Formulation and topical delivery of liposomes and proliposomes containing clofazimine

Abstract

The continual market increase in the transdermal and topical delivery of drugs makes cutaneous drug delivery exploration all the more attractive for scientists (Larraῆeta et al., 2016:62). The key in the delivery of drugs to the skin is bypassing its natural barrier, i.e. the stratum corneum, which functions as a protective skin layer against exogenous substances and poses as the fundamental obstacle for formulators. Although the stratum corneum constitutes a disadvantage for drug delivery, the skin provides numerous advantageous above the more common administration routes. The large surface of the skin is an appealing advantage by creating a much more accessible point for drug delivery with less patient compliance difficulty (Andrews et al., 2013:1099; Menon, 2002:S4). Although the skin has the barrier function to protect itself, it is still subject to diseases that could potentially damage the skin as seen in the variety of lesions developed form cutaneous tuberculosis (CTB). Tuberculosis (TB) is a bacterial disease of the lungs that originates form the M. tuberculosis bacterial organism. Only a small number (1 to 2%) of TB patients develop CTB exogenously or endogenously, the former being the most prevalent (Frankel et al., 2009:20-21, Sosnik et al., 2010:548). An intimidating challenge has emerged for scientists, as the TB bacterium has begun to generate resistance against the first-line anti-TB drugs (isoniazid and rifampicin), fostering a multidrug resistant-TB (MDR-TB) propagation (Dooley et al., 2013:1352). Recently the use of second-line drugs has been investigated more extensively to possibly alleviate the use of first-line drugs experiencing resistance. One of these second-line drugs has the characteristic properties to assist the first-line drugs against MDR-TB and forms part of the antibiotic riminophenazine family, namely clofazimine (CLF). Although its mechanism of action is unknown, it has been proven, by Yano et al., (2013:10276) that CLF has great potential against resistance from TB isolates, but illustrated issues regarding poor solubility. Naik et al. (2000:319) state for a drug to have optimum topical penetration, an aqueous solubility of < 1 mg/ml is required and since CLF has a solubility of 0.000225 mg/ml (Pubchem, 2015), it is presumed to be a highly unlikely candidate for topical delivery. Extensive studies have been performed in the field of particulate drug carrying systems (Prashar et al., 2013:130). These systems, such as liposomes, are capable of delivering drugs and improving the absorption and bioavailability of drugs (Drulis-Kawa & Dorotkiewicz-Jach, 2010:197; Prashar et al., 2013:130). Liposomes also possess an amphiphilic character making them ideal to encapsulate both hydrophobic and hydrophilic drugs (Madni, 2014:401). Hence, encapsulating CLF into the hydrophobic bilayer of liposomes may possibly enhance and improve the solubility of the drug and increase the chances of topical delivery. Liposomes have the added disadvantage of being prone to oxidative and hydrolytic degradation causing stability issues (Çağdaş et al. 2014:10), therefore employing proliposomes would safeguard the drug, due to stress caused by liposome instability, without changing the intrinsic character of vesicle (Xu et al., 2009:61). The principal aim of this study was to determine if the two vesicle systems, namely liposomes and proliposomes, would improve the topical diffusion of CLF by improving its solubility. Thus, CLF was encapsulated into liposomes ((CL2)) and proliposomes ((CPL2)) to evaluate the possible topical delivery that occurred. The vesicle systems were characterised according to their properties to verify an ideal dispersion for further transdermal/topical studies. A high performance liquid chromatography (HPLC) method for CLF was developed and validated for sample analysis throughout experiments. The (CL2) and (CPL2) dispersions both showed a successful release of CLF and yielded a similar level of release from both systems. The similar release is atoned to the vesicle systems being equivalent in nature due the same ingredients used during preparation. The skin diffusion studies of the (CL2) and (CPL2) dispersions showed no presence of the API in the Franz cells receptor phase, which in turn illustrates no systemic absorption. CLF was detected at low levels in the stratum corneum-epidermis and the epidermis-dermis from both vesicle systems, indicating a penetration and permeation of the API into the former and latter layers respectively, therefore supporting topical delivery of the API. It was expected that the lipophilic API would accumulate in the lipophilic stratum corneum. The presence of the lipophilic API in the hydrophilic dermis may be constituted to the use of the vesicle systems, which can theoretically improve the solubility, hence contributing to the permeation into the targeted layer. The in vitro cytotoxicity study on the toxic effect of the free drug (CLF) and vesicle dispersions ((PL2) and (CL2)) on immortalised human keratinocyte (HaCaT) cells illustrated that the vesicle system had a significant effect on the level of cytotoxicity. The result of the dispersions containing the vesicle system showed similar levels of cytotoxicity compared to the control (non-cytotoxic) samples regardless of their concentration, while the free drug exhibited a proportional increase from weak to strong cytotoxicity as the concentration of the free drug exposed to the cells increased. This highlighted the fact that liposomes provided a protective effect on the toxicity of the API and correlated to what literature suggested. These results only show the toxicity of CLF on a cell-to-cell basis and do not include the biotransformation and other affecting factors included in the skin’s physiology, therefore these results are not relatable to in vivo studies and are only deemed as a precursor study for future investigation