dc.description.abstract | Cutaneous tuberculosis (CTB), one of the forms of extra-pulmonary tuberculosis (TB), occurs
1.5% of the time when the Mycobacterium tuberculosis bacterium enters the skin, either through
direct contact or through airborne particles (Almaguer-Chávez et al., 2009:562-563; Arora et al.,
2006:344; VanderVen et al., 2015:2). Clinical presentations of CTB can vary, but commonly all
forms pose as ulcer-like infectious lesions (Bravo & Gotuzzo, 2007:174-177). Current CTB
treatment commences through oral TB regimens, which results in unfavourable patient
compliance. This is due to an extensive combination of drugs used over a period of months
(Van Zyl et al., 2015:634-635). Unfortunately, strains of M. tuberculosis are becoming greatly
resistant against available active pharmaceutical ingredients (APIs) (Van Zyl et al., 2015:634).
This resistance poses another problem during treatment, as patients need to receive therapy
they have not had before, which is difficult as only limited APIs are still effective (Almaguer-
Chávez et al., 2009:562; Dipiro, 2012:595; Van Zyl et al., 2015:630).
Artemether, a lipophilic derivative of artemisinin, is an existing anti-malarial drug currently being
investigated as a potential anti-TB treatment (Haynes, 2016; Miller et al., 2011:2076; Nneji et
al., 2013:2619). The effectiveness of artemisinin can be ascribed to the fact that the
endoperoxide bridge in their chemical structure leads to the production of free radicals (Nneji et
al., 2013:2619; Shrivastava et al., 2010:79). Consequently, artemether can be viewed as an
oxidant drug as it can lead to cytotoxic levels of reactive oxygen species (ROS), leading to
oxidative stress, proposing an oxidising environment to M. tuberculosis and resulting in cell
death within the parasitic cell (Ebrahimisadr et al., 2014:1; Haynes, 2015; Haynes, 2016;
McIntosh & Olliaro, 2010:2; Nneji et al., 2013:2619; Shahzad et al., 2013:197). Artemisinin
combinations have been found to lead to submicromolar activity against M. tuberculosis
(Shakya et al., 2012:702).
In this study, the aim was to formulate a novel CTB treatment through the topical delivery of
artemether. Resistance against existing APIs, together with the lack of topical CTB treatment,
presents an opportunity for the investigation thereof (Van Zyl et al., 2015:630). It can be
suggested that systemic TB treatment, in combination with topical treatment, could contribute to
better treatment of CTB lesions (Van Zyl et al., 2015:636; Wyrzykowska et al., 2012:297).
Firstly, on investigation of the physicochemical properties of artemether, it can be viewed as
topically favourable since it is a lipophilic API with an ideal molecular mass and melting point.
Secondly, artemether is highly metabolised through the liver when taken orally, hence topical
application will be more advantageous (Shahzad et al., 2013:197). Thirdly, as CTB is a
cutaneous disorder, it is proposed to be treated through a topical delivery system as direct contact between the CTB lesions and the API can be achieved. Topical drug delivery of
artemether is therefore aimed at keeping the API in the skin, following the direct application
thereof to the targeted site, i.e. the epidermis (Williams, 2013:676). The skin being by far the
largest and most easily accessible organ represents a great target site and many advantages
are proposed by topical drug delivery of which the most important is that it can be viewed as a
non-invasive drug delivery system (Williams, 2013:677). This is ascribed to an increase in
patient compliance and through the direct application of the API to the target, hence, bypassing
the hepatic system (Marrow et al., 2007:37; Naik et al., 2000:319). Although being applied
directly to the skin, the API needs to move and permeate through skin layers, but is initially
limited by the outer most layer – the stratum corneum (Williams, 2013:682). The lipids within
the stratum corneum control and regulate the movement of APIs through the skin and therefore
act as a drug flux regulator (Williams, 2003:10). The drug flux is consequently the quantity of an
API that can move across the layers of the skin; it is evident that the drug flux is also directly
dependent on the API’s physicochemical properties (Williams, 2003:28; Williams, 2013:680).
Therefore, to result in successful skin permeation, properties such as molecular mass, aqueous
solubility, partition coefficient (log P), diffusion coefficient and melting point should be ideal
(Allen et al., 2011:42; Williams, 2013:680-682).
Artemether presents with some ideal physicochemical properties for topical delivery, since its
molecular mass is less than 500 g/mol (298.37 g/mol) and its melting point is lower than 200 °C
(between 86 – 90 °C) (Naik et al., 2000:319; USP, 2013). An ideal octanol-buffer distribution
coefficient (log D) for an API to be delivered topically should range between 1 and 3, since this
value signifies that the API is soluble in both water and oil (Subedi et al., 2010:339; Williams,
2003:36). Experimental determination of the log D value was calculated to be 2.35 ± 0.1170,
which is ideal for topical drug delivery, whilst the aqueous solubility of artemether was found to
be 0.1053 ± 0.0022 mg/ml, which is significantly less than 1 mg/ml, hence, less than optimal for
topical delivery (Naik et al., 2000:319). It was proposed that less than optimal properties could
be overcome through the formulation of a successful delivery system.
Nano-emulsions can be viewed as a promising topical delivery system due to their small
droplets (20 – 200 nm) that can lead to better permeation and drug release, resulting in greater
concentration of the API accumulating within the skin (Abolmaali et al., 2011:139; Klang et al.,
2015:258; Lai et al., 2008:1; Lu et al., 2014:826). A nano-emulsion is generally constituted by
two phases, i.e. a water and an oil phase, dispersed within each other resulting in both
hydrophilic and lipophilic characteristics (Gaur et al., 2014:37; Klang et al., 2015: 258). A
greater surface area and larger interfacial area, combined with free energy, are contributors to a
nano-emulsion being a target site-specific drug delivery system that can result in localised
deposition, which is essential for successful CTB treatment (Clares et al., 2014:S91; Lai et al.,
2008:1; Lovelyn & Attama, 2011:626). Many approaches and techniques have been attempted to overcome the stratum corneum
properties (Williams, 2013:693). One approach has been the use of a nano-emulsion as a
delivery system, since it presents with enhanced penetration (Lovelyn & Attama, 2011:630;
Maruno & Da Rocha-Filho, 2010:17). Another successful approach is the incorporation of
penetration enhancers in topical formulations (Trommer & Neubert, 2006:108; Williams,
2013:694; Williams & Barry, 2012:129). Penetration enhancers are successful due to the fact
that they disrupt, modify and reduce the lipid barrier of the skin, resulting in increased
partitioning and absorption of the API (Babu et al., 2006:145; Trommer & Neubert, 2006:108;
Wang et al., 2003:1612; Williams, 2013:694). In this study, safflower oil, a natural oil, was
employed as chemical enhancer. C18-Unsaturated fatty acids, such as linoleic acid and
arachidonic acid, have been found to have near optimal enhancement effects (Williams & Barry,
2012:132). Since uncomplicated fatty acids constitute basic components of human skin, the
use thereof can be regarded as safe, therefore lowering the possibility of skin irritation (Boelsma
et al., 1996:729; Büyüktimkin et al., 1997:433; Gaur et al., 2014:1812; Menon, 2002:S9;
Vermaak et al., 2011:922). Safflower oil presents with a high concentration (± 75%) of linoleic
acid, which plays an important moisturising, healing and anti-inflammatory role when
incorporated within topical formulations (Van Wyk & Wink, 2009:81; Vermaak et al., 2011:922;
Wolters Kluwer Health, 2009).
Therefore, the aim was to formulate a topical oil-in-water (o/w) nano-emulsion containing 0.8%
(w/v) artemether and 5.0% (w/v) safflower oil. Consequently, an optimised nano-emulsion
obtained through pre-formulation was formulated within two semi-solid dosage forms, i.e. a
nano-emulgel and a conventional emulgel to contain 0.4% (w/v) artemether and 2.5% (w/v)
safflower oil. Through characterisation, it could be proposed that the three optimised
formulations presented with ideal properties for effective topical drug delivery. Hence, all three
formulations presented with small droplets, an ideal surface charge and stability for possible
successful permeation.
The effectiveness of each of the three formulations was evaluated by determining whether any
API release and/or permeation of the API through the skin had occurred, therefore in vitro
diffusion studies were conducted on each of the formulations (Wiechers, 2008:23; Williams,
2013:683). Franz cell diffusion studies are based on the employment of a vertical Franz cell
method, consisting of a two-chamber diffusion cell, which is separated by a membrane or a
piece of skin (Williams, 2013:683). Release of the API from the three different formulations was
evaluated through in vitro membrane release studies. Following the membrane release studies,
skin diffusion studies and tape stripping were done to determine whether any transdermal
and/or topical delivery were achieved, respectively. Experimental flux values of artemether, gained through membrane release studies proved that
artemether was released from all three formulations. During the skin studies, only the nanoemulsion
resulted in artemether being retained within the stratum corneum-epidermis and
although a small amount permeated into the receptor phase, the quantified values were lower
than the limit of detection (LOD) as well as the lower limit of quantification (LLOQ). Hence, as a
result of this it can be said that artemether was not found within in the systemic circulation and
only in the target site, i.e. the outermost layer of the epidermis. The formulated nano-emulgel
and conventional emulgel did also not result in any artemether in the skin or through the skin.
These non-existing artemether concentration values can possibly be ascribed to the formulation
itself and to the pH of the formulations averaging at a pH of 6.82 ± 0.03, 5.14 ± 0.02 and
5.86 ± 0.02, leading to only 0.11%, 5.44% and 1.09% being unionised, respectively. Low
unionised species could lead to low or no permeation, whilst high unionised species propose
effective permeation of the skin (Li et al., 2012:98; Williams, 2003:38). The low aqueous
solubility of artemether (0.1053 ± 0.0022 mg/ml) (water) and 0.090 ± 0.0030 mg/ml (phosphate
buffer solution (PBS) (pH 7.4)) as well as the low initial concentration of artemether within the
formulations, ranging between 0.4% and 0.8%, could also influence diffusion results.
To determine the safety of artemether and the optimised topical nano-emulsion on human skin,
in vitro cytotoxicity studies were conducted on normal immortalised human keratinocytes
(HaCaT) cells. Cytotoxicity evaluation on the HaCaT cells commenced through the conduction
of a methylthiazol tetrazolium (MTT) assay. Consequently, it was found that the optimised
nano-emulsion (with and without artemether) and artemether itself presented as non-cytotoxic
as there was less than 20% cell death when the cells were treated with a 0.5% and a 1.0%
treatment, respectively. Further in vivo experiments or in vitro efficacy studies against
M. tuberculosis would need to be conducted to evaluate the success of the formulation against
CTB. It can therefore be proposed that the nano-emulsion would not present toxic when
applied to the skin, in low concentrations.
It can be suggested that artemether could be delivered topically and that retention in the
epidermis could possibly be achieved. Throughout this study, very low concentrations of
artemether were found topically delivered through an optimised o/w nano-emulsion containing
artemether and safflower oil. Weak aqueous solubility could possibly explain these low
concentrations of artemether quantified. However, further investigations are required as it
appears that optimisation of the formula could possibly overcome the challenge of the topical
delivery of artemether as a novel CTB treatment | en_US |