The study on organic nitrates, part V. New derivatives of piperazine potential NO donors

We have obtained a series of non-symmetrical 1,4-disubstituted derivatives of piperazine, with the structure of organic nitrates, as potential NO donors. These compounds were obtained from respective hydroxyl derivatives of piperazine in an esterification reaction by fuming nitric acid. The obtained nitrates were tested in-vitro by reaction with a sulfhydryl compound. The structure of the most active nitrate and its hydroxyl analogue was used for the calculation of geometrical optimization with the determination of 3D-QSAR by a semi-empirical method PM3 using HyperChem 4.5.     

Effect of oral organic nitrates on expression and activity of vascular soluble guanylyl cyclase

Background and purpose:

The regulation of vascular soluble guanylyl cyclase (sGC) expression by nitric oxide (NO) is still under discussion. In vitro, NO has been shown to downregulate the expression of sGC but it is unclear if this mechanism is operative in vivo and occurs during nitrate treatment.

Experimental approach:

We investigated whether high dose isosorbide mononitrate (ISMN) or pentaerythrityl tetranitrate (PETN) treatment changes vascular sGC expression and activity in vivo. New Zealand White rabbits received a standard diet, 2 or 200 mg ISMN kg⁻¹ d⁻¹ for 16 weeks, and C57BL/6 mice received a standard diet, 6, 60 or 300 mg PETN kg⁻¹ d⁻¹ for four weeks. Absorption was checked by measuring the plasma levels of the drug/metabolite.

Key results:

Western blots of rabbit aortic rings showed similar protein levels of sGC α1- (P=0.2790) and β1-subunits (P=0.6900) in all groups. Likewise, ANOVA showed that there was no difference in the expression of sGC in lungs of PETN-treated mice (P=0.0961 for α1 and P=0.3709 for β1). The activities of isolated sGC in response to SNAP (1 μM–1 mM) were identical in aortae of ISMN-treated rabbits (P=0.0775) and lungs of PETN-treated mice (P=0.6348). The aortic relaxation response to SNAP slightly decreased at high ISMN but not at high PETN.

Conclusions and implications:

These data refute the hypothesis that therapeutic treatment with long acting NO donors has a significant impact on the regulation of vascular sGC expression and activity in vivo.     

The reaction between organic nitrates and sulfhydryl compounds. A possible model system for the activation of organic nitrates

The rate of loss of the sulfhydryl group, determined with the Ellman reagent, was used to derive second order rate constants for the reaction of a series of organic nitrates with a series of sulfhydryl compounds. For the organic nitrates, increases in the rate of reaction with cysteine, in general, ran parallel both with increases in pharmacological potency (flow in the Langendorff heart) and with increases in total clearance. Cysteine was the most active sulfhydryl compound examined, which is compatible with a possible role as an important nitrate receptor. Under some conditions the rate of loss of the sulfhydryl group was much greater than the rate of formation of nitrite ion. This indicates the presence of a reaction intermediate, probably a thionitrate. It is suggested that, in vivo, a thionitrate could function as an important intermediate in the activation of guanylate cyclase.     

地塞米松抑制兔内毒素休克进程中血浆ET 1和NO的异常升高

目的　探讨地塞米松（Ｄｅｘ）抗毒作用原理。方法　兔ｉｖ 内毒素６００ μｇ·ｋｇ－ １ 诱发休克，３０ ｍｉｎ 时分别ｉｖ 生理盐水或Ｄｅｘ 、Ｌ ＮＭＡ、ｐｈｏｓｐｈｏｒａｍｉｄｏｎ （ｐｈｏｓ）， 测平均动脉压（ＭＡＰ） 、每搏心输出量（ＳＶ）、和血浆ＥＴ １ 与ＮＯ－３ 浓度。结果　休克兔血浆ＮＯ－３ 、ＥＴ １ 浓度升高， 血流动力学进行性紊乱直至死亡。Ｌ ＮＭＡ 和Ｐｈｏｓ 分别抑制血浆ＮＯ－３ 或ＥＴ １浓度异常升高及基础分泌，短暂改善血流动力学指标。Ｄｅｘ 不影响ＮＯ、ＥＴ １ 基础水平，但抑制休克时兔血浆ＮＯ－３和ＥＴ １ 异常升高、纠正血流动力学紊乱、提高动物生存率。结论　Ｄｅｘ 抑制休克兔血浆ＮＯ、ＥＴ １ 异常升高， 可能是其抗内毒素休克的重要机制之一     

Actions of isosorbide dinitrate on smooth muscle cells of rabbit vascular tissues.

To investigate the mechanism of the anti-anginal actions of isosorbide dinitrate (ISDN), the effects of this agent on smooth muscle cells of intact and skinned preparations of the rabbit mesenteric artery and vein, and of the coronary artery were studied. ISDN (less than 10(-5) M) had no effect on the membrane potential or resistance of smooth muscle cells of the mesenteric artery and vein under resting conditions, nor when the membrane was depolarized by the presence of various concentrations of [K]o or noradrenaline (NA). The amplitude of spike evoked by outward current pulse after pretreatment with 10 mM tetraethylammonium (TEA) in the mesenteric artery was slightly inhibited by application of 10(-5) M ISDN. The K-induced and NA-induced contractions in the mesenteric artery were not affected by 10(-5) M ISDN, while those evoked in the mesenteric vein were inhibited in concentrations above 10(-6) M. The amplitude and facilitation of excitatory junction potentials evoked by perivascular nerve stimulation in the mesenteric artery were not affected by 10(-5) M ISDN. In skinned muscles, the free calcium concentration (pCa)-tension relationships observed in the mesenteric artery and vein were not affected by 10(-5) M ISDN. This agent had no effect on Ca accumulation into and Ca release from the stores in muscle cells of the mesenteric artery and vein, in skinned preparations. In the rabbit coronary artery, the membrane potential, resistance and spike evoked in the presence of 10 mM TEA were not affected by application of 10(-5) M ISDN. The contraction evoked by excess concentrations of [K]o was not affected. The contraction evoked by a low concentration of acetylcholine (3 X 10(-7) M) but by high concentrations (greater than 10(-6) M) was slightly inhibited by 10(-5) M ISDN. A tonic contraction induced in 39 mM [K]o was reduced by 10(-5) M nitroglycerine but not by 10(-5) M ISDN. Thus in rabbit vascular tissues, ISDN mainly acts on the venous system in vitro. The induced vasodilatation may lead to a reduction in the venous return and hence, reduce oxygen consumption in the cardiac muscles. This effect of ISDN may relate to the anti-anginal actions.     

丙溴磷对家兔组织中血管内皮活性物质的影响

目的　探讨丙溴磷对组织中血管内皮活性物质的影响及意义。方法　对不同染毒剂量、不同染毒时间家兔进行了全血胆碱酯酶 (ChE)活力 ,以及大脑、肝和肾组织中的内皮素 (ET)、一氧化氮 (NO)浓度的测定。结果　与染毒前和对照组比较 ,染毒后家兔全血ChE活力显著下降 (P <0 0 1) ;染毒后家兔组织中ET浓度 [( 9 2 1～ 12 65 )pg ml]较染毒前 [( 7 98～ 10 2 5 )pg ml]有明显增高趋势 ,而染毒后NO浓度 [( 10 3 8～ 17 3 6)nmol ml]较染毒前 [( 15 44～ 2 5 64 )nmol ml]有明显下降趋势 (P <0 0 5 ,P <0 0 1) ,且随染毒时间延长变化更明显。结论　丙溴磷所致的血管内皮活性物质的紊乱 ,可能是丙溴磷抑制ChE活力以外的毒性表现     

Correlation between nitric oxide formation during degradation of organic nitrates and activation of guanylate cyclase

Organic nitrates develop their vasodilating potency by stimulating the enzme guanylate cyclase. There are still several theories concerning the molecular mechanism of enzyme activation, the most likely of which sees nitric oxide (NO·) as the true modulator of the soluble guanylate cyclase. We therefore examined the release of nitric oxide from organic nitrates by means of difference-spectrophotometric method and found that our results correlated well with the extent of enzyme activation. The more NO· was liberated from the compounds in question, the higher was the enzyme activation observed. When the examined nitrates were used in a concentration which caused a half-maximal enzyme stimulation, the result was a NO· liberation of striking uniformity. This correlation also applied to SIN-1 for which it has been assumed up to now that the intact molecule itself is able to stimulate the enzyme and not the nitric oxide released from it. We found the reaction between organic nitrates and cysteine to be highly dependent on temperature, while the extent of the observed enhancement increased with the number of nitrate groups per molecule. We also studied the potential effects of certain compounds on non-enzymatic NO· release and found that, in addition to methylene blue, thionine and brilliantcresyl blue, but not ferricyanide were also effective inhibitors. So it seems likely that both an enzymatic and a non-enzymatic mode of inhibition of enzyme activity does exist. Since oxyhemoglobin is an effective scavenger of nitric oxide, its addition can inhibit enzyme activation by nitrovasodilators. Our results stress the important role of the non-enzymatic liberation of NO· from organic nitrates and related compounds as possible, perhaps even as the principal mode of activation of soluble guanylate cyclase by nitrovasditors.     

应激性大鼠不同组织中NO浓度的变化与血压的关系

目的 :研究应激性大鼠不同组织中NO浓度的变化与血压的关系。方法 :给SD大鼠间断性足底电刺激并噪声刺激 1 5d ,观察其尾动脉BP的变化和血浆、心耳、心室、血管、肾上腺等组织中NO的浓度变化。结果 :应激组经 1 5d应激后BP显著性升高 ;应激 L arg (精氨酸 )组应激前后BP变化不明显。应激后血浆中NO的浓度明显降低 ,心耳、心室、血管、肾上腺中的NO几乎难以测出 ,而应激 L arg组的大鼠经过 1 5d的应激刺激 ,血浆及血管组织中的NO浓度 ,明显升高。心耳、心室、肾上腺中NO的浓度与对照组相比变化不明显。结论 :应激刺激可使BP升高 ,同时血浆、心耳、心室、血管、肾上腺等组织中NO的浓度降低 ,而L arg可抑制BP升高 ,血浆及血管组织中NO浓度增加 ,心耳、心室、肾上腺等组织中NO浓度不降低。应激性大鼠血压的升高与血管内皮细胞及其他组织中NO合成功能障碍有关     

Inhibition by nicotine of the formation of prostacyclin-like activity in rabbit and human vascular tissue.

1 Rings of vascular tissue (from rabbit aorta or human peripheral vein) were incubated at room temperature in Tyrode solution in the absence or presence of nicotine or indomethacin. 2 Addition of portions of the incubates to human platelet-rich plasma (HPRP) elicited a decrease in adenosine 5'-diphosphate (ADP)-induced platelet aggregation in this plasma. Authentic prostacyclin (PGI2) also induced such a decrease. The decreased aggregation amplitudes that followed the addition of the vascular tissue incubates and of PGI2 were equally potentiated by theophylline (10(-4) M). 3 Both nicotine and indomethacin counteracted the formation of platelet anti-aggregatory activity in the vascular tissue incubates. The IC50S of nicotine and of indomethacin on the formation of platelet antiaggregatory activity were 2 X 10(-5) M and 6 X 10(-6) M, respectively. 4 Nicotine failed to affect the platelet anti-aggregatory effect induced by authentic PGI2 in HPRP. 5 It is concluded that nicotine counteracts the formation of platelet anti-aggregatory activity in rabbit aorta and human peripheral vein by eliciting an inhibitory effect on the bioformation of prostacyclin in these types of vascular tissue.     

Total prevention of the development of in vitro tolerance to organic nitrates. Experiments with antioxidants.

The nearly total inhibition of development of pharmacological tolerance to an organic nitrate is reported here for the first time. The development of in vitro tolerance in the rabbit aorta to isosorbide-5-mononitrate (CAS 87-33-2) was potently inhibited by five structurally unrelated antioxidants--diaminodurol, ascorbic acid, potassium sulphite, pyrogallol and quercetin. Diaminodurol, ascorbic acid and potassium sulphite decreased, but quercetin increased, the spasmolytic activity of isosorbide-5-mononitrate. Diaminodurol, potassium sulphite, quercetin and ascorbic acid potently inhibited the spasmolytic activity of nitric oxide (NO). Quercetin also inhibited the development of in vitro tolerance to glyceryl trinitrate. It is suggested that tolerance to organic nitrates is the result of biochemical damage caused by a reactive intermediate such as NO. To test this possibility directly the effect of pretreatment with NO on the spasmolytic activity of glyceryl trinitrate (CAS 55-63-0) was examined. This pretreatment produced a small but significant tolerance to glyceryl trinitrate and to SIN-1 (3-morpholinosydnone imine), which also acts through guanylate cyclase. There was no effect on the activity of the unrelated vasodilators nitrendipine and theophylline. It is concluded that the reaction between NO and soluble quanylate cyclase is a real but minor cause of tolerance to organic nitrates. Other possible mechanisms of tolerance development are discussed.     

Investigations into the development of tolerance to organic nitrates in conscious dogs

The role of doses and dose intervals in the development of tolerance towards organic nitrates was investigated. It was studied whether the decrease in systolic blood pressure (SBP) induced by i.v. injection of 30 micrograms/kg glyceryl trinitrate (GTN) is influenced by the simultaneous i.v. infusion of GTN or isosorbide-5-mononitrate (IS-5-MN) - two dose levels each - at doses that lower the SBP by 20-35 mmHg. Although infusion of the lower dose of IS-5-MN caused a constant plasma concentration that was about 100 times more than that required for the anti-anginal effects, the acute GTN response was not reduced. However, the acute response was diminished at the other groups. In additional investigations incremental doses of IS-5-MN were injected i.v. at different dose intervals. It was observed that the responsiveness was nearly identical at dose intervals of 24, 4 and 2 h (= 1.3 half-lives in the dog, which is equivalent to a t.i.d. dose schedule in man), whereas it was markedly reduced at intervals of 10 min.     

Selective expression of Kir6.1 protein in different vascular and non-vascular tissues

K(ATP) channels are composed of pore-forming subunits Kir6.x and auxiliary subunits SURx. These channels play important roles in modulating the contractility of vascular smooth muscle cells (SMCs) by altering membrane potentials. The molecular basis of K(ATP) channels in vascular SMCs is unclear and the expression of different K(ATP) channel subunits at protein level in various tissues still undetermined. In this study, using an anti-Kir6.1 antibody, we detected the expression of Kir6.1 proteins in rat vascular tissues including mesenteric artery, pulmonary artery, aorta, and tail artery. Kir6.1 proteins were also identified in heart and other non-vascular tissues including spleen and brain, but they were undetectable in liver and kidney. Immunocytochemical study revealed the expression of Kir6.1 proteins in cultured rat thoracic aortic SMCs. Using the whole-cell patch-clamp technique, it was found that the intracellularly applied anti-Kir6.1 antibody significantly inhibited K(ATP) channel currents in HEK-293 cells that were stably transfected with Kir6.1 cDNA. A better understanding of differential expression of Kir6.1 proteins in various vascular and non-vascular tissues may help discern different molecular basis and functions of K(ATP) channel complexes in these tissues.     

Effects of arachidonic acid in the rabbit pulmonary and systemic vascular beds in vivo

We studied the effects of arachidonic acid (AA) in the pulmonary and systemic circulations of rabbit under constant blood flow conditions in vivo, using a total heart bypass model. AA (100-150 micrograms/kg) injected into the pulmonary artery produced a dose-dependent increase in pulmonary arterial pressure (Ppa), without altering systemic arterial pressure (Psa). Conversely, injection of AA into the aorta reduced Psa in a dose-dependent fashion, but did not significantly alter Ppa. FPL55712, a leukotriene receptor antagonist, did not affect any of these responses to AA. Indomethacin, a cyclo-oxygenase inhibitor, totally prevented the pulmonary pressor response to intravenously administered AA and reduced the systemic depressor response to intra-arterially administered AA. The thromboxane A2 synthetase inhibitor, 7-(1-imidazolyl)heptanoic acid, totally abolished the increase in Ppa in response to intravenously administered AA, but did not alter the dose-dependent decrease in Psa in response to intra-arterially administered AA. These results suggest that in rabbits (1) AA produces pulmonary vasoconstriction and systemic vasodilation, (2) blood metabolites of AA mediate these effects and are then rapidly deactivated, and (3) AA-induced pulmonary vasoconstriction appears to be largely dependent on thromboxane A2.     

Bioavailability and bioequivalence of organic nitrates. Isosorbide dinitrate--a study of sustained-release preparations

Assessment of Bioavailability of Organic Nitrates/Comparative bioavailability study of sustained-release isosorbide dinitrate preparations. Relative bioavailabilities of isosorbide dinitrate (ISDN, CAS 87-33-2) and the metabolite isosorbide-5-mononitrate (IS-5-MN, CAS 16051-77-7) were studied after application of Maycor retard 40 (sustained-release capsules, multiple unit formulation, test preparation) in comparison to sustained-release tablets (single unit formulation, reference preparation) with 16 healthy male volunteers in a two-way crossover design. Test and reference formulations were previously characterised in vitro by dissolution tests. ISDN, IS-5-MN (IS-2-MN) plasma concentrations were determined using a selective and sensitive GLC-method with ECD-detection. As pharmacokinetic parameters AUC, Cmax and half value duration (HVD) were evaluated. Bioequivalence was assessed by calculating 90%-confidence intervals (ANOVA, ANOVAlog, Mann-Whitney-test) for ISDN and IS-5-MN. Bioequivalence was accepted if due to the inclusion rule one of the calculated intervals fulfill the requirements of 80 and 120% (AUC) or 70 and 130% (Cmax, HVD), respectively. Relative bioavailability of the test formulation was calculated as 94% (ISDN) and 96% (IS-5-MN). Maximum plasma concentrations of ISDN (IS-5-MN) were determined for the test preparation as 14.3 +/- 3.1 ng/ml (265 +/- 45 45 ng/ml) and as 22.8 +/- 12.6 ng/ml (287 +/- 59 ng/ml) for the reference product. HVD-values were for the test preparation 4.5 +/- 1.3 h (ISDN) and 8.5 +/- 1.3 h (IS-5-MN) and for the reference formulation 3.1 +/- 1.2 h (ISDN) and 8.1 +/- 1.4 (IS-5-MN).(ABSTRACT TRUNCATED AT 250 WORDS)     

Carbaryl distribution in rabbit tissues and body fluids.

After single po administration of 14C-naphthylcarbamate, liquid scintillation assays evaluated the distribution of carbaryl in rabbit serum, liver, kidneys, small and large intestine, spleen, heart, muscles of the thigh and lungs and its excretion in urine and feces at 2, 4, 6 and 8 h after dosing. At 2 and 8 h radioactivity was not observed in spleen, heart, muscle and lungs, while all other tissues had increased values up to 6 h. The main excretory pathway of carbaryl was the kidneys.     

Metabolism of nipradilol by liver homogenates from different species. I. Comparative studies on the denitration of nipradilol and other organic nitrates

Nipradilol (NP) was metabolized mainly in liver and denitrated and/or hydroxylated metabolites were formed. NP was not metabolized in heart or lung, and was stable in blood. Denitration of NP was catalysed mainly by glutathione-dependent organic nitrate reductase in liver cytosol, and the hydroxylation was catalysed by liver-microsomal enzymes. Denitration activity for NP was far lower than that for nitroglycerol and isosorbide dinitrate with liver homogenates of several animal species. A marked species difference was found in the denitration activity for NP; dog liver was especially low compared with rabbit, rat and mouse liver.