CGRP mediate the isosorbide-5-mononitrate cardiovascular response in healthy Chinese male volunteers through a XOR-independent pathway.

OBJECTIVES: This study was designed to determine whether xanthine oxidoreductase (XOR) is involved in Isosorbide- 5-mononitrate (IS-5-MN) metabolism, and to elucidate the role of the neuropeptide calcitonin gene-related peptide (CGRP) in the IS-5-MN response. METHODS: In 15 Chinese volunteers, we observed the relationship between baseline XOR-mRNA expression in peripheral blood mononuclear cells (PBMCs) and the response to 20 mg IS-5-MN. IS-5-MN pharmacokinetics profiles, changes in plasma concentrations of CGRP, and CGRPmRNA expression in PBMCs were assessed in vivo and in vitro. RESULTS: Individuals with a lower baseline XOR-mRNA expression showed lower plasma XOR activity and significantly greater changes in SBP (ΔSBP) after IS-5-MN administration. Individuals with a lower baseline XOR-mRNA expression also showed significantly greater increases in plasma concentrations of CGRP. There were no differences in IS-5-MN AUC between the two groups. IS-5-MN significantly up-regulated the expression of CGRP α- and CGRP ß-mRNA in PBMCs, which were not affected by the XOR inhibitor allopurinol. CONCLUSIONS: Our study suggests that CGRP may contribute to the response to IS-5 MN in a XOR-independent pathway.

A simple and sensitive gas chromatography method for determination of isosorbide dinitrate and its metabolites in human plasma: application to pharmacokinetics study on oral spray.

A sensitive method for the simultaneous determination of isosorbide dinitrate (ISDN) and its mononitrate metabolites, isosorbide 2-mononitrate and isosorbide 5-mononitrate (IS-2-MN and IS-5-MN), in human plasma was developed using capillary gas chromatography with electron-capture detection, whereas 1,2,4-butanetriol trinitrate was used as internal standard. The analytes were extracted with a simple liquid-liquid extraction from plasma and separated on a DB-1 column. The results of method validation demonstrated that the calibration curves were linear in range of 2-60 ng/mL for ISDN and IS-5-MN, 1-20 ng/mL for IS-2-MN, respectively. The precision (RSD%) was less than 15%, and the lower limit of quantitation was identifiable and reproducible at 2 ng/mL for ISDN and IS-5-MN, 1 ng/mL for IS-2-MN. The analytes in plasma were stable after being stored for more than 30 days and after 2 freeze-thaw cycles (-20 to 25°C). And then this method was successfully applied to a pharmacokinetic investigation on isosorbide dinitrate oral spray in healthy volunteers.

Determination of isosorbide-5-mononitrate in human plasma by high-resolution gas chromatography.

A method for the determination of isosorbide-5-mononitrate (5-ISMN) in human plasma by capillary gas chromatography with electron-capture detection was developed and applied to clinical samples. 9-Fluorenone was used as an internal standard, ethyl acetate was employed for liquid-liquid extraction. The advantage of the extraction procedure is the possibility of a direct injection of the plasma extract, without solvent removal/reconstitution of the sample. The precision and accuracy of the method were satisfactory in the concentration range 10-1600 ng/ml. The lower limit of quantification was 10 ng/ml.

Simultaneous determination of isosorbide dinitrate and its mononitrate metabolites in human plasma by capillary gas chromatography with electron-capture detection.

A method for the simultaneous determination of isosorbide dinitrate (ISDN) and its mononitrate metabolites (2- and 5-ISMN) in human plasma by capillary gas chromatography with electron-capture detection was developed. Two internal standards were used: isomannide dinitrate (IMDN) for the determination of ISDN and isomannide mononitrate (IMMN) for the determinations of 2- and 5-ISMN. After addition of the internal standards, the compounds were isolated from plasma by solid-liquid extraction. They were determined by gas chromatography using an electron-capture detector. The reproducibility and accuracy of the method were found suitable in the range of concentrations 2.5-83 ng/ml for ISDN, 2.6-208 ng/ml for 2-ISMN and 2.3-1010 ng/ml for 5-ISMN. The limit of quantitation (LOQ) was about 2.5 ng/ml for each compound. The method was applied to clinical samples.

Effect of organic nitrates on ex vivo platelet aggregation and fibrinolysis in man.

Previous in vitro studies have suggested that nitric oxide-releasing agents can increase the fibrinolytic capacity while platelet aggregability is decreased. We have therefore directly compared the influence of organic nitrates on ex vivo platelet aggregation and the fibrinolytic system in 16 healthy volunteers. Each subject received glyceryl trinitrate (GTN, 2 x 32 mg patches), isosorbide-dinitrate (ISDN, 120 mg p.o.), isosorbide-5-mononitrate (ISMN, 50 mg p.o.), and placebo in a randomized double-blind manner. Ex vivo platelet aggregation, activities and concentrations of tissue-type plasminogen activator (t-PA) and plasminogen activator inhibitor-1 (PAI-1) were measured before, 90 and 150 minutes after drug intake. GTN, ISDN, and ISMN were equipotent in respect to their hemodynamic responses. Platelet aggregation induced with a low (1 microM) but not with a high (4 microM) agonist concentration (adenosine diphosphate, ADP) was reduced after GTN and ISDN. ISMN had no influence on aggregation. An increase of t-PA activity was significantly reduced after GTN and ISDN (p<0.05), whereas t-PA concentrations were not affected by the nitrates. A decrease of PAI-1 activity after ISMN and placebo was not apparent after GTN and ISDN. In the case of GTN, the decrease of PAI-1 concentration was delayed. In conclusion, GTN and ISDN did not increase the fibrinolytic capacity but decreased the reversible phase of ADP-induced platelet aggregation, while ISMN had neither an effect on the fibrinolytic system nor on platelet aggregation.

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.

Gas chromatographic-mass spectrometric determination of isosorbide 5-mononitrate in human plasma.

A highly specific and precise method using gas chromatography-mass spectrometry was developed for the measurement of isosorbide 5-mononitrate in plasma using isomannide mononitrate as internal standard. With regard to the numerous analytical problems encountered when organic mononitrates were determined in plasma, such as thermal instability and adsorption, compounds were silylated before gas chromatography. In order to increase the specificity of the assay, two specific ions of the isosorbide 5-monitrate were simultaneously recorded. The accuracy of the assay was tested day to day with quality specimens spiked blind to the analyst.

[Gas chromatographic method with EC detection to determine isosorbide-5-mononitrate (IS-5-MN) in human serum and its pharmacokinetic parameters].

A simple, sensitive and precise capillary GC-ECD method was developed for the determination of isosorbide-5-mononitrate in human serum. Pharmacokinetic parameters of the drug was obtained from the human serum level-time curve measured. Serum samples were extracted with a mixture of ethyl ether-ethyl acetate (4:1), the upper phase was collected and evaporated to about 100 microliters under a gentle nitrogen stream. Isosorbide dinitrate was used as internal standard. With a human serum sample size of 200 microliters, the detection limit of IS-5-MN was found to be about 5 ng/ml, and the absolute recovery from 74% to 85%. The within-day and between-day relative standard deviation were less than 7% and 9%, respectively. This method was applied to the pharmacokinetic studies of IS-5-MN tablets from two different sources. Two sets of t1/2 (Ke), Tmax and AUC values obtained from 8 volunteers were tested statistically and no significant difference was found.

[Plasma levels of isosorbide dinitrate and its metabolites during intravenous infusion in surgical patients--effect of preoperative administration of isosorbide dinitrate].

Nineteen patients with ischemic heart disease were studied to determine plasma levels of isosorbide dinitrate (ISDN) and its metabolites, isosorbide-2-mononitrate (2-ISMN) and isosorbide-5-mononitrate (5-ISMN) for 6 hrs during intravenous administration of ISDN, using gas chromatography. Differences in plasma levels of these substances were also evaluated in patients with or without preanesthetic medication of ISDN. These patients ranging in ages from 42 to 80 years were administered ISDN intravenously at a rate of 1 micrograms.kg-1 x min-1 during anesthesia and surgery. Preanesthetic administration of ISDN consisted of transdermal application of 40 mg dose. Surgery included gastrectomy, pneumonectomy, extended cholecystectomy, radical mastectomy and so on under either enflurane anesthesia or neuroleptanesthesia. Plasma ISDN levels increased and reached a plateau 3 hrs after the start of intravenous infusion of ISDN in patients of both groups. Plasma 2-ISMN levels increased gradually and reached a plateau 5 hrs after the commencement of intravenous administration of ISDN in patients of both groups. Plasma 5-ISMN levels increased gradually reaching the peak 6 hrs after the start of intravenous infusion of ISDN in both groups. Plasma ISDN, 2-ISMN and 5-ISMN levels were slightly higher in patients who received preanesthetic ISDN than in those who did not receive preanesthetic ISDN. However, there were no statistical differences in plasma ISDN or 2-ISMN levels between the groups except prior to the infusion. On the contrary, plasma 5-ISMN levels were significantly higher in patients who received preanesthetic ISDN than in those who did not receive preanesthetic ISDN for 3 hrs after the start of the infusion.

Gas chromatographic analysis of isomeric organic mononitrates in plasma.

A specific, sensitive and precise capillary gas chromatographic method using electron-capture detection was developed for the determination of four isomeric vasodilating organic mononitrates, viz. L-isoidide mononitrate (L-IIMN), isosorbide-2-mononitrate (IS-2-MN), isomannide mononitrate (IMMN) and isosorbide-5-mononitrate (IS-5-MN), in rat plasma. With a sample size of 100 microliters of rat plasma, the detection limits were found to be between 0.5 and 2 ng/ml for these mononitrates, and the absolute recovery was found to range from 83 to 90%. The within-day coefficients of variation for the assay of the four isomers were less than 5%, while the between-day coefficients of variation were less than 10%. Because of the short retention times of these isomers in this assay, routine analyses of about sixty plasma samples per day can be carried out. The possibility of in vivo interconversion among these four isomers in rats was investigated after individual administration of each isomer. No interconversion was found based on examination of plasma samples. The gas chromatographic method was applied to the pharmacokinetic studies of these four isomers in rats; at an intravenous dose of 2 mg/kg, the biological half-lives of L-IIMN, IMMN, IS-2-MN and IS-5-MN were found to be 13.2, 25.2, 54.6 and 112 min, respectively.

Influence of standard and nicotine-reduced cigarette smoke on plasma concentrations of isosorbide dinitrate and its metabolites in rats.

Rats dosed orally with isosorbide dinitrate (1 mg kg-1) were exposed to smoke from standard and nicotine-reduced cigarettes for 8 min using a smoking machine. Plasma concentrations of isosorbide dinitrate and 5-isosorbide mononitrate, one of its major metabolites were approximately equal in the exposed groups, but were lower than in the non-smoking control group. The 2-isosorbide mononitrate concentration was also lower in the group exposed to smoke from standard cigarettes. Since the pharmacokinetics were influenced by smoke from both types of cigarette smoke, the effect may be attributed in large part to non-nicotine components of the smoke.

Assay of glyceryl trinitrate, isosorbide dinitrate, and their metabolites in plasma by large-bore capillary column gas-liquid chromatography.

Two large-bore capillary columns, one with dimethyl polysiloxane (HP-1) as the stationary phase and the other with phenyl (50 per cent) methyl (50 per cent) polysiloxane (DB-17), were used to develop gas-liquid chromatographic (GLC) assays for measuring isosorbide dinitrate (ISDN), glyceryl trinitrate (GTN), and their metabolites. ISDN, isosorbide-2-mononitrate (2-ISMN), and isosorbide-5-mononitrate (5-ISMN) in plasma, ranging in concentration from 1 to 300 nM, and GTN, glyceryl-1,2-dinitrate (1,2-GDN), and glyceryl-1,3-dinitrate (1,3-GDN), ranging in concentration from 3 to 60 nM in plasma, were analysed on both columns. GLC analysis yielded baseline resolution of the analytes. The method using the dimethyl polysiloxane column gave a lower limit of detectability for GTN of 0.75 nM (signal/noise (s/n) = 2), and the procedure using the phenyl-methyl column provided a lower limit of detectability for ISDN of 81 pM (s/n = 2). The large-bore column GLC procedures exhibited shorter retention times for both ISDN and GTN than those previously reported for capillary-column assays. The chromatographic resolution of analytes and column efficiency of the large-bore capillary columns were comparable to the results previously found using capillary-column GC. The assays for ISDN and GTN have been shown to be appropriate for pharmacokinetic studies in volunteers and patients. We determined that the HP-1 column is appropriate for the analysis of GTN and metabolites, and the DB-17 column is suitable for analysis of ISDN and its metabolites. We conclude that the use of large-bore capillary columns provides rapid and reliable GLC assays for organic nitrates.

Inhibition of platelet function during in vivo infusion of isosorbide mononitrates: relationship between plasma drug concentration and hemodynamic effects.

Isosorbide monitrates (IS-2-MN and IS-5-MN), hepatic metabolites of isosorbide dinitrate, inhibit platelet function in vitro very differently, with IS-2-MN being much more potent than IS-5-MN. To assess their antiplatelet properties in vivo and to compare time and dosage requirements, we infused both IS-2-MN and IS-5-MN for 30 minutes, on 2 separate days, into nine patients with stable coronary artery disease, at rates of 4 mg/hr (n = 4) and 8 mg/hr (n = 5). Two additional patients received IS-5-MN at 16 mg/hr. Platelet aggregation and thromboxane (TX) B2 generation in response to various agonists, drug plasma concentrations, and blood pressure were monitored throughout the study. A significant decrease in platelet aggregation and TXB2 production by adenosine diphosphate and adrenaline occurred in seven of nine patients receiving IS-2-MN and in 7 of 11 patients receiving IS-5-MN. Response was dose related, with more patients responding at 8 mg/hr to IS-2-MN (five of five) than to IS-5-MN (three of five), and was maximum at the end of the infusion time, corresponding to peak plasma levels. Patients responding to drug infusions with an inhibition of platelet function were characterized by a greater vascular responsiveness compared to nonresponders, since the decrease in systolic blood pressure (mean +/- SEM) was significantly greater in the former (15.4 +/- 3.2) than in the latter (2.5 +/- 2.1, p less than 0.05). Therefore both mononitrates, when administered at infusion rates between 8 and 16 mg/hr, are accompanied by a consistent inhibition of adenosine diphosphate- and adrenaline-induced aggregation and TX generation.(ABSTRACT TRUNCATED AT 250 WORDS)

Concentrations of isosorbide dinitrate, isosorbide-2-mononitrate and isosorbide-5-mononitrate in human vascular and muscle tissue under steady-state conditions.

The concentrations of isosorbide dinitrate (ISDN), isosorbide-5-mononitrate (IS-5-MN) and isosorbide-2-mononitrate (IS-2-MN) were determined in plasma (PL), saphenous vein wall (SV) and pectoral muscle (PM) from 8 patients undergoing coronary bypass surgery. The patients were pretreated for 2 days with ISDN 240 mg per day (standard release formulation) in 4 doses of 40 mg and one dose of 80 mg. The plasma and tissue samples were obtained during the operation, 10-12 h after the last dose. Isosorbide-2-mononitrate and isosorbide-5-mononitrate were present in plasma and tissues in the same concentration ranges with molar concentration ratios of 0.88 (IS-2-MN: PM/PL), 0.85 (IS-5-MN: PM/PL), 0.99 (IS-2-MN: SV/PL) and 1.06 (IS-5-MN: SV/PL). Mean ISDN concentrations in tissue were considerably higher than in plasma; the molar concentration ratios were 4.9 (SM/PL) and 7.21 (SV/PL). The accumulation of ISDN in vessel walls may contribute to its greater vascular action compared to the mononitrates, but it may also facilitate the development of tolerance during long-term treatment.

Isosorbide dinitrate ameliorates myocardial ischemia after development of collateral function in a canine model.

The effects of i.v. administered isosorbide dinitrate (ISDN) on the reduced level of regional myocardial function during coronary occlusion were studied in conscious dogs before and after collateral development and in the absence of persistent coronary stenosis. The animals were instrumented during sterile surgery with a miniature pressure gauge for measuring left ventricular pressure and a cannula for aortic pressure. Two pairs of piezoelectric crystals were placed in normal and ischemic areas to determine regional circumferential length. A hydraulic cuff occluder and a Doppler flow probe were placed around the left circumflex coronary artery. Collaterals were induced to develop by 2 min of coronary occlusion applied repetitively at an interval of 32 min for 2 to 9 days, until the regional dysfunction produced by coronary occlusion had disappeared. Collateral development was estimated according to percentage of systolic shortening and endsystolic length area (ESL area) during the occlusion. Before collateral development, ISDN in a dose of 100 micrograms/kg did not affect the level of regional dysfunction. The ESL area was 250 +/- 54 and 248 +/- 52 mm.sec before and after ISDN, respectively. After collateral development, the ESL area was 51 +/- 13 mm.sec and it decreased by 35% after the i.v. administration of ISDN. The improvement by ISDN of transient myocardial dysfunction was achieved even during electrical tachypacing. Accordingly, the beneficial effects of ISDN on regional wall motion rendered ischemic during transient coronary occlusion were appreciable after coronary collateral development.

[Comparative course of maintenance of the effects of nitrite derivatives and molsidomine after prolonged administration]. / Evolution comparative du maintien, lors d'une administration prolongée, des effets des dérivés nitrés et de la molsidomine.

Prolonged administration of nitroglycerin and its derivatives results in pharmacodynamic tolerance: although the dosage of these drugs is kept at the same level, their therapeutic effects decrease in amplitude. Experimental and clinical studies have shown that this escape phenomenon is due to depletion of cysteine in the vascular wall. This amino acid, which gives off SH radicals, is indispensable to the ultimate transformation of nitroglycerin enabling in to exert its vasodilator action. Molsidomine does not require any transformation to act on the vascular smooth muscle and should therefore remain insensitive to tolerance. The experimental and clinical data available at present seem to confirm that the effects of molsidomine administered for long periods in the treatment of coronary disease and heart failure are sustained. However, for the long-term effectiveness of molsidomine and nitrites to be compared objectively a prospective double-blind randomized trial would be needed, with both treatments being in optimal doses and intervals of administration.

Dose-dependent relaxation of human venous vessel strips with regard to chronic nitrate pretreatment.

Helical segments of human saphenous veins harvested at coronary bypass surgery were mounted in an organ-bath (Krebs-Henseleit buffer, pH 7.4; 37 degrees C; 95% O2/5% CO2 insufflation). After equilibration (60 min) and determination of basal tone the segments were depolarized and contracted by 24 mM potassium chloride. Then relaxation under isometric conditions was induced by cumulative concentrations of isosorbide dinitrate (ISDN) in a range of 10(-9) M to 10(-5) M. In vein segments from patients not pretreated with nitrates a concentration-response curve could be shown ranging from 8.4 +/- 4.5% to 71.9 +/- 8.6% (mean +/- SD). The relaxation pattern was not influenced by a 2-week pretreatment of patients with ISDN 20 mg twice daily or 40 mg four times daily even if the latter therapy was continued until 1 hour prior to surgery. Immersion of vessel strips in Krebs-Henseleit buffer containing 10(-6) M ISDN for 60 min prior to relaxation did not affect relaxation either. However, immersion of vessel segments in buffer medium containing 4.4 X 10(-4) M ISDN for 60 min led to a shift of the concentration-response curve by the factor 100 (EC50). Thus, chronic nitrate pretreatment of patients including high doses did not influence relaxation behaviour of isolated vessel segments. Induction of tolerance under in vitro conditions required concentrations exceeding the therapeutic limits. The most probable underlying mechanism is exhaustion of sulfhydril containing groups at the site of the smooth muscle cells. This hypothesis was supported by the finding that addition of cysteine into the organ-bath could widely reverse tolerance.(ABSTRACT TRUNCATED AT 250 WORDS)