Dinitrosyl Iron Complexes with Thiol-Containing Ligands Exist in Living Organisms Mainly in the Binuclear Form

The levels of the mononitrosyl iron complex with diethyldithiocarbamate that form in the liver of mice in vivo and in vitro after intraperitoneal injection of binuclear dinitrosyl iron complexes with N-acetyl-L-cysteine or glutathione, S-nitrosoglutathione, sodium nitrite, or the vasodilating drug isosorbide dinitrate (Isoket®) have been assessed by electron paramagnetic resonance (EPR). The levels of the complex in mice that received binuclear dinitrosyl iron complexes with thiol-containing ligands or S-nitrosoglutathione do not change after the treatment of liver preparations with the strong reducing agent dithionite, in contrast to those formed after nitrite or isosorbide dinitrate administration, whose levels sharply increase after the same treatment. It is inferred that in the latter case an EPR-active mononitrosyl iron complex with diethyldithiocarbamate is produced with the absence or presence of dithionite in the reaction of NO formed from nitrite with Fe²⁺-diethyldithiocarbamate and Fe³⁺-diethyldithiocarbamate complexes, respectively. In the former case, the mononitrosyl iron complex with diethyldithiocarbamate is produced by transition of iron-mononitrosyl fragments from already present iron-dinitrosyl groups of binuclear dinitrosyl complexes, whose content is three to four times higher than the content of the mononuclear form of these complexes in the tissue. The results we obtained indicate that when dinitrosyl iron complexes with thiol-containing ligands, either introduced into the body or produced with the participation of endogenous NO, appear in animal tissues in vivo, these complexes are presented in these tissues mainly in their diamagnetic, EPR-silent binuclear form.

     

Effect of isosorbide dinitrate,verapamil, and labetalol on portal pressure in cirrhosis.

The effects on portal pressure of the vasodilatory drugs isosorbide dinitrate and verapamil and of an alpha and beta blocking agent, labetalol, were assessed in 21 patients with cirrhosis and portal hypertension. The wedged hepatic venous pressure gradient (wedged minus free hepatic venous pressures) was used as an index of portal pressure and was not significantly changed by treatment with labetalol (n = 5) but was significantly decreased by verapamil (n = 6; p less than 0.05) and isosorbide dinitrate (n = 10; p less than 0.01). Long term administration of isosorbide dinitrate also had a significant effect (p less than 0.01).     

Inhibition of the morphine withdrawal syndrome by a nitric oxide synthase inhibitor,NG-nitro-L-arginine methyl ester

The hypothesis that an arginine-nitric oxide (NO) synthase-NO system mediates the morphine abstinence syndrome was tested in adult male rats implanted subcutaneosly for 3 days with one morphine (75 mg) pellet followed by naloxone-precipitated withdrawal (0.5 mg/kg). Injection with a NO synthase inhibitor, N^G-nitro-L-arginine methyl ester (NAME, 100 mg/kg subcutaneous), shortly before naloxone-induced withdrawal significantly inhibited abstinence signs by 25–80%. Continuous infusion of NAME via subcutaneous osmotic pumps during the development of morphine physical dependence and during naloxone-precipitated withdrawal also inhibited morphine abstinence signs. In addition, treatment with isosorbide dinitrate, a NO donor, induced a quasi morphine-abstinence syndrome (QMAS) that was significantly suppressed by implantation of a morphine pellet 3 days before isosorbide dinitrate treatment. These results indicate that NO mediates part of the expression of the morphine abstinence syndrome.     

Solid-phase extraction of isosorbide dinitrate and two of its metabolites from plasma for gas chromatographic analysis

A rapid, accurate and selective method for the determination of isosorbide dinitrate and its 2- and 5-isosorbide mononitrate metabolites in 1.0 ml of human plasma has been developed. Before chromatographic quantitation by gas-liquid chromatography with electron-capture detection, the compounds are subjected to solid-phase extraction, using ENVI 18 cartridges (Supelco). The intra-day and inter-day coefficients of variation are less than 10%, except the inter-day coefficient of variation for the assay of 5-isosorbide dinitrate which is less than 15%. Limits of quantitation are 10, 10 and 20 ng/ml for isosorbide dinitrate, 2-isosorbide mononitrate and 5-isosorbide mononitrate, respectively. Recoveries are in excess of 90% for isosorbide dinitrate and 70% for its two metabolites.     

Bioconversion of isosorbide dinitrate by various microorganisms

Summary A total of 19 microorganisms, selected from genera of bacteria, fungi and yeasts, were screened for their ability to hydrolyse isosorbide dinitrate (ISDN) to mononitrates. Cunninghamella echinulata and Cunninghamella elegans showed rates of bioconversion of ISDN of 74% and 88% respectively, measured after 73 h. However, the two strains exhibited opposite stereoselectivity, as reflected in the ratios of isosorbide 5-mononitrate (5-ISMN) to isosorbide 2-mononitrate (2-ISMN). These were 2.57 and 0.75 for C. echinulata and C. elegans, respectively.     

Improved method for the rapid determination of isosorbide dinitrate in human plasma and its application in pharmacokinetic studies

A rapid, accurate and highly sensitive method was developed for the determination of isosorbide dinitrate in human plasma. Concentrations in the lower nanogram and subnanogram range are determined by a one-step extraction of 2 ml plasma, containing 4 ng/ml nitroglycerine as internal standard, with 5.5 ml n-pentane. The extract is subjected to gas—liquid chromatography—electron capture detection analysis. The lower limit of quantitation is 200 pg/ml, but concentrations as low as 50 pg/ml are still detectable. The method allows the quantitative determination of isosorbide dinitrate plasma levels in man following a 5 mg sublingual administration up to four hours after application.     

Isoforms of cytochrome P450 on organic nitrate-derived nitric oxide release in human heart vessels.

Glutathione S-transferases and the cytochrome P450 system have been proposed for the vascular biotransformation systems in the metabolic activation of organic nitrates. The present study was designed to elucidate the role of human cytochrome P450 isoforms on nitric oxide formation from organic nitrates using lymphoblast microsomes transfected with human CYP isoforms cDNA. CYP3A4-transfected microsomes had the most effective potential of nitric oxide formation from isosorbide dinitrate. Anti-CYP3A2 antibody (which cross-reacts with CYP3A4) or ketoconazole (an inhibitor of the CYP3A superfamily) inhibited nitric oxide formation from isosorbide dinitrate in rat heart microsomes. Immunohistochemistry of human heart also showed intense bindings of CYP3A4 antibody in the endothelium of the endocardium and coronary vessels. These results suggest that the CYP3A4-NADPH-cytochrome P450 reductase system specifically participates in nitric oxide formation from isosorbide dinitrate.     

Hydralazine and Organic Nitrates Restore Impaired Excitation-Contraction Coupling by Reducing Calcium Leak Associated with Nitroso-Redox Imbalance

Although the combined use of hydralazine and isosorbide dinitrate confers important clinical benefits in patients with heart failure, the underlying mechanism of action is still controversial. We used two models of nitroso-redox imbalance, neuronal NO synthase-deficient (NOS1^−/−) mice and spontaneously hypertensive heart failure rats, to test the hypothesis that hydralazine (HYD) alone or in combination with nitroglycerin (NTG) or isosorbide dinitrate restores Ca²⁺ cycling and contractile performance and controls superoxide production in isolated cardiomyocytes. The response to increased pacing frequency was depressed in NOS1^−/− compared with wild type myocytes. Both sarcomere length shortening and intracellular Ca²⁺ transient (Δ[Ca²⁺]_i) responses in NOS1^−/− cardiomyocytes were augmented by HYD in a dose-dependent manner. NTG alone did not affect myocyte shortening but reduced Δ[Ca²⁺]_i across the range of pacing frequencies and increased myofilament Ca²⁺ sensitivity thereby enhancing contractile efficiency. Similar results were seen in failing myocytes from the heart failure rat model. HYD alone or in combination with NTG reduced sarcoplasmic reticulum (SR) leak, improved SR Ca²⁺ reuptake, and restored SR Ca²⁺ content. HYD and NTG at low concentrations (1 μm), scavenged superoxide in isolated cardiomyocytes, whereas in cardiac homogenates, NTG inhibited xanthine oxidoreductase activity and scavenged NADPH oxidase-dependent superoxide more efficiently than HYD. Together, these results revealed that by reducing SR Ca²⁺ leak, HYD improves Ca²⁺ cycling and contractility impaired by nitroso-redox imbalance, and NTG enhanced contractile efficiency, restoring cardiac excitation-contraction coupling.     

High-dosage isosorbide dinitrate therapy for angina.

We investigated the possibility that high dosages (480 mg/d) of isosorbide dinitrate might reduce the frequency of angina attacks in selected patients who had not responded to low dosages of the drug (40 mg/d), and that the patients could tolerate the high levels of medication and maintain their responsiveness over the long term. In the single-blind phases of this trial 24 patients with grade 3 stable angina pectoris were given a placebo for 4 weeks and then increasing doses of isosorbide dinitrate for a further 6 weeks. The 19 patients who both responded to and tolerated high doses of the drug kept taking 480 mg/d for an average of 1 year. The average weekly rate of angina attacks fell by 74%, from 6.05 in the placebo phase to 1.6 during long-term active treatment (p less than 0.01). Nitroglycerin consumption decreased accordingly. The patients'' assessments of their levels of activity and well-being and their angina thresholds showed improvement among most of them. The trend of angina frequency was stable in 12 cases, downward in 6 and upward in only 1 case. Exercise performance as evaluated by a graded treadmill test showed a small but nonsignificant improvement of 18%. It was concluded that some patients who do not respond to the antianginal action of low-dosage isosorbide dinitrate and cannot be given beta-blockers may respond to high dosages and tolerate them for over a year. Isosorbide dinitrate may be clinically useful in patients with coronary heart disease even though their exercise performance is not significantly improved.     

Bioconversion of isosorbide dinitrate into isosorbide mononitrate by the protozoan Tetrahymena thermophila: relationship to glutathione transferase levels

Summary Growing cells of Tetrahymena thermophila (T. t.) metabolized, after 72 h, 80% of isosorbide dinitrate (ISDN) to isosorbide 5-mononitrate (5-ISMN) and isosorbide 2-mononitrate (2-ISMN) in a ratio 5-ISMN/2-ISMN=2.6 as evaluated by HPLC. The level of glutathione S-transferases (GSH-ST) determined by following the reactions with of 1-chloro-2,4-dinitrobenzene (CDNB), o-dinitrobenzene (o-DNB) or ISDN, showed the inductive effect of ISDN (0.5 mg/ml) on the level of this enzyme. The enzymatic activity, evaluated at 72 h, showed a twofold increase compared with the control. The GSH-ST activity correlated well with the rate of ISDN bioconversion.     

硝酸异山梨酯片联合美托洛尔治疗老年冠心病心绞痛的疗效及对IL-18及hs-CRP水平的影响．

目的：观察硝酸异山梨酯片联合美托洛尔治疗老年冠心病心绞痛的临床效果,观察联合用药对患者血浆白介素．18（Inter．1eukin,IL-18）、超敏C反应蛋白（HighsensitivityCreactiveprotein,hs—CRP）的影响。方法：选择本院收治的老年冠心病心绞痛患者84例,随机分为研究组和对照组,各42例,研究组给予硝酸异山梨酯片20mg,3次／d,口服,美托洛尔12．5mg,1次／d,口服;对照组仅给予硝酸异山梨酯片20mg,3次／d,口服,疗程均为28d。观察两组用药后心绞痛改善情况,并观察两组治疗前后血清IL-8、hs-CRP变化。结果：治疗后,研究组总有效率为90．5％;对照组总有效率为69．0％,研究组总有效率明显高于对照组（P〈0．01）。治疗后两组心率、心绞痛发作频率明显降低,持续时间明显缩短（P〈0．05）;研究组心率、心绞痛发作频率明显低于对照组,持续时间也较对照组短（P〈0．05）。治疗前两组血浆IL-18、hs-CRP水平比较无统计学差异（P〉0．05）;治疗后研究组IL．18、hs．CRP水平明显低于对照组（P〈O．05）。结论：硝酸异山梨酯片联合美托洛尔治疗老年冠心病心绞痛能够进一步减轻患者IL．18、hs．CRP水平,对心绞痛治疗效果较单独应用酸异山梨酯片更佳。     

Dependence of a drop in blood pressure in pulmonary circulation and in the right heart on dosage of isosorbide dinitrate]

An effect of isosorbide dinitrate on blood pressure values in the pulmonary circulation and the right heart has been investigated in 25 patients with a history of the first transmural myocardial infarction. Group I including 12 patients has been given 5 mg isosorbide nitrate in a 60-minute intravenous infusion while group II of 13 patients has been given 10 mg of the drug in the same way. Both groups have been matched in clinical data and blood pressure value in the pulmonary circulation which has been normal. Pulmonary blood pressure has been measured with Swan-Ganz catheter prior to the administration of drug, and 15, 30, 45 and 60 minutes following an infusion. Isosorbide dinitrate in a dose of 5 mg did not decrease blood pressure in the pulmonary circulation statistically significantly. The differences in blood pressure falls did exceed 9%. Filling pressure in the right ventricle did not change either while systolic blood pressure decrease by 16.6%. A double dose of isosorbide dinitrate reduced blood pressure in the pulmonary artery by about 1/3 of the baseline value, and blood pressure in the right ventricle (mean right atrial pressure) by 57.2%. Both systolic and diastolic arterial pressures were reduced. Isosorbide dinitrate reduced blood pressure in the pulmonary circulation in patients who underwent myocardial infarction, and hypotensive effect has been dose-related. A reduction in the right ventricular filling pressure has been a one of important mechanisms decreasing pulmonary pressures.     

Crystal structure of the cyclomaltohexaose (alpha-cyclodextrin) complex with isosorbide dinitrate. Guest-modulated channel-type structure

The crystal structure of the 2:1 complex of cyclomaltohexaose (alpha-cyclodextrin, alpha-CD) with isosorbide dinitrate was determined by single-crystal X-ray analysis. In the crystal with the space group C2, two cyclomaltohexaose molecules form a head-to-head dimer with the secondary hydroxy-group sides facing each other. The dimer unit is stacked along the crystallographic c-axis to form a channel-type structure. The isosorbide dinitrate molecule is encapsulated in the cylindrical cavity of the cyclomaltohexaose dimer. The dimeric structure exhibits pseudo twofold symmetry, and the guest molecule is disordered on the local symmetry axis. The isosorbide moiety is located at the center of the dimer cavity, and the nitrate groups penetrate into the cyclomaltohexaose rings. The guest molecule modulates the dimer structure to attain the most stable accommodation into the cavity. The cyclomaltohexaose molecules are laterally shifted away from each other to create the cavity fitted to the shape of the guest molecule. As the result, the intermolecular hydrogen bonds between secondary hydroxy-groups are not fully formed, but the dimeric structure is stabilized by the interaction with the guest molecule.     

Effect of isosorbide dinitrate (sorbonit) on the exercise reaction in patients with coronary disease and in healthy individuals

The study aimed at evaluating an effect of a single dose of isosorbide dinitrate (Sorbonit) on the exercise reaction in the patients with coronary disease of various degree and in healthy individuals. The study involved 20 male patients of mean age 54.0 +/- 4.5 years with history of myocardial infarction and 12 healthy males of mean age 45.6 +/- 5.0 years. Ergometric test has been performed twice: prior to and 15 minutes after sublingual administration of isosorbide dinitrate in the dose of 10-15 mg. The first test has been interrupted when horizontal ST load exceeded 1 mm or contractions rate was 60% of the maximum value. Similar loads have been used after the administration of Sorbonit. The following parameters have been evaluated: heart rate (HR), systolic blood pressure (BPS), HR x BPS, lactate level (LA), and cardiac index. The value of the load has been measured with the aid of oxygen consumption (VO2). Significant depression of ST segment (less than 3 mm) in the exercise ECG has been noted in 8 patients following isosorbide dinitrate. Exercise tolerance has increased in these patients - CI increased during exercise following drug administration (VO2 the same as prior to the drug administration), and VO2/CI has became closer - physiological.     

Increased exercise tolerance with nitrates in beta-blockaded patients with angina.

In 14 beta-blockaded anginal subjects, 10 of whom had poor left ventricular function, sublingual isosorbide dinitrate significantly increased maximal exercise capacity on a standardized multistage treadmill test. This was associated with changes in heart rate and blood pressure suggestive of a fall in left ventricular work. The effect of isosorbide lasts for at least two hours and when taken before exercise may be a useful addition to beta-blockade in patients with angina.     

Assessment of dermal glyceryl trinitrate and isosorbide dinitrate for patients with angina pectoris.

Dermal nitrate preparations are claimed to be useful in the treatment of angina, as their slow absorption by-passing the liver leads to a sustained action. Ten patients with angina were exercised on a treadmill after dermal application of 16.64 mg glyceryl trinitrate or 100 mg isosorbide dinitrate or placebo. Exercise duration was significantly increased at one and three hours for both nitrate preparations but not at six hours after application. The calculated workload achieved was significantly increased (p less than 0.01) at one and three hours for both preparations and at six hours (p less than 0.05) for isosorbide dinitrate. Headaches were common with glyceryl trinitrate cream. The dermal nitrate preparations studied had a duration of antianginal action similar to that of oral nitrate tablets. Aside from their value when the oral route cannot be used or absorption may be delayed, dermal nitrate preparations have no advantage over oral preparations for angina pectoris.     

Isosorbide dinitrate bioconversion by Beauveria strains: implication of glutathione transferase levels

Summary The level of glutathione S-transferase (GSH0ST) activity was determined in growing cultures and in washed resting cells of Beauveria strains with and without addition of isosorbide dinitrate (ISDN), by following the reaction with o-dinitrobenzene (o-DNB). The level of GSH-ST varied according to the pH changes of the medium and decreased during culture. The enzymatic activity measured with o-DNB did not correlate with ISDN bioconversion carried out either with B. sulfurescens or B. tenella. Immediately after starting incubation of the resting cells with ISDN, the level of GSH-ST activity initially increased, but declined afterwards, whereas the bioconversion process continued and reached 500 mg/l isosorbide 5-mononitrate. When 1-chloro-2,4-dinitrobenzene was used as a substrate for the evaluation of GSH-ST activity using B. tenella, a conjugation product having a UV absorption at 410 nm was formed.     

Calcium-blocking effect of nitro compounds in human platelets: correlation with changes in the cyclic guanosine monophosphate level

The effects of nitrates on Ca2+ increase and cyclic nucleotide content in human platelets were studied. Nitroglycerin, isosorbide dinitrate and sodium nitroprusside were found to inhibit the intracellular Ca2+ increase induced by the platelet activating factor, ADP and a stable thromboxane A2 analog--U46619. The inhibiting effect of sodium nitroprusside manifested itself at lower concentrations than those of nitroglycerin and isosorbide dinitrate. Nitroglycerin suppressed the Mn2+ entry into the cells and caused a 2-fold increase of the cGMP content which correlates with the calcium blocking activity. Methylene blue, a guanylate cyclase and glutathione reductase inhibitor, decreased the calcium blocking effect of nitroglycerin and its influence on the cyclic nucleotide content but failed to suppress the inhibitory effect of sodium nitroprusside. The data obtained suggest that the effects of nitrates on platelets are mediated by their influence on guanylate cyclase which leads to a cyclic nucleotide content increase and to a calcium blocking effect.     

Use of simultaneous pressure and velocity measurements to estimate arterial wave speed at a single site in humans

It has not been possible to measure wave speed in the human coronary artery, because the vessel is too short for the conventional two-point measurement technique used in the aorta. We present a new method derived from wave intensity analysis, which allows derivation of wave speed at a single point. We apply this method in the aorta and then use it to derive wave speed in the human coronary artery for the first time. We measured simultaneous pressure and Doppler velocity with intracoronary wires at the left main stem, left anterior descending and circumflex arteries, and aorta in 14 subjects after a normal coronary arteriogram. Then, in 10 subjects, serial measurements were made along the aorta before and after intracoronary isosorbide dinitrate. Wave speed was derived by two methods in the aorta: 1) the two-site distance/time method (foot-to-foot delay of pressure waveforms) and 2) a new single-point method using simultaneous pressure and velocity measurements. Coronary wave speed was derived by the single-point method. Wave speed derived by the two methods correlated well (r = 0.72, P < 0.05). Coronary wave speed correlated with aortic wave speed (r = 0.72, P = 0.002). After nitrate administration, coronary wave speed fell by 43%: from 16.4 m/s (95% confidence interval 12.6-20.1) to 9.3 m/s (95% confidence interval 6.5-12.0, P < 0.001). This single-point method allows determination of wave speed in the human coronary artery. Aortic wave speed is correlated to coronary wave speed. Finally, this technique detects the prompt fall in coronary artery wave speed with isosorbide dinitrate.     

Topical treatment of erectile dysfunction: randomised double blind placebo controlled trial of cream containing aminophylline,isosorbide dinitrate,and co-dergocrine mesylate.

OBJECTIVE--To examine the effectiveness in treating impotence to topically applied cream containing three vasodilators--aminophylline, isosorbide dinitrate, and co-dergocrine mesylate--which act by different mechanisms. DESIGN--Randomised double blinded placebo controlled crossover trial over two weeks. SUBJECTS--36 men with erectile dysfunction randomly allocated to two equal groups. INTERVENTIONS--Active cream containing aminophylline 3%, isosorbide dinitrate 0.25%, and co-dergocrine mesylate 0.05% for one week and placebo for another. MAIN OUTCOME MEASURES--Patients'' reported experience of penile responses and side effects of treatment in questionnaires. Penile tumescence and arterial flow in the laboratory. RESULTS--21 patients reported full erection and satisfactory intercourse with the active cream. Three men reported full erection and satisfactory intercourse with either cream. The active cream was more effective in psychogenic than organic impotence (eight out of nine men with psychogenic impotence achieved a full erection upsilon four out of eight with neurogenic impotence and two out of seven with arterial insufficiency). No major side effects were reported. In the laboratory the active cream increased penile arterial flow (0.19 (SD 0.08) m/s upsilon 0.02 (0.15) m/s with placebo) and induced tumescence in 24 patients. CONCLUSIONS--Topical treatment with a cream containing three different vasodilators might be considered before intracavernous injection of vasoactive agents, particularly in psychogenic impotence.