Technology of stable, prolonged-release eye-drops containing Cyclosporine A, distributed between lipid matrix and surface of the solid lipid microspheres (SLM)

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Abstract

The aim of this study was to prepare solid lipid microspheres (SLM) with incorporated Cyclosporine A (Cs), suitable for ocular application. For this purpose, SLM were formulated by using different lipids and three different nonionic surfactants. The SLM were produced using a hot emulsification method. The SLM dispersions contained 10, 20 or 30% of lipid (w/w) and up to 2% (w/w) of Cs. The size of the microspheres with Cs ranged from 1 to 15 μm. Physically stable SLM with Cs were prepared using Compritol, as a lipid matrix, and Tween 80, as a surfactant. In contrast, dispersion with Precirol alone, formed semi-solid gels during storage, while in formulations with Precirol and Miglyol, crystals of Cs were observed. In vitro release profile of Compritol formulations showed that 40% of Cs is released within 1 h, while the release of the following 40% takes more time, depending on lipid content in the formulations. The large part of Cs, added to SLM formulations (from 45 to 80%), was found on the surface of microparticles, but no drug crystallization occurred during a long-term storage.

Introduction

Cyclosporine A (Cs) is a lipophilic, cyclic undecapeptide with a high molecular weight (1202.6 Da) used as an immunosuppressive agent (Gökce et al., 2009, Başaran et al., 2010). Over the past years, numerous studies have shown the potential applications of Cs in ophthalmology (Hingorani et al., 1999, Tang-Liu and Acheampong, 2005). It was reported that topical ocular administration of Cs could be used in treatment of the variety of immune-mediated ocular surface disorders and in the prevention of corneal allograft rejection (Perry et al., 2002, Gökce et al., 2008, Shen et al., 2010). Cs is also the first US Food and Drug Administration (FDA)-approved drug therapy for dry eye (Tang-Liu and Acheampong, 2005, Guzey et al., 2009).

Cs is practically insoluble in aqueous media (Lallemand et al., 2003). Various ophthalmic formulations of Cs, such as oily solutions (del Castillo et al., 1994), colloidal carriers (liposomes, nanoparticles, emulsions, micelles) (Milani et al., 1993, Van der Bijl et al., 2001, Lallemand et al., 2003), collagen particles and collagen shields (Reidy et al., 1990, Gebhardt et al., 1995) have been developed to enhance solubility and bioavailability of Cs (Gökce et al., 2009).

In hospitals, oily solutions are prepared using injectable or oral solution, which is solubilized by surfactants, and contain ethanol, which has to be evaporated before the oily eye drops are prepared. Oily solutions of Cs are poorly tolerated and provide a low ocular bioavailability (del Castillo et al., 1994, Shen et al., 2010). The only commercially available eye drops preparation with Cs in the United States is Restasis® (0.05% Cs), which is an oil-in-water emulsion. Unfortunately, Cs levels delivered by Restasis® are not sufficient to prevent rejection after corneal allograft. Optimmune®, 0.2% USP ophthalmic ointment, has been approved for veterinary use, but is not being used in humans, because of its poor tolerance by patients.

There is a growing interest in the use of lipid-based systems in drug discovery and product development to effectively overcome physical and biological barriers related to poor aqueous solubility and stability, membrane permeability and availability (Muller and Keck, 2004, Attama et al., 2009, Bunjes, 2010). The results of different studies show that solid lipid nanoparticles (SLN, mean size 200–500 nm) are promising systems (Gökce et al., 2008, Başaran et al., 2010). The particles are composed of triglycerides and/or fatty acids, as matrix lipids. It was reported that they can be administered to the eye and their use avoids blurred vision and is comfortable for the patient (Gökce et al., 2009). Although not studied so far as ocular drug carriers, solid lipid microspheres (SLM), which are in order of magnitude larger (generally from 200 nm to 50 μm in size) than SLN, are also developed (Reithmeier et al., 2001, Sanna et al., 2004).

SLM and SLN can be considered as physiologically compatible, physicochemically stable and allow for a large scale production at a relatively lower production cost than, for example, liposomes (Reithmeier et al., 2001, Muller and Keck, 2004, Sanna et al., 2004, Bunjes, 2010, Gökce et al., 2009). The release rate for the entrapped substance is controlled by the surfactant coating and the lipid carrier (Attama et al., 2009).

Cs was successfully incorporated into SLN, and such product was demonstrated to be a promising formulation to target the cornea (Ugazio et al., 2002, Gökce et al., 2008, Başaran et al., 2010, Shen et al., 2010). When Cs was administered in SLN, the corneal levels of Cs were shown to increase three to five times, compared to oily formulations.

Incorporation of Cs in SLM has not been reported up to date, although SLM, like SLN, can provide an alternative option for encapsulating lipophilic compounds. In recent years, biocompatible solid lipid microparticles (SLM) have been reported as potential drug carrier alternative to polymer microparticles, although they offer shorter release times. In contrast to numerous publications on SLN, limited data is currently available on SLM properties (Reithmeier et al., 2001, Pietkiewicz and Sznitowska, 2004, Sanna et al., 2004, Jaspart et al., 2005, He et al., 2006, Long et al., 2006, Pietkiewicz et al., 2006, Gökce et al., 2009, Shen et al., 2010, Nanjwade et al., 2011).

The aim of this work was to formulate Cs-loaded lipid microspheres, suitable for ocular application, for the purpose of sustained drug release, and will offer less frequent administration than emulsion-type eye drops and better comfort for the patient, in contrast to oily solutions.

Section snippets

Materials

Cyclosporine A (Cs) was obtained from IVAX Pharmaceutical (Czech Republic); Precirol ATO 5 (glyceryl palmitostearate) and Compritol 888 ATO (glyceryl behenate) were a gift sample from Gattefossé (France); Witepsol H15 was purchased from Sasol (Germany); Miglyol 812 (medium chain triglycerides) from Caelo Caesar and Loretz (Germany); Tween 80 (Polysorbate 80) from Sigma–Aldrich (USA). Methanol and acetonitrile were purchased from Merck (Germany); sodium lauryl sulfate (SLS), sodium hydroxide and

Solubility of Cs in lipids

The solubility of Cs in bulk-melted and solidified lipids (melting point of Precirol 52–55 °C and Compritol 65–77 °C) was 90–100 mg/g. Miglyol added to Compritol (1:4) markedly increased solubility of Cs to 210–220 mg/g, while in Precirol its effect was negligible (120 mg/g).

Preparation of SLM suspension

The composition and characteristics of placebo and Cs-loaded SLM formulations are listed in Table 1. The osmolarity of all SLM suspensions was 330–350 mOsm/kg. Each SLM composition was prepared in batches of 100 g.

The temperature

Discussion

Lipophilicity of Cs is responsible for its satisfactory solubility in oily formulations; it does not guarantee, however, good solubility in solid lipids. Determined Cs solubility (at least 100 mg/g in all tested formulations and twice as large in Compritol with Miglyol mixture) is, nonetheless, satisfactory and theoretically allows one to prepare 10%, 20% and 30% (w/w) of SLM suspensions containing 1%, 2% and 3% of Cs, respectively. Such Cs concentration, in the drug form, is considerably larger

Conclusion

The proposed method of SLM preparation is simple and the composition based on Compritol and Tween allows for autoclaving of SLM and ensures good physical stability in the long term storage. The size of SLM is in the range of 1–10 μm, which is suitable for ocular administration. The Cs concentration can be at least 2%, much higher than in the commercial ocular emulsion, and the solid lipid matrix offers the prolonged release of the drug which, in vitro, occurs at least for 48 h. Large fraction of

Acknowledgements

The authors wish to thank Mariusz Andrzejczuk from the Warsaw University of Technology, Faculty of Materials Science and Engineering (Poland), for performing SEM analysis. The authors would also like to thank Gattefossé for donating Precirol and Compritol.

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