Role of nitric oxide in zymosan induced paw inflammation and thermal hyperalgesia.

OBJECTIVE: To assess the involvement of spinal inducible nitric oxide synthase (iNOS) in inflammation and nociception. MATERIALS AND METHODS: The time course of iNOS mRNA expression in rat spinal cord and inflamed paw was assessed by means of quantitative real time RT-PCR. In addition, the effects of the iNOS inhibitor L-NIL on inflammatory paw edema and thermal hyperalgesia were studied in comparison to those of the NO-donor RE-2047. L-NIL (3, 9, 27 and 81 mg/kg) and RE-2047 (3, 9 and 27 mg/kg) or vehicle were administered orally 15 min prior to the intraplantar injection of 0.625 mg zymosan. RESULTS: Following zymosan injection, mRNA expression of iNOS increased in the inflamed paw and spinal cord with a maximum at 2.5 and 4 h, respectively. In the spinal cord iNOS mRNA started to decline at 10 h whereas it remained at maximum in the inflamed paw up to the end of the observation period of 24 h. As expected, RE-2047 had significant pronociceptive and proinflammatory effects. L-NIL significantly reduced paw inflammation at 27 and 81 mg/kg but failed to reduce hyperalgesia at the doses tested. CONCLUSIONS: The results show that iNOS is upregulated in the inflamed tissue and spinal cord with a similar time course. The effects obtained with L-NIL suggest that iNOS differently contributes to the inflammatory and nociceptive response induced by zymosan.

Suppressed injury-induced rise in spinal prostaglandin E2 production and reduced early thermal hyperalgesia in iNOS-deficient mice.

It is widely accepted that peripheral injury increases spinal inducible cyclooxygenase (COX-2) expression and prostaglandin E(2) (PGE(2)) formation as key mediators of nociceptive sensitization. Here, we used inducible nitric oxide synthase (iNOS) gene-deficient (iNOS-/-) mice to determine the contribution of iNOS-derived nitric oxide (NO) to this process. iNOS-/- mice exhibited reduced thermal hyperalgesia after zymosan injection. Spinal NO and PGE(2) formation both remained at baseline levels, in contrast to wild-type (wt) mice. In wt mice reduced hyperalgesia similar to that seen in iNOS-/- mice was induced by local spinal, but not by systemic treatment with the iNOS inhibitor l-NIL, suggesting that the reduced heat sensitization in iNOS-/- mice was attributable to the lack of spinal rather than peripheral iNOS. Two additional observations indicate that the antinociceptive effects of iNOS inhibition are dependent on a loss of stimulation of PG synthesis. First, intrathecal injection of the COX inhibitor indomethacin, which exerted pronounced antinociceptive effects in wt mice, was completely ineffective in iNOS-/- mice. Second, treatment with the NO donor RE-2047 not only completely restored spinal PG production and thermal sensitization in iNOS-/- mice but also its sensitivity to indomethacin. In both types of mice induction of thermal hyperalgesia was accompanied by similar increases in COX-1 and COX-2 mRNA expression. The stimulation of PG production by NO therefore involves an increase in enzymatic activity, rather than an alteration of COX gene expression. These results indicate that NO derived from spinal iNOS acts as a fast inductor of spinal thermal hyperalgesia.

New NO donors with antithrombotic and vasodilating activities, Part 27. Azide oximes and 1-hydroxytetrazoles.

Eleven azide oximes were prepared and tested for their antiplatelet (in vitro), antithrombotic, and blood pressure lowering activities. Nine of them inhibited the aggregation of blood platelets (Born test, inducer collagen) with IC50 values between 10 and 50 microM. The most active compounds i.e. azido-4-nitrophenylbenzaldoxime (2h) had an IC50 = 2 microM. Nine azide oximes exhibited significant antithrombotic properties. The most active compounds were 2h and 2c (azido-4-methylphenylbenzaldoxime) with an inhibition of thrombus formation above 20% in arterioles after a single p.o. dose of 60 mg/kg. Both compounds lowered the blood pressure in spontaneously hypertensive rats by 11% (2h) or 5% (2c), respectively. Seven azide oximes were rearranged to the title tetrazololes which however showed smaller antithrombotic effects. In separate in vitro experiments at 37 degrees C it could be demonstrated that azide oximes release nitric oxide (conversion rate approximately 10%.h-1) and nitrosohydrogen (conversion rate approximately 2%.h-1). This makes it appear probable that the above effects are mediated by these molecules.

New NO donors with antithrombotic and vasodilating activities, Part 29. N-(1-cyanocyclohexyl)-C-phenylnitrones and glyoxaldinitrones.

Six N-(1-cyanocyclohexyl)-C-phenylnitrones 4a-f (4b-f for the first time) and 22 glyoxaldinitrones 7a-v were prepared and tested for antithrombotic (p.o. administration to rats, 60 mg/kg) effects. Both classes of compounds exhibit considerable antithrombotic activities. Maximum inhibition of thrombus formation in arterioles (21%) was observed in N,N'-bis-2-phenylethylglyoxaldinitrone (7o) and N,N'-bis-4-nitrobenzylglyoxaldinitrone (7u). The compounds form only small amounts of nitric oxide in vitro by the addition of a Fe(3+)-porphyrine complex and an oxygen donor.

New NO donors with antithrombotic and vasodilating activities, Part 28. N-(1-cyanoalkyl)-N-hydroxyureas.

Nineteen N-(1-cyanoalkyl)-N-hydroxyureas comprising aliphatic (3a-i, 4a, b, and 5a) and aromatic (3j-n, 4c, 5b) compounds were prepared, fourteen of them for the first time, and tested for antithrombotic (p.o. administration to rats, 60 mg/kg) effects. The N-(1-cyanocyclohexyl)-N-hydroxy-N'-phenylurea (3j) was most potent and inhibited laser-induced (35 mW, 50 ms) thrombus formation in arterioles by 21% and that in venules by 15%. The compounds form nitric oxide in vitro by the addition of a Fe3(+)-porphyrin complex and an oxygen donor. Moreover, the most active compound 3j in vivo exhibits the highest NO formation in vitro. Furthermore, it was shown that the cyano group is essential for the desired activities and NO formation. These results suggest that the title compounds act as NO donors.

New NO donors with antithrombotic and vasodilating activities. Part 22. Nitrosation products of hexamethylenetetramine.

Two nitrosation products of hexamethylenetetramine, namely 1,3,5-trinitrosohexahydro-1,3,5-triazine (1) and 3,7-dinitroso- 1,3,5,7-tetrazabicyclo[3.3.1]nonane (2), were synthesized. It is shown that both compounds in vitro at 37 degrees C (1 h, pH 7.4) form nitric oxide at a rate of 3.1% (1) or 1.3% (2), respectively. In rats (60 mg/kg p.o.) both compound inhibit thrombus formation in arterioles (1: 20%; 2: 16%) and venules (1: 18%; 2: 9%). Compound 2 does not influence the blood pressure in spontaneously hypertensive rats.

Interaction of Viagra with the NO donors molsidomine and RE 2047 with regard to antithrombotic and blood pressure lowering activities.

After p.o. administration to rats in doses up to 30 mg/kg, Viagra showed no antithrombotic effect. However, it enhanced the inhibition of thrombus formation by RE 2047 from 9% to 17% (5 + 5 mg/kg) or 19% to 27% (10 + 10 mg/kg) in arterioles. This effect was even more obvious in venules where an inhibition of 9% (5 + 5 mg/kg) or 15% (10 + 10 mg/kg) was seen whereas the individual drugs had no effect. The antithrombotic activity of molsidomine was not altered. The blood pressure (b.p.) of spontaneously hypertensive rats was reduced by the combination of Viagra and RE 2047 (5 + 5 mg/kg) to 94% of normal after 2 h while the individual drugs had no effect at this dose. The coadminstration of 10 mg/kg of each drug reduced the b.p. to 87% of normal. The combination of Viagra with molsidomine decreased b.p. to 84% (5 + 5 mg/kg) or 79% (10 + 10 mg/kg), respectively.

Bromelain proteases reduce human platelet aggregation in vitro, adhesion to bovine endothelial cells and thrombus formation in rat vessels in vivo.

The thiol protease, bromelain, an extract from pineapple stem, was suggested to have antithrombotic and anticoagulant activities in vivo. We studied the effects of bromelain on cell size distribution of isolated human platelets in vitro by Coulter Counter measurements. Preincubation of platelets with bromelain (10 micrograms/mL) completely prevented the thrombin (0.2 U/mL) induced platelet aggregation. Papain was less active in preventing platelet aggregation. In vitro, bromelain (0.1 microgram/mL) reduced the adhesion of bound, thrombin stimulated, fluorescent labeled platelets to bovine aorta endothelial cells. In addition, preincubation of platelets with bromelain, prior to thrombin, activation, reduced the platelet adhesion to the endothelial cells to the low binding value of unstimulated platelets. On the basis of mass concentrations, the proteases papain and trypsin were as effective as bromelain. Using a laser thrombosis model, the in vivo effects of orally and intraveneously applied bromelain on thrombus formation in rat mesenteric vessels were studied. Bromelain, orally applied at 60 mg/kg body weight, inhibited the thrombus formation in a time dependent manner, the maximum being after 2 hours in 11% of arterioles and 6% of venoles. Intravenous application at 30 mg/kg was slightly more active in reducing thrombus formation in arterioles (13%) and venoles (5%), suggesting that orally applied bromelain is biologically active. These results may help to explain some of the clinical effects observed after bromelain treatment in patients with thrombosis and related diseases.

New NO donors with antithrombotic and vasodilating activities, Part 24. Hydrazine derivatives.

Suitable hydrazines like phenylhydrazine (1), N,N-dimethyl hydrazine (4), and N,N-diphenyl hydrazine (5) can be oxidized by hydrogen peroxide at pH 7.4 and 37 degrees C to nitrosohydrogen and further to nitrite and nitrate. The extent of this property is correlated with platelet aggregation inhibiting and antithrombotic effects of these compounds, suggesting that an NO mediated mechanism might be involved. All hydrazines tested and two N-ethoxycarbonyl prodrugs exhibited antihypertensive effects which were not correlated to the above properties. This is especially pronounced in hydralazine (6) and dihydralazine (7) which induced a strong decrease in blood pressure but exhibit neither antiplatelet nor antithrombotic effects. This indicates that the mechanism of the antihypertensive activity is different from that of the antiplatelet activity.

NO donors with antithrombotic and vasodilating activities, Part 23. Organic azides.

Twenty eight organic azides were synthesized and tested for their antithrombotic and blood pressure lowering activities in rats (60 mg/kg, p.o.). In fifteen compounds significant antithrombotic effects were observed. In thirteen cases a significant lowering of the blood pressure of spontaneously hypertensive rats (SHR) was seen. The peak activities in both systems were found for hexyl azide (4), 2-phenylethyl azide (14), and 4-pyridinecarboxylic acid azide (23). In these compounds the inhibition of thrombus formation in mesenteric arterioles was > 20%. The lowering of blood pressure was > 10% and long lasting (> 6 h) in 4 and 14 while 23 had a shorter duration of action (approximately 4 h). In two classes of azides, namely branched aliphatic azides (e.g. 2-azidopentane 9) and aliphatic carbonyl derivatives (e.g. benzoyl-azido-methane 17), only antithrombotic properties were observed. A lack of endothelial metabolism is suggested to be the reason for this therapeutically favorable behaviour.

New NO-donors with antithrombotic and vasodilating activities, Part 20. Azodioxides activated by electron acceptors in geminal or vicinal position.

Twenty-two nitroso compounds with cyano, acyloxy, or carbonyl groups in geminal position were prepared, eight of them for the first time. In the solid state these compounds dimerize to colorless azodioxides. Exceptions are the 4-nitrobenzoyloxynitroso compounds 7b, f, and g which form bright blue crystals. In vitro (Born test, collagen) considerable antiplatelet activity was observed in each class of compounds. Azodioxides with cyano groups in geminal position (3a, b) were most active (IC50 approximately 10 microM) suggesting the importance of strong electron withdrawing groups in geminal position to the azodioxide partial structure. When administered orally to rats (60 mg/kg) all compounds inhibited the thrombus formation in mesenteric arterioles and venules. The acetyloxy derivatives 5d and 5e were most active (18-21% inhibition in arterioles and 11-15% inhibition in venules). In aqueous media at 37 degrees C the cyanonitroso compound 3b and the benzoyloxynitroso compound 7a decomposed to nitric oxide and its reduced form nitrosohydrogen. This suggests that the above pharmacological effects are mediated by a NO dependent mechanism.

New NO-donors with antithrombotic and vasodilating activities, Part 21. Pseudonitrosites and other azodioxides with vicinal electron acceptors.

Twelve vicinally substituted nitro-nitroso compounds (pseudonitrosites) were synthesized, nine of them for the first time. In the solid state the dimeric azodioxides are present. In the class of the pseudonitrosites 2a-h, all compounds exhibited comparatively strong antiplatelet activity in vitro (Born test, collagen). Four of them showed an IC50 below 10 microM, 2a being the most active substance with an IC50 = 2.1 microM. When administered orally to rats (60 mg/kg) small antithrombotic effects were observed. The pseudonitrosite 6d was the most active compound (18% inhibition in arterioles). The in vitro decomposition of 2a at 37 degrees C gave NO and N2O, indicating that the above pharmacological effects were mediated by an NO-dependent mechanism. The replacement of the nitro group in the pseudonitrosite partial structure by other electron acceptors i.e. acetyl, carboxyl, or acetyloxy groups leads to inactive (10a) or less active compounds (10b, e).

New NO-donors with antithrombotic and vasodilating activities, Part 19. Pseudonitroles and their dimeric azodioxides.

Thirteen geminally substituted nitro-nitroso compounds (pseudonitroles) have been synthesized, four of them for the first time. In the solid state the pseudonitroles are dimerized to azodioxides. This is proved by IR spectroscopy, with the dimeric N-O valence vibration being observed between 1293 and 1306 cm-1. Only 1,3-diphenyl-2-nitro-2-nitrosopropane is monomeric even when solid. This is backed by its blue color and an IR band at 1574 cm-1. When dissolved in chloroform these azodioxides dissociate completely to the blue monomers (lambda max approximately 650 nm). Eight pseudonitroles inhibited the aggregation of blood platelets half-maximally at concentrations below 10 microM (Born test, collagen). When administered orally to rats (60 mg/kg) the thrombus formation in mesenteric arterioles and venules was inhibited up to 25 percent (k; 1-nitro-1-nitrosocyclohexane). When kept in aqueous media at 37 degrees C nitric oxide and its reduced from, i.e. nitrosohydrogen, are released. This suggests that the above biological effects arise from an NO dependent mechanism. The lack of influence on the blood pressure of spontaneously hypertensive rats, however, strongly suggests that an enzyme supported rather than a thermal formation of NO occurs in vivo.

New NO donors with antithrombotic and vasodilating activities, Part 26. Amidoximes and their prodrugs.

Seventeen amidoximes (2a-q) comprising aliphatic (2a-d), aromatic (2e-n), and bis compounds (2o-q) have been synthesized. In the Born test 4-chlorophenylethenecarboxamidoxime (21) was most active and inhibited the blood platelet aggregation induced by collagen with an IC50 = 3 microM. After oral administration to rats (60 mg/kg) fourteen compounds significantly inhibited the formation of thrombi in arterioles and venules. The strongest effect was observed with ethene-bis-carboxamidoxime (2q) (31% in arterioles and 18% in venules). The O-ethoxycarbonylderivatives 3 and the corresponding 1,2,4-oxadiazol-5-ones 4, which had been synthesized as prodrugs, showed smaller antithrombotic effects.

New NO donors with antithrombotic and vasodilating activities, part 25. Hydroxylamine derivatives.

Twelve ethoxycarbonyl or phenylsulfonyl derivatives as prodrugs of hydroxylamine or phenylhydroxylamine were prepared and tested for antiplatelet (in vitro, Born test) antithrombotic (in vivo thrombosis model), and antihypertensive (in vivo, SHR rats) effects. In the Born test N,N-bisphenylsulfonylhydroxylamine (10) was most active (IC50 = 11 mumol/L). The N-ethoxycarbonyl-phenylhydroxylamine (7) was the most potent antithrombotic compound. It inhibited the thrombus formation in mesenteric arterioles of rats by 39% after a single p.o. dose of 60 mg/kg. Compound 7 lacked any antihypertensive activity. It, therefore, had been possible to separate completely the antithrombotic activities from antihypertensive properties in suitable hydroxylamine derivatives.

2-Nitrosoimino-3,6-dihydro-2H-1,3,4-thiadiazines with antiplatelet and antithrombotic properties.

The successful synthesis of 17 nitrosimines of 2 H-1,3,4-thiadiazines is reported. They are best characterized by their electronic spectra (lambda max = 504-522 nm). Some of the compounds were able to inhibit the aggregation of blood platelets in the Born-test (inducer collagen). The most active compound was the 3-nonyl-5-phenyl-derivative 10c which showed an IC50 = 6.5 mumol.l-1. In mesenteric arterioles of rats after p.o. administration (60 mg/kg) a 13% inhibition of thrombus formation is observed. The reason for the rather small effects is the high chemical stability of the title compounds and the fact that decomposition occurs mainly by the evolution of molecular nitrogen. Only small yields of NO are formed.

New NO-donors with antithrombotic and vasodilating activities, Part 17. Arylazoamidoximes and 3-arylazo-1,2,4-oxadiazol-5-ones.

Seven arylazoamidoximes (3), six phenoxycarbonyl derivatives (4), and six 1,2,4-oxadiazol-5-ones (5) have been prepared and their structure and purity established by spectroscopy and elemental analysis. In the EI mass spectra ready elimination of NO from the title amidoximes was observed. A new addition reaction of 3a with hydrochloric acid to 4-chlorophenylhydro-azoamidoxime 7 is described. The compounds were tested for nitric oxide dependent biological properties, i.e. platelet aggregation, antithrombotic effects, and decrease in blood pressure. In arterioles of rats 5/19 compounds inhibited the formation of thrombi with a laser beam by > or = 20% 2 h after oral administration of 60 mg/kg. Among these are three amidoximes (3a, 3e, 3f), one phenoxycarbonyl derivative (4a), and one oxadiazolone (5a). With the 4-chlorophenylazoamidoxime 3c a long lasting (24 h) decrease of blood pressure in spontaneously hypertensive rats was observed. Microsomal fractions of rat liver oxidize arylazoamidoximes and generate nitric oxide (e.g. 3a and 3b). NO was measured by the oxyhemoglobin assay. The influence of SOD, pretreatment of the rats with dexamethasone, as well as kinetic parameters were determined. Type 3 compounds, therefore, are a new class of NO donors. Type 4 and 5 compounds function as their prodrugs.

New NO-donors with antithrombotic and vasodilating activities, Part 16. 3-Amino-1,2,4-oxadiazol-5-ones as prodrugs for hydroxyguanidines.

Nineteen 4-substituted 1,2,4-oxadiazol-5-ones (6a-s) were prepared as prodrugs for lipophilic hydroxyguanidines which should be metabolized in vivo to nitric oxide. This hypothesis was tested indirectly by measuring the antithrombotic properties of these compounds 2 h after oral administration to rats (60 mg/kg). In mesenteric arterioles seven compounds moderately (> or = 10%) inhibited the formation of thrombi by a laser beam. Maximum effects were observed in 6c (4-pentyl) and 6f (4-benzyl). The lack of activity in the corresponding 2-pentyloxadiazolone 10c, where no formation of nitric oxide seems possible, indirectly suggests that the antithrombotic properties of the title compounds could be mediated by the in vivo formation of nitric oxide.

New NO-donors with antithrombotic and vasodilating activities, Part 14. 1,3,4-Triazol-1-oles.

Five 1,3,4-triazol-1-oles (5a-f) with different alkyl, aryl, and arylalkyl substituents in 2,5-position were synthesized and tested for their antithrombotic properties. The 2,5-dimethyl derivative 5a was most active. 2 h after administration of 60 mg/kg to rats thrombus formation by a laser beam was inhibited by 42% in arterioles and by 33% in venules. At the same dose the blood pressure of SHR rats was slightly (5%) but significantly decreased even 4 h after application of 5a. This pattern of activities suggests a nitric oxide mediated mechanism of action. 1,1'-Azo-bis-ethanone oxime(7)-the synthetic precursor of 5a-inhibited the aggregation of blood platelet (Born test) with an IC50 = 15 mumol/L.

Platelet aggregation inhibiting and anticoagulant effects of oligoamines, Part 33. From tetradecane- to octacosanediamines.

Twenty alkanediamines were designed according to structure-activity relationships drawn from previous parts of this series and synthesized. Their general structure is CH3-(CH2)n-CHNH2-(CH2)m-CHNH2-(CH2)n-CH3, (n = 2-10; m = 3-6). Twelve of them inhibited the aggregation of human blood platelets in concentrations between 3-10 micromol/L halfmaximally (Born test, inducer collagen). With increasing m a decreasing n is necessary to achieve the optimum activity. In the most active compounds (7b, 7e, 7p) it is found that m + n = 9. When the nitrogen functions are hydroxyalkylated secondary amines with similar antiplatelet effects are obtained. The conversion of the amino groups into syndronimines is accompanied by the loss of activity. The bisethoxycarbonylderivatives of 7f and 7m (8f, 8m) exhibited antithrombotic effects in rats after oral administration.