New Organic Nitrite Therapy for Acute Experimental Pulmonary Embolism Pulmonary Hypertension

Hari Prasad Sonwani^* and Aakanksha Sinha
^*Correspondence: Hari Prasad Sonwani, Department of Pharmacy, Apollo College of Pharmacy, Anjora, Durg, C.G, India, Email:

Keywords

Alkyl nitrites • Chromatography • High pressure liquid • Hypertension • Inorganic nitrite

Abbreviations

CVP: Central Venous Pressure; ETCO₂: End-Tidal Carbon Dioxide; FENO: The Fraction of Nitric Oxide in Exhaled Gas; HR: Heart Rate; LAP: Mean Left Atrial Pressure; MAP: Mean Systemic Arterial Blood Pressure; mPAP: mean Pulmonary Arterial Pressure; MPE: Homogenized Skeletal Muscle Tissue Used for Induction of Pulmonary Embolism; NO: Nitric Oxide; PaCO₂: Arterial Partial Pressure of Carbon Dioxide; PaO₂: Arterial Partial Pressure of Oxygen; PE: Pulmonary Embolism; PVR: Pulmonary Vascular Resistance; RPP: Cardiac Rate-Pressure Product; Saline+NO: Saline Saturated with No Gas; SVR: Systemic Vascular Resistance

Introduction

When pulmonary vascular macro or micro-obstruction occurs together with active vasoconstriction brought on by released vasoconstrictive mediators and Nitric Oxide (NO) scavenging by cellfree hemoglobin, acute Pulmonary Embolism (PE) induces acute pulmonary hypertension [1,2]. Furthermore, acute PE significantly disrupts ventilation-perfusion matching, which results in hypoxemia [3]. One possible therapeutic option for acute PE is to use mediator antagonists or vasodilators to counteract the pulmonary hypertension. Since there are several vasoconstrictive mediators at play, mediator antagonist therapy is probably only going to be somewhat effective. NO acts as a pulmonary circulation vasodilator [4]. And discovered that in two types of experimental PE, endogenous NO generation provided protection, and that the fraction of NO in exhaled gas (FENO) rose in acute PE [5,6]. Sodium nitroprusside and nitroglycerin are the NO donors. have demonstrated some positive benefits in animal models of PE; nevertheless, the systemic circulation's vasodilation was more noticeable, and nitroglycerin may potentially impede gas exchange, making these medications less useful in cases of acute PE [7,8]. Additionally, in animal models of acute PE, sildenafil, inorganic nitrite, and a nitric oxide-releasing aspirin have demonstrated positive effects [9-13]. In acute PE, inorganic nitrite exhibited antioxidant properties and reduced the rise in matrix metalloproteinase. In cases of acute PE, L-arginine and the NO donor diethylenetriamine/nonoate were ineffective [14]. The results of inhaled NO in PE are inconclusive; there is a slight improvement in hemodynamics but little to no improvement in blood gases [15]. Nevertheless, a recent phase I single-center research found that inhaled NO was safe in submassive PE and recommended a clinical therapy regimen [16]. Unlike organic nitrates, which showed little to no tolerance, NO donors of the organic nitrite type are converted to NO in vivo [17-20]. Although ethyl nitrite has been proposed as an inhalation medication, it may cause a considerable amount of methemoglobin to develop. We recently synthesized novel organic nitrites and discovered that the relative selectivity of each organic nitrite molecule for vasodilation in the pulmonary and systemic circulations was dependent on its polarity. eighteen It is particularly interesting to investigate the two novel organic mononitrates, 2-hydroxy propyl nitrite and 2-hydroxy-1-methylethyl nitrite (henceforth referred to as PDNO, for molecular structure), in experimental acute PE because, when compared to nitroglycerin, for example, this composition exhibited increased vasodilator selectivity toward the pulmonary circulation. Furthermore, the vasodilatory effect showed no signs of cross-tolerance with nitroglycerin in the pulmonary and systemic circulations, suggesting bioactivation through an alternative route for PDNO. Our hypothesis was that intravenous PDNO may counteract pulmonary hypertension in acute experimental PE without causing methemoglobinemia, systemic adverse effects, or disruptions in gas exchange. We first conducted in vivo dose-response experiments with measurements of FENO, pulmonary and systemic hemodynamics, methemoglobin, and plasma nitrite in both naive animals and animals with pharmacologically induced pulmonary hypertension in order to separate the effects of the PDNO solution from inorganic nitrite and NO gas. We evaluated the effects of intravenous and left cardiac catheterization in order to examine if rapid elimination of PDNO from the circulation is one mechanism for the comparatively greater effects in the pulmonary compared with the systemic circulation ventricle injections of PDNO. We demonstrate that the NO donor PDNO is a short-lived efficient vasodilator in vivo, in contrast to inorganic nitrite, and that it effectively counteracts the pulmonary hypertension of acute PE without causing obvious adverse effects.

Materials and Methods

Systems under test

Animals: Since this species of rabbit has been employed in the past for studies on FENO and the pulmonary circulation, male veterinary science and animal husbandry white rabbits (body weight 2.5 3.5 kg, n=37) were used in this study. The study was carried out in compliance with Directive 2010/63/EU on the protection of animals used for scientific reasons, and the methods were as humane as feasible follows the ARRIVE reporting requirements for animal experiments. The rabbits were kept as singles in plastic cages at room temperature, with unrestricted access to water and lab-quality rabbit chow. They were maintained in a 12-hour day/night cycle. The regional committee for animal ethics (Stockholms Norra djurforsoksetiska nämnd, Stockholm, Sweden; registration numbers N400/03, N148/08, and N178/11) accepted the trials. Every study that uses animals is published in accordance with both anesthesia and surgical methods The rabbits were instrumented in accordance with Nilsson et al., given pentobarbital anesthesia, and ventilated with charcoal-filtered air (fraction of inspired oxygen, or FiO₂). To provide intravenous drug infusions and measure Central Venous Pressure (CVP) using a pressure transducer (PX600P, Edwards Lifesciences LLC, Irvine, CA, USA), a catheter was inserted via the right jugular vein to the right atrium's level. Following the tracheal cannula, an anesthetic monitor (AS/3, Datex, Helsinki, Finland) used a Pedi-lite +flow sensor and gas sampler (Datex) to detect respiratory gases, airway pressure, and tidal volume. Chemiluminescence was used to assess FENO in a mixed exhaled gas. The AS/3 used a pressure transducer (PX600P) to measure the Heart Rate (HR) and mean systemic arterial blood pressure (MAP) linked to a left common carotid artery catheter. For intracardiac medication infusions, a left carotid catheter was inserted into the left ventricle of certain animals (position verified by pressure monitoring). These animals had arterial blood drawn via a catheter inserted into the right femoral artery, and their MAP and HR were assessed. In the initial and subsequent sets of experiments with dosage responses. At this stage, the animals were given a 30 to 60 minute time without any interventions because the preparation was finished. The third set of dose-response experiments involved pharmacologically induced pulmonary hypertension, and the animals in the acute PE experiments were further instrumented, with mean Pulmonary Arterial Pressure (mPAP), mean Left Atrial Pressure (LAP), and cardiac output measured in accordance with Nilsson et al. The skeletal muscle of the right anterior tibia was produced as material for muscle tissue pulmonary embolization (MPE). The animals were given 30 to 60 minutes of intervention-free time following the procedure. Through an ear vein (STCâ?521 syringe pump, Terumo Corp., Tokyo, Japan), all animals received a continuous infusion of glucose (25.9 gL^-1), dextran 70 (Macrodex^®, 28.2 gL^-1), and NaHCO₃ (6.6 gL^-1). Within In order to conduct dose-response studies, sodium pentobarbital was added to the infusate (4.2 gL^-1), and the animals were given the infusion at a rate of 5 mL kg^-1 h^-1. To account for the increased fluid loss in the pulmonary hypertension and embolism studies, sodium pentobarbital (2.1 gL⁻¹) was added to the infusate, and the infusion rate was 10 mL kg^-1 h^-1. A computerized data acquisition system (MP150; BIOPAC Inc., Goleta, CA, USA) was used to gather respiratory and hemodynamic characteristics, including FENO. Periodically, carotid and mixed venous blood samples were obtained, and the following parameters were measured and analyzed: Plasma nitrite, methemoglobin and total hemoglobin (ABL 520, Radiometer A/S, Copenhagen, Denmark), blood gases, and acid–base status. The animals were put to death via pulmonary air embolization following the experiments under general anesthesia.

Innovative procedures

For practical reasons, the operator (author KFN) was not blinded to the interventions.

The exploratory dose-response trial protocol Rabbits were given the following intravenously in the first set of experiments:

• The PDNO solution (1.7 mmolL⁻¹ of dissolved NO gas and a total of 22 mmolL⁻¹ NO/nitrite).
• The PD+nitrite placebo solution (1,2-propanediol dissolved in saline, 25% v/v, supplemented with 20 mmolL⁻¹ inorganic nitrite).
• Saline+NO, which was isotonic saline treated with NO gas in the same manner as the PDNO solution (1.7 mmolL⁻¹ of dissolved NO gas and a total of 22 mmolL⁻¹ NO/nitrite).

The amount of inorganic nitrite present in a solution In order to get a comparable total NO/nitrite level in the control solution and the PDNO solution, 2 was used. The PDNO solution was administered at 25, 50, 100, 200, 400, 800, 1200, and 1600 nmolkg^-1 min^-1 for the delivery of organic mononitrites. The infusion rates of Saline+NO and PD+nitrite were reported and compared with the appropriate PDNO dose, and they were the same as with the PDNO solution. After being given multiple solutions, the animals were given a period without any interventions to establish stable baseline values in between the infusions. At least four independent animals received each dose. Since the studies in this set were exploratory, randomization was not used. In a further series of tests, four rabbits (n=4) were given the PDNO solution intravenously and through a catheter inserted into their left ventricle at concentrations of 100, 200, 400, 800, and 1600 nmolkg^-1 min^-1. The animals were given an intervention-free interval in between the infusions to establish stable baseline readings, and the order of the infusions-intravenous or left cardiac ventricle-was determined at random. To calculate the transit time to the pulmonary circulation, a 2.5 mL saturated sodium bicarbonate solution was bolus infused at the appropriate infusion site. In a third series of studies, a continuous intravenous infusion of U46619 (200, 1200 ng kg^-1 min^-1) was used to raise the Pulmonary Vascular Resistance (PVR) by 100%, 200%, therefore inducing pulmonary hypertension. Following that, one set of mice (n=4) got intravenous PDNO solution in increasing doses (200, 400, 800, and 1200 nmolkg^-1 min^-1) and three other animals (n=3) were given comparable doses and one higher dose of PD+nitrite placebo solution. A continuous saline carrier flow (864 Syringe Pump, Univentor LTD, Zejtun, Malta) was linked to a syringe pump (CMA/100, Carnegie Medicin AB, Stockholm, Sweden) for the purpose of making all infusions. Every dosage took 10 minutes to provide, and at the conclusion of each dosage, an arterial blood sample was drawn for plasma nitrite measurement and blood gas analysis. The effect of the infusion was assessed. In rabbits used for measuring the half-life of plasma nitrite, arterial blood samples were taken at 10, 20, 40, and 80 minutes after PDNO or PD+nitrite infusion.

Procedure for experiments: On pulmonary emboli An infusion of MPE (30 mg homogenized muscle kg^-1) was given to three groups of animals manually through a three-way stopcock into a saline carrier flow (864 Syringe Pump) at a rate of 150 μL kg^-1 min^-1 that entered the central venous catheter. The MPE dosage was selected to provide a notable, yet non-fatal, embolic challenge. After 20 minutes of MPE, the three groups (control, n=3) received saline, one group (PDNO, n=6) received 200 nmol kg^-1 min^-1 of PDNO, and one group (PD, n=6) received the same amount of 1,2-propanediol intravenously for 40 minutes in a random fashion. The PDNO dosage was the lowest that maximally reduced pulmonary arterial pressure following MPE as established by pilot studies. The control group that received MPE but saline intravenously responded similarly to the placebo group; data from this control group are not displayed. Samples of mixed exhaled gases were obtained at the exhalate mixing chamber's output concurrently with the collection of blood gases, and they were examined in the ABL520 increased cardiac output compared to PD +nitrite (n=3), which had little or no effects at corresponding doses.

Results

Investigative dose-response tests of nitrites in rabbits under anesthesia, as shown in Figure 1.

Impacts on plasma nitrite levels and the connection between biological impacts and plasma nitrite levels plasma nitrite concentrations were dose-dependently raised by PDNO (n=3) and PD +nitrite (n=3). On the other hand, at the higher doses, PD+nitrite considerably increased the plasma nitrite content more than PDNO did. In vivo, plasma nitrite had a half-life of 43 ± 1 minutes. When these studies were combined, they demonstrated that whereas PDNO and PD+nitrite both raised plasma nitrite concentrations, their biological consequences differed greatly. Plotting the effects of PDNO and PD+nitrite on PVR and SVR against the plasma nitrite content they induced made this clear. Comparable distinctions between plasma nitrite and its impacts on HR, MAP, and FENO (Figures 2 and 3).

Contrasting intravenous and left ventricular infusions

FENO and MAP (84 ± 1 mmHg vs. 86 ± 4 mmHg) were comparable prior to each pair of infusions. While PDNO infusions into the left ventricle (n=4) only marginally raised FENO and decreased MAP dose-dependently, PDNO intravenously (n=4) increased FENO and decreased MAP dose-dependently. When compared to left heart ventricle infusions, a noticeably greater increase in FENO was obtained at the higher intravenous dosages of PDNO. The left ventricular ventricle infusion reduced MAP more than the intravenous infusion did at PDNO 400 nmol kg^-1 min^-1. The estimated transit times to the pulmonary circulation from the left ventricle and intravenous infusion sites were roughly 1.5–2 seconds and 8 seconds, respectively, indicating a connection between the effects and travel time to the target locations (Figure 4).

Experiments on pulmonary embolism

The animals' condition prior to an acute pulmonary embolism Prior to MPE, all of the groups' assessed variables (n=6 in each group) were comparable. FENO was 10 ± 1 ppb and 12 ± 1 ppb; ETCO₂ was 4.4 ± 0.1% and 4.4 ± 0.1%; HR was 254 ± 12 beats min^-1 and 254 ± 4 beats min^-1; PVR was 36 ± 4 mm Hg min L^-1 and 32 ± 3 mm Hg min L^-1; and right ventricle RPP was 4000 ± 400 mm Hg beats min^-1 and 4300 ± 200 mm Hg beats min^-1, respectively, and the arterial pH in the PD and PDNO groups was 7.50 ± 0.03 and 7.52 ± 0.02, respectively (Figure 5).

Acute pulmonary embolism's effects

Following MPE infusion, there was a slight decrease in end-tidal carbon dioxide (to 4.0 ± 0.2% and 4.1 ± 0.1% in the PD and PDNO groups, respectively), an increase in mPAP, a 50% increase in PVR, an increase in right ventricle RPP, and a decrease in MAP. Additionally, there was an increase in FENO, which reached statistical significance in the PD group. Arterial pH marginally fell (to 7.45 ± 0.04 in the PD and PDNO groups, respectively), and PaO₂ decreased indicate that the MPE infusion had no effect on methemoglobin levels. Admixture, physiological dead space, and arterial partial pressure of carbon dioxide (PaCO₂) all rose. Heart output, LAP, HR, and drug infusion's effects on acute pulmonary embolism. Practically all of the negative hemodynamic consequences of acute PE were offset by PDNO. In comparison to PD PDNO enhanced FENO, lowered and normalized PVR, and right ventricular RPP. Additionally, PDNO reduced mPAP relative to pre-drug infusion values (P<0.05). After 40 minutes of drug infusion, the difference in mean arterial pressure (mPAP) between the PDNO and PD groups did not approach statistical significance (P=0.26). This could be attributed to the PD group's larger scatter, which prevented them from experiencing the same drop in mPAP as the PDNO group. When the drug was stopped abruptly, the effects of PDNO on mPAP, PVR, and right ventricular RPP persisted during infusion. without any rebound impact during nfusion (ventricular RPP, PVR, and mPAP were unaffected by PD and remained high over the monitoring period When PDNO was infused, MAP fell from baseline (however it did not drop as much as when PD was infused (5 mmHg difference, P=0.54 after 40 minutes of drug infusion). When the PDNO infusion was stopped, the decline in MAP was rapidly reversed without experiencing a rebound effect LAP, cardiac output, HR, blood-gas measurements (with the exception of PaCO₂ in the PD group), physiological dead space, and venous admixture were not affected by PDNO or PDAt the conclusion of PDNO, methemoglobin increased very slightly (by around 0.3%) infusion in contrast to baseline and PD. All of the animals had methemoglobin levels less than 1% during the study period, and once the PDNO infusion was stopped, the animals' levels began to decline toward normal levels (Figure 6).

Discussion

Without any serious side effects, this study demonstrated the pulmonary vasodilatory effects of the novel organic nitrite NO donor PDNO in pulmonary hypertension of acute PE. It was recently demonstrated that infusing sheep with PDNO for longer than six hours did not significantly increase the proportion of methemoglobin. The level of methemoglobin was less than 1%, which is significantly lower than the 2.5% threshold that has been deemed safe for inhaled NO. The PDNO group's mean systemic arterial blood pressure was roughly 5 mm Hg lower than the PD placebo group's (not significantly). Acute systemic hypotension PE would be harmful, leading to a reduction in the myocardium's perfusion, and PDNO appears to offer promising qualities in this regard as well. The rapid reversal of PDNO effects upon cessation of infusion demonstrated the remarkable controllability of this NO donor. When supplied intravenously, the PDNO presumably disappeared from the circulation much faster in the pulmonary than in the systemic circulations, as demonstrated by the comparison of intravenous and left heart ventricle infusions. When a NO donor is injected intravenously, its short half-life in the blood restricts its vasodilatory effects to the pulmonary circulation. The first doseresponse studies involving PDNO and inorganic nitrite demonstrated the effectiveness of PDNO as a vasodilator in the systemic and pulmonary circulations. And that the rise in FENO indicates that PDNO released NO. On the other hand, at the levels employed in this investigation, inorganic nitrite acted as a weak systemic vasodilator and had no effect on the pulmonary hypertension caused by a thromboxane A2 analogue (U46619). The concentration of plasma nitrite was raised by intravenous PDNO and inorganic nitrite infusions. It is possible to determine that PDNO's vasodilatory effects were mostly due to a mechanism other than increases in plasma inorganic nitrite by comparing the vasodilator characteristics of PDNO and inorganic nitrite with the plasma nitrite levels brought on by the corresponding infusion. Additionally, the brief half-life of PDNO's vasodilation (a few minutes) and the significant difference between intravenous and left heart ventricle infusions show that the fact that the active species was not inorganic nitrite was reinforced by PDNO from the circulation. On the other hand, the predicted half-life of inorganic nitrite is 43 minutes, falling within the same range as previously found in humans and rabbits. Most likely, the lung in the acute PE studies and the dose-response studies, PDNO caused vasodilation through the release of NO or a related active species. This study's findings regarding the limited vasodilatory efficiency of inorganic nitrite in the pulmonary circulation are consistent with research on newborn lambs and rats. Specifically, in rats with pulmonary hypertension, the traditional NO donor sodium nitroprusside was more than 100 times more powerful than inorganic nitrite. Large bolus injections of intravenous inorganic nitrite (10â?Â¥ 100 μmol kg^-1 and 145 μmol kg^-1) inhibited the pulmonary hypertension in rats caused by a thromboxane A2 analogue and the hypoxic pulmonary vasoconstriction in newborn lambs. Nebulized inorganic nitrite administered at levels of 4.3 μmol kg^-1 min^-1 produced lung vasodilatory effects in newborn lambs. pulmonary hypertension models. In these investigations, nebulized inorganic nitrite also marginally elevated FENO, which may have resulted from the ventilator circuit rather than the animal, whereas intravenous inorganic nitrite infusions had no effect on FENO. On the other hand, dogs appear to be more susceptible to intravenous inorganic nitrite, as evidenced by the fact that half of the PVR rise in acute PE was reduced when inorganic nitrite was administered intravenously at 450 nmol kg^-1 min^-1 for 15 minutes and then 280 nmol kg^-1 min^-1 for 105 minutes. Larger doses of inorganic nitrite were required for other species, such as humans, rabbits, sheep, and rats, but dogs appear to be more susceptible to the acute systemic blood pressure-lowering effects of the drug. Crucially, the amounts of inorganic nitrite needed to sharply reduce methemoglobin levels rose in response to increases in systemic and pulmonary blood pressure. The formation of methemoglobin in red blood cells can be easily explained by the interaction of inorganic nitrite with hemoglobin. However, a significant portion of the NO produced by inorganic nitrite in hypoxia originates from other tissues, which may account for the possible protective effects of inorganic nitrite in ischemia-reperfusion injuries. NO may help with acute PE in a number of ways. Initially, NO acts in the pulmonary circulation as a vasodilator, so 1 g kg^-1 day^-1 of 1,2- propanediol is regarded as safe when used as an addition in pharmaceutical intravenous preparations of lipophilic medications (such as lorazepam and diazepam). Treatment over several hours was possible because 1,2â?propanediol was delivered at a rate of about 320 mg kg^-1 h^-1 in the PE trials. Since the anticipated doselimiting toxicity of long-term intravenous inorganic nitrite infusion in humans was 108 nmol kg^-1 min^-1, it is also crucial to take the entire load of organic and inorganic nitrites into account (Figure 7).

Conclusion

Future PDNO research should focus on raising PDNO concentrations while lowering inorganic nitrite content in the solution. This will reduce the amount of 1,2-propanediol available and the overall nitrite load, respectively. Prospective applications of organic nitrites, such as PDNO, in acute In addition to the existing standard and non-traditional treatments, such as heparin, thrombolysis, embolectomy, or extracorporeal life support, PE may be used as a lifesaving measure to relieve the right heart's workload through pulmonary vasodilation. Since more than a century ago, organic nitrite donors known as NO donors have been utilized to treat anginal pain; but, to the best of the authors' knowledge, their effects have never been studied in PE. We conclude that in experimental acute PE, the new organic nitrite compounds had positive effects, i.e., decreased right ventricle pressure product and decreased pulmonary vascular resistance. There was no deleterious methemoglobin production, systemic hypotension, disrupted gas exchange, or development of tolerance with the organic nitrites that were administered. Thus, shorthalf- life organic nitrites appear as a new tentative living form preserving care for acute PE. Organic nitrite has a higher potency than inorganic nitrite to produce hemodynamic effects, while inorganic nitrite's hemodynamic effects in rabbits were linked to the development of methemoglobin. This study demonstrates the critical need to watch methemoglobin levels when utilizing both organic and inorganic nitrites, as there could be a dangerous side effect. Future research should examine the novel organic nitrite's effects in more pertinent clinical diseases linked to pulmonary hypertension.

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