Intra-tracheal Administration of Budesonide/Surfactant to Prevent Bronchopulmonary. Dysplasia. Online Data Supplement. Tsai, Hung C.

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1 Intra-tracheal Administration of Budesonide/Surfactant to Prevent Bronchopulmonary Dysplasia Online Data Supplement Tsu F. Yeh, Chung M. Chen,. Shou Y. Wu, Zahid Ullah, Tsai C. Li, Wu S. Hsieh, Chang H. Tsai, Hung C. Lin 1 Maternal Child Health Research Center, College of Medicine, Taipei Medical University, Taipei, Taiwan; 2 Department of Pediatrics, Children s Hospital, China Medical University, Taichung, Taiwan; 3 Department of Pediatrics, Taipei Medical University Hospital, Taipei, Taiwan; 4 Department of Pediatrics, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan; 5 Division of Neonatology, John Stroger s Hospital of Cook County, Chicago, USA; 6 Graduate Institute of Biostatistics, College of Public Health,China Medical University, Taichung, Taiwan; 7 Department of Healthcare Administration, College of Health Science, Asian University, Taichung, Taiwan; 8 Department of Pediatrics, College of Medicine, National Taiwan University and Hospital, Taipei, Taiwan; and 9 Department of Biotechnology, Asian University, Taichung, Taiwan. E1

2 Introduction Surfactant has been used as a vehicle to deliver steroid in animals (1, 2) and in preterm infants with RDS in a pilot study (3). However, the stability associated with administering a steroid/surfactant mixture have not been well studied. Budesonide (Pulmicort neubulising suspension, Astra Zeneca, Lund, Sweden), a dehalogenic glucocorticoid, and survanta (Abbott, Columbus, OH), a mixture of phospholipid and hydrophobic proteins, both are stable compounds. However, budesonide, (22R,S)-16α, 17α-butylenedioxy-11β, 21-dihydroxypregna-1,4-diene-3, 20-dione, having a ring structure with a certain degree of un-saturation and branching, may interfere with the structure of the surfactant monolayer at the air-liquid interface and, thus, may affect the surface-tension property of survanta. We, therefore, conducted an in vitro study if the mixture of survanta and budesonide is biophysically stable; and if the survanta and budesonide mixture is chemically stable. Method Biophysical and Chemical Stability of Survanta/Budesonide Mixture To test the biophysical stability of surfactant after addition of budesonide, a series of tests were performed using a surfactometer. Competitive absorption behavior of a survanta-budesonid suspension with different concentration ratios between these two compounds was conducted in a surfactometer (Amherst Electronics, Buffalo, NY). The dynamic surface tension behavior of survanta, budesonide, and their mixtures was evaluated E2

3 by pulsating the air-liquid interfaces at a rate of 20 cycles per minute at 37. To test the chemical stability of the survanta/budesonide mixture, high performance liquid chromatography (HPLC) was performed (SGS, Taoyen, Taiwan Ltd) at 0, 1, 4, 8, 12, and 24 hours after mixture of survanta/budesonide, with a concentration ratio of 25:1, 50:1, and 100:1. Animal Study To investigate if surfactant can be used as a vehicle to facilitate budesonide delivery, Sprague-Dawley rats were intratracheally injected with 50 μl surfactant/ 18 F-budesonidemixture (with a concentration ratio of 12.50:0.12 mg/ml by equal volume mixing, n = 3) or with 50 μl 18 F-budesonideonly (0.12 mg/ml, n = 3). The 18 F-budesonidebiodistribution and radioactivity was visualized and measured at 15, 30, 45, and 60 min after injection by a Nano/PET/CT digital scan. Statistical Analysis Data are expressed as means ± SD. Between-group comparisons were made using Student s t-tests. Differences were considered significant at P < Results Biophysical and Chemical Stability of Survanta/Budesonide Mixture When a survanta suspension was mixed with an equal volume of a budesonide suspension, at a concentration ratio of 12.50:0.25 mg/ml (50:1) or greater, the dynamic surface activity of E3

4 survanta suspension as shown in sufactometer was minimally affected (Table 1). Based on these results, we decided that the dosage between survanta/budesonide ratio for neonates should be 50:1. The results were reported previously (4). The HPLC analysis revealed that no new compounds were identified at any of the time points after mixing survanta and budesonide at various concentration ratios (Figure 1A to 1T). Thus, the mixture of budesonide and survanta appears to be chemically stable. Pulmonary Distribution of 18 F-Budesonide The radioactivityof 18 F-budesonide was most strongly detected near the trachea at 15 min after intra-tracheal injection (Figure S2A). Almost no radioactivity was seen in the lung region of the rats injected with 18 F-budesonide alone at 60 min. The radioactivity of 18 F-budesonide was distributed more in the peripheral lung and stayed longer in rats supplemented with surfactant than in the rats without surfactant. Rats injected with surfactant/ 18 F-budesonide mixture exhibited an approximately 200% increase in radioactivity compared with rats that received 18 F-budesonide alone during the study period (Figure 2B). The detail results will be published elsewhere. Conclusion Surfactant can be used as an effective vehicle to facilitate the delivery of budesonide into the lungs. With a concentration ratio of survanta/budesonide 50, the mixture is biophysically and chemically stable. E4

5 References 1. Fajardo C, Levin D, Garcia M, Adrams D, Adamson I. Surfactant versus saline as a vehicle for corticosteroid delivery to the lungs of ventilated rabbits. Pediatr Res 1998;43(4 pt 1): Chen CM, Fang CL, Chang CH. Surfactant and corticosteroid effects on lung function in a rat model of acute lung injury. Crit Care Med 2001;29: Yeh TF, Lin HC, Chang CH, Wu TS, Su BH, Li TC, Suma P, Tsai CH. Early intratracheal instillation of budesonide using surfactant as a vehicle to prevent chronic lung disease in preterm infants: a pilot study. Pediatrics 2008;121;e1310-e Chang DH, Chang CH, Lin YJ, Yeh TF. Influence of budesonide (B) on the dynamic surface tension behavior of surfactant (survanta) (s) at pulsating air liquid interface. Pediatr Res 2002;51:346A 5. Hvizdos KM, Jarvis B. Budesonide inhalation suspension: a review of its use in infants, children and adults with inflammatory respiratory disorders. Drugs 2000;60: E5

6 Table E1. Physical property of mixture of surfactant/budesonide Dynamic Surface Tension Behavior of Survanta, Budesonide, and Their Mixtures System Phospholipid Budesonide Ye, Ymin, Ymax, Concentration, Concentration, mn/m mn/m mn/m mg/ml mg/ml S suspension B suspension Mixed S/B suspension S indicates survanta; B, budesonide; mn/m, milli-newton/meter; Ye, equilibrium surface tension; Ymin, minimum surface tension; Ymax, maximum surface tension (from Yeh et al. Pediatrics 2008;121:e1310-e1318). E6

7 Figure Legends Figure 1. HPLC performed on budesonide, survanta, and survanta/budesonide mixture, comprising different concentration ratio of mixture (25:1, 50:1, and 100:1) at 0, 1, 4, 8, 12, and at 24 hours after mixing of the two drugs. There was no new compound identified during these tests, indicating that budesonide/survanta mixtures are chemically stable. Figure 2. The 18 F-budesonide bio-distribution (A) and radioactivity (B) in the Sprague-Dawley rats intra-tracheal injected with surfactant/ 18 F-budesonide (n = 3) or 18 F-budesonide alone (n = 3). The 18 F-budesonide was distributed more into the peripheral lungs and the accumulated 18 F-budesonide radioactivity was higher in the rats supplemented with S than in the rats without S during the study period. B = 18 F-budesonide, BS = surfactant/ 18 F-budesonide. E7

8 Fig-1 A Budesonide (B) 0.5 mg/ml Fig-1C S/B: 50/1 at 0 hr Fig-1B Survanta (S) 25 mg/ml Fig -1D S/B: 50/1 at 1 hr E8

9 Fig -1E S/B: 50/1 at 4 hr Fig -1G S/B: 50/1 at 12 hr Fig -1F S/B: 50/1 at 8 hr Fig -1H S/B: 50/1 at 24 hr E9

10 Fig -1I S/B: 25/1 at 0 hr Fig -1K S/B: 25/1 at 4 hr Fig -1J S/B: 25/1 at 1 hr Fig -1L S/B: 25/1 at 8 hr E10

11 Fig -1M S/B: 25/1 at 12 hr Fig -1O S/B: 100/1 at 0 hr Fig -1N S/B: 25/1 at 24 hr Fig -1P S/B: 100/1 at 1 hr E11

12 Fig -1Q S/B: 100/1 at 4 hr Fig -1S S/B: 100/1 at 12 hr Fig -1R S/B: 100/1 at 8 hr Fig -1T S/B: 100/1 at 24 hr E12

13 Figure 2 E13

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