Surfactant plus budesonide decreases lung and systemic inflammation in mechanically ventilated preterm sheep

Abstract

Mechanical ventilation with normal tidal volumes (V_T) causes lung and systemic inflammation in preterm sheep. Mechanical ventilation is associated with bronchopulmonary dysplasia (BPD) in preterm infants, and the addition of budesonide to surfactant decreases BPD in clinical trials. Budesonide with surfactant will decrease the lung injury from mechanical ventilation for 24 h in preterm sheep. Lambs at 126 ± 1 day gestational age were delivered and randomized to either: 1) surfactant (200 mg/kg) or 2) surfactant mixed with budesonide (0.25 mg/kg) before mechanical ventilation with V_T of 7–8 ml/kg for 2, 6, or 24 h (n = 6 or 7/group). Lung physiology and budesonide levels in the plasma and the lung were measured. Lung tissue, bronchoalveolar lavage fluid (BALF), liver, and brain tissues were evaluated for indicators of injury. High initial budesonide plasma levels of 170 ng/ml decreased to 3 ng/ml at 24 h. Lung tissue budesonide levels were less than 1% of initial dose by 24 h. Although physiological variables were generally similar, budesonide-exposed lambs required lower mean airway pressures, had higher hyperoxia responses, and had more stable blood pressures. Budesonide decreased proinflammatory mRNA in the lung, liver, and brain. Budesonide also decreased total protein and proinflammatory cytokines in BALF, and decreased inducible nitric oxide synthase activation at 24 h. In ventilated preterm lambs, most of the budesonide left the lung within 24 h. The addition of budesonide to surfactant improved physiology, decreased markers of lung injury, and decreased systemic responses in liver and brain.

INTRODUCTION

Lung inflammation is central to the pathophysiology of bronchopulmonary dysplasia (BPD) in very preterm infants (20). Systemic corticosteroids (dexamethasone and hydrocortisone), inhaled budesonide, and the combination of budesonide and surfactant was recently shown to decrease BPD (3, 4, 6, 23, 24). However, there is a risk of long-term adverse outcomes or death with steroids (3, 6). Budesonide (0.25 mg/kg) mixed with surfactant reduced the rate of BPD by 20% without increased mortality or adverse physical or cognitive outcomes on Bailey testing at 18 mo (23, 24). Although initial safety evaluations are encouraging, the infants in the studies by Yeh et al. (23, 24) required moderate ventilator support before budesonide therapy. Whether there will be similar benefits for less severe initial lung disease and normal tidal volume ventilation is unknown. Budesonide can conjugate with fatty acids in the lung tissue for delayed release and any systemic budesonide will be metabolized rapidly in the liver to 16-α-hydroxy prednisolone (2, 14, 21). Because budesonide has a 200-fold increased affinity for the glucocorticoid receptor relative to cortisol, an understanding of the pharmacology and systemic responses of the combination of surfactant and budesonide is crucial before its widespread use to decrease BPD (7).

Surfactant delivers budesonide to the more distal lung and reduces lung inflammation, improves gas exchange, and improves lung histology in RDS models (11, 13, 17, 23). In fetal lambs who received a high V_T-injurious ventilation, 0.25 mg/kg or 1 mg/kg budesonide mixed with surfactant caused lung maturation, decreased lung inflammation, decreased airway thickening, altered lung weights, and decreased inflammation and glycogen changes in the liver (11). To test the hypothesis that budesonide (0.25 mg/kg) mixed with surfactant will decrease lung inflammation and systemic responses (liver and brain) to gentler mechanical ventilation from birth, preterm sheep were ventilated with normal tidal volumes for 2, 6, or 24 h with evaluation of physiology, pharmacology, and markers of injury.

METHODS

Animal management.

With the approval of the Animal Ethics Committee of the University of Western Australia, date-mated Merino ewes at 126 ± 1 days gestational age (GA; term is ~150 days GA) were anesthetized with intravenous ketamine (8–10 mg/kg) and midazolam (0.25–0.5 mg/kg) and given spinal anesthesia with lidocaine (60 mg). The fetal head and neck were exposed, and the fetus was given ketamine 10 mg/kg intramuscularly. A 4.5-mm endotracheal tube was tightly secured in the trachea, followed by gentle aspiration of fetal lung fluid. The lamb then was delivered, dried, weighed, and placed under a radiant warmer.

Surfactant and budesonide treatment.

Prior to delivery, lambs were assigned to receive either 1) 200 mg/kg surfactant (poractant alfa, Curosurf 2.5 ml/kg; Chiesi Farmaceutici, Parma, Italy) + saline 0.5 ml/kg or 2) surfactant + 0.25 mg/kg budesonide (0.5 mg/ml; Pulmicort, AstraZeneca, Wilmington, DE) and either 1) 2 h, 2) 6 h, or 3) 24 h of mechanical ventilation. The number of lambs used per group (n = 6 or 7) was determined from previous experiments that demonstrated minimal markers of injury or inflammation in unventilated controls (10). A 3-kg birth weight was used for surfactant and budesonide dosing. Surfactant was gently mixed with budesonide and then administered through the endotracheal tube with body positioning to assist with distribution to the lungs (11).

Mechanical ventilation.

Ventilation was with Fabian ventilators (Acutronic, Bubicon, Switzerland) with a peak inspiratory pressure (PIP) of 30 cmH₂O, a positive end-expiratory pressure of 5 cmH₂O, a rate of 50 breaths/min, an inspiratory time of 0.5 s, with 40% heated and humidified oxygen. The PIP (max 40 cmH₂O) was adjusted not to exceed a tidal volume (V_T) of 8 ml/kg. Oxyhemaglobin saturations were maintained greater than 90% using continuous pulse oximetry. $\text{P}{\text{a}}_{\text{O}}{}_{{}_2}$ values on arterial blood gases were also used to adjust oxygen concentrations. The lambs received ketamine and did not breathe spontaneously. Immediately following birth, the lamb received a 10 ml/kg transfusion with placental blood. Each lamb was continuously monitored for temperature, heart rate, and blood pressure. Plasma samples for budesonide measurements were collected at 15 and 30 min and then at 1, 2, 4, 6, 12, 18, and 24 h. At the end of the ventilation period, $\text{P}{\text{a}}_{\text{O}}{}_{{}_2}$ was measured after 2 min of 100% oxygen. The endotracheal tube was then clamped for 2 min to permit lung collapse by oxygen absorption, followed by euthanasia with pentobarbital sodium (100 mg/kg iv). Unventilated control lambs were euthanized at delivery.

Tissue sampling.

Postmortem inflation and deflation pressure-volume curves were measured to a maximum pressure of 40 cmH₂O (11). Three saline lavages of the left lung were pooled for bronchoalveolar lavage fluid (BALF) for total protein, cytokine ELISA, and budesonide measurements. Tissues from the right lower peripheral lung, liver, and periventricular white matter (PVWM) of the brain were snap frozen for RNA isolation (12). The right upper lobe was inflation-fixed at 30 cmH₂O with 10% formalin and then paraffin embedded (12). Lung tissue was dried at 65°C for 72 h, and the ratio of dry-to-wet weight was determined.

Quantitative RT-PCR.

Messenger RNA (mRNA) was extracted from the right lower peripheral lung, liver, and the PVWM, and cDNA was used for quantitative RT-PCR on a CFX Connect (Bio-Rad, Hercules, CA) (10, 11). Custom TaqMan primers (Life Technologies, Carlsbad, CA) for ovine epithelial sodium channel (ENaC), epiregulin (EREG), IL-1β, IL-6, IL-8, monocyte chemoattractant protein-1 (MCP-1), serum amyloid A3 (SAA3), surfactant protein B (SFTPB), and TNF-α were used. 18S primers (Life Technologies) were the internal loading controls.

Immunohistochemistry.

Paraffin sections (4 µm) of formalin-fixed right upper lobe tissue were used for hematoxylin-and-eosin (H&E) staining and immunohistochemistry with MsαHuman inducible nitric oxide synthase (iNOS) (BD Biosciences) 1:250. Blinded iNOS slides had 10 random regions/animal (×40 on Zeiss Axioskop 40) scored as 0 = no positive cells, 1 = occasional positive cells, and 2 = large number of cells. H&E-stained slides of lungs were blinded and evaluated for airway epithelial injury, edema, hemorrhage, and inflammation (19).

BALF cytokine analysis.

Ovine MCP-1 [GpαOv MCP1 1:300, RbαOv MCP1 1:300 (Seven Hills)] and IL-8 [MsαOv IL8 1:1,000, RbαOv IL8 1:1,000 (Chemicon)] were measured using sandwich ELISA on BALF (19).

Budesonide measurements.

Budesonide was measured in the plasma, lung tissue, and bronchoalveolar lavage fluid using a previously published protocol (11). Hydrolysis of lung tissue was performed with bovine pancreas cholesterol esterase in 0.1 M K₂HPO₄ buffer, pH 7.5, at a concentration of 0.0125 mg/ml with 10 mM taurocholate for 15 min before extraction.

Data analysis and statistics.

Physiologic variables are presented as means ± SE, with calculations of the ventilation efficiency index (VEI) [3800/(PIP × rate × $\text{P}{\text{a}}_{\text{C}\text{O}}{}_{{}_2}$)] and oxygenation index (OI) [${\text{F}\mathrm{I}}_{{\text{O}}_{\text{2}}}$ × mean airway pressure)/$\text{P}{\text{a}}_{\text{O}}{}_{{}_2}$] (16). mRNA and cytokines are reported as fold increase over unventilated controls, with control values set at 1. Statistics were analyzed with Prism 6 (GraphPad, La Jolla, CA) using the Student’s t-test or Mann-Whitney nonparametric tests as appropriate. Significance was accepted as P < 0.05.

RESULTS

There were no differences in gestational age (126 ± 0.5 days), sex, birth weight, or initial blood gases between groups. The animals were ventilated with normal tidal volumes (7 to 8 ml/kg for sheep) for 2, 6, or 24 h without difficulty (Table 1).

Table 1.

Physiology, oxygenation, and ventilation

	2 h Surf + Saline	2 h Surf + Bud	6 h Surf + Saline	6 h Surf + Bud	24 h Surf + Saline	24 h Surf + Bud
n per group	6	6	6	6	7	6
Birth weight, kg	3.0 ± 0.1	3.2 ± 0.2	3.1 ± 0.2	3.1 ± 0.2	3.4 ± 0.2	3.4 ± 0.1
Sex (M/F)	4/2	1/5	3/3	3/3	1/6	3/3
Average VT, ml/kg	7.6 ± 0.2	7.9 ± 0.4	7.6 ± 0.4	7.9 ± 0.4	7.4 ± 0.1	7.6 ± 0.1
At end of ventilation period
Cdyn, ml·cmH2O−1·kg−1	0.32 ± 0.02	0.34 ± 0.02	0.30 ± 0.02	0.35 ± 0.02	0.35 ± 0.03	0.35 ± 0.03
VEI	0.12 ± 0.02	0.12 ± 0.01	0.07 ± 0.01	0.10 ± 0.01	0.11 ± 0.02	0.10 ± 0.01
VEI	0.12 ± 0.02	0.12 ± 0.01	0.07 ± 0.01	0.10 ± 0.01	0.11 ± 0.02	0.10 ± 0.01
OI	12.7 ± 2.3	9.9 ± 1.1	49.0 ± 21.5	19.5 ± 6.6	5.8 ± 1.2	5.4 ± 0.8
MAP, cmH2O	13.7 ± 0.4	12.8 ± 0.6	14.8 ± 1.0	12.2 ± 0.4†	12.4 ± 10.6	11.8 ± 0.6
$\text{P}{\text{a}}_{\text{O}}{}_{{}_2}$ on 100%, mmHg	97 ± 25	275 ± 52*	124 ± 89	159 ± 66	359 ± 43	366 ± 38

Values are means ± SE. Bud, budesonide; C_dyn, dynamic compliance; F, female; M, male; MAP, mean airway pressure; OI, oxygenation index; Surf, surfactant; VEI, ventilation efficiency index; V_T, tidal volume.

^*P < 0.05 vs. 2 h Surf + Saline.

^†P < 0.05 vs. 6 h Surf + Saline.

Physiology.

Although the physiology results tended to favor budesonide groups, the dynamic compliance VEI and OI were not different throughout the ventilation period (Table 1). The mean airway pressures required to maintain oxygenation and ventilation were lower in the animals receiving budesonide at 6 h, but were not different at 2 h or 24 h. When animals were tested with 100% oxygen at the end of ventilation, animals receiving budesonide had higher $\text{P}{\text{a}}_{\text{O}}{}_{{}_2}$ values at 2 h. Mean blood pressures were higher for budesonide-treated animals (44 ± 1 mmHg) than for saline-treated animals (35 ± 1 mmHg) over the last 6 h of ventilation (P < 0.01).

Budesonide levels.

Plasma budesonide levels 15 min after delivery were 171 ± 45 ng/ml, with a rapid decrease to 3 ± 0.2 ng/ml at 24 h (Fig. 1A, left axis). On the basis of a blood volume of 80 ml/kg and the hematocrit measured for each blood sample, the amount of budesonide in the plasma at 15 min was 3.6 ± 0.9% of the initial dose (Fig. 1A, right axis) and decreased across time. The amount of unesterified budesonide in the lung tissue also decreased over time (Fig. 1B). The free budesonide level in the lungs at 2 h (28 ± 6 ng/100 mg lung tissue, 43 ± 7 μg total) was 14 times higher than for the 24-h animals (1.9 ± 0.6 ng/100 mg lung tissue, 2.1 ± 1.5 μg total). The lung tissue was treated with an esterase to deesterify budesonide, which increased budesonide by ~40% at the three time points. The total budesonide in the lung was equal to 7.3 ± 0.6% of the initial dose at 2 h, 3.1 ± 0.3% at 6 h, and 0.4 ± 0.1% at 24 h (Fig. 1B right axis). The budesonide levels in the bronchopulmonary lavage fluid were extremely low at all time points, with average values of 3.8 μg at 2 h, 1.0 μg at 6 h, and 0.3 μg at 24 h.

Fig. 1.

Budesonide in the plasma and lung. A: plasma budesonide over time in nanograms per milliliter (left axis) and as a percentage of the 0.75-mg dose (0.25 mg/kg dosed at 3 kg) given by tracheal instillation with surfactant (right axis). Percentage of dose was calculated on the basis of Hct of lamb and the assumption of a 80 ml/kg of blood volume. B: budesonide in lung in milligrams at 2 h, 6 h, and 24 h (left axis) and percentage of original 0.75-mg dose (right axis). Budesonide was extracted from lung tissue without (gray bars) and with (striped bars) deesterification to measure total budesonide in the lungs. Budesonide increased by 40% after deesterfication. n = 5 or 6 animals per time point and condition. Values are expressed as means ± SE. *P < 0.05 vs. the previous time point. #P < 0.05 vs. nonhydrolyzed.

Markers of lung injury.

The total protein recovered in the BALF was higher at 6 h in lambs that received only surfactant vs. lambs that received budesonide (Table 2). The lung gas volume measured at 40 cmH₂O at necropsy tended to be increased at 6 h in the Bud + Surf Group (P = 0.07). The dry-to-wet ratios increased with time of ventilation as the lung tissue lost water (P < 0.05 vs. previous time point), but these ratios were not changed with budesonide. The lung injury scores were similar between groups by histology. There were more iNOS-activated cells in the 24-h Surfactant and Saline Treatment Group than the 24-h Surfactant and Budesonide Group (Table 2).

Table 2.

Markers of injury

	2 h Surf + Saline	2 h Surf + Bud	6 h Surf + Saline	6 h Surf + Bud	24 h Surf + Saline	24 h Surf + Bud
Total protein, g/kg	1.6 ± 0.3	1.2 ± 0.2	2.1 ± 0.6	0.8 ± 0.2†	0.6 ± 0.1	0.5 ± 0.2
V40, ml/kg	37 ± 2	41 ± 3	27 ± 4	36 ± 3	30 ± 3	30 ± 3
Wet/dry weight	0.08 ± 0.01	0.11 ± 0.02	0.10 ± 0.01	0.13 ± 0.01	0.15 ± 0.01	0.16 ± 0.01
Injury score (out of 8)	2.6 ± 0.1	3.2 ± 0.3	5.1 ± 0.8	3.7 ± 0.3	5.8 ± 0.7	3.9 ± 0.6
iNOS+ cells (out of 2)	0.0 ± 0.0	0.0 ± 0.0	0.7 ± 0.3	0.3 ± 0.3	1.3 ± 0.3	0.2 ± 0.2‡
Bronchioalveolar lavage cytokine protein (fold increase over unventilated controls)
BAL IL-8, fold increase	16 ± 4	1.1 ± 0.7*	84 ± 31	75 ± 34	87 ± 24	45 ± 35
BAL MCP-1, fold increase	27 ± 3	4 ± 1*	27 ± 3	12 ± 3†	21 ± 3	12 ± 2

Values are means ± SE. BAL, bronchioalveolar lavage; iNOS, inducible nitric oxide synthase; MCP-1, monocyte chemoattractant protein-1; V₄₀, volume at 40 cmH₂O.

^*P < 0.05 vs. 2 h +Saline;

^†P < 0.05 vs. 6 h + Saline;

^‡P < 0.05 vs. 24 h + Saline.

Lung and systemic mRNA response.

Mechanical ventilation increased proinflammatory cytokine mRNA in the lungs at all time points relative to unventilated controls (Fig. 2, A–C). Compared with surfactant-only animals, budesonide decreased IL-1β mRNA at 2 h and 24 h (Fig. 2A), IL-6 mRNA at 6 h (Fig. 2B), and MCP-1 mRNA at 2, 6, and 24 h (Fig. 2C). In budesonide animals, IL-8 protein in BALF was lower at 2 h, and MCP-1 protein levels were lower at 2 and 6 h (Table 2). ENaC mRNA increased with time in all groups compared with unventilated controls. Surfactant and budesonide further increased ENaC mRNA at all time points relative to surfactant alone (Fig. 2D). SFTPB mRNA also increased about twofold in all groups compared with unventilated controls (P < 0.05), but budesonide did not change SFTPB mRNA relative to surfactant alone (data not shown).

Fig. 2.

Select mRNA levels in the lung, liver, and brain relative to unventilated controls. A–D: lung mRNA levels for the proinflammatory cytokines IL-1β (A), IL-6 (B), and monocyte chemoattractant protein-1 (MCP-1; C) decreased with budesonide and surfactant (Surf + Bud). IL-8 mRNA was not significantly different (D). E: epithelial sodium channel (ENaC) mRNA increased in the lungs with budesonide exposure. F: liver mRNA for MCP-1 was decreased by budesonide. G and H: periventricular white matter mRNA levels for IL-1β (G) and MCP-1 (H) were also decreased in the brains of budesonide animals. Individual animals (n = 6 or 7 per group) are represented. Values are means ± SE. *P < 0.05 vs. surfactant-only animals at 2, 6, or 24 h.

Budesonide decreased liver MCP-1 mRNA compared with surfactant at 2 h, and the ventilation-associated increase at 6 h was blocked by budesonide (Fig. 2F). The acute phase gene SAA3 mRNA increased similarly in the liver at 6 and 24 h in both groups with no effects of budesonide (data not shown). In the periventricular white matter of the brain, the mRNA for IL-1β was decreased compared with unventilated controls and surfactant-only animals at 2 and 6 h (Fig. 2G). The increase in MCP-1 mRNA with mechanical ventilation at 2 and 6 h were prevented by budesonide (Fig. 2H). There were no differences in mRNA for IL-6, IL-8, or TNF-α in the brains of any groups compared with unventilated controls.

DISCUSSION

We evaluated surfactant with budesonide relative to surfactant alone in a preterm sheep model using gentle ventilation to minimize injury and to model budesonide plus surfactant in the “normal” preterm ventilated lung. The combination of budesonide and surfactant decreased markers of lung injury and inflammation and improved some physiological variables in preterm lambs ventilated for 24 h. Budesonide also decreased the systemic effects of mechanical ventilation in the liver and brain. Budesonide levels were measurable in plasma for 24 h, which was consistent with the systemic effects.

A 0.25 mg/kg budesonide dose was used by Yeh et al. (23, 24) in their clinical trials. Yeh et al. found that greater than 0.5 mg/kg of budesonide decreased the surface tension lowering the ability of the surfactant. We also previously demonstrated no differences in anti-inflammatory effects with budesonide dose of 0.25 mg/kg or 1 mg/kg (11). The combination of budesonide and surfactant is stable at room temperature without effects on surface tension measurement and is easy to mix and administer at the bedside (2, 17, 24).

Although in many lung models the budesonide remains in the lungs, the ventilated lambs had budesonide measured in the plasma by 15 min, with higher plasma levels than previously reported (18, 24). The average 1-h plasma budesonide levels reported by Roberts et al. (18) in sheep were 25 ng/ml, somewhat lower than the 50 ng/ml found in this study. The lambs in this study were 132 days gestational age, when the lamb may have endogenous surfactants, which will mitigate injury from mechanical ventilation (9, 18). In preterm infants, the plasma budesonide was ~20 ng/ml at 30 min, which is one fourth the level that we found at 30 min (87 ± 20 ng/ml) (24). Plasma and lung tissue budesonide levels at 24 h were low in sheep and humans (11, 18, 24). The low levels are consistent with the half-life in the human fetal lung of ~4 h, with systemically released budesonide rapidly metabolized by the liver to 16-α-hydroxy prednisolone (2, 14, 24). Similar systemic release of radioactive dexamethasone or budesonide administered intratracheally occurred in rats and rabbits, where only 32 to 60% remained in the lungs after 30 min of ventilation (8, 15). The 40% increase in budesonide with deesterification supports the premise that preterm lungs can esterify budesonide that is released over time (2). Although we did not measure budesonide levels in liver and brain tissues, previous studies did not identify budesonide in the brain (18). Changes in mRNA in the brain are more sensitive for detecting responses to steroids than measurement of drug levels.

The improved oxygenation measured with 100% oxygen, and the decreased mean airway pressure to maintain oxygenation and ventilation in sheep treated with budesonide parallel the findings from other animals and human trials. In surfactant-depleted piglets, budesonide with surfactant improved $\text{P}{\text{a}}_{\text{O}}{}_{{}_2}$ and decreased lung injury and inflammation at 24 h (22). Similarly, infants had lower mean airway pressures on the first day of life when given budesonide with surfactant (23, 24). There were also trends in the other physiological variables (VEI and OI) and injury markers (V₄₀, the volume at 40 cmH₂O; injury scores; and total protein) that favor the budesonide and surfactant combination.

Budesonide had multiple anti-inflammatory effects in these preterm lambs. As increased cytokines in tracheal aspirates have been associated with the development of BPD (5), the decreased inflammation soon after delivery could affect the development of BPD. There were also fewer iNOS-positive inflammatory cells in the budesonide exposed lung. Similar to our findings, budesonide decreased the release of IL-8 and MCP-1 proteins and suppressed mRNA for 40 inflammatory genes in fetal lung explants (2). In studies by Yeh et al. (23, 24), budesonide decreased IL-1β, IL-6, and IL-8 in tracheal aspirates at 12 h, and IL-8 remained decreased at 8 days. Along with decreasing proinflammatory cytokines, budesonide had maturational effects on the lungs, with increases in SFTPB and ENaC mRNA. Similarly, expression of surfactant proteins increased in human fetal lung explants exposed to budesonide and in fetal rabbits given intratracheal budesonide with surfactant (2, 13). The epithelial sodium channel is important for clearance of fluid near the time of birth and is low in preterm fetuses. ENaC increases toward term and is steroid-responsive. Postnatal budesonide increased ENaC, which should improve respiratory transition at birth. Budesonide mixed with surfactant decreased lung inflammation and triggered lung maturation in ventilated preterm sheep.

The decrease in proinflammatory cytokines in the liver and brain could result from direct effects of the systemic levels of budesonide or indirectly from suppression of lung inflammation. Prolonged mechanical ventilation alone was linked to abnormal MRI findings and poor neurologic outcomes (5). In preterm sheep, even short periods of ventilation cause brain inflammation and MRI changes (1, 10). Serum protein profiles, including multiple proinflammatory cytokines (MCP-1, IL-8), are predictive of white matter injury in ventilated preterm infants. A direct effect of budesonide is likely because it has 10 times the affinity of dexamethasone for the glucocorticoid receptor. Infants treated with budesonide and surfactant had similar neurodevelopmental outcomes to other infants in small randomized trials (23, 24). Infants randomized to inhaled budesonide also had similar neurological outcomes to controls, but mortality was increased (3). Further evaluation of the effects of budesonide on the premature brain is warranted.

The experiment has some limitations from the animal group size of 6 to 7. Small differences between treatment groups for individual markers of injury could be missed. By using multiple markers of injury, we demonstrate an overall protective effect response from budesonide. The lambs received the same dose of budesonide and surfactant based on an estimated weight, with an average dose 0.23 mg/kg budesoinde. Alternative ventilation strategies (higher positive end-expiratory pressure) or antenatal steroid therapy may change the results but were not tested. There was a waning effect of the budesonide by 24 h, and thus, additional doses would be of interest.

Conclusions.

Budesonide, when mixed with a surfactant, decreased markers of inflammation and injury in preterm sheep ventilated for up to 24 h. The majority of the budesonide was released from the lung and cleared over the 24 h of mechanical ventilation, with a significant portion being lost from the lungs by 2 h of mechanical ventilation. Budesonide also decreased proinflammatory markers in the liver and brain, demonstrating systemic effects. Budesonide mixed with surfactant has promise for decreasing the lung inflammation that contributes to the development of BPD in preterm infants, but the systemic effects should be recognized.

GRANTS

This work was supported by National Institutes of Health National Institute of Child Health and Human Development Grant R01-HD-072842 (A. H. Jobe) and and Chiesi Farmaceutici (A. H. Jobe and N. H. Hillman). M. W. Clarke is affiliated to Metabolomics Australia, University of Western Australia, which is supported by infrastructure funding from the Western Australian and Australian Federal Government, through Bioplatforms Australia and the National Collaborative Research Infrastructure Strategy.

DISCLOSURES

Fabrizio Salomone is employed by Chiesi Farmaceutici. Chiesi Farmaceutici provided funding for the project, but the experiments were performed and analyzed by the other authors. Chiesi approved the manuscript. None of the other authors has any conflicts of interest, financial or otherwise, to disclose.

AUTHOR CONTRIBUTIONS

M.W.K., F.S., G.C.M., A.H.J., and N.H.H. conceived and designed research; T.B.K., M.W.K., A.F.S., E.R., F.S., G.C.M., A.H.J., and N.H.H. performed experiments; T.B.K., M.W.K., A.F.S., E.R., M.W.C., G.C.M., A.H.J., and N.H.H. analyzed data; T.B.K., M.W.K., A.F.S., E.R., M.W.C., G.C.M., A.H.J., and N.H.H. interpreted results of experiments; T.B.K. and N.H.H. prepared figures; T.B.K., M.W.K., A.F.S., E.R., G.C.M., A.H.J., and N.H.H. drafted manuscript; T.B.K., M.W.K., A.F.S., E.R., F.S., M.W.C., G.C.M., A.H.J., and N.H.H. edited and revised manuscript; T.B.K., M.W.K., A.F.S., E.R., F.S., M.W.C., G.C.M., A.H.J., and N.H.H. approved final version of manuscript.