An α4β1 integrin antagonist decreases airway inflammation in ovalbumin-exposed mice

Abstract

Inhibition of the α4 subunit of both the α4β1 and α4β7 integrins has shown promise in decreasing airway inflammation and airway hyperresponsiveness in various animal models. We hypothesized that a novel, high-affinity α4β1 antagonist (LLP2A) would decrease the migration of eosinophils to the lung and ameliorate the airway hyperresponsiveness in a mouse model of ovalbumin-induced airway inflammation. To test this hypothesis, we administered LLP2A, or scrambled LLP2A (a negative control), prior to exposure of sensitized BALB/c mice to ovalbumin aerosol. We can partially prevent, or reverse, the airway inflammatory response, but not airways hyperresponsiveness, by treatment of mice with LLP2A, a synthetic peptidomimetic α4β1 antagonist. Specifically engineered, PEGylated (PEG) formulations of this antagonist further reduce the airway inflammatory response to ovalbumin, presumably by improving the circulating half-life of the drug.

Introduction

Human asthma is a spectrum of diseases characterized by persistence of airway inflammation that contributes to structural airway changes and alterations in airway mechanics, including increased airway resistance and airway hyperreactivity. The key effector cells that dictate these changes in the airways include eosinophils, mast cells, and Th-2 CD4 lymphocytes. While we believe that the primary integrins, chemokines, and cytokines that govern the migration of these cells into the lung have been identified, it is not clear which targets in which pathways will lead to future asthma therapies.

Integrins are a family of cell surface receptors involved in numerous intercellular and cell-matrix trafficking functions. Integrins exist as αβ heterodimers; the α4 subunit has proven an attractive target for blocking organ-specific inflammation. Inhibition of the α4 subunit of both the α4β7 and the α4β1 (very late antigen 4, VLA-4) integrins has shown promise in decreasing airway inflammation and airway hyperresponsiveness. Whether such an inhibitor could improve the care of patients with asthma or other inflammatory airway disorders is now being studied (Ravensberg et al., 2006).

We have previously published a series of studies describing the synthesis and activity of LLP2A (Fig. 1), a novel, high-affinity α4β1 integrin peptidomimetic antagonist (Liu et al., 2006, Peng et al., 2008, Peng et al., 2006). For example, we have shown that LLP2A binds specifically to α4β1-expressing lymphomas (Peng et al., 2008, Peng et al., 2006). The purpose of the present study was to elucidate the role of the α4β1 integrin in the recruitment of eosinophils and lymphocytes to the lung in a common mouse model of allergic airway inflammation and hyperresponsiveness. We hypothesized that there is a direct linkage between the expression and activation of the α4β1 integrin on eosinophils and lymphocytes and increased airway inflammation and hyperresponsiveness caused by exposure to ovalbumin lbumin. To test this hypothesis, we administered LLP2A, or scrambled LLP2A (S-LLP2A, Fig. 1), prior to the ovalbumin exposures. We determined the response of this antagonist on the development of airway inflammation, airway hyperresponsiveness, and goblet cell hyperplasia. We further hypothesized that the bioavailability and efficacy of the synthetic α4β1 antagonist could be improved by constructing different PEGylated (PEG) formulations of the α4β1 antagonist, i.e., LLP2A conjugated to a linear 30 kDa polyethylene glycol (L-PEG-LLP2A), a branch 40 kDa PEG (B-PEG-LLP2A) and a tetravalent 4-arm 10 kDa PEG (T-PEG-LLP2A) (Fig. 1). LLP2A, S-LLP2A and the three PEGylated antagonists were tested in subsequent in vivo experiments.