Intravenous L-Citrulline to Treat Children Undergoing Heart Bypass Surgery : Revised Protocol

Citrulline Blood Levels: 1. Baseline sample in OR prior to Cardiopulmonary Bypass prior to administration of first bolus of Citrulline or Placebo 2. Immediately after Bolus 1 administered in Operating Room. 3. 30 minutes after separation from Cardiopulmonary Bypass; immediately prior to administration of Bolus 2 and start of continuous infusion of Citrulline or Placebo. 4. Six hours after after start of infusion. 5. 12 hours after start of infusion. 6. 24 hours after start of infusion. 7. 48 hours after start of infusion; or whenever infusion is discontinued if prior to 48 hours.

Time frame: Measured in seven blood sample time points from the beginning of surgery until end of IV Citrulline Infusion; at either 48 hours postoperatively or at extubation, whichever comes first.

Population: All randomized patients that received surgery, were treated with L-citrulline, and had at least 1 citrulline concentration measurement

The composite endpoint comprised the longer of the duration of positive pressure ventilatory support or of inotrope therapy. Since inotrope use was only documented until Hour 48 after surgery (end of study medication treatment), patients with inotrope use continuing until Hour 48 and with mechanical ventilation duration of ≤48 h were censored at this time point. If mechanical ventilation was continued beyond the 48-hour time point, the duration of mechanical ventilation was used in the analysis.

Time frame: Until cessation of positive pressure ventilation and of inotrope therapy or Day 30, whichever occurred first

Time frame: Measured in hours from the end of surgery until removal of chest tubes or Day 30, whichever occurred first

There were insufficient data to analyze ECHO measurements in summary statistics. Additionally, most images were of very low quality and the data from ECHO evaluations were insufficient to determine whether the affected patients had pulmonary hypertension.

Time frame: Measured in hours from the end of surgery until extubation or Day 30, whichever occurred first

Population: It was not possible to reliably determine the presence of PVT in most patients due to low quality ECHO images. Additionally, no patients were identified with sufficient ECHO measurements to detect the presence of sustained mean pulmonary artery pressure greater than 20 mm Hg for at least 2 hours.

Time frame: Measured from the day of surgery until discharge from hospital or Day 30, whichever occurred first

Duration of ICU stay was analyzed once as the total number of postoperative hours spent in the ICU and once as the total number of postoperative hours that a patient required postoperative mechanical ventilator or continuous intravenous inotrope or vasodilator support. The latter combination of parameters represents another surrogate endpoint for ICU stay

Time frame: Measured in hours from the end of surgery to discharge from ICU or Day 30, whichever occurred first

Age specific mean arterial blood pressure (MAP) limits compared between the citrulline and placebo groups will be used to determine significant hypotension. Defined as MAP below a specific age-based value (infants and age 1 year, 40; ; age 2 years, 44; age 3 years, 47; age 4 years, 50; age 5 years, 52; age 6 years, 53), that lasted greater than 30 minutes and was unresponsive to therapeutic interventions such as fluid administration (volume bolus) and increasing inotropic support.

Time frame: Mean Arterial Blood pressure as continuously monitored postoperatively in the PCCU (Hour 0 to Hour 24) during Citrulline or Placebo infusion.

The length of time on IV inotropes was documented from the time of first use after surgery until completion of the study medication at Hour 48 (i.e., duration of inotrope use was maximally 48 hours). Patients still receiving inotropes at Hour 48 were censored.

Time frame: Measured at 48 hours

Duration of postoperative invasive mechanical ventilation was derived as the time in hours from separation from cardiopulmonary bypass until endotracheal extubation. If a patient required reintubation within 24 hours after extubation, the reintubation time was added in the main analysis. In a second analysis, the reintubation time was not included.

Time frame: Measured in hours from the end of surgery until extubation, or Day 30, whichever occurs first

Duration of postoperative invasive mechanical ventilation was derived as the time in hours from separation from cardiopulmonary bypass until endotracheal extubation. If a patient required reintubation within 24 hours after extubation, the reintubation time was added in the main analysis. In a second analysis, the reintubation time was not included.

Time frame: Measured in hours from the end of surgery until extubation or Day 30, whichever occurred first

28-day postoperative survival and survival to discharge

Time frame: Measured at 28 days post surgical repair

The total number of hours on vasoactive medications, including nitroglycerin, nitroprusside and vasopressin, was calculated from the end of surgery until the discontinuation of vasoactive medications or end of study medication (Hour 48), whichever occurred first

Time frame: Hour 0 to Hour 48

Analysis included any invasive and non-invasive respiratory support required during the study period

Time frame: Baseline to discharge or Day 30, whichever occurs first

Population: All randomized patients that received surgery

The inotrope dose was calculated each hour postoperatively from the time of PICU admission until the completion of study drug using the following scoring system: Dopamine (μg/kg/min) x 1 plus Dobutamine (μg/kg/min) x 1 plus Milrinone (μg/kg/min) x10 plus Epinephrine (Adrenaline) (μg/kg/min) x 100 plus Phenylephrine (μg/kg/min) x 100 plus Norepinephrine (Noradrenaline) (μg/kg/min) x 100 = Total inotrope score An ANOVA was performed. Additionally a repeated measures analysis of variance was used to compare total inotrope score between placebo and citrulline.

Time frame: PICU admission until Hour 48

Vasoactive score is reflective of the pharmacological support required by the cardiovascular system and is a good predictor of mortality and morbidity in patients undergoing bypass surgery. A lower vasoactive score represents less pharmacological support and would indicate a lower risk for a poor clinical outcome. Therefore, any treatment effect would be indicated by a lower score in the citrulline group when compared to the placebo group. In this study, the score was calculated post-operatively from the time of separation from bypass until the completion of study drug. Vasoactive score was calculated using the following formula: Total Vasoactive Score = nitroglycerin dose + nitroprusside dose + vasopressin dose The minimum value is zero (i.e., no vasoactive drugs administered), but it is not possible to define a maximum as this is wholly dependent upon the dose of each vasoactive drug administered.

Time frame: Hour 0 to Hour 48

Time frame: Measured in milliliters from the end of surgery until removal of chest tubes or Day 30, whichever occurred first

Citrulline is the precursor of arginine and nitric oxide. Nominal sampling times were Baseline, Post-bolus 1, Pre-bolus 2, and Hours 2, 6, 12, 24 and 48.

Time frame: Baseline to Hour 48

Population: All randomized patients that received surgery, were treated with L-citrulline, and had at least 1 arginine concentration measurement

Citrulline is the precursor of arginine and nitric oxide. Nominal sampling times were Baseline, Post-bolus 1, Pre-bolus 2, and Hours 2, 6, 12, 24 and 48.

Time frame: Baseline to Hour 48

Population: All randomized patients that received surgery, were treated with L-citrulline, and had at least 1 arginine concentration measurement