Stability of amorphous forms of roxithromycin when encapsulated in liposomes
Acne vulgaris is a common chronic inflammatory disease, which affects the pilosebaceous units in the dermal layer of the skin (Krautheim & Gollnick, 2004:398; Williams et al., 2012:361). Several factors are involved during the formation of acne, with the most important being the accumulation of the Propionibacterium acnes organisms in the sebaceous and sweat glands, thus causing inflammation to the skin (Ramanathan & Hebert, 2011:332). A number of oral antibacterial agents, i.e. erythromycin and clindamycin, have been successfully used in the treatment of acne vulgaris (Jeong et al., 2017:243), however, antibiotics used today are reported to be up to 60% resistant, leading to poor patient compliance due to an increase in side-effects as the result of continuous use of these antibacterial entities (Jeong et al., 2017:243; Scheinfield et al., 2003:43). Hence, roxithromycin is one of the newer antibiotics, which might be used to treat acne, especially in a topical dosage form (Csongradi et al., 2017:100; Ostrowski et al., 2010:83). The main purpose of this research study was to conclude whether the excipients used during the formulation of liposomes had an effect on the solid-state nature of the three forms of roxithromycin. Secondly, it was to determine which liposome dispersion had the highest concentration of roxithromycin delivered topically to the site of action. The target area for the active pharmaceutical ingredient (API) was the epidermis-dermis (ED), as this area is favoured by the bacterium P. acnes (Gollnick, 2003:1585). During the investigation, the API (roxithromycin) was used to prepare the two amorphous forms by means of the well-known quench cooling of the melt method and re-crystallisation of the crystalline raw material from chloroform, in an attempt to overcome the low solubility of roxithromycin monohydrate ((RM); aqueous solubility of 0.0335 mg/ml in water at 25 °C)) (Aucamp et al., 2013:26). The preparation method proved successful in rendering the quench cooled (QC) and the chloroform desolvated (CD) amorphous forms (Aucamp et al., 2012:468; Aucamp et al., 2013:18; Craig et al., 1999:181). The crystalline form and the two amorphous forms of roxithromycin were characterised using different analytical techniques to determine the crystallinity or amorphicity of the API in each sample. These techniques include the following: differential scanning calorimetry (DSC), Fourier-transform infrared spectroscopy (FT-IR) as well as x-ray powder diffraction (XRPD) techniques, while the purity of each sample was confirmed through high performance liquid chromatography (HPLC). To explore the effect of the different liposome excipients, lipid films (precursors for liposomes) were prepared to determine the physical stability of the three solid-state forms when formulated within lipid films. The concentration of the three solid-state forms of roxithromycin remained constant (2% w/w) and was formulated within varying concentrations of cholesterol and egg phosphatidylcholine (n = 3), which resulted in the formulation of nine lipid films (three lipid films per solid-state form). After the crystallinity or amorphicity of the API in the lipid films were determined, it was discovered that the crystalline RM converted to an amorphous form in the lipid films. It also led to the discovery that the two amorphous forms (QC and CD) remained amorphous, also being stabilised in the lipid film. Thus, the aim was reached in proving that the excipients had a stabilising effect on the different solid-state forms of roxithromycin. The study progressed to the formulation of nine different liposomes, each formulated with the three solid materials of roxithromycin in different excipient concentrations. These liposomes were characterised by means of their morphology (microscopic evaluations), droplet size and distribution, pH measurements, surface charge (zeta-potential) and the entrapment efficiency (%EE) to determine the APIs physicochemical properties and to establish whether it adheres to the requirements for successful topical drug delivery. All nine dispersions revealed small, spherically shaped and stable vesicles, with an ideal surface charge and a high entrapment of the API within the vesicles. Thus, the study progressed towards release studies. Membrane release experiments were performed on the different dispersions to evaluate if the vesicle systems were successful in the release of the API through the synthetic membrane, before skin diffusion studies were conducted. Skin diffusion studies followed, to determine if any transdermal delivery of the API was possible. Another technique used during skin diffusion studies was the tape stripping method and this was to prove if the API would have topical delivery to the stratum corneum-epidermis (SCE) and/or the target-site, namely the ED. The experimental flux values of roxithromycin, obtained after the membrane release studies, showed that the three different solid-state forms of roxithromycin were released from all nine formulations, with formula RM2 presenting with the highest average flux of 28.322 ± 5.340 μg/cm2.h. Skin diffusion studies revealed that some transdermal delivery of the API was reached, with RM2 having the highest average %diffused and average amount per area diffused (149.184 ± 169.397 μg/cm2). Tape stripping results also show that RM2 had the highest average concentration in both the SCE (371.260 ± 95.486 μg/ml) and ED (179.265 ± 88.364 μg/ml). This concludes that topical delivery of the API is possible for the treatment of acne. The aims set out for this study were reached because the preparation method and the excipients used during the formulation of the liposomes rendered the crystalline form (RM) of the API into an amorphous form, whilst preventing the amorphous forms (QC and CD) from re-crystallising to the more stable crystalline form. Characterisation results proved the nine dispersions adhered to the requirements to be formulated within a topical drug preparation. In addition, quantifiable concentrations of the API were delivered to the target area, i.e. ED, which led to successful topical drug delivery. From the data gathered for the three solid-state forms of roxithromycin, it became evident that liposomes consisting of the RM form displayed the best results. It became evident that the delivery of the API was not dependent on the solid-state form within the formulations, but rather the ratios of the excipients used in the formulation of liposomes. During this study, it was found that irrespective of the solid-state; diffusion of roxithromycin when incorporated into liposomes is possible into and through the skin.
- Health Sciences