This was a prospective, multicenter, double-blind, placebo-controlled, randomized, phase II study of participants with SCD presenting with VOC. The study was approved by the National Heart, Lung and Blood Institutes (NHLBI) Institutional Review Board and by each participating center’s Institutional Review Boards. An NHLBI Data Safety and Monitoring Board (DSMB) monitored the study conduct and safety and an unblinded independent biostatistician (W. Blackwelder) reported interim methemoglobin safety results for children and adults to the DSMB. This was requested by the DSMB to ensure that children on inhaled NO in this trial were not subjected to increased risk of methemoglobinemia. Eleven sites (National Institutes of Health, Johns Hopkins University, Boston Children’s Hospital, University of Alabama at Birmingham, Howard University Hospital, St. Christopher’s Hospital for Children, Children’s Hospital Oakland/Alta Bates Medical Center, University of Colorado Health Science Center, Brigham and Women’s Hospital, Cleveland Case Western University, Children’s Hospital of Pittsburgh) participated between October 5, 2004 and December 22, 2008.
Participants with known SCD age 10 years and older were identified during presentation with VOC to the Emergency Department/Emergency Clinic (ED/EC) or other appropriate unit. Written informed consent was obtained, with patients under age 18 providing assent along with parental consent. Participants were also recruited in the outpatient setting while not in pain, signing a final consent form at the time of VOC presentation. Exclusion criteria included: HbSC disease; exposure to therapeutic NO within the previous 12 hours; use of phosphodiesterase 5 inhibitors, L-arginine, nitroprusside or nitroglycerine within the previous 12 hours; previous ED/EC or other appropriate unit treatment for a VOC within 48 hours, or hospitalization within14 days of presentation with VOC; ED/EC visits or hospitalizations for VOC >10 times in the preceding year; clinically diagnosed bacterial infection at presentation; current enrollment in any other investigational drug study, except for hydroxyurea studies; current pregnancy or nursing; chronic transfusion or exchange transfusion in the preceding 30 days; suspected splenic sequestration; new pulmonary infiltrate at presentation; or previous participation in the study.
Participants were randomized using block randomization by site and age at entry (age 10–15 and >15 years), in blocks of 4, in a 1:1 ratio of placebo to inhaled NO. Randomization was defined at the time a set of study placebo or NO gas cylinders was assigned and a cylinder was opened.
NO for inhalation (Ikaria - formerly INO Therapeutics, Port Allen, LA) was supplied at a concentration of 800 ppm balanced with nitrogen (99.92% grade 5 nitrogen, 0.08% pharmaceutical grade NO). Placebo study gas was 100% Grade 5 nitrogen gas. Either NO or placebo was delivered with air and mixed with oxygen to achieve a constant FiO2 of 24%. Participants were treated via facemask using a continuous flow delivery system. Those randomized to inhaled NO received 80 ppm for 4 hours, followed by 4 hours of 40 ppm. For participants remaining in the hospital after the initial 8 hour dose, study gas was administered through a pulsed flow delivery system with 1L continuous oxygen via nasal cannula at a dose of 6 mL/pulse/breath of 800 ppm NO for body weight ≥27 kg, or 3 mL/pulse/breath if <27 kg, up to a maximum of 72 hours total study gas administration. This is the first clinical trial to use a pulse delivery system, which delivers a lower dose of NO gas to the circulation, equivalent to approximately 5 ppm depending on minute ventilation. The use of pulse nasal canula delivery was considered necessary to facilitate the practical use of prolonged gas therapy for inpatients. The oxygen flow was increased as required to maintain SaO2 ≥ 85%, with a maximum of 4L permissible to continue in the study. If study gas was interrupted for more than 1 hour, it was not restarted. If the gas was stopped or a patient withdrew from treatment, the time to resolution of the VOC was still collected.
Coded labels were applied to the study cylinders at the manufacturing site. A “blinded” version of the face mask NO delivery systems blanked out and covered the NO and NO2 monitor displays. The placebo gas was administered in the same way and over the same time to ensure that participants and investigators were blinded.
Pain associated with the VOC was measured on a scale of 0–10 using a visual analogue scale (VAS), which consisted of a 10 cm horizontal line with the ends representing the extreme limits of “no pain” (0) and “worst pain” (10). The participant was asked to make a mark along the line to indicate the intensity of pain at baseline and at hours 2, 4, 6 and 8 after the start of the study drug and then at 4 hour intervals. Each participant’s score was the measurement in cm from 0 of his mark, to the nearest 0.1 cm. Starting with hour 12, sleeping participants were not woken to complete the VAS and a missing value was assigned. Participants were not shown their previous response. Demographic, clinical and laboratory variables were collected by the site coordinators from source documents and recorded on study case report forms.
Primary Efficacy Variable
The primary efficacy variable was the time to VOC resolution, modified from the poloxomer-188trial,42
defined by all of the following criteria being met: freedom from parenteral opioid use for at least 5 hours; pain relief [VAS scores ≤6 cm maintained during 2 consecutive readings obtained at least 2 hours apart, each at least 3 hours after the last dose of parenteral opioids]; ability to walk (except for chronically non-ambulatory participants); and agreement of the physician, and patient and parent or guardian that residual pain was low enough to be manageable at home. Time to VOC resolution for participants who were discharged with missing endpoint data or with incomplete criteria for VOC resolution was censored at the actual time of discharge from the hospital. Death before discharge without meeting the definition for VOC resolution was censored at a time later than the latest time of censoring or VOC resolution in participants who did not die before discharge. For participants in the hospital longer than 30 days without crisis resolution, the duration of crisis was determined by the time of the discharge order.
Secondary Efficacy Variables
The following secondary efficacy variables were evaluated: length of hospitalization from admission to discharge (time of discharge order); VAS score over time; total dose of opioids in the first 8 hours after enrollment into the study and during the entire hospitalization; rate of ACS or pneumonia requiring blood transfusion; proportion discharged in the first 24 hours; proportion returned to ED or hospital within 30 days;; change in nitrate/nitrite levels and methemoglobin levels as measures of NO metabolism and reactions in the blood (). Secondary evaluation of possible pain relapse was determined by the proportion of participants treated again for pain in the ED/EC, hospital or other appropriate unit within 24 hours and within 30 days after hospital discharge.
Effect of Inhaled NO Gas on Secondary Outcomes
Since the primary known toxicity of inhaled NO is methemoglobinemia, venous methemoglobin was monitored at baseline and every 2 hours for the first 8 hours, then every 24 hours for the rest of the study. Bedside personnel and site investigators were not allowed access to methemoglobin levels, which were reported by designated laboratory personnel through an interactive voice response system at each site to a central safety monitor, who notified site investigators if dose change was indicated. Methemoglobin values were only accessible to the DSMB and an unblinded monitoring statistician reporting to the DSMB. If any value was ≥ 5%, the treatment dose was to be decreased by 50%. If any value was >7.5%, the investigational therapy was to be discontinued. Other stopping rules included: assessment of the investigator, treating physician or participant that discontinuation of the inhalation therapy was in the patient’s best interest; any serious adverse event thought to be related to the investigational therapy; clinically significant hypotension; sepsis or septic shock; or sustained pulse oxygen saturation below 85% for >15 minutes while on supplemental oxygen up to 4L by nasal cannula or 35% oxygen by mask. Participants who were discontinued from therapy remained in the study and continued with all data collection, unless consent to do so was withdrawn.
Serious adverse events were recorded during study gas inhalation and defined as any event that at any dose required hospitalization or resulted in disability or death (). As is standard in clinical trials in the SCD field, ACS was considered a serious adverse event during active treatment with NO because it is one of the major complications that occurs during hospitalizations for pain crisis. ACS requiring blood transfusion was also captured as a secondary outcome measure both on and off NO or placebo treatment ().
Number and Percentage of Participants with Serious Adverse Events
Statistical and Analytic Plans
At the end of the study, the dataset was analyzed by the steering committee, independently of the sponsor, using pre-specified analyses, endpoints and subgroups. Interim analysis of efficacy was not planned or performed.
Sample size was estimated based on data from a previous study of NO therapy in children9
predicting a mean length of stay approximating 106 hrs. For the purposes of the calculation, time to crisis resolution, which was selected to minimize effects of factors unrelated to medical condition that impact discharge time, was assumed to approximate length of stay. The study was designed to have 80% power to find a significant decrease in duration of crisis if the true decrease was 24 hours. Sample size was based on a difference in log10
(duration), since the logarithm of duration was more normally distributed than untransformed values. Assuming a 2-sided Wilcoxon rank-sum test at the 5% significance level, normal distributions for log10
(duration) with difference 0.11 (log10
(106) − log10
(82)) and common standard deviation 0.22, we obtained a sample size of 68 per group, or a total of 136. Allowing for 9% of participants withdrawing or censored, the sample size became 75 participants per group, or a total of 150. Sample size and power calculations were done using PASS 2005 software (Number Cruncher Statistical Systems, Kaysville, Utah).
All analyses were intention to treat and 2-sided P-values less than 0.05 were considered significant. Clinical and laboratory characteristics were compared between the inhaled NO treatment group and the placebo group by the nonparametric Wilcoxon rank sum test for continuous variables and Pearson’s chi-square statistic for categorical variables. Treatment efficacy was evaluated by calculating Kaplan-Meier survival curves for duration of crisis over time, using the log-rank test to determine significant differences in time to resolution between the 2 groups. The effect of inhaled NO vs. placebo was also examined in pre-defined subgroups by age (10–15 years and >15 years) and hydroxyurea use, as suggested by the poloxomer-188 trial in which young patients and those on hydroxyrea therapy appeared to respond better to treatment.42
The effect of treatment on length of hospital stay and opioid use, both total and cumulative, were examined using the Wilcoxon 2-sample test. Treatment related changes in pain score and NO metabolites were evaluated using an unpaired 2-tailed t-test to examine differences at specific points in time and repeated measures ANOVA to examine changes over time. The frequency of dichotomous secondary outcomes and serious adverse events was examined using either the Pearson’s chi-square or the Fisher’s exact test.
Cox Proportional Hazards regression models were used to examine the association of treatment with duration of crisis while adjusting for significant covariates. The likelihood ratio test was used to determine the significance of individual regression coefficients. As a post-hoc analysis, associations of potential confounders, including study site, gender, baseline VAS score, age and hydroxyurea use, with time to crisis resolution were examined using Kaplan-Meier analysis. The effect of study site on length of hospitalization, total opioids and baseline VAS score was performed using either the Kruskal-Wallis test or ANOVA.
In a post-hoc analysis, comparisons of time to resolution with other measures of disease severity were examined using the Spearman rank order correlation coefficient and the Wilcoxon 2-sample test. All analyses were performed using SAS version 9.1.3 (SAS Institute Inc, Cary, NC) and Stata version 9.0 (StatCorp LP, College Station, TX).