Electrochemical evidence of nitrate release from the nitrooxy compound 4-((nitrooxy) methyl)-3-nitrobenzoic acid and its antinociceptive and anti-inflammatory activities in mice.

Considering the many biological activities of nitric oxide (NO), some lines of research focused on the modulation of these activities through the provision of this mediator by designing and synthesizing compounds coupled with an NO donor group. Thus, the objectives of the present study were to carry out an electrochemical investigation of the nitrooxy compound 4-((nitrooxy) methyl)-3-nitrobenzoic acid (1) and evaluate its activities and putative mechanisms in experimental models of pain and inflammation. Voltammetric studies performed in aprotic medium (mimetic of membranes) showed important electrochemical reduction mechanisms: nitroaromatic reduction, self-protonation, and finally reductive elimination, which leads to nitrate release. Systemic administration of the nitrooxy compound (1) inhibited the nociceptive response induced by heat and the tactile hypersensitivity and paw edema induced by carrageenan in mice. The activities in the models of inflammatory pain and edema were associated with reduced neutrophil recruitment and production of inflammatory cytokines, such as interleukin (IL)-1ß, IL-6, tumor necrosis factor-α and CXCL-1, and increased production of IL-10. Concluding, electrochemical analysis revealed unequivocally that electron transfer at the nitro group of the nitrooxy compound (1) results in the cleavage of the organic nitrate, potentially resulting in the generation of NO. This electrochemical mechanism may be compared to a biochemical electron-transfer mediated nitrate release that, by appropriate in vivo bioreduction (enzymatic or not) would lead to NO production. Compound (1) exhibits activities in models of inflammatory pain and edema that may be due to reduced recruitment of neutrophils and production of inflammatory cytokines and increased production of IL-10. These results reinforce the interest in the investigation of NO donor compounds as candidates for analgesic and anti-inflammatory drugs.

A Phase 1 Randomized Study of Single Intravenous Infusions of the Novel Nitroxyl Donor BMS-986231 in Healthy Volunteers.

Nitroxyl (HNO) is a reactive nitrogen molecule that has potential therapeutic benefits for patients with acute heart failure. The results of the first-in-human study for BMS-986231, a novel HNO donor, are reported. The aim of this sequential cohort study was to evaluate the safety, tolerability, and pharmacokinetic profile of BMS-986231 after 24- and 48-hour intravenous infusions in healthy volunteers. Eighty subjects were randomized and dosed. Seven cohorts (stratum A) received BMS-986231 0.1, 0.33, 1, 3, 5, 10, and 15 µg/kg/min or placebo, infused over 24 hours. An additional cohort (stratum B) received 10 µg/kg/min or placebo, infused over 48 hours. Adverse events (AEs) were reported for 30 days after completion of infusion. Blood/urine samples were collected at regular intervals; other parameters (blood pressure, heart rate/rhythm, cardiac index) were also assessed. Headaches were the most commonly reported drug-related AE (48%) in those who received BMS-986231, although their severity was reduced by hydration. No other significant drug-related AEs were noted. BMS-986231 was associated with dose-dependent and well-tolerated reductions in systolic and diastolic blood pressure versus baseline; cardiac index, as measured noninvasively, was increased. BMS-986231 had no clinically significant effect on heart rate/rhythm or laboratory parameters. Its mean elimination half-life was 0.7-2.5 hours. BMS-986231 was safe and well-tolerated for up to 24 hours (15 µg/kg/min) or 48 hours (10 µg/kg/min), with a favorable hemodynamic profile observed. Ongoing studies continue to evaluate the potential benefit of BMS-986231 in patients with acute heart failure.

In vivo and in silico evaluation of a new nitric oxide donor, S,S'-dinitrosobucillamine.

PURPOSE: In a previous work, we have synthetized a new dinitrosothiol, i.e. S,S'-dinitrosobucillamine BUC(NO)2 combining S-nitroso-N-acetylpenicillamine (SNAP) and S-nitroso-N-acetylcysteine (NACNO) in its structure. When exposed to isolated aorta, we observed a 1.5-fold increase of •NO content and a more potent vasorelaxation (1 log higher pD2) compared to NACNO and SNAP alone or combined (Dahboul et al., 2014). In the present study, we analyzed the thermodynamics and kinetics for the release of •NO through computational modeling techniques and correlated it to plasma assays. Then BUC(NO)2 was administered in vivo to rats, assuming it will induce higher and/or longer hypotensive effects than its two constitutive S-mononitrosothiols. METHODS: Free energies for the release of •NO entities have been computed at the density functional theory level assuming an implicit model for the aqueous environment. Degradation products of BUC(NO)2 were evaluated in vitro under heating and oxidizing conditions using HPLC coupled with tandem mass spectrometry (MS/MS). Plasma from rats were spiked with RSNO and kinetics of RSNO degradation was measured using the classical Griess-Saville method. Blood pressure was measured in awake male Wistar rats using telemetry (n = 5, each as its own control, 48 h wash-out periods between subcutaneous injections under transient isoflurane anesthesia, random order: 7 mL/kg vehicle, 3.5, 7, 14 µmol/kg SNAP, NACNO, BUC(NO)2 and an equimolar mixture of SNAP + NACNO in order to mimic the number of •NO contained in BUC(NO)2). Variations of mean (ΔMAP, reflecting arterial dilation) and pulse arterial pressures (ΔPAP, indirectly reflecting venodilation, used to determine effect duration) vs. baseline were recorded for 4 h. RESULTS: Computational modeling highlights the fact that the release of the first •NO radical in BUC(NO)2 requires a free energy which is intermediate between the values obtained for SNAP and NACNO. However, the release of the second •NO radical is significantly favored by the concerted formation of an intramolecular disulfide bond. The corresponding oxidized compound was also characterized as related substance obtained under degradation conditions. The in vitro degradation rate of BUC(NO)2 was significantly greater than for the other RSNO. For equivalent low and medium •NO-load, BUC(NO)2 produced a hypotension identical to NACNO, SNAP and the equimolar mixture of SNAP + NACNO, but its effect was greater at higher doses (-62 ± 8 and -47 ± 14 mmHg, maximum ΔMAP for BUC(NO)2 and SNAP + NACNO, respectively). Its duration of effect on PAP (-50%) lasted from 35 to 95 min, i.e. shorter than for the other RSNO (from 90 to 135 min for the mixture SNAP + NACNO). CONCLUSION: A faster metabolism explains the abilities of BUC(NO)2 to release higher amounts of •NO and to induce larger hypotension but shorter-lasting effects than those induced by the SNAP + NACNO mixture, despite an equivalent •NO-load.

Hypotensive Effect and Accumulation of Dinitrosyl Iron Complexes in Blood and Tissues after Intravenous and Subcutaneous Injection.

Subcutaneous injection of Oxacom with glutathione-bound dinitrosyl iron complex as the active principle produced a slower drop of mean BP and longer accumulation of protein-bound dinitrosyl iron complexes in whole blood and tissues than intravenous injection of this drug, while durations of hypotensive effect in both cases were practically identical. In contrast to intravenous injection of the drug, its subcutaneous administration was not characterized by a high concentration of protein-bound dinitrosyl iron complexes in the blood at the onset of experiment; in addition, accumulation of these NO forms in the lungs was more pronounced after subcutaneous injection than after intravenous one.

Nitrones: not only extraordinary spin traps, but also good nitric oxide sources in vivo.

Free radicals are involved in the development of reperfusion injuries. Using a spin trap, the intensity of such lesions can be reduced. Nitrones (effective in vivo spin traps) were tried in this work as in vivo nitric oxide donors. Nitrite and nitrate concentration values (rabbit blood) were used as biomarkers of nitric oxide production. Most nitrones did not increase plasma concentrations of nitrite and nitrate; on the contrary, reduced plasma concentrations of these indicators were noted. However, glyoxal isopropyldinitrone, in a dose of 50 mg kg-1, was highly effective in increasing nitric oxide production. At the same time, nitrones do not react with hepatic homogenates, proving that the release of nitric oxide takes place in the tissues and is not related to hepatic metabolism. Before using nitrones in vivo, they were tested in vitro for the ability to release nitric oxide following a reaction with the hydroxyl radical.

Computational analysis of nitric oxide biotransport to red blood cell in the presence of free hemoglobin and NO donor.

Red blood cells (RBCs) modulate nitric oxide (NO) bioavailability in the vasculature. Extracellular free hemoglobin (Hb) in the vascular lumen can cause NO bioavailability related complications seen in pathological conditions such as pancreatitis, sickle cell disease and malaria. In addition, the role of extracellular free Hb has been critical to estimate kinetic and transport properties of NO-RBCs interactions in 'competition experiments'. We recently reported a strong dependence of NO transport on RBC membrane permeability and hematocrit. NO donors combined with anti-inflammatory drugs are an emergent treatment for diseases like cancer, cardiovascular complications and wound healing. However, the role of RBCs in transport NO from NO donors is not clearly understood. To understand the significance of extracellular free Hb in pathophysiology on NO availability and estimation of the NO-RBC interactions, we developed a computational model to simulate NO biotransport to the RBC in the presence of extracellular free Hb. Using this model, we studied the effect of hematocrit, RBC membrane permeability and NO donors on NO-RBC interactions in the presence and absence of extracellular free Hb. The plasma NO concentration gradients and average plasma NO concentrations changed minimally with increase in extracellular free Hb concentrations at the higher hematocrit as compared to those at the lower hematocrit irrespective of the NO delivery method, indicating that the presence of extracellular free Hb affects the NO transport only at a low hematocrit. We also observed that NO concentrations increased with NO donor concentrations in the absence as well as in the presence of extracellular free Hb. In addition, NO donor supplementation may increase NO availability in the plasma in the event of loss of endothelium-derived NO activity.

Physicochemical characterization and an injection formulation study of water insoluble ZCVI4-2, a novel NO-donor anticancer compound.

ZCVI(4)-2 was a novel nitric oxide-releasing glycosyl derivative of oleanolic acid that displayed strong cytotoxicity selectively against human hepatocellular carcinoma in vitro and in vivo. In this study, ZCVI(4)-2 was characterized by FT-IR spectroscopy, differential scanning calorimetry, powder X-ray diffractometry, Raman spectroscopy, hygroscopicity and stability. A high performance liquid chromatography method was also established for the quantitative determination of solubility and additional stability profile of ZCVI(4)-2. ZCVI(4)-2 was found to be an amorphous and stable solid with low solubility of less than 10 µg/mL. Based on the solubilization tests that included methods of cosolvency and micellization, the solution mixture of 5% Solutol HS-15, 5% 1, 2-propylene glycol and 5% anhydrous ethanol was determined to be the system for the preparation of the ZCVI(4)-2 early injection solution. The effect of pH, temperature, light and injectable isotonic glucose or NaCl solution on ZCVI(4)-2 injection was also investigated. Good stability was observed at all testing conditions. Under the conditions studied, the NO-releasing rate and amount of ZCVI(4)-2 from the early injection solution in rat plasma demonstrated a promising therapeutic efficacy while maintaining a good safety profile.

Identification of circulatory and excretory metabolites of a novel nitric oxide donor ZJM-289 in rat plasma, bile, urine and faeces by liquid chromatography-tandem mass spectrometry.

ZJM-289, [2-(1-diethylaminoacetoxy)pentyl] benzoic acid-{2-methoxy-4-[2-(4-nitrooxybutoxy carbonyl)-vinyl]}phenyl ester hydrochloride, is a novel nitric oxide-donating derivative of 3-n-butylphthalide synthesised on the hypothesis that it may be hydrolysed in vivo into 3-n-butylphthalide, ferulic acid and nitric oxide in hope that the three components may exert effects on the platelets as well as on central nervous system synergistically. In this study, ZJM-289 was extensively metabolised in rats. Eight major metabolites were identified by liquid chromatography (LC)-mass spectrometry (MS)/MS in rat plasma, bile, urine and faeces after intravenous administration. Metabolites M1, M2, M3, M4 and M5 were hydrolytic products of ZJM-289, M6 and M7 was a hydroxylation product of M5, and M8 was a glucuronide of M1. The pharmacologically active metabolite ferulic acid (M3) was a major metabolite in all the biological matrixes examined. 3-n-Butylphthalide was also present at a moderate level in the circulation. And along with the previous research, the anti-platelet activity of ZJM-289 was more potent than that of 3-n-butylphthalide both in vivo and in vitro. All these findings validated the theory of drug design.

Association of GSTM1 null polymorphism with isosorbide-5-mononitrate cardiovascular response and involvement of CGRP in healthy Chinese male volunteers.

OBJECTIVES: To determine whether functional polymorphisms of glutathione S-transferase µ type 1 (GSTM1) and aldehyde dehydrogenase-2 (ALDH2) affect the isosorbide 5-mononitrate (IS-5-MN) response, and the role of the calcitonin gene-related peptide (CGRP) in IS-5-MN response in healthy volunteers. METHODS: A two-phase, placebo-controlled study was carried out in 24 healthy Chinese volunteers with their ALDH2 and GSTM1 genotypes known. During each phase, either 20-mg IS-5-MN tablet or placebo was orally administered; blood pressure (BP), heart rate, and plasma concentration of CGRP was determined before and at several time points after drug administration. Pharmacokinetic parameters of IS-5-MN were determined. RESULTS: GSTM1 null individuals showed significantly lower systolic BP (SBP) and diastolic BP (DBP), and higher degree of decreases in SBP (ΔSBP) and DBP (ΔDBP) after IS-5-MN administration. GSTM1 null individuals showed significantly decreased IS-5-MN area under the plasma concentration-time curve than GSTM1 wild-type individuals (P<0.05). Plasma concentration of CGRP was increased significantly at 0.5 (P<0.01), 1 (P<0.05), and 2 h (P<0.05) after IS-5-MN administration in GSTM1 null individuals but not wild-type individuals. GSTM1 null individuals also showed significantly higher degree of percentage increase in the plasma concentration of CGRP than GSTM1 wild-type individuals at 1 h after IS-5-MN administration (P<0.05). IS-5-MN upregulated CGRP I and CGRP II mRNA expressions in cultured peripheral blood mononuclear cells, and the IS-5-MN-induced CGRP II mRNA expression was inhibited by GSTs inhibitor, ethacrynic acid. No difference in the IS-5-MN response was observed between ALDH2 genotypes. CONCLUSION: We suggest that GSTM1, but not ALDH2, may interfere with the bioactivation of IS-5-MN, and CGRP contributes to the IS-5-MN response in a GSTM1 genotype-dependent manner.

Isosorbide-based aspirin prodrugs: integration of nitric oxide releasing groups.

Aspirin prodrugs and related nitric oxide releasing compounds hold significant therapeutic promise, but they are hard to design because aspirin esterification renders its acetate group very susceptible to plasma esterase mediated hydrolysis. Isosorbide-2-aspirinate-5-salicylate is a true aspirin prodrug in human blood because it can be effectively hydrolyzed to aspirin upon interaction with plasma BuChE. We show that the identity of the remote 5-ester dictates whether aspirin is among the products of plasma-mediated hydrolysis. By observing the requirements for aspirin release from an initial panel of isosorbide-based esters, we were able to introduce nitroxymethyl groups at the 5-position while maintaining ability to release aspirin. Several of these compounds are potent inhibitors of platelet aggregation. The design of these compounds will allow better exploration of cross-talk between COX inhibition and nitric oxide release and potentially lead to the development of selective COX-1 acetylating drugs without gastric toxicity.

Nitric oxide inhibits platelet adhesion to collagen through cGMP-dependent and independent mechanisms: the potential role for S-nitrosylation.

Nitric oxide (NO)-mediated inhibition of platelet function occurs primarily through elevations in cGMP, although cGMP-independent mechanisms such as S-nitrosylation have been suggested as alternative NO-signaling pathways. In the present study we investigated the potential for S-nitrosylation to act as a NO-mediated cGMP-independent signaling mechanism in platelets. The NO-donor, S-nitrosoglutathione (GSNO), induced a concentration-dependent inhibition of platelet adhesion to immobilized collagen. In the presence of the soluble guanylyl cyclase inhibitor, ODQ, NO-mediated activation of the cGMP/protein kinase G signaling pathway was ablated. However, ODQ failed to completely abolish the inhibitory effect of NO on collagen-mediated adhesion, confirming that cGMP-independent signaling events contribute to the regulation of platelet adhesion by NO. Biotin-switch analysis of platelets demonstrated the presence of several S-nitrosylated proteins under basal conditions. Treatment of platelets with exogenous NO-donors, at concentrations that inhibited platelet adhesion, increased the number of S-nitrosylated bands and led to hyper-nitrosylation of basally S-nitrosylated proteins. The extent of S-nitrosylation in response to exogenous NO was unaffected by platelet activation. Importantly, platelet activation in the absence of exogenous NO failed to increase S-nitrosylation beyond basal levels, indicating that platelet-derived NO was unable to induce this type of protein modification. Our data demonstrate that S-nitrosylation of platelet proteins in response to exogenous NO may act as a potentially important cGMP-independent signaling mechanism for controlling platelet adhesion.

(Nitrooxyacyloxy)methyl esters of aspirin as novel nitric oxide releasing aspirins.

A series of (nitrooxyacyloxy)methyl esters of aspirin were synthesized and evaluated as new NO-donor aspirins. Different amounts of aspirin were released in serum from these products according to the nature of nitrooxyacyloxy moiety present. In the aromatic series, there is a rather good linear correlation between the amount of aspirin released and the potencies of the products in inhibiting platelet aggregation induced by collagen. Both the native compounds and the related nitrooxy-substituted acid metabolites were able to relax rat aorta strips precontracted with phenylephrine, in keeping with a NO-induced activation of the sGC as a mechanism that underlies the vasodilator effect. The products here described are new improved examples of NO-donor aspirins containing nitrooxy groups. They could represent an alternative to the use of aspirin in a variety of clinical applications.

Central insulin regulates heart rate and arterial blood flow: an endothelial nitric oxide synthase-dependent mechanism altered during diabetes.

OBJECTIVE: Central neural insulin regulates glucose homeostasis, but less is known about its cardiovascular effects. Endothelial nitric oxide synthase (eNOS)-derived nitric oxide (NO) represents a molecular link between metabolic and cardiovascular disease. Its role in the central nervous system remains to be determined. We studied the effects of central insulin infusion on femoral arterial blood flow and heart rate in normal chow-fed, high-fat diet-fed diabetic, and eNOS-null mice. RESEARCH DESIGN AND METHODS: We recorded heart rate and femoral blood flow (ultrasonic flow probe) during 3-h central insulin infusion in conscious, freely moving mice. To study the role of NO in this setting, we assessed total and phosphorylated eNOS in the hypothalamus and examined the effects of brain infusion of NO donors/NOS inhibitors on cardiovascular responsiveness to central insulin in these experimental mouse models. RESULTS: In normal mice, central insulin rapidly increased heart rate by 30% and more progressively increased blood flow by 40%. In high-fat diet-fed mice, the cardiovascular effects of insulin were blunted and associated with a 50% reduction of the total and phosphorylated eNOS expression in the hypothalamus, suggesting a causal link. In line with this hypothesis, in eNOS-null mice and central N(G)-monomethyl-L-arginine-infused normal mice, the cardiovascular effects of insulin were abolished, whereas central NO donor infusion restored these effects in eNOS-null mice. In high-fat diet-fed mice, central NO donor infusion mimicked the cardiovascular responses evoked by central insulin in normal mice. CONCLUSIONS: Central insulin has cardiovascular effects in conscious, freely moving mice that are mediated, at least in part, by central neural eNOS. These effects are impaired in insulin-resistant high-fat diet-fed mice.

Chemosensitization of cancer in vitro and in vivo by nitric oxide signaling.

PURPOSE: Hypoxia contributes to drug resistance in solid cancers, and studies have revealed that low concentrations of nitric oxide (NO) mimetics attenuate hypoxia-induced drug resistance in tumor cells in vitro. Classic NO signaling involves activation of soluble guanylyl cyclase, generation of cyclic GMP (cGMP), and activation of cGMP-dependent protein kinase. Here, we determined whether chemosensitization by NO mimetics requires cGMP-dependent signaling and whether low concentrations of NO mimetics can chemosensitize tumors in vivo. EXPERIMENTAL DESIGN: Survival of human prostate and breast cancer cells was assessed by clonogenic assays following exposure to chemotherapeutic agents. The effect of NO mimetics on tumor chemosensitivity in vivo was determined using a mouse xenograft model of human prostate cancer. Drug efflux in vitro was assessed by measuring intracellular doxorubicin-associated fluorescence. RESULTS: Low concentrations of the NO mimetics glyceryl trinitrate (GTN) and isosorbide dinitrate attenuated hypoxia-induced resistance to doxorubicin and paclitaxel. Similar to hypoxia-induced drug resistance, inhibition of various components of the NO signaling pathway increased resistance to doxorubicin, whereas activation of the pathway with 8-bromo-cGMP attenuated hypoxia-induced resistance. Drug efflux was unaffected by hypoxia and inhibitors of drug efflux did not significantly attenuate hypoxia-induced chemoresistance. Compared with mice treated with doxorubicin alone, tumor growth was decreased in mice treated with doxorubicin and a transdermal GTN patch. The presence of GTN and GTN metabolites in plasma samples was confirmed by gas chromatography. CONCLUSION: Tumor hypoxia induces resistance to anticancer drugs by interfering with endogenous NO signaling and reactivation of NO signaling represents a novel approach to enhance chemotherapy.

Pharmacokinetics and pharmacodynamics of a nitric oxide-releasing derivative of enalapril in male beagles.

1. Pharmacological compounds that release nitric oxide (NO) have been useful tools in the evaluation of the broad role of NO in physiopathology and therapeutics. The present study compared the pharmacokinetics and pharmacodynamics of enalapril and an NO-releasing enalapril molecule (NCX899) in conscious male beagles. The effects of both enalapril and NCX899 in the arterial hypertension and bradycardia induced by acute NO inhibition in anaesthetized dogs were also investigated. 2. Dogs received either NCX899 (4 micromol/kg, i.v.) or enalapril (4 micromol/kg, i.v.), after which plasma concentrations of the analytes and metabolites were quantified by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). 3. In the NCX899 group, the area under the time-course curve (AUC(0-24h)) was 29.18 +/- 4.72, 229.37 +/- 51.32 and 5159.23 +/- 514.88 microg.h/L for the analytes nitro-enalapril, enalapril and enalaprilat, respectively. In the enalapril group, the AUC(0-24h) was 704.53 +/- 158.86 and 4149.27 +/- 847.30 microg.h/L for the analytes enalapril and enalaprilat, respectively. Statistical analysis of data from both groups showed a significant difference for the analyte enalapril, but not for enalaprilat. Moreover, NCX899 and enalapril were equally effective in inhibiting the activity of serum angiotensin-converting enzyme. 4. In anaesthetized dogs, i.v. administration of the NO synthase (NOS) inhibitor N(G)-nitro-l-arginine methyl ester (l-NAME; 0.1-10 mg/kg) significantly elevated arterial blood pressure, with concomitant bradycardia. The compound NCX899 significantly attenuated both arterial hypertension and bradycardia, whereas enalapril had no significant effect. 5. In conclusion, the present results showed that the NO-releasing derivative of enalapril NCX899 presents a pharmacokinetic/pharmacodynamic relationship similar to its parent compound enalapril. Moreover, NCX899 (but not enalapril) was effective in protecting against the cardiovascular changes induced by acute NOS inhibition.

Effect of aspirin, paracetamol and their nitric oxide donating derivatives on exudate cytokine and PGE2 production in zymosan-induced air pouch inflammation in rats.

Effects of different doses of aspirin, compared to equimolar doses of nitric oxide (NO)-donating aspirin (NCX 4016), and of a single dose of paracetamol, compared to an equimolar dose of NO-donating paracetamol (NCX 701) were investigated in acute zymosan-induced air pouch inflammation in rats. Treatments were administered by orogastric route, and interleukin-1beta (IL-1beta), tumor necrosis factor-alpha (TNF-alpha) and prostaglandin E(2) (PGE(2)) levels in the exudates were analysed 4 h after zymosan injection by enzyme immunoassay (EIA). Aspirin, at 10, 30 and 100 mg/kg doses, increased IL-1beta levels in exudates, however, only the highest dose lead to a significant increase when compared to control, whereas a significant increase in TNF-alpha level was observed at all doses tested. NCX 4016, at equimolar doses for aspirin, i.e., 18.6, 55.8 and 186 mg/kg, respectively, did not cause any changes in exudate IL-1beta or TNF-alpha levels. These effects were significantly different, when aspirin was compared with the corresponding NCX 4016 group. Nevertheless, the ability of aspirin and NCX 4016 to inhibit PGE(2) synthesis in the exudate where comparable. Although paracetamol significantly increased exudate TNF-alpha level compared to the control group and NCX 701 group, neither paracetamol, nor NCX701 treatments changed the levels of exudate IL-1beta significantly. As expected, paracetamol and NCX 701 showed poor PGE(2) inhibition. At high doses, aspirin and NCX 4016 decreased the number of polymorphonuclear leukocytes in the exudate. However, this inhibition was not significantly different from the control group. Paracetamol and NO-paracetamol did not cause any change in the number of polymorphonuclear leukocytes in exudate. These results indicated that aspirin and NCX 4016 possessed different effects on cytokine production or release, despite the fact that both drugs inhibited the synthesis of PGE(2) in a similar way. Unlike paracetamol, which increased exudate TNF-alpha level, NCX 701 had no effect on TNF-alpha level in the exudates.

High performance liquid chromatography-electrospray ionization mass spectrometric determination of isosorbide 5-mononitrate in human plasma.

A novel, selective and sensitive high performance liquid chromatography-mass spectrometric (HPLC-MS) method has been developed for the determination of isosorbide 5-mononitrate (5-ISMN) in human plasma. With acetaminophen as internal standard, sample pretreatment involved one-step extraction with diethyl ether of 0.5 mL plasma. Analysis was performed on an ACQUITY UPLC BEH C(18) column (100 mm x 2.1mm, 1.7 microm) with mobile phase consisting of acetonitrile-water (20:80, v/v). The detection was carried out by means of electrospray ionization mass spectrometry in negative ion mode with selected ion recording (SIR). Standard curves were linear (r(2)> or =0.99) over the concentration range of 1.04-1040 ng/mL. The lower limit of quantification (LLOQ) was 1.04 ng/mL. The intra- and inter-day precisions (RSDs) were less than 8.6% and 13.4%, respectively, and the accuracy (RE) was within +/-0.45%. The method herein described was fully validated and successfully applied to the pharmacokinetic study of 5-ISMN in compound extended-release tablets in 18 healthy male volunteers after oral administration.

An extensive study on isosorbide-5-mononitrate acid adducts for quantification in human plasma using liquid chromatography/electrospray ionization tandem mass spectrometry and its application to bioequivalence sample analysis.

A rapid and sensitive liquid chromatography/electrospray ionization tandem mass spectrometry (LC/ESI-MS/MS) method for the determination of isosorbide-5-mononitrate (5-ISMN), used in the treatment of angina pectoris, in human plasma is described. The quantification of 5-ISMN was performed via stable acetate adduct formation with a high relative abundance. The plasma filtrate obtained after solid-phase extraction (SPE), using a polymer based, hydrophilic-lipophilic balanced (HLB) cartridge, was submitted directly to reversed-phase high-performance liquid chromatography separation followed by ESI and detection of the resulting ions using triple-quadrupole mass spectrometry in selected reaction monitoring (SRM) mode. There was no significant matrix effect on the analysis. For validation of the method, the recovery of the free analyte response was compared to that obtained from an optimized extraction method. The analyte stability was examined under conditions mimicking the sample storage, handling, and analytical procedures. The extraction procedure yielded extremely clean extracts with a recovery of 95.51% and 93.98% for iossorbide-5-mononitrate and topiramate (internal standard (IS)), respectively. The calibration curves were linear for the dynamic range of 10.0 to 1000.0 ng/mL with a correlation coefficient r > or = 0.9985. The intra-assay and inter-assay precision for the samples at the lower limit of quantification (LLOQ) were 9.02 and 13.30%, respectively. The intra-assay accuracies at LLOQ, LQC, MQC and HQC levels varied from 98.13 to 118.15, 102.34 to 105.21, 100.69 to 109.68, and 95.76 to 102.92%, respectively, while the inter-assay accuracies ranged from 93.10 to 118.15, 93.03 to 107.04, 86.97 to 109.68 and 86.18 to 105.85%, respectively, at these levels. The method is rugged and fast with a total run time of 2 min. The method was successfully applied for a bioequivalence study in 24 human subject samples after oral administration of 60 mg extended release (ER) formulations.

Pharmacological characterization of 2NTX-99 [4-methoxy-N1-(4-trans-nitrooxycyclohexyl)-N3-(3-pyridinylmethyl)-1,3-benzenedicarboxamide], a potential antiatherothrombotic agent with antithromboxane and nitric oxide donor activity in platelet and vascular preparations.

Thromboxane (TX) A(2), prostacyclin (PGI(2)), and nitric oxide (NO) regulate platelet function and interaction with the vessel wall. Inhibition of TXA(2), implemented synthesis of PGI(2), and supply of exogenous NO may afford therapeutic benefit. 2NTX-99 [4-methoxy-N(1)-(4-trans-nitrooxycyclohexyl)-N(3)-(3-pyridinylmethyl)-1,3-benzenedicarboxamide], a new chemical entity related to picotamide, showed antithromboxane activity and NO donor properties. 2NTX-99 relaxed rabbit aortic rings precontracted with norepinephrine or U46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxy-prosta-5Z,13E-dien-1-oic acid; EC(50), 7.9 and 17.1 microM, respectively), an effect abolished by 10 microM 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (ODQ). 2NTX-99 inhibited arachidonic acid (AA)-induced washed platelet aggregation (EC(50), 9.8 microM) and TXB(2) formation (-71% at 10 microM), and its potency increased in the presence of aortic rings (EC(50), 1.4 microM). In whole rabbit aorta incubated with homologous platelets, AA caused contraction and TXA(2) formation, reduced by 2NTX-99 (10-40 microM): contraction, -28 and -47%, TXA(2) formation, -37 and -75.4%, respectively, with concomitant increase in PGI(2). 2NTX-99 (20-40 microM) inhibited U46619-induced aggregation in rabbit platelet-rich plasma (PRP) (-74 +/- 6.7 and -96 +/- 2.4%, respectively) and inhibited collagen-induced aggregation in human PRP (-48.2 +/- 10 and -79.2 +/- 6%), whereas ozagrel was ineffective. In human embryonic kidney 293 cells transfected with the TXA(2) receptor isophorm alpha receptor, 2NTX-99 did not compete with the ligand, [(3)H]SQ29,548 ([(3)H][1S-[1alpha,2beta(5Z),3beta,4alpha]]-7-[3-[[2-(phenylamino)-carbonyl]hydrazino]methyl]-7-oxabicyclo[2,2,1]-hept-2-yl]-5-heptanoic acid), or prevent inositol phosphate accumulation. After oral administration (50-250 mg/kg), 2NTX-99 inhibited TXA(2) production in rat clotting blood (-71 and -91%); at 250 mg/kg, an area under the curve, 0 to 16 h, of 149.5 h/microg/ml and a t(1/2) of 6 h were calculated, with a C(max) value of 31.8 +/- 8.2 microg/ml. An excellent correlation between plasma concentrations and TXA(2) inhibition occurs. 2NTX-99 controls platelet function and vessel wall interaction by multifactorial mechanisms and possesses therapeutic potential.

In vitro metabolism of (nitrooxy)butyl ester nitric oxide-releasing compounds: comparison with glyceryl trinitrate.

We investigated the in vitro metabolism of two (nitrooxy)butyl ester nitric oxide (NO) donor derivatives of flurbiprofen and ferulic acid, [1,1'-biphenyl]-4-acetic acid-2-fluoro-alpha-methyl-4-(nitrooxy)butyl ester (HCT 1026) and 3-(4-hydroxy-3-methoxyphenyl)-2-propenoic acid 4-(nitrooxy)butyl ester (NCX 2057), respectively, in rat blood plasma and liver subcellular fractions compared with (nitrooxy)butyl alcohol (NOBA) and glyceryl trinitrate (GTN). HCT 1026 and NCX 2057 undergo rapid ubiquitous carboxyl ester hydrolysis to their respective parent compounds and NOBA. The nitrate moiety of this latter is subsequently metabolized to inorganic nitrogen oxides (NOx), predominantly in liver cytosol by glutathione S-transferase (GST) and to a lesser extent in liver mitochondria. If, however, in liver cytosol, the carboxyl ester hydrolysis is prevented by an esterase inhibitor, the metabolism at the nitrate moiety level does not occur. In blood plasma, HCT 1026 and NCX 2057 are not metabolized to NOx, whereas a slow but sustained NO generation in deoxygenated whole blood as detected by electron paramagnetic resonance indicates the involvement of erythrocytes in the bioactivation of these compounds. Differently from NOBA, GTN is also metabolized in blood plasma and more quickly metabolized by different GST isoforms in liver cytosol. The cytosolic GST-mediated denitration of these organic nitrates in liver limits their interaction with other intracellular compartments to possible generation of NO and/or their subsequent availability and bioactivation in the systemic circulation and extrahepatic tissues. We show the possibility of modulating the activity of hepatic cytosolic enzymes involved in the metabolism of (nitrooxy)butyl ester compounds, thus increasing the therapeutic potential of this class of compounds.