Nitric Oxide Prodrug Research Articles

Synthesis, hydrolysis kinetics and anti-platelet effects of isosorbide mononitrate derivatives of aspirin

Two isomeric aspirin derivatives of isosorbide-5-mononitrate (ISMN) were prepared and evaluated as potential mutual prodrugs of aspirin and nitric oxide. The hydrolysis of both compounds was studied in pH 7.4 phosphate buffer solution, buffered α-chymotrypsin solution and 10% buffered rabbit plasma. The benzodioxin-4-one derivative was hydrolysed to salicylic acid and ISMN acetate in buffer solution ( t 1/2 32.1 h), 10% buffered rabbit plasma ( t 1/2 25.7 min) and α-chymotrypsin ( t 1/2 86.6 min). The carboxylic acid ester derivative ISMNA was hydrolysed via the salicylate ester in buffer solution ( t 1/2 48.5 h) but was rapidly and almost exclusively hydrolysed to aspirin and ISMN in plasma solution ( t 1/2 2.8 min). The hydrolysis appeared to be enzyme mediated as it was suppressed by co-incubation with eserine. ISMNA was evaluated for its ability to inhibit platelet aggregation in rabbit PRP in response to the following agonists: arachidonic acid (AA) (100 μM), ADP (1.2 μM), phorbol ester (0.5 μM), platelet activating factor (PAF) (5 nM) and the thromboxane mimic U46619 (1.5 μM). ISMNA suppressed platelet response to AA at 1 μM whereas 10 μM aspirin showed no inhibitory effects.

Role of Denitration in Pharmacokinetics and Pharmacodynamics of Organic Nitrates.

Role of denitration in the pharmacokinetics (PK) and pharmacodynamics (PD) of organic nitrates was reviewed. The PK/PD relationship following i.v. bolus injection of stereoisomeric organic nitrates can be described using simple compartment and sigmoidal Emax models. Because the nitrates are prodrugs of nitric oxide and eliminate mainly as denitrated metabolites, both potency and duration of vasodilation depend on the extent of denitration. At present, the mechanism of nitrate tolerance remains poorly understood. However, the most of the important hypotheses are counter-regulative, so that the denitration may play an important role in the development of tolerance. It is desired to establish the PK/PD relationship of organic nitrates involving tolerance period and improve nitrate-therapy based on the relationship.

Role of inhibition of nitric oxide production in monocrotaline-induced pulmonary hypertension.

Monocrotaline (MCT)-induced pulmonary hypertension (PH) is associated with impaired endothelium-dependent nitric oxide (NO)-mediated relaxation. To examine the role of NO in PH, Sprague-Dawley rats were given a single subcutaneous injection of normal saline [control (C)], 80 mg/kg MCT, or the same dose of MCT and a continuous subcutaneous infusion of 2 mg.kg-1.day-1 of molsidomine, a NO prodrug (MCT+MD). Two weeks later, plasma NO3- levels, pulmonary arterial pressure (Ppa), ratio of right-to-left ventricular weights (RV/LV) to assess right ventricular hypertrophy, and pulmonary histology were evaluated. The plasma NO3- level in the MCT group was reduced to 9.2 +/- 1.5 microM (n = 12) vs. C level of 17.7 +/- 1.8 microM (n = 8; P < 0.02). In the MCT+MD group, plasma NO3- level was 12.3 +/- 2.0 microM (n = 8). Ppa and RV/LV in the MCT group were increased compared with C [Ppa, 34 +/- 3.4 mmHg (n = 6) vs. 19 +/- 0.8 mmHg (n = 8) and 0.41 +/- 0.01 (n = 9) vs. 0.25 +/- 0.008 (n = 8), respectively; P < 0.001]. In the MCT+MD group, Ppa and RV/LV were not different when compared with C [19 +/- 0.5 mmHg (n = 5) and 0.27 +/- 0.01 (n = 9), respectively; P < 0.001 vs. MCT]. Medial wall thickness of lung vessels in the MCT group was increased compared with C [31 +/- 1.5% (n = 9) vs. 13 +/- 0.66% (n = 9); P < 0.001], and MD partially prevented MCT-induced pulmonary vascular remodeling [22 +/- 1.2% (n = 11); P < 0.001 vs. MCT and C]. These results indicate that a defect in the availability of bioactive NO may play an important role in the pathogenesis of MCT-induced PH.

Inhibition of the action of nitric oxide prodrugs by pyocyanin: mechanistic studies

Previously, we reported on the antagonism by pyocyanin (PYO) of the relaxant effects of nitrovasodilators such as glyceryl trinitrate, S-nitroso-N-acetylpenicillamine (SNAP), and 3-morpholinosydnonimine (SIN-1). The purpose of the present study was to elucidate the mechanism of this action of PYO by examining its effect on the steps considered to be necessary for nitrovasodilator-induced relaxation of blood vessels. PYO (10 microM) attenuated the accumulation of guanosine-3',5'-cyclic monophosphate (cGMP) in rabbit aorta induced by nitrovasodilators SIN-1, SNAP, and GTN, 65, 81, and 67%, respectively. Additionally, PYO (1 or 10 microM) interfered with in vitro activation of soluble guanylyl cyclase. PYO did not inhibit vascular relaxation induced by 8-bromo-cyclic guanosine monophosphate. PYO (10 microM) also decreased the quantity of nitric oxide measured in the headspace above intact vascular tissue incubated with glyceryl trinitrate in the presence of oxygen. These observations are consistent with the interpretation that PYO interfered with the nitrovasodilator action of glyceryl trinitrate by inactivation of NO or by inhibition of enzymatic biotransformation of GTN; this would result in decreased guanylyl cyclase activation and thus lowering cellular levels of cGMP. NO chemiluminescence studies with SIN-1 (10 microM) revealed that this NO donor produced NO in a time-dependent manner and PYO (10 microM) caused no inhibition of NO production, but in fact, potentiated NO release after 10 min of incubation (1395 +/- 179 pmol NO compared with 1088 +/- 154 pmol NO). NO production from 10 microM SNAP was similarly potentiated by PYO after 0.5, 2, 5, and 10 min of incubation. Therefore, it is likely that PYO acts as an inhibitor of guanylyl cyclase with respect to NO donors, SIN-1 and SNAP, but it also appears that PYO can exert additional inhibitory effects in the case of vascular relaxation by GTN. Such differences in relaxant effects may reflect inhibition of enzymatic biotransformation that is unique to GTN or that PYO may complex with an alternative redox form of NO (perhaps NO+) that is generated by vascular metabolic activation of GTN.

Quantification of Diazeniumdiolate Mutagenicity in Four Differentin VitroAssays

Diazeniumdiolates are under investigation as possible prodrugs of the multifaceted bioregulatory agent nitric oxide. This study was undertaken to assess further the mutagenic potential of two diazeniumdiolates, DEA/NO (Et2N[N(O)NO]Na) and SPER / NO ( [ H2N ( CH2)3NH ( CH2)4N [ N ( O ) NO−] ( CH2)3NH+3]), which generate NO spontaneously with half-lives at 37°C and pH 7.4 of 2 and 39 min, respectively. The genotoxic potential of these compounds was investigated with the Ames bacterial reverse mutation assay, two mammalian cell gene mutation assays (CHO/HGPRT and L5178Y TK+/−), and an assay for sister chromatid exchange (SCE) using Chinese hamster ovary (CHO) cells. Both diazeniumdiolates had previously been shown to be mutagenic in the AmesSalmonellaplate assay. In the experiments reported here,Salmonella typhimuriumstrain TA1535 was exposed to the compounds in a liquid incubation assay for either 15 min or 48 h without an S-9 fraction. With the 15-min exposure, DEA/NO was mutagenic at concentrations of 0.625 mm(3.5 × control) and greater, while SPER/NO was mutagenic at 0.5 mm(2.7 × control) and above. In the CHO/HGPRT assay, DEA/NO was weakly mutagenic only at the highest concentration used, 20 mm, inducing a mutant frequency per survivor that was 2.5 × control, while SPER/NO was mutagenic at 0.5 mmwith a mutant frequency of 2.5 × control. When the CHO cells were given 10 repetitive 20 mmDEA/NO exposures (3 min each), HGPRT mutant frequency was 4.1 × control. In the L5178Y mouse lymphoma cell TK+/−assay, DEA/NO doubled the mutation rate at 1.82 mm, while SPER/NO's mutation frequency was more than twice that of control at 0.63 mm. DEA/NO was positive in the SCE assay without metabolic activation, yielding significant SCE at 1.25, 2.5, and 5 mmthat was 1.8, 2.2, and 2.6 times control, respectively. SPER/NO increased the SCE by 1.2, 1.4, and 1.3 times at 1.5, 2.0, and 2.5 mm. The results suggest that the two diazeniumdiolates, although mutagenic in the bacteria, are much weaker mutagens in mammalian cells.

Inhibition of the action of nitric oxide prodrugs by pyocyanin: mechanistic studies

Inhibition of the action of nitric oxide prodrugs by pyocyanin: mechanistic studies

Toxicology of Selected Nitric Oxide-Donating Xenobiotics, with Particular Reference to Azide

Nitric oxide (NO) has been discovered recently to be a ubiquitous, endogenous mediator, which is responsible for a variety of normal physiological functions. However, NO also has been implicated in several pathophysiological processes. For example, the pulmonary toxicity of various nitrogen oxides, including NO, found in photochemical smog has been studied for decades; endogenous NO also is associated with bleomycin-induced lung damage, as well as other adverse effects. Recently, a variety of xenobiotics have been shown to owe their biological activity in vivo to their biotransformation to NO. Thus, the therapeutic vasodilatation produced by drugs such as nitroglycerin and sodium nitroprusside is now believed to result from their release of NO, which then mimics the effects of endogenously synthesized NO. The toxic effects of NO prodrugs are, therefore, a matter of concern, especially the extent to which, if any, NO contributes to their toxicity. As reviewed here, NO does not appear to contribute importantly to the toxicity of the NO donors nitrite, hydroxylamine, or nitroprusside. However, it is by no means clear whether or not the NO generated in vivo from sodium azide contributes in a major way to its toxicity. Azide is almost as acutely toxic as cyanide, with which it shares a number of biological effects; yet, azide also has certain cardiovascular actions in common with nitrite. Unlike either cyanide or nitrite, some evidence suggests a tendency for azide to produce low-grade cumulative toxicity. In laboratory animals, azide frequently produces nonasphyxial convulsions, whereas most human deaths appear to be the result of cardiovascular collapse. Neither of these azide-induced syndromes appears to be due to the inhibition of cytochrome c oxidase. Azide is widely used as a preservative in aqueous laboratory reagents and as the propellant in automobile air bags and aircraft escape chutes. Both of these inflable systems are generally safe, and will prevent untold numbers of injuries and deaths. However, to protect workers who handle these devices and others who may come into contact with the sodium azide propellant in these systems, our rudimentary knowledge of azide toxicity needs to be expanded.

A novel method for detection of nitric oxide binding sites by using a chemiluminescence-headspace gas technique

Nitric oxide (NO) is important in many physiological, pharmacological, and pathological processes. According to current concepts, guanylyl cyclase is considered to be a receptor for NO in vascular and nonvascular smooth muscle and other tissues. Since there are no suitable radioisotopes of oxygen and nitrogen available for conventional radioligand-receptor binding studies for NO, a novel method was developed to identify NO binding site(s). A chemiluminescence-headspace gas assay was utilized to measure the sequestration of NO in biological systems, and this was used as an index of NO binding. In the present report, myoglobin (a hemoprotein, Mb) was used as a prototype macromolecule to develop the binding assay for subsequent application to studies of putative NO receptors. Solutions containing various concentrations of Mb were incubated with NO in sealed micro-Fernbach flasks at 37°C in an argon atmosphere for 30 min; NO remaining in the headspace gas was analyzed by means of the chemiluminescence assay. The magnitude of NO sequestration was dependent on Mb concentration, and 5 nM Mb was the lowest Mb concentration for which NO sequestration was measurable. Application of the method to the measurement of NO sequestration by bovine serum albumin (BSA) and pulmonary artery medial layer homogenate (BPA-M) revealed that the lowest BSA concentration at which NO sequestration was measurable was 1.6 μM, which was 320 times greater than that for Mb. Applicability of the method to address the question of putative NO receptors was indicated by significant NO sequestration after incubation with 20% (w/v) homogenate of BPA-M, which is responsive to NO and putative NO prodrugs.

The Biochemical Pathways of Nitric Oxide Formation from Nitrovasodilators

The endogenous formation of nitric oxide (NO) plays a key role in many bioregulatory systems inclunding the control of vascular tone, platelet aggregation, neutrotransmission, and macrophage cytotoxicity. Due to the instability and inconvenient handling of aqueous solutions of authentic NO, there is increasing interest in using compounds capable of generating NO in situ. Nitrovasodilators are prodrugs of NO and thus principally suited to mimic endogenous NO activities.