Beneficial effects of nitric oxide breathing in adult patients with sickle cell crisis
Authors: C. Alvin Head, Paul Swerdlow, William A. McDade, Ratan Mani Joshi, Tohru Ikuta, Melanie L. Cooper, James R. Eckman
Journal: American Journal of Hematology (2010)

Pain from vaso-occlusive crisis (VOC) is the major cause of hospitalization in patients with sickle cell disease (SCD). The beneficial therapeutic effects of inhaled nitric oxide (NO) on the pathophysiology of SCD have been reported. A double-blind, randomized, placebo-controlled clinical trial was conducted to determine whether NO breathing reduces acute VOC pain in adult patients and to study the safety of inhaled NO. Twenty-three patients experiencing acute VOC were enrolled. After randomization but before treatment, five were found to not meet final eligibility criteria. Nine patients were assigned to inhaled NO (80 ppm) and nine to placebo (21% O2). Primary outcome was the mean change in pain scores after 4 hr of inhalation, measured on a 10-cm visual analog scale (VAS). Both groups had similar baseline VAS pain scores but inhaled NO significantly reduced pain scores compared with placebo (P = 0.02) at the end of NO inhalation. Secondary outcome was parenteral morphine use at baseline, 4, and 6 hr. Parenteral morphine use was lower in the inhaled NO group, but the difference was not statistically significant. Safety assessments included systolic blood pressure measurements, pulse oximetry readings, concentration of delivered nitrogen dioxide, and concentration of methemoglobin (metHb). None of these NO toxicities was observed. Pulmonary complications including pulmonary hypertension [1] and acute chest syndrome [2] have serious influences on the morbidity and mortality of sickle cell disease (SCD) patients. Although previous studies have shown successful application of inhaled nitric oxide (NO) for animal models of pulmonary disorders as well as for adult patients with respiratory distress syndrome [3-5], the therapeutic potential of NO inhalation for pulmonary complications in SCD had not been scrutinized until recently. We previously investigated physiologic effects of inhaled NO on sickle hemoglobin in adult SCD patients [6]. Subsequently, several lines of experimental evidence have shown that NO promptly downregulates the expression of adhesion molecules that are assumed to be involved in vaso-occlusive crisis (VOC) [7-9]. These led us to speculate that inhaled NO may inhibit cell interactions between multiple types of cells and thereby resolve VOC. Weiner et al. [10] have shown that inhaled NO decreases pain scores in pediatric SCD patients with VOC. There are some differences in the pathophysiologies between adult and pediatric patients [11, 12]. In this study, we evaluated the safety and efficacy of inhaled NO in the treatment of acute VOC in adult patients with SCD (Supplementary Fig. 1). The baseline clinical characteristics of the patients from the inhaled NO and placebo groups are shown in Supplementary Table I. Five patients found to be ineligible after randomization but before receiving treatment were removed from the study and excluded from the analysis, suggesting that initial treatment intent did not influence the outcome of this clinical trial. We found a significant reduction in the pain scores of these adult patients with SCD after 4 hr of inhaled NO, indicating efficacy of inhaled NO in relieving pain (see Fig. 1). It is still unclear whether prolonged exposure to NO breathing would result in continued benefit or produce any harmful outcomes. In this study, the difference in mean visual analog scale (VAS) pain scores between the groups widened during the inhalation period, reaching a maximum at 4 hr and persisting for the subsequent 2-hr post-inhalation period (see Fig. 1). In the placebo group, the pain scores did not change during the post-inhalation period in either the adult or the pediatric study [10]. However, although the pain scores of NO-treated pediatric patients increased during the 2-hr post-inhalation period to equal those of the placebo group [10], the pain scores of our adult patients did not increase. We thought that this difference would be attributable to a waning effect of NO after inhalation, but the mechanism underlying this phenomenon remains unclear. Another interesting observation was the use of narcotics in the adult patients (see Fig. 2). Although the pain scores were reduced in adult patients in response to inhaled NO, the mean narcotic use was equivalent in the inhaled NO and placebo groups, separating narcotic use and pain sensation. This may reflect a level of narcotic tolerance in the adult sickle cell population. There were no apparent safety or toxicity concerns in the patients enrolled in this study (Table I). Reduced pain scores were observed in the INO group. Statistical analysis was performed by 2-way analysis of variance using GraphPad Prism software. Significant changes in pain scores were observed after 3 hr of treatment with INO vs. placebo (P = 0.0376). Cumulative mean narcotic use >6 hr. The total amount of morphine used by the INO group was slightly less than the control group, but the difference was statistically and clinically insignificant (P = 0.26). Because there is no diagnostic or predictive laboratory test for acute painful episodes, pain assessment relies on patient's self-report [13]. Pain intensity may be assessed by any of several available scales, including the VAS, verbal scale, or Wong- Baker FACES Pain Rating Scale for children [14]. The VAS is reliable, easy to use, and used extensively in patients with SCD. To date, few studies have defined a clinically significant change in the magnitude of VAS pain scores, although some studies in adult emergency department (ED) patients with acute pain or acute exacerbations of chronic pain suggest that a decrease of 1.3 cm or greater may be clinically significant. Here, we report decreases in pain scores of 0.7 to 1.9 cm at each time point in the inhaled NO group as compared with decreases of 0.2 to 1.3 cm in the placebo group (P = 0.002, calculated by 2-way analysis of variance; see Fig. 1). After 4 hr of NO treatment, the total reduction was 6.3 ± 2.2 cm in the INO group vs. 2.97 ± 2.1 cm in the placebo group (P = 0.02). NO spontaneously forms NO2 in the presence of oxygen. Although NO2 rather than NO is primarily responsible for the acute lung injury in earlier reports of NO toxicity [15], the patients in the inhaled NO group showed very low and clinically insignificant levels of NO2 (∼0.13 ppm, Table I). NO combines with hemoglobin to form nitrosyl-hemoglobin and then metHb. The levels of metHb have not been shown to be clinically significant (usually <2%), while breathing low concentrations of NO [6]. All patients in this study were measured for their metHb levels, which were found to be much lower than the clinically significant limit (Supplementary Table I). Although the current study and a study by Weiner et al. [10] showed reductions in the pain scores of SCD patients who inhaled low concentrations of NO, the possible mechanisms by which NO reduces pain scores in SCD patients remain unclear. Weiner et al. concluded that the mechanisms for NO-induced pain reduction depends on the changes of vascular tone by inhaled NO but not on the modulation of gene expression in blood cells or endothelial cells within a 4-hr period of NO inhalation [10]. This view is supported by a recent study demonstrating a role for nitrite in the formation of nitrosylated hemoglobin and resulting vasodilation [16]. However, a number of previous studies have clearly shown that NO could rapidly regulate the expression of adhesion molecules in a variety of cells such as platelets and endothelial cells. The expression of P-selectin, a major adhesion molecule expressed on platelets and endothelial cells [17], is rapidly downregulated by INO or NO donor molecules [7, 8]. It is thus possible that the expression of adhesion molecules on blood cells including platelets as well as endothelial cells could be quickly downregulated by inhaled NO, especially in the lungs, which would help resolve vaso-occlusions in venules by inhibiting cell adhesion. In addition, our in vitro studies have found that NO inhibits the polymer formation of sickle hemoglobin and disrupts even preformed polymers (T.Ikuta et al., paper submitted). It is possible that NO may disrupt sickle hemoglobin polymers, help restore the shape of red blood cells, and thereby improve blood flow in the periphery. Also, regional lung perfusion is likely to be optimized by selective vasodilation of ventilated lung regions during NO breathing, thus improving ventilation-perfusion matching [5]. Collectively, these may have relevance to the mechanisms by which inhaled NO reduces pain scores in SCD patients. A recent clinical trial with the PDE5 inhibitor sildenafil for SCD patients with pulmonary hypertension has reported several serious adverse events including increased rates of VOC, headache, and visual disturbance [18]. Because both sildenafil and NO are assumed to exert their physiologic activities through the cGMP-dependent pathway [19], their finding is in contrast with the clinical benefits of inhaled NO for SCD patients demonstrated by this study. Mochado et al. concluded a potential involvement of the cGMP-dependent pathway in the pathogenesis of VOC. Because no evidence has been obtained regarding whether the cGMP-dependent pathway was indeed activated in several types of cells including smooth muscle cells in the lung or blood cells in SCD patients who received sildenafil, it is uncertain whether their data support their conclusion. In contrast, several case reports have clearly demonstrated that clinical manifestations in SCD patients with pulmonary complications were significantly improved by inhaled NO [20-22], indicating that activation of the cGMP-dependent pathway in the lung by inhaled NO benefits SCD patients with pulmonary complications. This view is supported by a previous study showing that inhaled NO activates the cGMP-dependent pathway [8]. Importantly, NO gas has greater physiologic effects than vasodilation alone allows, including reduced cellular adhesion and decreased P50 changes. We did not measure P50 values of sickle hemoglobin in SCD patients enrolled in this study. Participating institutions had varying degrees of access to the Hemox analyzer to measure P50 values. Rather, we focused on whether inhaled NO reduces pain scores as well as narcotics use in adult SCD patients. However, our recent in vitro studies demonstrated that 80 ppm NO inhibits the polymer formation of sickle hemoglobin and disrupts even preformed sickle hemoglobin polymers (T. Ikuta et al., paper submitted). NO shares a number of common biological activities with carbon monoxide [23], which multiple studies found is known to depolymerize sickle hemoglobin polymers [24]. It is possible that low-dose NO depolymerizes sickle hemoglobin polymers in vivo and improves access of oxygen to sickle hemoglobin. Further studies are under way in an in vivo setting. Despite the small sample size and other limitations, this study fully supports the safety and efficacy findings of Weiner et al. in the use of NO gas in acute VOC [10]. Further work is required to determine the optimal dose, timing and duration of inhalation, and molecular basis of the beneficial effects of NO in patients with SCD. Participating hospitals received institutional review board approval. An investigational new drug application for INO was obtained from the US Food and Drug Administration Division of Cardio-Renal Drug Products. Patients with homozygous HbS experiencing uncomplicated severe acute VOC (score, >6 cm on a 10-cm VAS and total hemoglobin concentration >6.0) were enrolled (Supplementary Fig. 1). Acute VOC pain was defined as pain in the chest, back, abdomen, or extremities that could not be explained by any other complication of SCD or by any cause other than SCD. Exclusion criteria included significant respiratory compromise, new focal neurological changes, acute priapism, known pregnancy or positive urine pregnancy test, other sickle hemoglobin variants, blood transfusion within 30 days, exposure to therapeutic NO, enrollment in other clinical studies, significant cardiac dysfunction, fever greater than 38.5°C, recent tobacco use, and chronic pain or treatment for VOC in ED within the previous 12 hr. When a potential subject was admitted to the hospital, the physician-investigator was paged. All interested patients were appropriately enrolled with written consent forms. Patients meeting eligibility criteria received standard ED treatment with pain medication consisting of intravenous morphine sulfate (initial dose up to 0.3 mg/kg body weight) and fluids (isotonic sodium chloride solution, 10 mL/kg over 30 min). After initial evaluation, patients were randomized to receive INO (80 ppm with 21% final concentration of inspired oxygen) or placebo (21% inspired oxygen). Demographic information was obtained and an intravenous catheter was placed to draw baseline blood samples and administer fluids and medications. A single dose of intravenous diphenhydramine (25-50 mg) was given to reduce narcotic side effects (e.g., itching), if necessary. Additional intravenous morphine was delivered on demand by a patient-controlled analgesia (PCA) device. After patients were stabilized, they were transferred to the general clinical research center where NO or placebo inhalation therapy was given by facemask. Additional morphine was administered intravenously by PCA pump at a dose of 1-4 mg delivered on demand with a lockout period to prevent overdose. The attending physician was blinded to type of inhalation therapy administered (INO or placebo). The study gas was given within 60 min after the initial intravenous morphine injection. Although the study gas was administered for 4 hr, the patient was monitored for 2 additional hr after the study gas was stopped. The PCA-delivered morphine doses and VAS pain scores were recorded at baseline (before study gas was given) and at each hour during the 6-hr study. Vital signs including blood pressure and oxygen saturation (measured by pulse oximetry) were monitored continuously during the 6-hr study and recorded at baseline and hourly before each blood sample was taken. The patient was allowed a 5-min break per hour to remove the mask immediately after the data/blood collection were obtained. Medical grade NO, in nitrogen (Datex-Ohmeda) packed as NO (0.08%) and N2 (99.92%) gas for 800 ppm, (INO-Therapeutics, Clinton, NJ) was mixed with oxygen before administration to deliver 80 ppm of NO using the INOvent delivery system. The concentration of NO and NO2 in the gas mixture was continuously monitored using an alarm-equipped analyzer and delivery system (INOvent, INO-Therapeutics, Clinton, NJ). Early termination from the study was based on patient safety and included NO2 delivery of >3 ppm, oxygen saturation decreasing >2% from baseline by pulse oxymetry reading, mean systolic blood pressure of less than 80 mm Hg, bradycardia (heart rate <50%) or tachycardia (heart rate >150 bpm), metHb increase of >5% from baseline, increase in pain score of >2 cm during the inhalation period, and patient or family request for termination. The primary outcome was reduction in pain scores at 4 hr of inhalation as measured by an unmarked VAS with labels from 0 for "no pain" at one end to 10 for "worst pain" at the other. The secondary outcome was the amount of PCA-administered narcotic used at baseline, 4 hr, and 6 hr. Safety assessments included systolic blood pressure, pulse oximetry, concentration of delivered NO2, and concentration of metHb. GraphPad Prism Version 4 software (GraphPad Software, La Jolla, CA) was used to perform the 2-way analysis of variance on the pain score data to determine the effect of two independent factors, time and treatment. The authors thank INO Therapeutics for providing NO gas. Additional Supporting Information may be found in the online version of this article. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. C. Alvin Head*, Paul Swerdlow , William A. McDade , Ratan Mani Joshi§, Tohru Ikuta*, Melanie L. Cooper¶, James R. Eckman**, * Department of Anesthesiology and Perioperative Medicine, Medical College of Georgia, Augusta, Georgia, Department of Internal Medicine, Wayne State University, Detroit, Michigan, Pritzker School of Medicine, The University of Chicago, Chicago, Illinois, § Dean McGee Eye Institute, Oklahoma Health Science Center, Oklahoma City, Oklahoma, ¶ Family Health Center, Lilburn, Georgia, ** Georgia Comprehensive Sickle Cell Center, Emory University, Atlanta, Georgia.