Although there are well-established therapeutic advantages with use of iNO in infants, there are some mixed results on how far these applications reach. Some reports say it does not reduce mortality, length of hospitalization, or accompanying neurological complications [31]. In a study involving older pre-term infants (7-21 days) post birth, when they received 20ppm of iNO for 24 days, there was a 23% increase in survival rates [41]. A 1-year post treatment follow-up showed patients were less likely to require supplemental oxygen, bronchodilators, inhaled/systematic steroids, or diuretics [42]. There still requires further investigations into the short- and long-term advantages of iNO therapy. There appears to be a crucial time window after birth that treatment needs to be initiated, as well as proper dosage and length of application.
Acute Respiratory Distress Syndrome
Acute respiratory distress syndrome (ARDS) results in patients that experience a period of hypoxia, that is not resolved via administration of O2 [43]. The resulting consequences are acute lung inflammation with infiltration of activated lymphocytes, increased permeability of alveolar-capillary membrane, and resulting surfactant abnormalities [43]. A meta-analysis of 12 trials including 1237 patients found that there were no significant improvement for patients who receiving iNO therapy that experienced acute lung injury, or ARDS [44]. Oxygenation improved in the first 24 hours by approximately 13% measured via PaO2/FiO2 ratio, and some data suggests that these results last up to 96 hours. There was no observed improvement for mortality in ARDS patients. Observed renal dysfunction remains a concern due to NO modulating glomerular function and renal vascular tone, and alterations in mitochondrial and enzymatic function being a potential contributor [44, 45]. The use of iNO in patients with ARDS is therefore not recommended as a treatment option.
Bacterial Applications
The bactericidal nature of NO has been exhibited endogenously in bacterial pulmonary
infections and suggested to be an important pathological mediator [46, 47]. In a case study of a patient with severe pneumococcal pneumonia, administration of 15-40ppm of iNO resulted in improved gas exchange, reduced pulmonary vasoconstriction and rapid and complete disappearance of bilateral pulmonary infiltration [48]. Although this was an isolated incident, the application of iNO contributed to the resolution of infection and recovered pulmonary function.
Production of NO by iNOS within the lungs is thought to be responsible for host response against mycobacteria, and supplementation via iNO could contribute to host defense against the pathogen M. tuberculosis [49]. Long et al. [46] investigated if it was possible to accelerate airway disinfection in patients with positive sputum smears that were being treatment for M. tuberculosis by co-administering iNO. Therapies of 80ppm were given for 72 h and deemed safe, however increased airway disinfection was not exhibited. The results from this study were preliminary and further testing should be considered due to the modeled link between NO and M. tuberculosis pathology. Impaired pulmonary NO bioavailability has been associated with increased severity of disease and delayed clearance [50], therefore alterations in dosing and duration of treatment may still have therapeutic potential for patients with M. tuberculosis.
Cystic fibrosis (CF) is one of the most common lethal hereditary diseases, where mortality often occurs due to persistence of P. aeruginosa infections within the lungs [51]. P. aeruginosa’s capability to form a biofilm leads to persistent infections in patients with CF, where proper immune clearance is lacking [52]. Endogenous NO induces P. aeruginosa biofilm dispersal by increasing bacteria phosphodiesterase activity and decreasing c-di-GMP levels, making treatment with antibiotics more achievable [53, 54]. In a randomized clinical trial, low doses of 10ppm of iNO were administered to CF patients (n=12) via nasal cannula for 8 h ON for 5-7 days in combination with I.V. antibiotic therapy in an attempt to reduce P. aeroginosa biofilm [52]. In an ex vivo proof of concept model, fluoresce in situ hydrbization (FISH) of sputum samples from 5 patients modeled significant reduction in mean biofilm thickness (p = 0.02). When clinical isolates from the biofilms were treated with solely antibiotics, tobramycin or tobramycin/ceftazidime, biofilm biomasses increased 243% and 155%, respectively. Combinational application of <500nM iNO and antibiotics resulted in significant killing of biofilm mass and viable planktonic cells, resulting in almost visable no bacterial presence. After iNO treatments in patients of 10ppm, significant reduction in P. aeruginosa biofilm aggregates were observed after 7 days of treatment compared to placebos. Collectively, this study models that iNO would be an advantageous application in patients with biofilm bacterial infections to increase antibiotic efficiency, especially when physiological host defenses are lacking.
Reperfusion Injury
The pulmonary applications of iNO are generally straightforward, as the active molecule is directly administered within the lungs and subsequent vasodilation can aid to increase oxygen acquisition. Oxygen deficit during diseases with hypoxic episodes can result in detrimental effects. In patients who experiences cardiac arrest, 60% exhibited moderate to severe cognitive defects 3 months post-resuscitation [55]. Hypoxic conditions can occur in diseases including hypertension, atherosclerosis, peripheral artery diseases, myocardial infarction, and cardiac arrest [56]. Following a period of ischemia, additional complications occur where the recirculation of blood results in reperfusion injury. The reintroduction of blood to starved cells results in generation of ROS, calcium overload, proteolysis of myofibrils, pH imbalances, and inflammation [57]. These events contribute to neutrophil recruitment increasing cellular adhesion molecule (CAM) expression on endothelial cells that can result in capillary plugging and edema [58, 59]. Ultimately, these events can lead to increased morbidity in patients; however administration of iNO before, during, and/or after procedures can reduce these risks.
Supplementation of iNO has shown to reduce this reperfusion injury damage during surgical procedures requiring restricted blood flow. Mathru et al. [60] tested if this application could reduce inflammation associated with reperfusion in patients undergoing elective knee surgery requiring a tourniquet. Patients were administered 80 ppm of iNO upon intubation, and for the duration of the surgery. A pilot study of 20 ppm, and 40 ppm (n = 3) were not significant, therefore a higher concentration of 80ppm iNO was chosen. Classical inflammatory markers were up-regulated in the reperfusion control patients including NF-κB measured in quadriceps muscle biopsies, and CD11B/CD18 and soluble P-selectin in drawn blood. Nearly all effects were attenuate to baseline levels when iNO was administered. This study was limited due to a small sample size in the treatment group (n = 9); however its effects are clinically relevant. The ability to reduce inflammation and injury when extremities are being depleted of blood is significant to improve successful surgical outcomes. This application can expand for potential use during surgeries with tissue transplantations, and reconnections.
The above study was reproduced by Hållström et al. [61] in patients (n = 45) requiring knee arthroplasty, under spinal anesthesia. This involved 3 groups (n =15) where a placebo, partial NO (piNO), and iNO at 80 ppm where administered during surgery. There were no significant findings and no inflammatory response in the treated vs. untreated groups. The differences between both studies could be attributed to the choice of anesthesia. Mathru et al. [60] used general anesthesia, with induction via thiopental and fentanyl, while Hållström et al. [61] used EudraCTnr as choice of anesthetic method. There is evidence that different anesthetics can influence inflammation and myocardial injury in patients depending on compound of choice and surgery being performed [62, 63].
Cardiac Diseases
Liu et al. [64] used a porcine model to show that iNO improved microvascular flow, and decreased infarction size after myocardial ischemia and reperfusion. The mid-portion of the left anterior descending coronary artery was occluded to model ischemia for 50 min, followed by 4 h of reperfusion. Ten minutes prior to reperfusion, 80ppm of iNO was administered for the duration of the 4 h reperfusion. Infarct size measured in the left ventricle was lower in iNO pigs (31% iNO treated vs. 58% in control), with less creatine phosphokinase-MB release. Endocardial and epicardial blood flow measured by microsphere migration was improved. There was decreased leukocyte infiltration and cardiomyocyte apoptosis indicating less inflammation and cellular damage. Collectively, these findings show that iNO is protective from many angles and reduces injury from reperfusion.
Even brief periods of 80 ppm iNO for <15min have shown to increase NO metabolites in tissue lead to reduction in cardiac ishchemia reperfusion injury [65]. In mice the uptake of iNO models a linear relationship with time, and is able to be measure via NO metabolites in the tissue. From the total iNO absorbed, 53% was recovered via RSNO, RNNO, NO-heme, nitrite, and nitrate; however many other metabolites may have not been detected. Breathing of 80 ppm iNO 5 or 60 min prior to reperfusion decreased the area at risk by 31% and 32% respectively. In a similar study, breathing of 40 ppm and 80 ppm, beginning 20 minutes prior to reperfusion and 24 h following significantly decreased myocardial infarction size and increased left ventricular function [66]. In the iNO at 80 ppm group, myocardial infarction size:area at risk ratio was reduced by 53%, 51%, and 45% after 30, 60, and 120 min of ischemia, respectively [66].
Cardiopulmonary Bypass Surgery
During cardiopulmonary bypass surgery (CPB) with periods of ischemia, the resulting inflammatory response can lead to postoperative complications. Reperfusion injuries can results in reduced reflow due to leukocyte recruitment and CAM increase, myocardial necrosis and apoptosis, and reduction in myocardial contractibility [67]. Gianetti et al. [59] tested if applications of 20 ppm during and 8 h post CPB operation could mitigate complications. Measurements of myocardial injury and left ventricular dysfunction post surgery were used to quantify effectiveness [59]. There was a significant decrease in observed myocardial injury 24 h post surgery, as measure by creatine kinase (p = 0.04), creatine kinase-muscle/brain (p = 0.03), and troponin I (p = 0.04). Endothelial activation measured via P-selectin in blood samples was also decreased. Although specific dosage has yet to be determined for iNO, supplementation during CPB could be advantageous for patient populations susceptible to complications including inflammation.
A case report of a 39-year-old female that presented with acute right heart syndrome secondary to chronic pulmonary artery hypertension received 5 ppm and 10 ppm of iNO as a rescue treatment and responded positively [68]. After treatment she had a decrease in pulmonary artery systolic pressure and was able to be weaned off ventilation. The selected case shows how iNO can be used as a tool to rescue patients from hypo-perfused state when an immediate therapy is required.
Transplantation Surgery
Liver Transplant
Following liver transplantation the subsequent reperfusion leads to cellular and systemic injury, with patients ultimately having decreased function of the newly acquired graft. Cells responsible for injury include polymorphonuclear cells, T cells, Kupffer cells, endothelial cells, and ROS/RNS species [69-71]. Decreased hepatic NO production has been modeled in hepatic cells undergoing transplantation [72]. To test if iNO supplementation could reduce damage with liver transplant, patients undergoing orthotopic liver transplantation surgery were administered 80 ppm of iNO for the duration of surgery [73]. Biopsies and liver function were measured to observe beneficial effects [73]. Length of hospital stay for patients receiving iNO was reduced by 10%. Liver function recovery increased at a faster rate measured by Serum AST and ALT, and hepatocyte apoptosis was decreased 75%. Additionally, the volume of platelets required during surgery between iNO treated and placebo were significantly decreased by approximately 50%, likely due to NO’s attribute of preventing platelet activation. There were no negative effects observed, such as changes is cardiopulmonary performance or toxicity from administration of iNO.
Heart Transplant
Patients receiving heart transplants are at risk for serious adverse effects, especially if they have a predisposition to disease. These complication surgeries involve occluding blood sources, altering oxygen availability to surrounding tissue. Upon receiving a heart transplant, the resulting pulmonary hypertension from chronic heart failure can cause ventricular dysfunction. Serious risks are increased in patients receiving heart transplants exhibiting dysfunction including increased morbidity and mortality [74]. Patients (n = 16) modeling low PAP undergoing heart transplantation were chosen to receive 20 ppm of iNO during the post-operative period to see if PVR and right ventricle work could be improved [75]. Patients receiving iNO treatment had a 30-day survival of 100%, compared to 81% survival of historically treated transplants. Six patients in the control group developed right ventricle dysfunction following the surgery, while there was only one in the iNO treated group (p < 0.05). There was also a decrease in PVR for the iNO treated group. Collectively, these findings suggest iNO is beneficial for patients in postoperative phase of heart transplant that have pulmonary hypertension.
Brain
The effects that occur during ischemia are not limited to the localized tissue or heart, but occur most importantly in the brain. The current therapeutic application used to restore blood flow in patients who have experience ischemia is recombinant tissue plasminogen activator (rtPA). The window of administration is within 3 – 4.5 hours after ischemia, however when administered to patients with hemorrhagic stroke presenting as ischemic stroke, it can be fatal [76]. In a cerebral ischemic mouse model, and later confirmed ischemic stroke sheep model, the administration of iNO at 5-50ppm showed significant effect in preventing tissue death in the brain [77]. Histological scores 72 h post hypoxic ischemia were significantly better in animals treated with iNO in the cortex, hippocampus, striatum and thalamus. Infarct volume was significantly decreased, with increased functional outcome measured by Neurobehavioral Severity Scale (NSS) during the 7 days following ischemia. No adverse side effects were observed, including no increase in tail bleeding time or alterations in blood pressure.
Approximately 25-50% of very premature births exhibit cognitive impediments attributed to damage to white matter caused by hypoxia, inflammation, infection, ROS excitotoxicity and deficiencies in growth factors [78-80]. To model the neuronal injury seen in newborns, P5 rat pups were intracranially injected with glutamate agonists to induce neuronal injury, and the protective effects of 5 and 20ppm of iNO was investigated [81]. At P10, a dose dependent neuroprotective effect was modeled against i.c. ibotenate administration, with significance in 20ppm administration in white matter (p <0.001) and the cortical plate (p < 0.05). Astrocyte activation in the white matter and cortical plate were also significantly decreased (p < 0.001). The observed effects seem to be a result of down-regulation of pCREB and up-regulation of pAkt, leading to decreased gene expression of multiple glutamate receptor subunits. The neuroprotective effect is likely due to the anti-oxidant nature that proper dosing of NO can provide.
Subarachnoid Hemorrhage
In type of stroke, known as subarachnoid hemorrhage (SAH), the resulting outcome is poor for patients even when treatment is received. Approximately half of patients that experience SAH die within the first 48 h [82]. A mouse model of SAH was used by performing an endovascular perforation technique, to determine if administration of 50 ppm of iNO could be prevented death/damage seen in SAH patients [83]. Measurements were taken 3-7 days post SAH induction, with significantly lower brain edema observed in iNO treated groups. Microvasospasms were reduced by approximately 80%. Neuronal survival in CA3 hippocampal regions of the iNO treated mice was also significantly higher (p <0.05). The 72 h survival rate of untreated animals was 70%, whereas the iNO treated subjected has 100% survival rate. It hard to predict if these effects would translate in a clinical setting, however due to the poor outcome of patients with SAH, it could be advantageous.
Hemodynamics
One of the concerns for administrating iNO to patients is NO’s hemodynamic influences. NO is known to decrease platelet activation and subsequent aggregation, a mechanism required for clotting [84]. Decreased platelet aggregation has been observed in patients being treated with iNO [85-87], while some authors have reported no effects [88, 89]. The bleeding time results are varied as well; with some cases reporting increased bleeding times, no significant change, or a possible correlation with iNO exposure time. To test this relationship, Goldstein et al. [90] carried out a randomized, controlled, blinded study on the hemostasis in healthy adults after being administered iNO [90]. Healthy patients received 80ppm of iNO or medical air as a control for 30 minutes, and a placebo (0.9% sodium chloride), aspirin (600mg), or 5 ml heparin (5,000 i.u.). Activated clotting time, prothrombin time, and activated partial thromboplastin time increased only in groups receiving heparin, which was expected. Bleeding and platelet aggregation alterations were only observed in aspirin groups. There was no significant effect on administration of iNO on bleeding or platelet aggregation. The study was able to control for multiple known influencers of blood hemodynamics, and were able to support no significant observed effects by iNO on the blood.
Blood Transfusions
The storage of red blood cells (RBCs) for transfusions leads to multiple alterations in the integrity of the samples. Increased storage time leads “RBC storage lesion” including loss of integrity, morphology, membrane components, and alterations in metabolism of the RBCs [91]. After storing RBCs 30 days, hemolysis leads to free hemoglobin that scavenges endogenous NO upon transfusion [92]. Loss of NO availability within the endothelium can lead to vasoconstriction and hypertension. The vascular homeostasis that NO provides can be lost due to the NO deficient state. Consequential downstream effects can lead to complications in diseases such as sickle cell disease, malaria, and hemolysis-associated smooth muscle dystonia [93].
Lei et al. [94] observed a mouse model with endothelial dysfunction and investigated the effects of resuscitation after hemorrhagic shock for 90 min with stored erythrocytes (2 weeks) or fresh erythrocytes (24 h). The HFD-mouse model had exacerbated effects, with increased lactate, plasma hemoglobin, inflammation and oxidative stress when administered stored erythrocytes. Interestingly, administration of 80 ppm iNO, 10 minutes prior to transfusion reduced lactate, inflammation, and improved survival rates. Short-term survival rates improved in HFD-mice receiving stored erythrocytes from 30% in control groups, to 80% in iNO groups. This relationship has been modeled as well ex vivo, when stored erythrocytes (40 days) were exposed to 300 ppm NO prior to transfusion in lambs, and had profound effects at prevent transfusion associated pulmonary hypertension (p <0.0001) [95].
Hemoglobin (Hb)-based oxygen carriers (HBOC) are being developed as an alternative therapeutic to blood supply, when traditional blood is not readily available. One of the biggest challenges in administration is vasoconstriction leading to coronary and cerebral vasospasm [96]. Clinical trials have also attributed nausea, vomiting, and chest/abdominal pain to likely be linked to the resulting vasoconstriction [97]. Yu et al. [98] induced hypertension using tetrameric Hb (HBOC) (0.48 g/kg) in mice, and then used a KO NOS3-/- to show that the hypertension was dependent on NOS3 (eNOS). The pretreatment with 80ppm iNO 1 h prior to HBOC administration completely prevented an increase in systolic blood pressure seen in controls, 10 minutes after HBOC administration (117±1 versus 141±4 mmHg). There was no noted metHb toxicity. The authors followed up with testing HBOC-201 (cross-linked bovine hemoglobin) in awake lambs, where pulmonary and systemic vasoconstriction occurred, along with decreased cardiac output upon transfusion. In order to mitigate observed effects pretreatment with 80ppm 1 h before transfusion followed by 5ppm was necessary [99].
Challenges of iNO Delivery Systems
Licensed pharmaceutical grade NO is distributed solely by INOmax, where NO supplementation runs at a metered cost of approximately $100-150/h [100-102]. The yearly cost to have it institution wide was reported approximately $1.8 million [101]. The average duration of treatment in patents is 72-96 h [101, 102]. These costs can come at a heavy burden for hospitals due to the availability of medical grade NO being available by a sole distributor. When a collaborative effort is made to implement a nitric oxide program at a treatment facility; however, clinical, social, and economic advantages are seen for patients, families, and the health care system [102]. Since 1999, Mallinckrodt Pharmaceuticals has held patents dominating the field of iNO treatment and blocking generic distribution.
There have been attempts to develop alternative methods of iNO machinery and alterations to current systems. Many alterations have been made in hopes providing a more controlled delivery of NO gas, create portable machines, and reducing costs. When iNO is delivered to a patient, proper testing along the breathing circuit is required to ensure adequate gas mixing [103]. Gas mixing can create complications when extended residence times within the breathing circuit is allowed [104]. Inadequate mixing of gas can occur due to reduced volume of delivery method, such as when using high flow nasal cannula [103]. Martin et al. [103] investigated novel adaptors for NO injection into an iNO system and required mixing lengths for a 5% desired range. The adapters resulted in ranges of approximately 7.8cm, 23cm, 27cm and 47cm needed for mixing. This study highlights the potential to improve iNO delivery devices, however limitations remain, such as controlling flow rates of air/O2, that would need to be proportionally adjusted based on individual [103]. Further development could contribute to safer administration, ease of use, and compatibility when using various types equipment.
Some researchers have attempted to develop entirely new iNO delivery devices that offer portability and stability. Qin et al. [105] investigated NO production via electrochemical reduction of nitrite ions using a copper(II)-ligand electron transfer mediator complex. The extracted NO molecules can be purged from the system with nitrogen or air, or pass through gas extraction silicone fiber-based membrane-dialyzer. Gas production can be control proportional to a constant cathode current, with ability to tune a range of 5 – 1000 ppm. This NO generator offers low costs and rapid production time. The application was tested in pigs undergoing CPB with extracorporeal circulation. The addition of 500ppm of NO to sweep gas of the oxygenator prevented activation of granulocytes and monocytes [105].
Similar NO generation devices are making an appearance, that use pulsed electrical discharges in air to produce NO in hopes of being used for inhalation therapy [106]. This mini-NO generator comes equipped with an NO 2 scavenger consisting of 0.8 g of Ca(OH)2, HEPA filters to remove any dispersed metal particles, a mini air pump, and even a Bluetooth controller. Airflow rates of 70 ml/min have shown to produce NO at levels relevant for clinical use. The device only weighs 14 g makes it light and portable.
Conclusion
There is still further investigation into the potential clinical applications of iNO. Establishing more thorough guidelines for dosing, duration of treatment, and time of administration after disease onset could help regulate treatments. Additionally, improving current devices to decrease toxic NO2 production, gas mixing times, and fine-tuned dosing could be valuable [15, 103]. It’s hard to determine if specific guidelines for treatment with iNO would be beneficial, as bioavailability of NO within the patient, and pre-existing conditions can effect patient outcomes. Regardless, iNO has proved to be beneficial for many clinical applications, being used for over 20 year to treat pulmonary diseased infants [107]. It is exciting to see how far the therapeutic effects of iNO will reach in years to come.