relationship between the relaxant response elicited by nitroglycerin (NTG) and NO formation in the rabbit isolated aorta. Furthermore, the effect of isolated cytochrome P450 on NO formation from organic nitrates was investigated. 2. Rabbits received diethyldithiocarbamate (DETC; 200 mg kg-1 initial bolus i.p. and 200 mg kg-1 during 20 min, i.v.) and either saline, or one of the following organic nitrates: nitroglycerin (NTG, 0.5 mg kg-1), isosorbide dinitrate (ISDN), N-(3-nitratopivaloyl)-L-cysteine ethylester (SPM 3672), S-carboxyethyl-N-(3-nitratopivaloyl)-L-cysteine ethylester (SPM 5185), at 10 mg kg-1 each. After 20 min the animals were killed, blood vessels and organs were removed, and subsequently analyzed for spin-trapped NO by cryogenic electron spin resonance (e.s.r.) spectroscopy. 3. In the saline-treated control group, NO remained below the detection limit in all vessels and organs. In contrast, all of the nitrates tested elicited measurable NO formation, which was higher in organs (liver, kidney, heart, lung, spleen) (up to 4.8 nmol g-1 20 min-1) than in blood vessels (vena cava, mesenteric bed, femoral artery, aorta) (up to 0.7 nmol g-1 20 min-1). Classical organic nitrates (NTG, ISDN) formed NO preferentially in the mesenteric bed and the vena cava, while the SPM compounds elicited comparable NO formation in veins and arteries. 4. Using a similar spin trapping technique, NO formation was assessed in vitro in phenylephrine-precontracted rabbit aortic rings. The maximal relaxation elicited by a first exposure (10 min) to NTG (0.3 to 10 microM) was positively correlated (r = 0.8) with the net increase (NTG minus basal) of NO spin-trapped during a second exposure to the same concentration of NTG in the presence of DETC. 5. Cytochrome P450 purified from rabbit liver enhanced NO formation in a NADPH-dependent fashion from NTG, but not from the other nitrates, as assessed by activation of purified soluble guanylyl cyclase. 6. We conclude that the vessel selective action of different organic nitrates in vivo reflects differences in vascular NO formation. Thus, efficient preload reduction by classical organic nitrates can be accounted for by higher NO formation in venous capacitance as compared to arterial conductance and resistance vessels. In contrast, NO is released from cysteine-containing nitrates (SPMs) to a similar extent in arteries and veins, presumably independently of an organic nitrate-specific biotransformation. Limited tissue bioavailability of NTG and ISDN might account for low NO formation in the aorta, while true differences in biotransformation seem to account for differences in NO formation in the other vascular tissues.     

Effects of R-102444, an orally active 5-HT2A receptor antagonist, in rat models of peripheral vascular disease

R-102444 is a prodrug that is metabolized into R-96544, a potent and selective 5-hydroxytryptamine2A (5-HT2A) receptor antagonist. The effects of R-102444 on peripheral vascular disease were examined using two different rat models: one induced by lauric acid and the other by ergotamine plus epinephrine. R-96544 (0.3-30 nM) relaxed the 5-HT (3 microM)-precontracted rat caudal artery in a concentration-dependent manner. The intravenous administration of R-96544 (0.3-3 microg/kg) to anesthetized rats inhibited the pressor response to 5-HT (50 microg/kg i.v.) dose dependently. The oral administration of R-102444 (1 mg/kg) to rats resulted in a marked inhibition of platelet aggregation induced by 5-HT plus ADP, and statistically significant inhibition was still evident 8 h after the dosing. In contrast, sarpogrelate, at a dose of 100 mg/kg p.o., produced only a moderate antiplatelet effect. Oral administration of R-102444 (1 mg/kg/day, o.d.) significantly prevented the progression of peripheral vascular lesion induced by the injection of lauric acid into a rat femoral artery, whereas sarpogrelate (100 mg/kg/day) showed only a minimal effect. Both 5-day treatments with R-102444 (1-30 mg/kg/day p.o., o.d.), one commenced 1 h before the injection of epinephrine plus ergotamine and one just after injection, resulted in the prevention of rat tail gangrene in a dose-dependent manner, whereas sarpogrelate (100 mg/kg) produced a minimal protection in this model. Based on these results, we conclude that 5-HT2A receptor activation is involved in peripheral vascular disease in the rat and that R-102444 is a useful oral agent for the investigation of diseases involving 5-HT2A receptor activation.     

Inhibition of bronchospasm and ozone-induced airway hyperresponsiveness in the guinea-pig by CDP840, a novel phosphodiesterase type 4 inhibitor.

1. The activity of CDP840, a novel, potent and selective cyclic nucleotide phosphodiesterase type 4 (PDE 4) inhibitor, was evaluated in guinea-pig models (in vitro and in vivo) of bronchospasm, ozone-induced airway hyperresponsiveness (AHR) and non-cholinergic bronchoconstriction. Comparisons were made with (i) other PDE 4 inhibitors: CT1731 (S-enantiomer of CDP840), rolipram, RP73401 and (ii) the clinically used agents salbutamol and theophylline. 2. CDP840 relaxed isolated trachea, under basal tone (EC50 4.5 +/- 1.1 microM) being 17 fold less potent than rolipram (EC50 0.26 +/- 0.13 microM) but attaining the same Emax (83 +/- 6% of the response to 300 microM papaverine). 3. CDP840 relaxed tracheae pre-contracted with carbachol (IC25 39 +/- 9 microM) and histamine (IC25 4 +/- 1 microM) producing monophasic curves. Stereoselectivity was not observed with CT1731 against either carbachol (IC25 33 +/- 11 microM) or histamine (IC25 17 +/- 10 microM). Aminophylline was 1.6 fold (carbachol) and 11 fold (histamine) less potent than CDP840. Rolipram and RP73401 produced tri-phasic relaxation curves but were of similar potency (at the IC25 level) to CDP840 against carbachol (rolipram 18 +/- 5 microM, RP73401 39 +/- 1 microM) whereas against histamine they were approximately 20 fold more potent (rolipram 0.2 +/- 0.1 microM, RP73401 0.2 +/- 0.1 microM). In producing > 30% (carbachol) and > 60% (histamine) relaxation these inhibitors had similar potency and were poor compared to salbutamol. 4. Pre-incubation with CDP840 (10 microM) did not antagonize histamine-induced contraction of isolated trachea; however, it did cause a slight potentiation of the subsequent relaxation to salbutamol (IC50 23 +/- 1 to 15 +/- 2 nM). 5. Pretreatment (1 h) with either CDP840 (1 mg kg-1, i.p. or 3 mg kg-1, i.v.) or rolipram (1 mg kg-1, i.p.) did not bronchodilate or antagonize bronchospasm due to inhaled histamine in anaesthetized, ventilated guinea-pigs. Salbutamol (1 mg kg-1, i.p.) did not bronchodilate but caused a parallel 7 fold rightward shift in the histamine dose-response curve. 6. Stimulation of the vagus nerve in the presence of atropine resulted in a frequency-related bronchoconstriction. CDP840 and rolipram (i.v.) inhibited the response being approximately equipotent (EC50 approximately 10 micrograms kg-1). Neither drug inhibited bronchospasm to inhaled substance P. 7. CDP840 (1-10 micrograms kg-1 i.p.) dose relatedly inhibited ozone-induced bronchoconstriction. CT1731 (1 mg kg-1), rolipram (1 mg kg-1), RP73401 (10 micrograms kg-1) and aminophylline (10 mg kg-1) had no effect. Ozone-induced AHR to inhaled histamine was inhibited by CDP840 in a dose-related manner, 10 micrograms kg-1 abolishing the AHR. This effect was stereoselective as CT1731 was approximately 30 fold less potent than CDP840. Rolipram was approximately 100 fold less potent and RP73401 and aminophylline had no effect. CDP840 was orally active being approximately 10 fold less potent compared to i.p. administration. 8. CDP840 is a poor spasmolytic and anti-spasmogenic agent in response to exogenous mediators; however, it potently inhibits vagally mediated non-cholinergic bronchoconstriction and ozone-induced AHR to histamine. It is possible that regulation of cyclic AMP by PDE 4 contributes to neuronal sensitivity in the airways. Furthermore, CDP840 may suppress AHR without being an overt bronchodilator. Such a profile of activity may have therapeutic benefit in airways diseases such as asthma.