Enhancement of anti-inflammatory property of aspirin in mice by a nano-emulsion preparation

Abstract

Aspirin, a non-steroidal anti-inflammatory drug, widely used for its anti-inflammatory properties is associated with several systemic side effects including gastro-intestinal discomfort. Inflammation can be mediated by pro-inflammatory cytokines and, along with various other host factors eventually give rise to edema at the inflamed site. Because of the adverse side effects oftentimes associated with systemic exposure to aspirin, the aim of the present study was to investigate whether the anti-inflammatory property of aspirin would enhance if delivered as nano-emulsion preparation. Nano-emulsion preparations of aspirin prepared with a Microfluidizer® Processor were evaluated in the croton-oil-induced ear edema CD-1 mouse model using ear lobe thickness and the accumulation of specific in situ cytokines as biomarkers of inflammation. The results showed that particle size (90 nm) populations of nano-emulsion preparations of aspirin compared to an aspirin suspension (363 nm), significantly decreased (p < 0. 05) ear lobe thickness approximately 2 fold greater than the aspirin suspension. In addition, the aspirin nano-emulsion further reduced the auricular levels of IL-1α (− 37%) and TNFα (− 69%) compared to the aspirin suspension preparation (p < 0.05). The reductions in ear lobe thickness were also significantly associated with accumulated tissue levels of IL-1α (r = 0.5, p < 0.009) and TNFα (r = 0.7, p < 0.0004), respectively. In conclusion, these studies indicate that a nano-emulsion preparation of aspirin significantly improved the anti-inflammatory properties of an aspirin suspension in a CD-1 mouse model of induced inflammation.

Introduction

Aspirin (acetylsalicylic acid) belonging to a class of compounds called non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for their antipyretic, anti-inflammatory, analgesic and platelet anti-aggregation properties [1], [2]. The underlying mechanism(s) for these activities are linked to aspirin's ability to impede the induction of the cyclo-oxygenase (COX-2) enzyme and nuclear factor kappa B (NFкB) activity [3], [4], [5]. Similar to other drugs, the beneficial effects of aspirin can also be accompanied by adverse side effects including gastro-intestinal discomfort and blood loss [6], [7], [8]. Furthermore, long-term usage of high-dosage levels of aspirin may increase the risk for dyspepsia, chronic nephritis, angio-edema, Reye's syndrome and intra-cranial bleeding [9], [10], [11], [12]. Despite efforts to replace aspirin with other systemic anti-inflammatory drugs such as rofecoxib (registered as Vioxx) and Celecoxib (registered as Celebrex) or certain derivatizations [11], [13], [14], [15], the established efficacy of aspirin continues to make it as one of the most recommended over-the-counter medications. A possible approach to reducing the incidence and severity of drug-related adverse effects has been to develop delivery systems that would convert the water insoluble drugs to water-soluble drugs and increase bioavailability and efficacy thereby reducing the required dosage levels [16], [17].

It has also been suggested that emulsion formulations are an attractive alternative as delivery systems for poorly water-soluble drugs such as palitaxel and difluprednate [18], [19]. Nano-emulsions, defined as oil-in-water or water-in-oil emulsions, can be prepared with a Microfluidizer® Processor or with a conventional high-speed homogenizer, with particle sizes averaging ≤ 100 nm [20], [21]. Their long-term stability also referred to as “Approaching thermodynamic stability” and nanoscale particle sizes make them unique and appealing for drug delivery systems [22], [23]. In support of these findings, our laboratory recently reported that a nano-emulsion of an antioxidant synergy formulation (ASF) dramatically increased tumor reduction compared to ASF alone [24].

It is well established in the CD-1 mouse model that inflammation induced by substances such as croton oil is mediated by increased production of various host proteins including pro-inflammatory cytokines such as Interleukin-1α (IL-1α), and Tumor necrosis factor-α (TNFα), which, in turn are associated with increased swelling at the inflamed site [25], [26], [27]. In contrast, the Balb/c or C57BL6/J mice do not produce significant cutaneous inflammation in response to croton oil [28], [29], [30]. Furthermore, a subpopulation of T cells called as T4 or T helper cells that are known to orchestrate various immune response and signal other immune cells as part of natural anti-inflammatory defense mechanisms is not present in CD-1 mouse strains, which facilitate its response to exogenous agents for the study of the efficacy of anti-inflammatory substances in cutaneous inflammation [31].

This communication describes a nano-emulsion preparation produced with a Microfluidizer® Processor which contains the surfactant polysorbate 80, soybean oil and HPLC-grade water as a drug delivery formulation for aspirin. This nano-emulsion preparation significantly improved the anti-inflammatory effect of aspirin in a croton oil-induced CD-1 mouse model of inflammation using ear lobe thickness and the accumulation of auricular cytokine levels as biomarkers for inflammation [26].