nature publishing group Articles Translational Investigation Antenatal dexamethasone vs. betamethasone dosing for lung maturation in fetal sheep Augusto F. Schmidt1, Matthew W. Kemp2, Paranthaman S. Kannan1, Boris W. Kramer3, John P. Newnham2, Suhas G. Kallapur1 and Alan H. Jobe1 BACKGROUND: Dexamethasone-phosphate (Dex-PO4) and glucocorticoid usually given in four doses of 6 mg each, 12 h the combination betamethasone-phosphate (Beta-PO4) + apart, for a total dose of 24 mg, but other dosing regimens betamethasone-acetate (Beta-Ac) are the most used antena- are also frequently used such as two doses of 12 mg, 24 h tal corticosteroids to promote fetal lung maturation. We com- apart. Betamethasone is usually given as a combination of pared fetal lung maturation induced by Beta-Ac+Beta-PO , 4 equal parts betamethasone phosphate (Beta-PO4) and beta- Dex-PO4, or Beta-PO4 alone. methasone acetate (Beta-Ac) in two doses 24 h apart of 12 mg METHODS: Pregnant ewes received two intramuscular doses each for a total dose of 24 mg. Beta-PO4 has a short biological 24h apart of 0.25mg/kg/dose of Beta-Ac+Beta-PO , Dex-PO4 or 4 half-life, similar to Dex-PO4, while Beta-Ac is a slow-release Beta-PO4; or 2 doses of 0.125 mg/kg/dose of Beta-PO4 at 6, 12, suspension with a longer half-life (3,4). In clinical practice, or 24h intervals. Fetuses were delivered 48h after the first dose different formulations, such as Beta-PO4 alone, and dosing and ventilated for 30 min. We assessed ventilatory variables, vital intervals are used despite limited data from animal or human signs, and blood gas. After ventilation pressure-volume curves studies (1,4). were measured and lungs were sampled for analysis. A meta-analysis of the clinical trials comparing dexametha- RESULTS: All treatments improved lung compliance and sone to betamethasone showed no difference in respiratory ventilation efficiency. Only Beta-Ac + Beta-PO4 required lower distress syndrome or neonatal mortality, but a decreased inci- positive inspiratory pressure compared with control. Beta-Ac + dence of intraventricular hemorrhage (IVH) with Dex-PO4, Beta-PO4 and Beta-PO4 alone, but not Dex-PO4, increased the mostly influenced by the trial from Elimian et al. (4,5). Subtil mRNA of surfactant proteins compared with control. Low-dose et al. compared the combination of Beta-PO4 + Beta-Ac to Beta-PO did not increase mRNA of surfactant proteins. There 4 Beta-PO4 alone in 69 infants and observed no difference in the were no differences among Beta-PO4 treatment intervals. outcomes of neonatal death, respiratory distress syndrome, or CONCLUSION: Beta-Ac + Beta-PO4 given as two doses 24 h IVH (6). A large randomized controlled trial of betamethasone apart was more effective in promoting fetal lung maturation combination drug vs. dexamethasone using a 2 dose 12 mg and than Dex-PO4 or Beta-PO4 alone, consistent with a prolonged 24-h treatment interval with long term follow up is currently exposure provided by the Beta-Ac + Beta-PO4. These results underway in Australia (7). support the clinical use of combined Beta-Ac + Beta-PO prep- 4 A recent trial of antenatal corticosteroids using Dex-PO4 arations over phosphate corticosteroids alone for fetal lung in low resource environments including almost 100,000 live- maturation. births found no benefit for small infants but increased mor- tality for large infants exposed to prenatal Dex-PO4 (8). The ability to distinguish between treatment responses to different ntenatal corticosteroids decrease the incidence of respira- corticosteroids, doses, and treatment intervals is impractical Atory distress syndrome and other neonatal morbidities in given the number of variables and large number of patients preterm infants and are considered standard of care for women needed for multiple trials. The sheep model of preterm birth at risk of preterm delivery (1). Despite widespread clinical use and lung maturation allows the analysis of gas exchange and there is minimal experimental evidence regarding the formu- ventilatory variables in a stage of lung development similar to lation or dosing regimen, thus multiple different treatments preterm newborns (9). To provide guidance about maturation are used worldwide (2). responses we compared the fetal lung maturation from clini- Dexamethasone and betamethasone are the fluorinated cally used doses of antenatal Beta-PO4+Beta-Ac to Dex-PO4 or steroids most commonly used for maternal treatment (2). Beta-PO4 alone, as well as a lower dose of Beta-PO4 alone with Dexamethasone phosphate (Dex-PO4) is a short acting different dosing intervals, in preterm sheep. 1Division of Neonatology and Pulmonary Biology, Cincinnati Children’s Hospital Medical Center, Cincinnati, Ohio; 2School Women’s and Infants’ Health, University of Western Australia, Perth, Australia; 3Department of Pediatrics, University of Maastricht, Maastricht, Netherlands. Correspondence: Alan H. Jobe ([email protected]) Received 16 May 2016; accepted 26 September 2016; advance online publication 11 January 2017. doi:10.1038/pr.2016.249 496 Pediatric REsEarcH Volume 81 | Number 3 | March 2017 Copyright © 2017 International Pediatric Research Foundation, Inc. Steroid formulation for lung maturation Articles METHODS compliance was recorded as measured by the ventilator. The ventila- Antenatal Corticosteroid Treatments tion efficiency index (VEI) was calculated using the formula VEI = The animal ethics committee of The University of Western Australia 3,800/(respiratory rate (PIP − PEEP) × PCo2 (mm Hg)). approved the animal studies (RA/3/100/1378). All animals received Lung Assessment an i.m. injection of 150 mg medroxyprogesterone acetate (Depo- After ventilation for 30 min lambs were disconnected from the ven- Provera, Pfizer, New York, NY) at 115 ± 1 d gestation to decrease the tilator and the endotracheal tube was clamped for 2 min to achieve risk of steroid-induced premature labor. Time-mated Merino ewes atelectasis by oxygen absorption. The lambs received a lethal dose with singleton fetuses received maternal i.m. injections based on of pentobarbital, were weighed and the chest was opened for visual maternal weight of normal saline or corticosteroid treatments with evaluation of gross lung injury–pulmonary hemorrhage, pulmonary the first dose given on 120 ± 1 d of gestation (80% of gestation, term interstitial emphysema, gas pockets within the lung or subpleural = 150 d). A composite group of control ewes received two doses of dissection—performed by the same investigator. A deflation pres- normal saline i.m. 6, 12, or 24 h apart. Treatment groups received: two sure-volume curve was measured after air inflation of the lungs to a doses of Beta-PO4+Beta-Ac (Celestone Chronodose, Merck Sharp pressure of 40 cmH O (8). & Dohme, Australia) 0.25 mg/kg/dose i.m. 24 h apart; two doses of 2 Quantitation of mRNA Dex-PO4 (DBL Dexamethasone sodium phosphate, Hospira NZ, Total RNA was isolated from frozen lungs after homogenization with New Zealand) 0.25 mg/kg/dose i.m. 24 h apart; two doses of Beta-PO4 (Betnesol, Focus Pharmaceuticals, UK) 0.25 mg/kg/dose i.m. 24 h TRIzol (Invitrogen, Carlsbad, CA). Reverse transcription was per- formed using Verso cDNA kit (Thermo Scientific, Waltham, MA) apart. Other groups received two doses of Beta-PO4 0.125 mg/kg/dose i.m. 6, 12, or 24 h apart. We adopted the standard dose of 0.25 mg/kg/ to produce single-strand cDNA. The genes for surfactant protein A (SPA), surfactant protein B (SPB), surfactant protein C (SPC), sur- dose of Beta-PO4 + Beta-Ac to maintain dosing regimens comparable with previous studies from our group. All animals were delivered 48 h factant protein D (SPD), ATP-binding cassette subfamily A member after the first i.m. injection at 122 ± 1 d of gestation. 3 (ABCA3)——expressed by type 2 alveolar cells, and aquaporin 5 (AQP5)—expressed by type 1 alveolar cells, were amplified using Ventilatory Assessment the cDNA template and sheep-specific primers along with Taqman For delivery pregnant ewes received i.v. midazolam (0.5 mg/kg) probes (Applied Biosystems, Foster City, CA). The mRNA expression and ketamine (10 mg/kg) for sedation. Then, 3 ml of 2% (20 mg/ml) for each gene was normalized to the mRNA for the ribosomal protein lidocaine was given for spinal anesthesia for delivery of the fetus. 18s as internal standard. Final data are expressed as fold increase over The head of the fetus was delivered through abdominal and uterine the control value. incisions and the fetal skin over the trachea was infiltrated with lido- caine. A 4 mm endotracheal tube was secured by tracheostomy. After Statistical Analysis delivery of the fetus the ewe was killed with pentobarbital. The lamb Data are presented as mean ± SEM. Statistical tests were performed was weighed and dried, and temperature maintained with a radiant with Prism software 6.0 (GraphPad Software, San Diego, CA). Initial warmer (Cozy Cot, Fisher & Paykel Healthcare, New Zealand) and comparisons were performed with ANOVA followed by multiple a plastic cover (Neowrap, Fisher & Paykel, NZ). Mechanical venti- groups comparison with Tukey’s post-hoc test, with separate ANOVA lation (Fabian HFO, Accutronic Medical Systems AG, Switzerland) tests performed for the comparison among Beta-PO4 + Beta-Ac, was immediately started with the following settings: peak inspira- Dex-PO4, and Beta-PO4 alone, among Beta-PO4 + Beta-Ac, standard and low dose Beta-PO , and among the low-dose Beta-PO intervals. tory pressure (PIP) of 40 cmH2O, positive end expiratory pressure 4 4 Selected group comparisons were performed by t-tests where appro- (PEEP) of 5 cmH2O, respiratory rate of 50 breaths per minute, inspi- ratory time of 0.6 s, and 100% heated and humidified oxygen. The priate and are indicated in the results section. Significance was attrib- standard use of 100% oxygen allows the comparison of oxygenation uted for P-values < 0.05.