AP301, a synthetic peptide mimicking the lectin-like domain of TNF, enhances amiloride-sensitive Na(+) current in primary dog, pig and rat alveolar type II cells

Abstract

Pulmonary permeability oedema is a frequent complication in a number of life-threatening lung conditions, 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(+) 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(+) 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(+) 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(+) 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(2+) or l-cis-diltiazem prior to AP301. These results provide strong evidence that AP301 activates the amiloride-sensitive Na(+) 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.

Figure 1

Effect of AP301 on Na⁺ current in a dog alveolar epithelial type II cell patched in the whole cell mode. Representative original recording from a cell clamped at a holding potential of - 100 mV during control phase and following addition of AP301 (240 nM) to the bath solution.

Figure 2

Effect of AP301 on Na⁺ current in a dog, pig and rat alveolar epithelial type II cells patched in the whole cell mode. Inward current was elicited by application of a pulse of −100 mV. Addition of AP301 (240 nM) and amiloride (1 mM) to the bath solution was performed in each case at the time point indicated by the arrow.

Figure 3

Mean values of inward Na⁺ currents in dog (n= 3), pig (n=5) and rat (n=3) primary alveolar epithelial type II cells and A549 cells (n=5), clamped at −100 mV in a whole cell patch, during control phase, following addition of AP301 (240 nM) and after final addition of amiloride (1 mM) to the bath solution. Values are mean +/−SE.

Figure 4

Concentration response curves for nebulised and untreated AP301 in A549 cells; mean values +/−SE for three to five experiments are given.

Figure 5

Effect on AP301-induced current of replacing NaCl in the bath solution with equimolar concentration of NMDG-Cl (140 mM) in a whole cell patch clamp assay using A549 cells (n=5). Mean values (+/−SE) of inward currents in cells clamped at −100 mV.

Figure 6

Effect of inhibitors of CNG channels on amiloride-sensitive Na⁺ current induced by AP301 in A549 cells patched in whole cell mode. Inward current elicited by a pulse of −100 mV. a) Mean values of inward currents during control phase, following sequential addition of Zn²⁺ (1 mM), AP301 (120 nM) and final addition of amiloride (100 µM) to the bath solution (n=5). b) Mean values of inward currents during control phase, following sequential addition of L-cis-diltiazem (300 µM), AP301 (120 nM) and final addition of amiloride (100 µM) to the bath solution (n=3). Values are mean +/−SE.

Figure 7

Multiple sequence alignment of TIP domain in human, pig, dog, rat and mouse TNF, mTIP peptide and hTIP peptide/AP301. The human and pig sequences are identical, the dog differs at one position, and the mouse and rat sequences, which are identical, differ at four positions from the human TNF sequence. Three residues which have been shown to be essential for the ENaC-enhancing activity of the TIP domain are conserved throughout all species (highlighted and underlined); in AP301 these residues are T6, E8 and E11 [8]. Both human and mouse TIP peptides are derived from the corresponding native sequences by replacing the N-terminal (P) and C-terminal (E) residues with cysteine residues and replacing the native cysteine with a glycine residue. Cyclisation of the TIP peptides is effected by oxidation of the terminal cysteine residues [8]. Alignment carried out manually using Accelrys DS Visualiser 2.0.