Topical delivery of different ketoconazole and acyclovir formulations
Acquired immunodeficiency syndrome (AIDS) has shown a rapid increase in incidence over the past 25 years. Many clinical manifestations occur in patients infected with human immunodeficiency virus (HIV) due to compromised immunity caused by this virus. Dermatological disorders are almost inevitable for individuals suffering from HIV/AIDS and are seen in approximately 90% of all infected patients (Cedeno-Laurent et al., 2011:5; Dlova & Mosam, 2004:12). Vulnerability of the skin causes impaired life quality by causing low self-esteem, depression or even suicide. The skin is regarded as the most visible organ due to its location and large surface area (Cedeno-Laurent et al., 2011:5). Cutaneous manifestations, including viral, fungal and bacterial pathogens, can serve as markers in HIV/AIDS progression or as indicators for commencing HIV/AIDS treatment (Vusadevan et al., 2012:20). Acyclovir is an anti-viral agent showing activity against herpes simplex virus type 1 and type 2, varicella-zoster virus and cytomegalovirus to a certain extent (King, 1988:176; Beers, 2006:1061). The anti-fungal agent, ketoconazole, shows activity against the majority of pathogenic fungal infections seen in HIV/AIDS including Candida spp, Cryptocococcus neoformans and Histoplasma capsulatum (Bennet, 2006:1225). Ketoconazole has shown to have in vitro inhibitory activity against certain Staphylococcus spp (Pottage, 1986:217). According to Bickers (1994:89), ketoconazole shows a synergistic anti-viral activity when used in combination with acyclovir. Using the mucosal route of administration may be beneficial for these compounds due to the location of occurrence as many of these diseases are found on mucosal surfaces such as the labial and vaginal areas. Compounds are mainly delivered via passive diffusion across epithelium membranes (Patel et al., 2011:107). In mucosal skin, the principle barrier function is removed by the absence of the keratinised stratum corneum as found in normal skin and is, thus, more permeable (Farage & Scheffler, 2011:117). In this study three different formulations containing acyclovir (5% w/w) and ketoconazole (2% w/w) were formulated for topical delivery on mucus membranes, which included a cream, gel and lip balm. Topical delivery is used to target specific sites on the skin by penetration of the skin layers, but has a minimal requirement for systemic effect (Dayan, 2005:67). The aim in this study was to formulate a stable product containing acyclovir and ketoconazole that would provide an efficient flux of both compounds when applied on mucosal membranes. In vitro studies were performed to determine skin permeation of acyclovir and ketoconazole by using a flow-through diffusion system. The formulated products were compared to Acitop® and Ketazol®, which are two products available on the South African market, containing acyclovir and ketoconazole, respectively. However, no product is yet available containing both acyclovir and ketoconazole. Results obtained for acyclovir released from the different formulations during the permeation studies depicted no statistical significant differences between the different formulations in the average cumulative amount of acyclovir released (p > 0.05). The cream, gel and lip balm formulations depicted a decreased average cumulative acyclovir amount released through the mucosa when compared to Acitop®. The following rank order could be established: Acitop® > gel > cream > lip balm. Furthermore, the gel formulation and Acitop® produced a relatively similar percentage of acyclovir diffused. A linear relationship (r2 = 0.9977) existed between the flux and the release rate of acyclovir from the different formulations, indicating that as the acyclovir was released, the flux increased correspondingly. Using the Higuchi model, the average cumulative amount of acyclovir released that permeated the mucosa per unit surface area was constructed against the square root of time (h½). All formulations depicted a correlation coefficient (r2) of 0.9644 – 0.9914 for acyclovir, indicating that the release of acyclovir from the different formulations could be described by the Higuchi model. No statistical significant differences could be obtained for acyclovir between any of the formulations for % diffused, apparent release constant (ARC), release rate (RR) and lag times. The amount of ketoconazole that permeated the mucosa from the gel and cream formulations exhibited a smaller average cumulative amount that permeated the mucosa when compared to Ketazol®. The lip balm was the only formulation that showed a statistically significant (p < 0.05) increase in permeation through the mucosa in comparison to Ketazol®. A rank order for the average cumulative amount of ketoconazole that permeated through the mucosa could be established namely: lip balm >>> Ketazol® > gel > cream. A linear relationship (r2 = 0.9991) was depicted between the average release constant and the average release rate from each of the different formulations for ketoconazole. This indicated that as the compound was released, the flux increased correspondingly which was in accordance with the acyclovir release tendency. The only statistically significant difference (p < 0.05) was seen for the release rate of ketoconazole from the lip balm formulation compared to that of the cream and gel formulations. Release rate and flux of ketoconazole was the highest from the lip balm formulation. The rate of ketoconazole released from all of the different formulations obeyed the Higuchi model as the amount of compound released from each formulation was a linear function of the square root of time (r2 = 0.9584 - 0.9899). Statistically significant (p < 0.05) differences were furthermore noted between the lip balm and both the cream and gel formulations when % diffused, ARC and RR were compared. The lip balm depicted the highest percentage diffused, the highest ARC as well as the fastest RR. However, no statistical differences were obtained between the cream and gel formulation even though the gel formulation performed slightly better. Considering the lag time, all the formulations presented with a relatively shorter initial time of release (less than an hour). Shorter lag time values indicate that the ketoconazole was preferentially released by the base of the formulations. Statistically significant differences (p < 0.05) were depicted between the lag times of Ketazol® and lip balm formulation, as well as between the lip balm and the cream and gel formulations. The stability of the formulated products was examined over a period of three months according to the standards of the International Conference of Harmonisation (ICH) Tripartite Guidelines (2003:12) and the Medicines Control Council (MCC) of South Africa (2011:12). Each of the formulated products was stored at three different temperatures and humidities. Stability tests conducted included visual appearance, mass variation, assay, pH determination, viscosity, particle size observation, and zeta potential. Due to the different properties of the formulations, some tests could not be conducted on the gel and lip balm formulations. The outcomes of the stability tests showed that all three formulations presented acceptable results for some of the tests conducted. No significant changes were noted in the visual appearance, mass variation and pH values of all tested formulations at the specified storage conditions. Acyclovir is slightly soluble in water and has a solubility of 1.3 mg/ml at 25 ºC according to Bethesda (2010). Low solubility often causes crystal formation in products. All of the formulations developed in this study presented crystals on the surface. Due to non-homogenous sample preparation differences in concentrations could be obtained as the amount and size of crystals may differ. Ketoconazole did, however, not depict any significant changes in concentration for any of the formulations at all storage conditions. The cream depicted variable changes in viscosity over the three months, showing no clear trend, whereas, the viscosity measurement results of the gel formulation depicted a definite trend. The sodium carboxymethylcellulose (Na-CMC) used as the thickening agent in this formulation was responsible for this trend obtained in the results, due to the effects of pH, hydration and temperature on this excipient (Aqualon, 1996:10). Results obtained from zeta potential determination for the cream formulation depicted no significant change and the values remained below 25 mV. Zeta potential values below 25 mV present the risk of coalescence due to the lower repelling forces between particles (Jelvehgari et al., 2010:1240). The average size of the particles in dispersion was also observed and could be linked to zeta potential values. The cream depicted an increase in particle size over the three months stability testing. Due to the low zeta potential depicted in the cream formulation it was expected that coalescence would occur over time. From results obtained in this study it was clear that manufacturing different formulations containing both acyclovir and ketoconazole proved difficult due to the significant differences between their physicochemical properties, which in turn influenced the stability of the formulation. Furthermore, it was evident that formulation at specific pH values, as well as the incorporation of certain excipients, played a significant role in the stability of formulations.
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