Diclofenac acid nanocrystals as an effective strategy to reduce in vivo skin inflammation by improving dermal drug bioavailability

Highlights

• Wet media milling produced nanocrystals retain the polymorphic form of starting powder.

• Poloxamer 188 produced a slight oedema reduction and myeloperoxidase inhibition.

• Nanosuspensions enhance drug skin accumulation compared to a commercial gel.

• Nanosuspensions inhibit in vivo myeloperoxidase activity and oedema in damaged tissues.

Abstract

In this work a diclofenac acid nanosuspension formulation was produced as a novel approach for the treatment of skin inflammation. Drug nanocrystals, prepared by the wet media milling technique and stabilized using Poloxamer 188, were characterized by different techniques: scanning electron microscopy, differential scanning calorimetry, X-ray powder diffractometry, Fourier transform infrared spectroscopy and photon correlation spectroscopy. The ability of nanocrystals to improve dermal drug bioavailability was investigated ex vivo by using Franz diffusion vertical cells and mouse skin, in comparison with both diclofenac acid coarse suspensions and a commercial formulation. The topical anti-inflammatory activity of the drug nanosuspension was assessed in vivo by testing its effect compared to common inflammatory endpoints: i.e. the inhibition of chemically induced oedema and leucocyte infiltration (reflected in myeloperoxidase activity). Following the milling procedure, diclofenac nanocrystals exhibited a mean diameter of approximately 279 nm, a low polydispersity index (∼0.17) and maintained the same polymorphic form of the starting bulk powder. When the drug nanosuspension was applied on the mouse skin it produced a higher accumulation of diclofenac in the skin compared to both the coarse suspensions and the commercial formulation, as demonstrated by ex vivo transdermal delivery experiments. Moreover, the nanosuspension provided an in vivo oedema inhibition of 50%, which was not statistically different from the commercial formulation. On the contrary, the nanosuspension showed a higher inhibition of myeloperoxidase activity in the damaged tissue (86%) than the commercial formulation (16%).

Introduction

Inflammation is an essential protective process to preserve the integrity of the body against physical, chemical and infective agents as well as autoimmune responses. However, the inflammatory response to such attacks may erroneously lead to the damaging of normal tissues, due to an overproduction of reactive oxygen species (ROS), nitric oxide (NO) and cytokines, such as tumor necrosis factor-alpha (TNF-ɑ) [10], [19], [29]. Therapeutic medication involves the treatment of symptoms by interrupting the inflammatory process and, if possible, the native cause. Usually, inflammation is localized in a specific organ or apparatus, such as the skin, subcutaneous tissue, muscular or osteoarticular apparatus, and it can often become chronic. Long-term systemic treatments with steroidal or non-steroidal anti- inflammatory drugs can cause the onset of several side effects as well as a low drug bioavailability at the site of action [18]. Topical administration may represent a valid alternative to the systemic treatment of local inflamed skin or muscle. In particular, nanosized formulations of anti-inflammatory drugs, which are usually poorly soluble in an aqueous media, can be used to improve the drug’s local bioavailability and efficacy, minimizing the side effects [1].

Nanocrystals can be defined as nanoparticles made of pure drug, with no matrix material, and an average diameter below 1 μm (typically in the range of 200–500 nm). They are prepared as a suspension (nanosuspension) in a liquid dispersion medium, usually water, stabilized by a suitable surfactant and/or polymer. Nanosuspensions can be delivered to the local inflamed skin or muscle by various routes of administration, the most common and developed being the oral route [20]. The dermal administration of nanocrystals of poorly soluble actives is a recent application. Indeed, the dermal delivery of nanocrystals to enhance the local efficacy of actives was completely neglected, up to 10 years ago, both in the pharmaceutical and cosmetic field [25], and relatively little has been published, thus far, concerning nanocrystal topical administration [4], [14], [15], [26], [27], [30].

Recent in vitro studies have demonstrated that nanocrystals can increase dermal drug bioavailability by enhancing its dissolution velocity and saturation solubility, thus, leading to an increased concentration gradient with a consequent improved skin penetration of drugs [21], [27], [28]. However, to the best of our knowledge, no in vivo studies concerning the biological activity of nanocrystal drugs applied to the skin have been published, thus far.

Diclofenac acid (2-(2-[(2,6-dichlorophenyl)amino]phenyl) acetic acid, DCF) is one of the most powerful and commercially successful non-steroidal anti-inflammatory drugs (NSAID) that presents three different polymorphic forms: two monoclinic forms, HD1 (space group P21/c) and HD2 (space group C2/c) [2], and an orthorhombic form (HD3, space group Pcan) [12]. Due to several drawbacks when given orally, DCF is preferably administered topically, in spite of its low skin permeability, in long-term treatments, such as in the treatment of local muscle inflammation. Currently the available commercial topical preparations contain DCF in salt form, because of DCF’s free acid very low aqueous solubility [7]. However, the lipophilicity of the DCF acid allows a better penetration than hydrophilic DCF salts.

The aim of this work was to produce DCF acid nanocrystals as a novel approach for the treatment of skin inflammation. The characterization of DCF nanosuspensions, prepared using the wet media milling technique, was carried out via different techniques: scanning electron microscopy (SEM), differential scanning calorimetry (DSC), X-ray powder diffractometry (XRPD), Fourier transform infrared (FTIR) spectroscopy and photon correlation spectroscopy (PCS).

In addition, we also investigated the diffusion ex vivo of DCF through the mouse skin and assessed the topical anti-inflammatory activity of the drug nanosuspension in vivo by testing its effect against common inflammatory endpoints: i.e. the inhibition of chemically induced oedema and leucocyte infiltration (reflected in myeloperoxidase–MPO–activity).

The efficacy of the nanosuspension was compared both to a DCF coarse suspension and a commercial topical preparation containing diclofenac diethylamine, Voltaren Emulgel^®.