A Randomized Clinical Trial Testing the Anti-Inflammatory Effects of Preemptive Inhaled Nitric Oxide in Human Liver Transplantation

<div><p>Decreases in endothelial nitric oxide synthase derived nitric oxide (NO) production during liver transplantation promotes injury. We hypothesized that preemptive inhaled NO (iNO) would improve allograft function (primary) and reduce complications post-transplantation (secondary). Patients at two university centers (Center A and B) were randomized to receive placebo (n = 20/center) or iNO (80 ppm, n = 20/center) during the operative phase of liver transplantation. Data were analyzed at set intervals for up to 9-months post-transplantation and compared between groups. Patient characteristics and outcomes were examined with the Mann-Whitney U test, Student t-test, logistic regression, repeated measures ANOVA, and Cox proportional hazards models. Combined and site stratified analyses were performed. MELD scores were significantly higher at Center B (22.5 vs. 19.5, p<0.0001), surgical times were greater at Center B (7.7 vs. 4.5 hrs, p<0.001) and warm ischemia times were greater at Center B (95.4 vs. 69.7 min, p<0.0001). No adverse metabolic or hematologic effects from iNO occurred. iNO enhanced allograft function indexed by liver function tests (Center B, p<0.05; and p<0.03 for ALT with center data combined) and reduced complications at 9-months (Center A and B, p = 0.0062, OR = 0.15, 95% CI (0.04, 0.59)). ICU (p = 0.47) and hospital length of stay (p = 0.49) were not decreased. iNO increased concentrations of nitrate (p<0.001), nitrite (p<0.001) and nitrosylhemoglobin (p<0.001), with nitrite being postulated as a protective mechanism. Mean costs of iNO were $1,020 per transplant. iNO was safe and improved allograft function at one center and trended toward improving allograft function at the other. ClinicalTrials.gov with registry number 00582010 and the following URL:<a href="http://clinicaltrials.gov/show/NCT00582010" target="_blank">http://clinicaltrials.gov/show/NCT00582010</a>.</p></div

iNO therapy did not affect hospital or ICU length of stay.

<p>Kaplan-Meier plots shown and with P-values from Cox proportional hazards models adjusting for MELD, cold ischemic time, weight, BMI, warm ischemic time or donor age within each center. Placebo = ○, iNO = ▪.</p

The Distribution, Frequency and Type of Hepatobiliary Complications.

<p>Center A.</p><p>Placebo: allograft dysfunction (1), primary graft non-function (1), reduced portal vein flow (1), hepatic artery bleeding (2).</p><p>iNO: allograft dysfunction (1), hepatic artery bleeding (1).</p><p>Center B.</p><p>Placebo: 1-month:primary graft dysfunction (1), reduced hepatic artery blood flow (1), biliary leak (1), 6-months:biliary stricture (2), 9-months:rejection (1), hepatic artery stenosis (1), death (1).</p><p>iNO: 9-months:hepatic artery stenosis (1), death (1).</p

Effects of iNO on reperfusion induced injury and PMN accumulation.

<p>Liver biopsy samples were collected pre- (LB1, □) and 1 hr post dual reperfusion (LB2, ▪) and assessed for injury by histopathologic evaluation (Panel A, B) and infiltration of PMN (panel C, D). Data from both UAB and UW cohorts are combined (n = 38). P-values indicated on graph are by paired t-test for panels A and C or by * unpaired t-test (panels B and D).</p

Rates of post-operative hepatobiliary complications.

<p>A. Data are presented as ‘no’ or ‘yes’ indicating the absence or presence of hepatobiliary complications respectively within 9 months of surgery.</p

Patient and Surgery Demographics for Center A Cohort.

<p>Values show median (range). P-values calculated from unpaired t-test for placebo vs. iNO. N = 20 except An = 19. Bn = 18.</p

Effects of iNO on liver nitrite levels pre- (LB1) and post-reperfusion (LB2).

<p>Nitrite was measured in paired liver biopsies and data normalized to protein. No significant differences in nitrite levels were observed pre- vs. post-reperfusion or between placebo and iNO treatments.</p

Effects of iNO on TUNEL staining pre- and post-reperfusion.

<p>Hepatic cell death was assessed by TUNEL staining in central vein and triad regions pre- (LB1) and 1 hr post-dual reperfusion (LB2) in placebo and iNO treated patients. Panel A and B show representative immunofluorescence images for TUNEL staining (green = TUNEL positive cell, blue is Hoechst 33342 staining of nuclei, magnification 40×). Panel C and D compare the magnitude of increase in TUNEL staining between placebo and iNO groups for Center A and Center B cohorts respectively. Indicated P-values calculated by Mann-Whitney U-test.</p

Patient and Surgery Demographics for Center B Cohort.

<p>Values show median (range). P-values calculated from unpaired t-test for placebo vs. iNO.</p

Changes in metHb and nitrogen dioxide (NO₂) as a function of blood draw (BD) in placebo (○) or iNO (▪) groups.

<p>Data are mean ± SEM (n = 20 for each group, except UAB placebo where n = 19 and UW iNO where n = 17–20. *P<0.0001 by 2-way ANOVA with Bonferroni multiple comparisons post-test.</p