1. Introduction
Nitric oxide (NO) is a volatile, multifunctional free radical with a short life span. It is synthesized by one of three isoforms of nitric oxide synthase and exerts its effect by the activation of the soluble glunylate cyclase (sGC), which culminates in the production of cyclic guanosine monophosphate (cGMP) and the activation of the cGMP-dependent kinase (PKG) [
1,
2]. It has been described that a reduction in NO bioavailability is involved in the pathophysiology of many cardiovascular diseases (CVD). In this context, the use of drugs capable of releasing NO is an effective approach while dealing with CVD [
3].
Organic nitrates represent the oldest class of NO donors applied clinically. Among them, glyceryl trinitrate (GTN) is the main representative of the class, which also includes isosorbide dinitrate (ISDN), isosorbide 5-mononitrate (ISMN) and pentaerythritol tetranitrate (PETN) [
4]. Despite the benefits of thesemolecules in treating CVD like
angina pectoris, pulmonary hypertension and heart failure, their continued use may cause tolerance, which is, in fact, the main limitation to the use of organic nitrates [
5,
6]. Here we briefly discuss the past and the present perspectives in organic nitrates and highlight some preclinical tests performed by our group with a novel organic nitrate.
2. Historical Perspective: The Past
Glyceryl trinitrate, the first organic nitrate, was discovered in 1847 by Ascanio Sobrero. At that time, it was already described that headacheswere an unpleasant side effect of thissubstance [
7]. In 1879, the English physician William Murrel described, for the first time, the beneficial effects of the GTN against
angina pectoris [
8]. Since then, GTN was established as a treatment for the relief of chest pain, although the exact mechanism of action of this compound remained obscure for about 100 years [
9]. Murad and colleagues described, in 1977, that nitrates needed to release NO to present physiological effects [
10]. Nitric oxide was only described as an endothelium-derived relaxing factor in middle 80s [
11].
After to the introduction of GTN as a therapeutic agent for the treatment of angina pectoris, other nitro compounds with similar chemical properties have been developed. Most recent studies show that, in addition to angina, GTN and other organic nitrates such as ISDN and ISMN are able to improve left ventricular function in patients with congestive heart failure and pulmonary hypertension. Also, they show favorable effects on left ventricular remodeling after myocardial infarction and silent ischemia, in addition to reducing blood pressure alone or in combination with other drugs [
12,
13].
3. Mechanism of Action
Organicnitrates are potential NO donors in biological systems. In general, they do not exert their effect in cell-free systems and act as pro-drugs that need to be bioactivated by either enzymatic or non-enzymatic pathways to release NO. Enzymatic bioactivation of high-potency nitrates like GTN and PETN (tri- and tetranitrates, respectively) depends on the activity of cytosolic and/or mitochondrial aldehyde dehydrogenase (ALDH2), which converts them into nitrite and the denitrated metabolite. Non-enzymatic bioactivation of GTN involves its reaction with thiols from cysteine and cysteine derivatives or with ascorbate, which promote NO release. In contrast, low-potency nitrates such asISMN and ISDN (mono and dinitrates, respectively) undergo activation through a mechanism independent of ALDH2, which usually it occurs in endoplasmic reticulum via P450 enzymes [
13,
14,
15].
Nitric oxide binds to soluble guanylate cyclase (sGC), leading to an increase in the intracellular concentration of cGMP, which in turn activates a specific cGMP-dependent protein kinase (PKG). Once PKG is active, it promotes the phosphorylation of diverse substrates like myosin light chain kinase (MLCK), sarco/endoplasmic reticulum Ca
2+-ATPase (SERCA), plasma membrane Ca
2+-ATPase and Na
+/Ca
2+ exchanger. All these events result in vasorelaxation and reduction in peripheral vascular resistance [
16,
17,
18,
19]. Several authors have presented evidence that the vascular relaxation mediated by NO can occur also by a mechanism independent of cGMP, due to direct activation of K
+ channels [
20].
5. New Perspectives: The Future
Nitrate tolerance is characterized by a reduction in the vasodilator effect of NO donors and the requirement of higher doses. Experimental approaches revealed that organic nitrates such as GTN, ISMN and ISDN induce tolerance. This phenomenon is not yet completely understood and may have several causes such as impaired bioactivation of the drug, desensitization of sGC/cGMP pathway and increase in reactive oxygen species (ROS), which inactivate both endogenous NO and NO released from nitrovasodilators. Furthermore, the process of tolerance is associated with the appearance of unfavorable cardiovascular changes such as increase in sympathetic activity and endothelial dysfunction. Apparently, the only organic nitrate in clinical used which does not induce tolerance or endothelial dysfunction is PETN. In fact, the antioxidant properties of this drug may be responsible, at least in part, for this advantageous characteristic [
13,
32,
33,
34].
Considering that tolerance is the major limiting factor to clinical use of this class of drugs and that there is only one organic nitrate clinically available that does not induce tolerance, the search of new compounds unable to induce this undesirable effect has been increasing. Our group has recently evaluated new organic nitrates obtained from glycerin, which molecular structures can be observed in
Figure 1.
Figure 1.
Structural formula of the new organic nitrates obtained from glycerin [
35].
Figure 1.
Structural formula of the new organic nitrates obtained from glycerin [
35].
Our first goal was to evaluate if these molecules could induce vascular relaxation, the main effect of organic nitrates. For this purpose, we performed concentration-response curves of the new NO donors in resistance rings of superior mesenteric artery from rats. We observed that all four compounds were able to induce vasorelaxation in a dose-dependent manner both in the presence or absence of functional endothelium, as shown in
Table 1 and
Figure 2.
Table 1.
Maximum effect (ME) and sensibility (pD
2) values regarding the vasorelaxant effect of the new organic nitrates derived from glycerin in superior mesenteric artery isolated from rats precontracted with phenyleprine, in the presence or absence of functional endothelium (n = 6 for each group) [
35].
Table 1.
Maximum effect (ME) and sensibility (pD2) values regarding the vasorelaxant effect of the new organic nitrates derived from glycerin in superior mesenteric artery isolated from rats precontracted with phenyleprine, in the presence or absence of functional endothelium (n = 6 for each group) [35].
Compound | Intact Endothelium | Denuded Endothelium |
---|
(%) ME ± SEM | pD2 ± SEM | (%) ME ± SEM | pD2 ± SEM |
---|
NDMP | 88.5 ± 11.2 | 4.7 ± 0.13 | 93.8 ± 11.7 | 4.4 ± 0.07 |
NDEP | 94.1 ± 6.7 | 4.6 ± 0.08 | 108.8 ± 5.4 | 4.8 ± 0.06 |
NDPP | 96.4 ± 8.3 | 5.5 ± 0.10 | 111.1 ± 8.5 | 5.4 ± 0.08 |
NDBP | 89.5 ± 3.4 | 5.8 ± 0.10 | 105.4 ± 2.7 | 5.9 ± 0.06 |
Apparently, the vasodilator response tended to increase as the length of the organic chain added to glycerin increased. Because of that, we have focused our work on 1,3-dibutoxy-2-propyl nitrate (NDBP), which has a molecular formula of C
11H
23NO
5 and a molecular weight of 249.3.
Scheme 1 shows the steps of NDBP synthesis.
Figure 2.
Concentration-response curves to new organic nitrates synthetized by our group (10
−8–10
−4 M or 3 × 10
−4 M) in rat mesenteric artery rings (n = 6 per group). The vasorelaxant effect is expressed as a percentage of relaxation of phenylephrine-induced contraction [
35].
Figure 2.
Concentration-response curves to new organic nitrates synthetized by our group (10
−8–10
−4 M or 3 × 10
−4 M) in rat mesenteric artery rings (n = 6 per group). The vasorelaxant effect is expressed as a percentage of relaxation of phenylephrine-induced contraction [
35].
Scheme 1.
Synthesis of 1,3-dibutoxy-2-propyl nitrate [
36].
Scheme 1.
Synthesis of 1,3-dibutoxy-2-propyl nitrate [
36].
Using an
in vitro pharmacological approach based on simultaneously use of NDBP and blockage of diverse steps of NO/sGC/cGMP pathway, we identified that vasorelaxation induced by NDBP is dependent of NO release, activation of sGC, generation of cGMP and activation of potassium channels [
34], which is in accordance to what is expected from a NO-releasing drug. It was observed that NDBP is capable of increasing NO bioavailability in cultured vascular smooth muscle cells like other NO donors, such as GTN (
Figure 3).
Figure 3.
Nitric oxide generation by NDPB in vascular smooth muscle cells. * p < 0.05 versus basal fluorescence; # versus NDBP (10−6 M) and NDBP (3 × 10−5 M). Values are shown as mean ± S.E.M.
Figure 3.
Nitric oxide generation by NDPB in vascular smooth muscle cells. * p < 0.05 versus basal fluorescence; # versus NDBP (10−6 M) and NDBP (3 × 10−5 M). Values are shown as mean ± S.E.M.
Considering that NDBP could be an alternative option to existing organic nitrates, our second goal was to analyze the effects of NDPB
in vivo. Preclinical trials using five different doses of NDBP showed that the new compound presented hypotensive and bradicardic effects in a dose-dependent manner in normotensive and hypertensive non-anesthetized rats.These effects are of interest in a potential new NO donor, once the classical drugs of this class like GTN and sodium nitroprusside (SNP) can cause reflex tachycardia as a side effect. Parasympathetic blockage with atropine or vagotomy suggests that the reduction in blood pressure observed after NDPB administration depends not only to peripheral vasodilation but also to reduction in cardiac output due to increase in parasympathetic tone to the heart [
36]. Further studies revealed that cardiovascular responses evoked by NDBP
in vivo are dependent of NO release.When a NO-scavenger was used, both bradycardia and hypotension were attenuated [
37].
Tolerance is one of the most important undesirable effects evoked by organic nitrates and is responsible for limiting the clinical use of this class of drugs. Thus, our third goal was to evaluate the ability of NDBP to induce tolerance, as previously described [
35,
38]. Preclinical
in vitro approaches revealed that exposition of mesenteric artery rings to NDBP (10 µM or 100 µM) for 30 min prior to concentration-responses curves to this substance did not alter the vasorelaxant response, suggesting that NDBP did not induced tolerance [
35].
Studies by our research group showed that intravenous treatment with NDBP (5 mg/kg) for three days did not affect vascular reactivity of superior mesenteric artery isolated from normotensive rats to cumulative addition of acetylcholine or SNP (unpublished data), suggesting that continuous exposure to NDBP, at least in the experimental conditions used, does not cause endothelial dysfunction or desensibilization of vascular smooth muscle to NO. From this data we can infer that treatment with NDBP for three days does not induce tolerance in the vascular preparation used, although more studies are needed to elucidate this phenomenon. One possible mechanism is that NDBP, like PETN, presents antioxidant properties.
6. Conclusions
Since deficiency in NO/sGC/cGMP pathway is involved in many pathological features of cardiovascular system, the use of NO-releasing drugs can figure as an option to treat these conditions. Nitric oxide donors are widely applied in clinical practice despite their undesirable effects, like tolerance. Recently, our group has developed a new organic nitrate with potential to be used clinically in cardiovascular disorders. Preclinical studies confirmed NDBP ability to release NO, leading to vasodilation. Besides, NDBP appears to also act in central nervous system to increase parasympathetic drive to the heart, culminating in reduced blood pressure. This new organic nitrate does not seem to induce tolerance neither endothelial dysfunction in vitro, suggesting that this molecule can figure as a new potential therapeutic approach for cardiovascular disorders. Further studies in vivo are required to investigate the effect of continuous treatment with NDBP on vascular oxidative stress in normotensive and hypertensive conditions and the ability of the compound to prevent pseudotolerance, other common effect of organic nitrates.