Plasma levels of glyceryl trinitrate and dinitrates during application of three strengths of a new glyceryl trinitrate transdermal patch

The pharmacokinetic characteristics and the bioavailability of glyceryl trinitrate (GTN, CAS 55-63-0) and of its main metabolities 1,2-glyceryl dinitrate (1,2-GDN, CAS 621-65-8) and 1,3-glyceryl dinitrate (1,3-GDN, CAS 623-87-0) during a single 24-h application of three strengths of a newly developed GTN transdermal patch (Epinitril) were investigated. The three strengths coded in this paper EPI-5, EPI-10 and EPI-15 have a nominal release rate of GTN of 5, 10 and 15 mg, respectively, in 24 h. The study was an open, randomized balanced cross-over study on 18 healthy male volunteers, to whom the patches were applied for 24 h to the antero-lateral part of the thorax in three periods, separated by an at least 3-day wash-out. Blood samples were collected before administration, during the 24-h patch application and 1, 3 and 6 h after patch removal. Assayed in plasma were GTN, 1,2-GDN and 1,3-GDN using validated GC/MS methods with stable isotope labeled internal standards (15N3-GTN, 15N2-1,2-GDN, and 15N2-1,3-GDN). GTN and its two metabolites reached the plateau already 3 h after application of the patches and remained on extended and fairly constant levels during the whole 24-h application. The plasma levels of the three nitrates were proportional to the strengths of the patches. The plasma levels of 1,2-GDN were about 6 times higher than those of GTN. The plasma levels of 1,3-GDN were similar to those of GTN. Upon removal of the patches the concentrations of the three nitrates fell to negligible values within 3 h, an important property when an intermittent GTN therapy is needed in order to avoid tolerance to the drug. The patches were well tolerated at the application site. For their good tolerability, small size and transparency, the new GTN patches are very patient friendly, a quality that improves compliance with the therapeutic regimen.     

Different effects of ascorbate deprivation and classical vascular nitrate tolerance on aldehyde dehydrogenase-catalysed bioactivation of nitroglycerin

Background and purpose:

Vascular tolerance to nitroglycerin (GTN) may be caused by impaired GTN bioactivation due to inactivation of mitochondrial aldehyde dehydrogenase (ALDH2). As relaxation to GTN is reduced but still sensitive to ALDH2 inhibitors in ascorbate deficiency, we compared the contribution of ALDH2 inactivation to GTN hyposensitivity in ascorbate deficiency and classical in vivo nitrate tolerance.

Experimental approach:

Guinea pigs were fed standard or ascorbate-free diet for 2 weeks. Reversibility was tested by feeding ascorbate-deficient animals standard diet for 1 week. Nitrate tolerance was induced by subcutaneous injection of 50 mg·kg⁻¹ GTN 4 times daily for 3 days. Ascorbate levels were determined in plasma, blood vessels, heart and liver. GTN-induced relaxation was measured as isometric tension of aortic rings; vascular GTN biotransformation was assayed as formation of 1,2-and 1,3-glyceryl dinitrate (GDN).

Key results:

Two weeks of ascorbate deprivation had no effect on relaxation to nitric oxide but reduced the potency of GTN ∼10-fold in a fully reversible manner. GTN-induced relaxation was similarly reduced in nitrate tolerance but not further attenuated by ALDH inhibitors. Nitrate tolerance reduced ascorbate plasma levels without affecting ascorbate in blood vessels, liver and heart. GTN denitration was significantly diminished in nitrate-tolerant and ascorbate-deficient rings. However, while the ∼10-fold preferential 1,2-GDN formation, indicative for active ALDH2, had been retained in ascorbate deficiency, selectivity was largely lost in nitrate tolerance.

Conclusions and implications:

These results indicate that nitrate tolerance is associated with ALDH2 inactivation, whereas ascorbate deficiency possibly results in down-regulation of ALDH2 expression.     

Vascular and anti-platelet actions of 1,2- and 1,3-glyceryl dinitrate.

1. The aim of this study was to investigate whether two metabolites of glyceryl trinitrate (GTN), 1,2 and 1,3-glyceryl dinitrate (1,2-GDN and 1,3-GDN) could account for the pharmacological effects of GTN. To this end the formation of nitric oxide (NO) from 1,2- and 1,3-GDN in the presence of bovine aortic smooth muscle cells (SMC) or endothelial cells (EC) was studied. The effects of various thiols on NO formation from these dinitrates was also evaluated. 2. 1,2-GDN or 1,3-GDN (10(-10)-10(-5) M) caused a dose-dependent relaxation of rabbit aortic strips denuded of endothelium and precontracted with phenylephrine. The dinitrates were less than one tenth as potent as GTN. 3. Incubation of 1,2-GDN or 1,3-GDN (75-2400 microM) with SMC for 30 min led to a concentration-dependent increase in nitrite (NO2-) formation but this increase was less than that produced from GTN. Likewise incubation of 1,2-GDN or 1,3-GDN with N-acetylcysteine (NAC), glutathione (GSH) or thiosalicylic acid (TSA) (all at 1 mM) for 30 min at 37 degrees C produced a concentration-dependent increase in NO2- formation. 4. Platelet aggregation induced by thrombin (40 mu ml-1) was not modified by high concentrations of 1,2-GDN or 1,3-GDN (175-700 microM). However, aggregation was inhibited when platelets were exposed to 1,2-GDN or 1,3-GDN (700 microM) in the presence of SMC (0.24-1.92 x 10(5) cells) or EC (0.8-3.2 x 10(5) cells). These effects were abrogated by co-incubation with oxyhaemoglobin (OxyHb, 10 microM) indicating that they were due to NO release.(ABSTRACT TRUNCATED AT 250 WORDS)     

A new class of organic nitrates: investigations on bioactivation,tolerance and cross-tolerance phenomena

Background and purpose:

The chronic use of organic nitrates is limited by serious side effects including oxidative stress, nitrate tolerance and/or endothelial dysfunction. The side effects and potency of nitroglycerine depend on mitochondrial aldehyde dehydrogenase (ALDH-2). We sought to determine whether this concept can be extended to a new class of organic nitrates with amino moieties (aminoalkyl nitrates).

Experimental approach:

Vasodilator potency of the organic nitrates, in vitro tolerance and in vivo tolerance (after continuous infusion for 3 days) were assessed in wild-type and ALDH-2 knockout mice by isometric tension studies. Mitochondrial oxidative stress was analysed by L-012-dependent chemiluminescence and protein tyrosine nitration.

Key results:

Aminoethyl nitrate (AEN) showed an almost similar potency to glyceryl trinitrate (GTN), even though it is only a mononitrate. AEN-dependent vasodilatation was mediated by cGMP and nitric oxide. In contrast to triethanolamine trinitrate (TEAN) and GTN, AEN bioactivation did not depend on ALDH-2 and caused no in vitro tolerance. In vivo treatment with TEAN and GTN, but not with AEN, induced cross-tolerance to acetylcholine (ACh)-dependent and GTN-dependent relaxation. Although all nitrates tested induced tolerance to themselves, only TEAN and GTN significantly increased mitochondrial oxidative stress in vitro and in vivo.

Conclusions and implications:

The present results demonstrate that not all high potency nitrates are bioactivated by ALDH-2 and that high potency of a given nitrate is not necessarily associated with induction of oxidative stress or nitrate tolerance. Obviously, there are distinct pathways for bioactivation of organic nitrates, which for AEN may involve xanthine oxidoreductase rather than P450 enzymes.     

Nitroglycerin and isosorbide dinitrate stimulation of glutathione disulfide efflux from perfused rat liver

Nitroglycerin (GTN) and isosorbide dinitrate (ISD) are metabolized by glutathione S-transferase to nitrite with production of GSSG from GSH. Infusion of organic nitrates into perfused rat liver led to efflux of GSSG in the bile and nitrite in the perfusate. Biliary GSSG increased more rapidly than did nitrite release as GTN infusion rate was increased, indicating that GSSG reducing capacity was being exceeded. Rapid GTN-induced oxidation of GSH may be the mechanism of tissue GSH depletion by GTN and other alkylnitrates. Such depletion of glutathione may reduce nitrite production from organic nitrates and underlie tolerance to these drugs.     

Oxidative stress and mitochondrial aldehyde dehydrogenase activity: a comparison of pentaerythritol tetranitrate with other organic nitrates

Mitochondrial aldehyde dehydrogenase (ALDH-2) was recently identified to be essential for the bioactivation of glyceryl trinitrate (GTN). Here we assessed whether other organic nitrates are bioactivated by a similar mechanism. The ALDH-2 inhibitor benomyl reduced the vasodilator potency, but not the efficacy, of GTN, pentaerythritol tetranitrate (PETN), and pentaerythritol trinitrate in phenylephrine-constricted rat aorta, whereas vasodilator responses to isosorbide dinitrate, isosorbide-5-mononitrate, pentaerythritol dinitrate, pentaerythritol mononitrate, and the endothelium-dependent vasodilator acetylcholine were not affected. Likewise, benomyl decreased GTN- and PETN-elicited phosphorylation of the cGMP-activated protein kinase substrate vasodilator-stimulated phosphoprotein (VASP) but not that elicited by other nitrates. The vasodilator potency of organic nitrates correlated with their potency to inhibit ALDH-2 dehydrogenase activity in mitochondria from rat heart and increase mitochondrial superoxide formation, as detected by chemiluminescence. In contrast, mitochondrial ALDH-2 esterase activity was not affected by PETN and its metabolites, whereas it was inhibited by benomyl, GTN applied in vitro and in vivo, and some sulfhydryl oxidants. The bioactivation-related metabolism of GTN to glyceryl-1,2-dinitrate by isolated RAW macrophages was reduced by the ALDH-2 inhibitors benomyl and daidzin, as well as by GTN at concentrations >1 microM. We conclude that mitochondrial ALDH-2, specifically its esterase activity, is required for the bioactivation of the organic nitrates with high vasodilator potency, such as GTN and PETN, but not for the less potent nitrates. It is interesting that ALDH-2 esterase activity was inhibited by GTN only, not by the other nitrates tested. This difference might explain why GTN elicits mitochondrial superoxide formation and nitrate tolerance with the highest potency.     

Bioavailability and pharmacokinetic profile of glyceryl trinitrate and of glyceryl dinitrates during application of a new glyceryl trinitrate transdermal patch

The pharmacokinetics and bioavailability of glyceryl trinitrate (GTN, CAS 55-63-0) and of its main metabolites, i.e. 1,2-glyceryl dinitrate (1,2-GDN, CAS 621-65-8) and 1,3-glyceryl dinitrate (1,3-GDN, CAS 623-87-0), were compared during a single 24-h application of a new GTN transdermal patch (Epinitril 10, hereinafter called EPI-10) or a reference patch (hereinafter called ND-10) releasing 10 mg GTN in 24 h. The study was an open, randomized balanced cross-over study on 24 healthy male volunteers to whom the patches were applied to the antero-lateral part of the thorax in two periods separated by a 3-day wash-out. Blood samples were collected before administration, during the 24-h patch application and at 0.5, 2 and 3 h after patch removal. Assayed in plasma were GTN, 1,2-GDN and 1,3-GDN using validated GC/MS methods with stable isotope-labeled internal standards (15N3-GTN, 15N2-1,2-GDN, and 15N2-1,3-GDN). The ratios of the AUCs of GTN, 1,2-GDN and 1,3-GDN measured during application of EPI-10 or of ND-10 were within the 0.85-1.25 limits required to assess equivalence of the extent of bioavailability. The ratios of the Cmax were within said limits for the signal metabolite 1,2-GDN and only slightly below (0.78-1.16) for the parent GTN. EPI-10 can therefore be considered equivalent to ND-10 also with regard to the rate of bioavailability. Under both patches GTN reached steady-state levels after 3-6 h of patch application and remained on sustained levels during the whole 24-h application. The plasma levels of 1,2-GDN were about 6 times higher than those of GTN. The plasma levels of 1,3-GDN were similar to those of GTN. Upon removal of the patches the concentrations of the three nitrates fell to negligible values within 3 h. Both patches were well tolerated at the application site. For its small size, thinness and transparency, EPI-10 is very patient friendly, a quality that improves compliance with the therapeutic regimen.     

Clinical pharmacokinetics and pharmacodynamics of glyceryl trinitrate and its metabolites

This review discusses the pharmacokinetics and pharmacodynamics of glyceryl trinitrate (nitroglycerin; GTN) pertinent to clinical medicine. The pharmacokinetics of GTN associated with various dose regimens are characterised by prominent intra- and inter-individual variability. It is, nevertheless, important to clearly understand the pharmacokinetics and characteristics of GTN to optimise its use in clinical practice and, in particular, to obviate the development of tolerance. Measurements of plasma concentrations of GTN and of 1,2-glyceryl dinitrate (1,2-GDN), 1,3-glyceryl dinitrate (1,3-GDN), 1-glyceryl mononitrate (1-GMN), and 2-glyceryl mononitrate (2-GMN), its four main metabolites, remain difficult and require meticulous techniques to obtain reliable results. Since GDNs have an effect on haemodynamic function, pharmacokinetic analyses that include the parent drug as well as the metabolites are important. Although the precise mechanisms of GTN metabolism have not been elucidated, two main pathways have been proposed for its biotransformation. The first is a mechanism-based biotransformation pathway that produces nitric oxide (NO) and contributes directly to vasodilation. The second is a clearance-based biotransformation or detoxification pathway that produces inorganic nitrite anions (NO(2) -). NO(2) - has no apparent cardiovascular effect and is not converted to NO in pharmacologically relevant concentrations in vivo. In addition, several non-enzymatic and enzymatic systems are capable of metabolising GTN. This complex metabolism complicates considerably the evaluation of the pharmacokinetics and pharmacodynamics of GTN. Regardless of the route of administration, concentrations of the metabolites exceed those of the parent compound by several orders of magnitude. During continuous steady-state delivery of GTN, for instance by a patch, concentrations of 1,2-GDN are consistently 2-7 times higher than those of 1,3-GDN, and concentrations of 2-GMN are 4-8 times higher than those of 1-GMN. Concentrations of GDNs are approximately 10 times higher, and of GMNs approximately 100 times higher, than those of GTN during sustained administration. The development of tolerance is closely related to the metabolism of GTN, and can be broadly categorised as haemodynamic tolerance versus vascular tolerance. Efforts are warranted to circumvent the development of tolerance and facilitate the use of GTN in clinical practice. Although this remains to be accomplished, it is likely that, in the near future, regimens will be developed based on a full understanding of the pharmacokinetics and pharmacodynamics of GTN and its metabolites.     

Tissue disposition of glyceryl trinitrate, 1,2-glyceryl dinitrate, and 1,3-glyceryl dinitrate in tolerant and nontolerant rats.

The tissue distribution of glyceryl trinitrate (GTN) and its two dinitrate metabolites 1,2-glyceryl dinitrate (1,2-GDN) and 1,3-glyceryl trinitrate (1,3-GDN), was studied in GTN-tolerant and nontolerant male Sprague-Dawley rats. The concentrations of GTN, 1,2-GDN, and 1,3-GDN were measured in plasma, heart, brain, liver, aortic tissue, and adipose tissue at various time points after a subcutaneous dose of GTN (50 mg/kg). At the first time point (5 hr), concentrations of GTN, 1,2-GDN, and 1,3-GDN in plasma were equal for tolerant and nontolerant rats, but the elimination rate was altered for the tolerant rats as compared with nontolerant rats. In adipose tissue, the concentration of GTN was significantly higher as compared with concentrations of the dinitrate metabolites. In contrast, the other tissues studied showed significantly higher concentrations of the GDNs when compared with GTN. The 1,3-GDN/1,2-GDN ratio decreased with time for both tolerant and nontolerant rats. This study indicates that long-term GTN administration results not only in tolerance development, but also in altered pharmacokinetics of GTN, 1,2-GDN, and 1,3-GDN. The results also show that the 1,3-GDN/1,2-GDN ratio is dependent on the GTN concentration.     

Cutaneous Metabolism of Nitroglycerin in Vitro. I. Homogenized Versus Intact Skin

The metabolism of nitroglycerin (GTN) to 1,2- and 1,3-glyceryl dinitrate (GDN) by hairless mouse skin in vitro has been measured. In the first set of experiments, GTN was incubated with the 9000g supernatant of fresh, homogenized tissue in the presence and absence of glutathione (GSH), a cofactor for glutathione-S-transferase. After 2 hr of incubation with GSH, 30% of the initially present GTN had been converted to 1,2- and 1,3-GDN; without GSH, less than 5% of the GTN was metabolized. The ratio of 1,2-GDN to 1,3-GDN produced by the homogenate was 1.8– 2.1. In the second series of studies, GTN was administered topically to freshly excised, intact hairless mouse skin in conventional in vitro diffusion cells. The concurrent transport and metabolism of GTN was then monitored by sequential analysis of the receptor phase perfusing the dermal side of the tissue. Three topical formulations were used: a low concentration (1 mg/ml) aqueous solution, a 2% ointment, and a transder-mal delivery system. Delivery of total nitrates (GTN + 1,2-GDN + 1,3-GDN) into the receptor phase was similar for ointment and patch formulations and much greater than that from the solution. The percentage metabolites formed, however, was greatest for the solution (61% and 2 hr, compared to 49% for the patch and 35% for the ointment). As has been noted before, therefore, the relative level of skin metabolism is likely to be greatest when the transepidermal flux is small. Distinct from the homogenate experiments, the 1,2/1,3-GDN ratios in the penetration studies were in the range 0.7– 0.9. It would appear that homogenization of the skin permits GTN to be exposed to a different distribution of enzymes than that encountered during passive skin permeation.     

Nitroglycerin metabolism in subcellular fractions of rabbit liver. Dose dependency of glyceryl dinitrate formation and possible involvement of multiple isozymes of glutathione S-transferases

The hepatic transformation of glyceryl trinitrate (GTN), commonly known as nitroglycerin, was studied in subcellular fractions prepared from rabbit livers. Both the cytosolic and microsomal fractions show activity toward GTN metabolism. Moreover, the formation of glyceryl dinitrates (GDNs) seems to be governed by different enzymatic processes in the two fractions. 1,2-GDN was preferentially formed in cytosolic fractions, whereas in microsomal fractions, 1,3-GDN was the predominant product. In cytosolic fractions, increasing starting concentrations of GTN led to a decrease in both the GTN degradation rate and the GDN ratio (1,2-GDN/1,3-GDN), which was mainly accounted for by saturation of the 1,2-GDN formation pathway. Various glutathione S-transferase (GST) inhibitors affected the rate of GDN formation differentially. In cytosolic fractions, 1-chloro-2,4-dinitrobenzene and iodomethane caused no change in the GDN ratio, while sulfobromophthalein, ethacrynic acid, and p-nitrobenzyl chloride decreased the GDN ratio, suggesting that different GST isozymes are inhibited by these agents. In microsomal fractions, no dose-dependent GTN metabolism and related change in the GDN ratios could be observed. With the exception of ethacrynic acid, addition of GST inhibitors did not decrease GDN metabolite production, and even in this case, no change in the GDN ratio was observed. The results suggest that different GTN metabolic pathways are present in the liver, most likely involving different GST isozymes.     

The enigma of nitroglycerin bioactivation and nitrate tolerance: news, views and troubles

Nitroglycerin (glyceryl trinitrate; GTN) is the most prominent representative of the organic nitrates or nitrovasodilators, a class of compounds that have been used clinically since the late nineteenth century for the treatment of coronary artery disease (angina pectoris), congestive heart failure and myocardial infarction. Medline lists more than 15 000 publications on GTN and other organic nitrates, but the mode of action of these drugs is still largely a mystery. In the first part of this article, we give an overview on the molecular mechanisms of GTN biotransformation resulting in vascular cyclic GMP accumulation and vasodilation with focus on the role of mitochondrial aldehyde dehydrogenase (ALDH2) and the link between the ALDH2 reaction and activation of vascular soluble guanylate cyclase (sGC). In particular, we address the identity of the bioactive species that activates sGC and the potential involvement of nitrite as an intermediate, describe our recent findings suggesting that ALDH2 catalyses direct 3-electron reduction of GTN to NO and discuss possible reaction mechanisms. In the second part, we discuss contingent processes leading to markedly reduced sensitivity of blood vessels to GTN, referred to as vascular nitrate tolerance. Again, we focus on ALDH2 and describe the current controversy on the role of ALDH2 inactivation in tolerance development. Finally, we emphasize some of the most intriguing, in our opinion, unresolved puzzles of GTN pharmacology that urgently need to be addressed in future studies.     

Nitroglycerin-patch induced tolerance is associated with reduced ability of nitroglycerin to increase exhaled nitric oxide

Nitroglycerin (GTN), used in the treatment of ischemic heart disease, acts through the liberation of nitric oxide (NO). However, its clinical use is limited due to tolerance development. Expired NO was used as an indicator of GTN-bioactivation and was measured together with plasma nitrite and mean arterial pressure (MAP) during GTN indicator infusions. The model was applied in rabbits subjected to various time periods of low-dose GTN pretreatment by patch application for 1, 24 and 72 h. Pretreatment with GTN-patch resulted in significant attenuation of expired NO from the GTN indicator infusion in the 24 h and 72 h pretreatment groups compared to placebo (72 h). Dose-response curves with increasing GTN infusions after 24 h GTN-patch pretreatment revealed a significant attenuation of the MAP decrease compared to placebo. GTN-induced changes in plasma nitrite correlated to increases in expired NO and decreases in MAP. This indicates that expired NO could serve as an indicator of NO generation from GTN in the vascular system. We conclude that GTN tolerance is associated with reduced capacity to generate NO from GTN. Care should be taken in using MAP-reduction to evaluate tolerance since high indicator doses could liberate sufficient amounts of NO to elicit maximal MAP decrease even in tolerant animals.     

Dissociation of sympathetic compensation from glyceryl trinitrate tolerance

The hypothesis that changes in sympathetic function play an integral part in the development of tolerance to the vasodepressor effects of organic nitrates was tested. Glyceryl trinitrate (GTN) tolerant rats showed increased norepinephrine turnover but no changes in myocardial norepinephrine levels or in uptake of exogenous norepinephrine. GTN tolerant animals did not have altered sensitivities to sympathomimetics. Marked changes in sympathetic tone did not change the animals' sensitivity to glyceryl trinitrate (GTN) and did not alter the development of tolerance to GTN. No changes in sympathetic compensation to direct administration of a vasoconstrictor to the carotid sinus were observed. It is concluded that changes in sympathetic function play no rule in the development of tolerance to GTN.     

Glutathione S-Transferase-Mediated Metabolism of Glyceryl Trinitrate in Subcellular Fractions of Bovine Coronary Arteries

The possible role of glutathione S-transferases (GTSs) in vascular glyceryl trinitrate (GTN) metabolism was investigated. GTN degradation to form its dinitrate metabolites (GDNs) in the 9000g (9k) supernatant fraction of bovine coronary arteries (BCA) was examined. BCAs were homogenized with a 3x volume of phosphate buffer, and the 9k fraction was obtained by centrifugation. GTN (40 ng/ml; 1.76 x 10^–7 M) was incubated for 2 hr in the 9k fraction of BCA in the presence of reduced glutathione (2 x 10^–3 M). Samples were taken at 10, 20, 40, 60, and 120 min. GTN was observed to degrade readily, exhibiting a half-life of 26 min in the incubate. While both 1,2- and 1,3-GDNs were generated from GTN, formation of 1,3-GDN was predominant (GDN ratio, as 1,2/1,3-GDN, = 0.7–0.8). Coincubation with 2 x 10^–5 Mconcentrations of two GST inhibitors, sulfobromophthalein (SBP) and ethacrynic acid (ECA), decreased the rate of GTN loss. The GTN half-lives in SBP- and ECA-treated incubations were 66 and 84 min, respectively. In addition, the pattern of GDN formation was also altered. The resultant GDN ratios exceeded unity in the presence of these inhibitors, indicating that 1,3-GDN formation was attenuated to a greater extent than that of 1,2-GDN. These data suggest that vascular GTN metabolism in BCA is carried out by cytosolic GST isozymes which possess a preference for C-2 denitration of GTN.     

Dexamethasone inhibits the inducible bioconversion of glyceryl trinitrate to nitric oxide

The aim of this study was to assess the effects of dexamethasone (DEX) on the inducible bioconversion of glyceryl trinitrate (GTN) into nitric oxide in cultured smooth muscle cells, endothelial cells, and the J774 macrophage cell line as well as in vivo and ex vivo in rats either untreated or pretreated with Escherichia coli lipopolysaccharide. In vitro, an increased bioconversion of GTN to nitrite and an elevation of cyclosine guanosine 3,5;-monophosphate (cGMP) levels occurred after treatment with lipopolysaccharide (LPS) (0.5 microg/ml, 18 h). This effect was ablated by co-incubation with DEX (10 microM, 18 h). Rats treated with an intraperitoneal (IP) injection of LPS (4 mg/kg) 18 h beforehand showed enhanced hypotensive responses to GTN (1 mg/kg, intravenously [IV]) and this was prevented when DEX (4 mg/kg, IP) was given together with LPS. Progesterone (50 mg/kg, IP) had no effect on GTN-induced hypotensive response. Conversely, exposure of rat aortic strips obtained from animals pretreated with LPS produced an enhanced vasorelaxant response in LPS-treated rats. Also, this effect was inhibited by pretreatment with DEX. Thus, the induction of the pathway leading to the formation of nitric oxide from GTN is blocked by DEX both in vitro and in vivo, and this may represent a useful tool in the assessment of the enhanced bioconversion of organic nitrates into nitric oxide occurring via inflammatory mechanisms.     

Biotransformation of organic nitrate esters in vitro by human liver, kidney, intestine, and blood serum

The biotransformation of glycerol trinitrate (GTN), isosorbide dinitrate (ISD), pentaerythritol tetranitrate (PETN), erythritol tetranitrate (ETN), and mannitol hexanitrate (MHN) by extracts from human liver, small intestine mucosa, kidney, and blood serum was investigated. The glutathione-dependent organic nitrate ester reductase activity of the intestinal mucosa was 21, 4, 4, and 2 times higher than the liver activity for ISD, PETN, GTN, and ETN, respectively. The liver enzymatic activity for MHN was 35% higher than the intestinal activity and 56% higher than kidney enzyme activity. The order of increasing enzymatic rates was: ISD = PETN less than GTN less than ETN less than MHN in the intestinal mucosa; ISD less than PETN less than GTN less than ETN less than MHN in the liver; and ISD less than PETN = GTN less than ETN less than MHN in the kidney. Human serum also metabolized these organic nitrates at lower rates than the studied organs. Thus, the serum specific activities were 1/5 for MHN, 1/30 for ETN, 1/40 for GTN, 1/44 for ISD, and 1/2000 for PETN of the activity present in kidney. On the other hand, the activity of human albumin was lower than that of blood serum. The serum and albumin activities were not modified by reduced glutathione or sulfhydryl inhibitors. These results suggest that small intestine may play an important role in the biotransformation of these drugs at their absorption site, after oral administration. They also demonstrate the possible participation of various human tissues in the overall metabolism of organic nitrate esters.     

Bioactivation of pentaerythrityl tetranitrate by mitochondrial aldehyde dehydrogenase

Mitochondrial aldehyde dehydrogenase (ALDH2) contributes to vascular bioactivation of the antianginal drugs nitroglycerin (GTN) and pentaerythrityl tetranitrate (PETN), resulting in cGMP-mediated vasodilation. Although continuous treatment with GTN results in the loss of efficacy that is presumably caused by inactivation of ALDH2, PETN does not induce vascular tolerance. To clarify the mechanisms underlying the distinct pharmacological profiles of GTN and PETN, bioactivation of the nitrates was studied with aortas isolated from ALDH2-deficient and nitrate-tolerant mice, isolated mitochondria, and purified ALDH2. Pharmacological inhibition or gene deletion of ALDH2 attenuated vasodilation to both GTN and PETN to virtually the same degree as long-term treatment with GTN, whereas treatment with PETN did not cause tolerance. Purified ALDH2 catalyzed bioactivation of PETN, assayed as activation of soluble guanylate cyclase (sGC) and formation of nitric oxide (NO). The EC(50) value of PETN for sGC activation was 2.2 ± 0.5 μM. Denitration of PETN to pentaerythrityl trinitrate was catalyzed by ALDH2 with a specific activity of 9.6 ± 0.8 nmol · min(-1) · mg(-1) and a very low apparent affinity of 94.7 ± 7.4 μM. In contrast to GTN, PETN did not cause significant inactivation of ALDH2. Our data suggest that ALDH2 catalyzes bioconversion of PETN in two distinct reactions. Besides the major denitration pathway, which occurs only at high PETN concentrations, a minor high-affinity pathway may reflect vascular bioactivation of the nitrate yielding NO. The very low rate of ALDH2 inactivation, presumably as a result of low affinity of the denitration pathway, may at least partially explain why PETN does not induce vascular tolerance.     

Protective effect of DL-alpha-lipoic acid in cyclophosphamide induced oxidative injury in rat testis

The aim of the present study was to evaluate the protective effect of DL-alpha-lipoic acid on the biochemical changes, tissue peroxidative damage and abnormal antioxidant levels in the rat testis during cyclophosphamide (CP)-induced injury. Adult male Wistar rats were divided into four treatment groups: (I) control, (II) 15 mg/kg CP once a week for 10 weeks by gavage, (III) 35 mg/kg lipoic acid once a week for 10 weeks by intraperitoneal injection, and (IV) CP plus lipoic acid (24 h prior to CP administration). Testicular toxicity, assessed by decreased enzymatic activities of lactate dehydrogenase and glucose-6-phosphate dehydrogenase, was reversed with lipoic acid pretreatment. CP-exposed rats (group II) showed abnormal levels of enzymes (superoxide dismutase, catalase, glutathione peroxidase, glutathione-S-transferase and glutathione reductase) and antioxidants (reduced glutathione, ascorbate and alpha-tocopherol) along with high malondialdehyde levels. In contrast, rats pretreated with lipoic acid (group IV) showed normal lipid peroxidation and antioxidant defenses. These findings indicate a cytoprotective role of lipoic acid in this experimental model of testicular toxicity.