Anti-inflammatory activity of CKC-containing cationic emulsion eye drop vehicles

Preservative-free cationic emulsion-based artificial tears (ATs) or drug vehicles are innovative eye drop formulations with tear film stabilization and drug delivery properties, and valuable in vivo anti-inflammatory and wound healing properties. These ATs have recently reached the market as ATs for the management of dry eye disease (DED) symptoms (i.e., Cationorm) or as a drug vehicle for cyclosporine (Ikervis). The aim of the present study was to explore the mechanism of action underlying the intrinsic anti-inflammatory and wound-healing efficacies harbored by the cationic emulsions of cetalkonium chloride (CE-CKC).

The anti-inflammatory activity of two CE-CKC (0.002% and 0.005% CKC) emulsions was evaluated by assessing the expression of proinflammatory genes and the secretion of various markers in the following human cell types stressed by different agents: peripheral blood mononuclear cells (PBMCs; stimulation with anti-CD3/anti-CD28 or lipopolysaccharide (LPS)), CD4⁺ T lymphocytes (TCD4; stimulation with anti-CD3/anti-CD28), and a human corneal epithelial cell line (HCE-2; stimulation with LPS). The cells were incubated for 30 min with a 10% dilution of CE-CKC emulsions and then cultured without the emulsions for 24 h or 72 h in the presence of the various challenging agents. The supernatant was collected, and the secreted markers quantitated with flow cytometry or an enzyme-linked immunosorbent assay (ELISA). Gene expression of inflammatory markers was evaluated only in the PBMCs and HCE-2 cells stimulated with LPS. The in vitro protein kinase C (PKC) binding assay for IC₅₀ determination was performed using standard procedures.

The CE-CKC emulsions decreased inflammatory gene expression in LPS-stimulated PBMCs (IFN-γ, IL-17A, CXCL-9, and TNFα) and LPS-stimulated HCE-2 cells (THBS1 and CCL2). Both CE-CKC emulsions inhibited the secretion of IL-17 (from anti-CD3/anti-CD28-stimulated TCD4), TNFα, IFN-γ, and IL-2 (from anti-CD3-/anti-CD28-stimulated PBMCs), and IL-6 and IL-8 (from LPS-stimulated HCE-2). The in vitro PKC binding assay revealed that CKC, the cationic agent, is a specific PKCα inhibitor. In addition, tyloxapol, another excipient, showed some anti-inflammatory activity on IL-6 and IL-8 in the LPS-stimulated HCE-2 cells.

This study indicates that the CE-CKC emulsions are able to directly modulate the secretion and expression of proinflammatory cytokines and chemokines. The results also suggest that CKC and tyloxapol are pharmacologically active excipients with potentially beneficial effects in vivo. These data shed new light on the efficacy observed on the DED signs of these CE-CKC emulsions in clinical trials.