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-β-D-ribofuranoside (AICAR), an inhibitor of ENaC, but enhancement was unaffected by addition of cyclic nucleotide-gated (CNG) channel inhibitors Zn2+ 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.

A Novel Tumor Necrosis Factor–mediated Mechanism of Direct Epithelial Sodium Channel Activation

“…In previous studies, our research group and other investigators have demonstrated that the lectin-like domain of TNF represents a potent stimulator of ALC in several mammalian species and involves activation of ENaC via an unknown mechanism (21,24,(27)(28)(29)(30)(31)38) . This study was designed to determine the mechanism by which the lectin-like domain of TNF can promote ENaC activity.…”

“…Because both Na (29) and ENaC (27,28,38) can be activated by the TIP peptide, we investigated in more detail which of these Na 1 transporters is the primary target in a homologous cell system and whether the peptide has a direct or indirect effect on their activity.…”

“…These activities are most likely mediated by the lectin-like domain of the cytokine (24), which is spatially distinct from the receptor binding sites (25,26). The lectin-like domain of TNF is mimicked by the 17-amino acid circular TIP peptide (26), which increases amiloride-sensitive Na 1 uptake in type II AECs (27,28). The TIP peptide has been shown to activate ALC in several animal models of hydrostatic and permeability edema (21,(29)(30)(31).…”

Mechanism of Action of Novel Lung Edema Therapeutic AP301 by Activation of the Epithelial Sodium Channel

“…The current-enhancing effect of AP301 is not inhibited by CNG channel blockers, suggesting that AP301 activates Na 1 current flowing through ENaC (Tzotzos et al, 2013).…”

“…AP301 [Cyclo(CGQRETPEGAEAKPWYC)], a cyclic peptide comprising the human TIP sequence and currently being developed as a treatment for lung edema (phase II clinical trials), has been shown to reduce extravascular lung water and improve lung function in a pig model of acute lung injury (Hartmann et al, 2013) and to enhance the amiloride-sensitive Na 1 current in freshly isolated ATII cells from dog, pig, and rat lungs (Hamacher et al, 2010;Tzotzos et al, 2013). The current-enhancing effect of AP301 is not inhibited by CNG channel blockers, suggesting that AP301 activates Na 1 current flowing through ENaC (Tzotzos et al, 2013).…”