AP301, a synthetic peptide mimicking the lectin-like domain of TNF, enhances amiloride-sensitive Na þ current in primary dog, pig and rat alveolar type II cells Q1 Susan Tzotzos a, * , Bernhard Fischer a , Hendrik Fischer a , Helmut Pietschmann a , Rudolf Lucas b , Gilles Dupré c , Rosa Lemmens-Gruber d , Parastoo Hazemi d , Victoria Prymaka d , Waheed Shabbir d a Apeptico Research and Development GmbH, Mariahilferstrasse 136,1150 Vienna, Austria b Vascular Biology Center, Georgia Health Sciences University, Augusta, GA 30912, USA c Clinic for Small Animal Surgery, University of Veterinary Medicine Vienna,1210 Vienna, Austria d Department of Pharmacology and Toxicology, University of Vienna, Althanstrasse 14,1090 Vienna, Austria article info Article history: Received 18 May 2012 Received in revised form 11 December 2012 Accepted 31 December 2012 Keywords: AP301 ENaC Synthetic cyclic peptide Lectin-like domain Alveolar fluid clearance Type II alveolar cells Patch clamp abstract Pulmonary permeability oedema is a frequent complication in a number of life-threatening lung condi- tions, such as ALI and ARDS. Apart from ventilation strategies, no specific therapy yet exists for treatment of these potentially fatal illnesses. The oedema-reducing capacity of the lectin-like domain of TNF (TIP) and of synthetic peptides, mTIP and hTIP, which mimic the TIP domain of mouse and human TNF, have been demonstrated in various studies in rodents. Cell-based electrophysiological studies have revealed that the alveolar fluid clearing capacity of TNF and the TIP peptides is due to activation of the amiloride-sensitive Na þ current in alveolar epithelial cells and that the primary site of action is on the apical side of these cells. AP301, a synthetic cyclic peptide mimicking the TIP domain of human TNF is currently undergoing clinical trials as a therapy for pulmonary permeability oedema. AP301 has been shown to improve alveolar liquid clearance and lung function in a porcine model of ALI. For non-clinical regulatory assessment, dog, pig and rat are standard animal models; accordingly, pre- clinical toxicological and pharmacological safety studies have been conducted with AP301 in dogs and rats. Hitherto, no studies have assessed the pharmacodynamic effect of AP301 on primary canine or porcine type II AEC. The current study describes the effect of AP301 on the amiloride-sensitive Na þ current in type II AEC isolated from dog, pig and rat lungs. In whole cell patch clamp experiments with dog type II AEC, an increase in the amiloride-sensitive Na þ current from 3.7 pA to 49.4 pA was observed in the presence of AP301; in pig type II AEC, an increase from 10.0 pA to 159.6 pA was observed, and in rat AEC, from 6.9 pA to 62.4 pA. In whole cell patch clamp experiments in A549 cells, AP301-induced enhancement of the amiloride-sensitive current was eliminated when Na þ in the bath solution was replaced with N-methyl-D- glucamine (NMDG), and when the cells were pre-incubated with 5-aminoimidazole-4-carboxamide-1-ß- D-ribofuranoside (AICAR), an inhibitor of ENaC, but enhancement was unaffected by addition of cyclic nucleotide-gated (CNG) channel inhibitors Zn 2þ or L-cis-diltiazem prior to AP301. These results provide strong evidence that AP301 activates the amiloride-sensitive Na þ current through ENaC in type II AEC from dog, pig and rat. To our knowledge, this is the first cell-based analysis of the oedema-clearing effect of AP301 observed in the porcine model of pulmonary oedema. Furthermore, the results validate the dog and pig models in non-clinical assessment of AP301. Ó 2013 Published by Elsevier Ltd. 1. Introduction Pulmonary permeability oedema is a frequent complication in a number of severe and life-threatening lung conditions, such as ALI, ARDS or ischaemia reperfusion injury, the latter of which can cause primary graft failure following lung transplantation. In ALI/ ARDS patients, higher rates of alveolar fluid clearance (AFC) have Abbreviations 1 : AEC, alveolar epithelial cell; AFC, alveolar fluid clearance; ALI, acute lung injury; ARDS, acute respiratory distress syndrome; ENaC, epithelial so- dium channel; EVLWI, extravascular lung water index; TIP, lectin-like domain of TNF; hTIP, synthetic peptide mimicking the TIP domain of human TNF; mTIP, synthetic peptide mimicking the TIP domain of murine TNF. * Corresponding author. Tel.: þ43 664 88504559; fax: þ43 125330337795. E-mail address: s.tzotzos@apeptico.com (S. Tzotzos). 1 Amino acids and designation of peptides according to the IUPAC-IUB Joint Commission on Biochemical Nomenclature Recommendations 1983 (IUPAC-IUB, 1984). Contents lists available at SciVerse ScienceDirect Pulmonary Pharmacology & Therapeutics journal homepage: www.elsevier.com/locate/ypupt 1094-5539/$ e see front matter Ó 2013 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.pupt.2012.12.011 Pulmonary Pharmacology & Therapeutics xxx (2013) 1e8 Please cite this article inpress as: Tzotzos S, et al., AP301, a synthetic peptide mimicking the lectin-like domain of TNF, enhances amiloride- sensitive Na þ current in primary dog, pig and rat alveolar type II cells, Pulmonary Pharmacology & Therapeutics (2013), http://dx.doi.org/ 10.1016/j.pupt.2012.12.011 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102 103 104 105 106 107 108 109 110 YPUPT1251_proof ■ 12 January 2013 ■ 1/8