Protection Against Acute Lung Injury by Intravenous or Intratracheal Pretreatment with EPI-HNE-4, a New Potent Neutrophil Elastase Inhibitor

Abstract

Excessive accumulation of active neutrophil elastase (NE) in pulmonary fluids and tissues of patients with cystic fibrosis (CF) is thought to act on the lungs, compromising their structure and function. The aim of this study was to investigate the in vitro and in vivo protective effect of a new, rapidly acting, potent (Ki = 5.45 x 10(-12) M and Kon = 8 x 10(6) M(-1) s(-1)) and specific human NE inhibitor, EPI-HNE-4, engineered from the Kunitz domain. The results demonstrated that this inhibitor was able to (i) effectively inhibit in vitro the high levels of active NE present in a medium as complex as sputum from children with CF, with a measured IC(50) equal or close to the calculated IC(50) in 60% of cases, and (ii) almost completely block (91%) the N-formyl-methionine-leucine-phenylalanine-induced migration of purified human neutrophils across a Matrigel basement membrane. Intratracheal administration (250, 175, or 100 microg per rat) of the inhibitor 5 min before instillation of pure human NE (HNE) (150 microg per rat) to rats induced effective, dose-dependent protection of the lungs, 4 h later, from hemorrhage, serum albumin leakage, residual active NE, and discrete neutrophil influx in air spaces induced by instillation of pure HNE. Intravenous administration (3 mg per rat) of EPI-HNE-4, 15 min before instillation of the soluble fraction of pooled sputum (delivering 120 microg of active NE per rat) from children with CF, effectively reduced (64%), 4 h later, the massive neutrophil influx induced by sputum instillation. Overall, these data strongly suggest that associated aerosol and systemic administration of EPI-HNE-4 would be beneficial in the treatment of CF.

Structural Characterization of Mouse Neutrophil Serine Proteases and Identification of Their Substrate Specificities

“…Another example is the recently engineered hNE inhibitor, EPI-hNE4. This molecule initially designed as a putative therapeutic for lung inflammatory diseases does not inhibit mNE (23,31). It must be emphasized that crystal/x-ray structures of mouse neutrophil serine proteases need to be determined to ascertain our findings and gain in-depth insights on the structural differences with their human counterparts.…”

Neutrophil Elastase Modulates Cytokine Expression

“…induction of TNF-␣, MIP-2, and IL-6 transcript expression) will be best defined using mice that are deficient in all these proteases. Regarding cell culture studies, the concentration of NE used in this study is relevant because it can be reached or even exceeded in the lungs in diseased conditions (26). In fact, these suggested estimates do not even take into account possible evanescent quantum bursts of pericellular NE activity, local microenvironment, or the half-life of NE in tissues, which might increase the effective concentration of the enzyme (7).…”

Venular basement membranes contain specific matrix protein low expression regions that act as exit points for emigrating neutrophils

“…Because the serine protease neutrophil elastase is known to be expressed on the cell surface of transmigrating neutrophils both in vitro (39) and in vivo (32) and has been implicated in neutrophil migration through the BM (17,32,39,40), the role of this protease in disruption of the BM in IL-1β-stimulated tissues was investigated. Data presented here implicate neutrophil-derived serine proteases such as NE in increased size of laminin 10 LE regions during cytokine-induced neutrophil transmigration.…”

“…It is of course highly probable that NE may have indirect roles in mediating neutrophil transmigration, for example, via activation of other leukocyte proteases such as MMP-9 (17) or generation of chemotactic fragments by selective cleavage of BM constituents (42)(43)(44) or cellular receptors. However, NE may also play a direct role in mediating neutrophil transmigration, possibly via cleavage of BM constituents (17,40). In this context, although at present it is unclear whether NE can directly cleave the vascular laminins, laminin 8 and 10, evidence for this possibility was suggested in experiments showing that neutrophils transmigrating through laminin-1-coated fi lters carried laminin on their cell surface in an NE-dependent manner.…”