Local anaesthetics are used regularly in the medical world for a variety of different procedures. Topical anaesthetics are used largely in minor skin breaking procedures, laceration repair and minor surgical procedures such as laryngoscopy, oesophagoscopy or urethroscopy (Franchi et al., 2008:186e1). The topical means of application of a local anaesthetic is non-invasive and painless that results in a good patient acceptability profile (Little et al., 2008:102). An existing commercial topical anaesthetic product contains a eutectic mixture of the amide-type local anaesthetics lidocaine hydrochloride (HCl) and prilocaine hydrochloride (HCl). This commercial product takes up to an hour to produce an anaesthetic effect. This is considered as a disadvantage in the use of topical anaesthetics, an hour waiting time is not always ideal in certain medical circumstances (Wahlgren & Quiding, 2000:584). This study compared the lag times, transdermal and topical delivery of lidocaine HCl and prilocaine HCl from four different semi-solid formulations with the inclusion of a current commercial product. One of the formulated semi-solid formulations included Pheroid™ technology, a novel skin-friendly delivery system developed by the Unit for Drug Research and Development at the North-West University, Potchefstroom Campus, South Africa. The skin is the body’s first line of defence against noxious external stimuli. It is considered the largest organ in the body with an intensive and complex structure. It consists of five layers with the first outer layer, the stratum corneum, the most impermeable (Williams, 2003:1). The stratum corneum has excellent barrier function characteristics and is the cause for the time delay in the transdermal delivery of active pharmaceutical ingredients (API) (Barry, 2007:569). Local anaesthetics need to penetrate all the epidermal skin layers in order to reach their target site, the dermis. Skin appendages as well as blood vessels and skin nerve endings are located in the dermis. Local anaesthetics have to reach the free nerve endings in the dermis in order to cause a reversible block on these nerves for a local anaesthetic effect (Richards & McConachie, 1995:41). Penetration enhancement strategies for the transdermal delivery of lidocaine and prilocaine have been investigated and include methods like liposomal entrapment (Franz-Montan et al., 2010; Müller et al., 2004), micellisation (Scherlund et al., 2000), occlusive dressing (Astra Zeneca, 2006), heating techniques (Masud et al., 2010) and iontophoresis (Brounéus et al., 2000). The Pheroid™ delivery system has improved the transdermal delivery of several compounds with its enhanced entrapment capabilities. Pheroid™ consists mainly of unsaturated essential fatty-acids, non-harmful substances that are easily recognised by the body (Grobler et al., 2008:285). The morphology and size of Pheroid™ is easily manipulated because it is a submicron emulsion type formulation which provides it with a vast flexibility profile (Grobler et al., 2008:284). Vesicular entrapment was used to entrap lidocaine HCl and prilocaine HCl in the Pheroid™ and incorporated into an emulgel formulation. An emulgel without the inclusion of Pheroid™ was formulated for comparison with the Pheroid™ emulgel as well as with a hydrogel. Pheroid™ solution was prepared and compared to a phosphate buffer solution (PBS) without Pheroid™, both containing lidocaine HCl and prilocaine HCl as APIs. Franz cell type transdermal diffusion studies were performed on the four semi-solid formulations (emulgel, Pheroid™ emulgel, hydrogel and the commercial product) and two solutions (PBS and Pheroid™). The diffusion studies were performed over a 12 h period followed by the tape stripping of the skin after each diffusion study. Caucasian female abdominal skin was obtained with consent from the donors. The skin for the diffusion cells were prepared by using a Zimmer Dermatome®. PBS (pH 7.4) was prepared as the receptor phase of the diffusion studies. The receptor phase was extracted at certain pre-determined time intervals and analysed with high performance liquid chromatography (HPLC) to determine the amount of API that had traversed the skin. Stratum corneum-epidermis samples and epidermis-dermis samples were prepared and left over night at 4 °C and analysed the next day with HPLC. This was done to determine the amount of API that accumulated in the epidermis-dermis and the amount of API that were left on the outer skin layers (stratum corneum-epidermis). The results from the Franz cell diffusion studies indicated that the emulgel formulation without Pheroid™ shortened the lag time of lidocaine HCl and that the emulgel formulated with Pheroid™ shortened the lag time of prilocaine HCl, when compared to the commercial product. Pheroid™ did not enhance the flux of lidocaine HCl and prilocaine HCl into the skin. The hydrogel formulation demonstrated a high transdermal flux of prilocaine HCl due to the hydrating effect it had on the stratum corneum. The commercial product yielded high flux values for both APIs but it did not result in a high concentration of the APIs delivered to the epidermis-dermis. Pheroid™ technology did, however, enhance the epidermal-dermal delivery of lidocaine HCl and prilocaine HCl into the skin epidermis-dermis. The stability of the emulgel formulation, Pheroid™ emulgel formulation and the hydrogel formulation was examined over a 6 month period. The formulations were stored at 25 °C/60% RH, 30 °C/60% RH and 40 °C/75% RH. The API concentration, mass, pH, zeta potential, particle size, viscosity and visual appearance for each formulation at the different storage conditions were noted and compared at month 0, 1, 2, 3 and 6 to determine if the formulations remained stable for 6 months. The results obtained from the stability study demonstrated that none of the formulations were stable for 6 months. The emulgel remained stable for the first 3 months. At 6 months, large decreases in API concentration and pH occurred which could cause a loss of anaesthetic action in the formulations. The Pheroid™ emulgel formulation did not remain stable for 6 months. / Thesis (MSc (Pharmaceutics))--North-West University, Potchefstroom Campus, 2013.

HIV/AIDS is the most threatening and challenging infectious diseases of our time, with the highest increase of newly infected cases reported. This infectious disease was discovered in the early eighties under homosexual men and was later to be discovered in heterosexuals. HIV is a systemic immunosuppressive disorder which causes a depletion of CD4+ T cells and develops into the acquired immunodeficiency syndrome - AIDS. Africa is the continent most affected by HIV/AIDS with the southern parts of Africa having the highest prevalence rates compared to the rest of Africa. Statistics indicate that AIDS is responsible for 3% of deaths in children worldwide - one in seven people dying of an HIV-related illness is a child under the age of 15 years. It was stated by the WHO that countries should develop improved antiretrovirals regimes for the prevention of mother-to-child transmission. Difficulties in administering antiretrovirals (ARVs) to patients (especially children) are the strict dosage regimes and the severe adverse reactions. These factors complicate patient adherence. The list of problems in treating patients is endless and includes the distribution, stability as well as the low efficacy of these drugs. Most of the above mentioned problems and obstacles related to ARVs and ARV treatment could be minimized or eliminated by the use of a stable and effective drug delivery system. Enhancing ARV treatment may be accomplished by the use of the Pheroid™ drug delivery system. Pheroids™ consists mainly of fatty acids and sterile nitrous oxide gassed water. Pharmacological active substances are entrapped into submicron and micron sized structures called Pheroids™. Research showed promising results and advantages in delivering drugs through oral and transdermal routes using Pheroid™ technology. The focus of this study was to test the possible enhancement of the efficacy of antiretrovirals using Pheroid™ technology. The assays used to study this possible enhancement were a modified neutral red and a modified 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) assay. These assays confirmed and illustrated the toxic and protective properties of the tested ARVs (stavudine, lamivudine and nevirapine). An MT-2 cell line was used and infected with an HIV-1 strain, SW7-TCL. Applying Pheroid™ technology in these assays resulted in massive cell death, due to increased ARV toxic levels within the cells. Viability tests proved that Pheroids™ had no effect on the viability of cells at the concentration typically used. This confirmed the enhancing properties of Pheroids™ in the delivery of drugs into the cells. The MTT assay was further adapted from a seven day incubation period to a three day incubation period. By using a low concentration series and a three day incubation period the loss of cells through toxicity was partially overcome. One of the problems that arose form this study was the non-reproducibility of the results. Absorbance levels fluctuated at specific concentrations of the same ARV, which cause difficulties in comparing results. This result was repeatedly confirmed in this syncytium forming infection model. In conclusion, Pheroid™ technology enhanced the delivery of ARVs into the cells although it resulted in cell death. Both the neutral red and MTT assays were found to be inaccurate but further development, research and assay optimization could result in improved in vitro studies. The article format was used for this thesis, as described in the general academic rules in section A.13.7.3 of the North West University. Chapter 1 deals with HIV/AIDS related problems, statistics and treatment obstacles. Chapter 2 is a summary of the cell viability assays used in this study. Pheroid™ technology and its application to ARV treatment are dealt with in chapter 3. The proposed article for submission in the journal Cell Death and Differentiation has been included in chapter 4. Some of the results from the study are reported in the article and annexures, whilst other results are shown and discussed in Chapter 5. Chapter 6 gives a conclusion and final summary of this study. All other experimental methods and results are enclosed in the annexures, as is the "Guide for authors" for the article. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2008.

The aim of this in vitro study was to investigate the feasibility of the transdermal delivery of isoniazid (INH) and rifampicin (RMP) by means of the novel PheroidTM technology system. 'The application of the latter is being investigated in combination with various actives such as peptides (insulin, human growth hormone), anti-malarial drugs (chloroquine), anti-fungals (ketoconazole), local anaesthetics (lidocaine, prilocaine) as well as tuberculostatics (ethambutol, pyrazinamide etc.) via different administration routes at the North- West University. PheroidTM, a stable skin-friendly carrier, comprises of a submicron (200 nm - 2 m) emulsion type formulation for which previous studies have confirmed the ability to penetrate keratinised tissue, skin, intestinal linings, the vascular system, fungi, bacteria and even parasites. Studies involving an oral PheroidTM formulation containing the current approved regime of four anti-tuberculosis drugs showed improved efficacy results whilst an in vitro analysis of bacterial growth indicated a reduction in drug resistance in multidrug resistant tuberculosis (MDR-TB) strains. Therefore we thought it prudent to ascertain whether or not the PheroidTM system would be able to improve the transdermal delivery of a combination of INH and RMP as a possible treatment against cutaneous tuberculosis (tuberculosis involving the skin). The latter refers to pathological lesions of the skin caused by any one of the following: Mycobacterium tuberculosis, Mycobacterium bovis or the bacilli Calmette- Guerin (BCG) vaccine. Demonstration of M. tuberculosis within the infected tissues by traditional acid-fast bacilli (AFB) staining, culture or polymerase chain reaction (PCR) confirms the diagnosis. CTB lesions are associated with various degrees of one or more of the following ulceration, plaque formation, hyperkeratosis or the presence of necrotic matter. Seeing as C-TB is mostly associated with systemic involvement, current treatment comprises of the standard three/four drug regimens used for pulmonary 'TB in general. Cases of CTB usually show improvement within 1 month of therapy with anti-TB drugs, but complete resolution is only attained after 4 - 6 months. 'The major drawback to current therapy is that patients not only remain a source of infection (viable organisms can still be demonstrated in the lesions), but they also suffer from constant embarrassment due to the disfiguring nature of CTB until these lesions have healed completely. No evidence of an already existing topical formulation of this kind could be found. Therefore in vitro permeation studies were conducted using vertical Franz diffusion cells and female abdominal skin as permeation membrane over a period of 12 hours. Concentrations of 5 mg/ml and 10 mg/ml for isoniazid( INH) and rifampicin (RMP) respectively, were applied to the donor phase suspended in either phosphate buffered saline (PBS) or entrapped in PheroidTM. Permeation studies were conducted at pH 5.5. In vitro penetration of INH and RMP were assayed directly by HPLC. Particle size distribution for rifampicin and entrapment of actives within the PheroidTM carrier system was determined by polarized light and laser scanning microscopy (CLSM) respectively and revealed definite entrapment. Permeation profiles obtained for INH in PheroidTM indicated a biphasic character, whilst that obtained for RMP in PheroidTM showed a triphasic character. The PheroidTM delivery system proved more efficacious for delivery of both anti-tubercular drugs and resulted in greater percentage yield as well as flux values than that for a PBS solution. Furthermore, the PheroidTM formulation was able to deliver, the entrapped INH and RMP in concentrations sufficient to exceed their respective minimum inhibitory concentrations (MIC). / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2008.

Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2009.

Extra pulmonary tuberculosis makes up 10% of all tuberculosis cases and cutaneous tuberculosis (CTB) only a fraction of this 10%. CTB is caused by mainly Mycobacterium tuberculosis and can lead to scarring and deformities. The disease presents in different forms, from superficial granulomas to deeper ulceration and necrosis. Tissue cultures, polymerase chain reactions or purified protein derivative staining is used for the diagnosis of CTB (Barbagallo etal., 2002:320). Since the current treatment for CTB is oral anti-tubercular regimens and no topical treatment is available yet (Barbagallo et a!., 2002:320), this study aims to provide a topical preparation of isoniazide and rifampicin which will prevent the deformities and scarring caused by CTB and deliver quicker healing. This topical preparation is to be used in addition to oral treatment. Isoniazide and rifampicin are powerful first-line anti-tubercular drugs, active against both intra- and extracellular bacteria (SAMF, 2005:293). Human skin is a resistant and protective barrier against the external environment and the stratum corneum is the main barrier against diffusion of compounds through the skin (Williams, 2003:9). The physicochemical characteristics (lipophilicity and molecular size) of neither isoniazide nor rifampicin are optimal for penetration of the stratum corneum and the skin-friendly Pheroid™ delivery system was incorporated in two of the formulations to investigate the possibility of improving drug delivery. In this study the transdermal delivery of isoniazide and rifampicin was studied after formulation into four different topical preparations. The stability of these formulations were determined over a six month period under three different conditions of temperature and humidity (25°C/60% RH (relative humidity), 30°C/60% RH and 40°C/75% RH). Isoniazide and rifampicin were formulated into two Pheroid™ and two non-Pheroid™ spray formulations: lotion, Pheroid™ lotion, emulgel and Pheroid™ emulgel. Micrographs were taken with a confocal laser scanning microscope and it was seen that the formulations were homogenous and oil droplets were smaller than 10 urn, allowing permeation through skin. Vertical Franz diffusion cells were used for in vitro permeation studies, with cellulose acetate membranes, for 12 h periods at pH 7.4, to determine drug release. The donor phase was the formulation, with 5 mg/ml of isoniazide and 10 mg/ml of rifampicin. The actives were released from the formulations and small concentrations penetrated the membranes. Release for isoniazide was best from the Pheroid™ emulgel and for rifampicin from the Pheroid™ lotion. Thus it can be concluded that the Pheroid™ improved drug release. The diffusion study was repeated, substituting the membranes with female abdominal skin in order to investigate transdermal delivery. Isoniazide and rifampicin failed to permeate the skin from any of the formulations and no isoniazide or rifampicin could be found in the skin by means of tape stripping after 12 h. Stability tests performed at 4, 8, 12 and 24 weeks was the determination of drug concentrations, pH, weight loss, viscosity, particle size, physical appearance and colour change tests. In these emulsion-type formulations, rifampicin proved to be more stable than isoniazide and after 24 weeks minimal concentrations of isoniazide (20.2 ug/ml) was left. The Pheroid™ formulations were proven to be more stable than the non-Pheroid™ formulations. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2010.

The Southern parts of Africa have the highest prevalence of HIV-infected people and South Africa is the country with the highest number of infections in the world. There is still no cure for AIDS, but anti-HIV medicine can prolong and enhance the quality of life of an HIV infected person. Patient adherence with antiretroviral therapy is extremely low due to difficult dosing intervals, problematic dosage forms, instability of the antiretrovirals (ARVs) and the severe side-effects caused by these drugs; this leads to resistance of HIV to these drugs. Pheroid™ technology is a patented delivery system. Pheroid™ vesicles were used during this study. The entrapment of an active within the Pheroid™ would generally provide a safer, more effective formulation than the active alone. This could mean that the amount of drug needed for treatment of HIV can be decreased while producing fewer adverse effects and reducing the price of treatment. The main objectives of this study were to optimise and validate the cell viability and viral replication assays that can be used in an in vitro viral infection model. The MTT assay was used to asses the viability of the cells and to determine the toxicity of the antiretroviral drugs and Pheroid™ on the cells. HIV-1 assays were evaluated and used to determine the viral replication in the cells. Two different continuous cell lines were chosen for this study, an anchorage dependent GHOST cell line and suspended M7-Luc cells. Both these cell lines were best infected with the SWl virus. SWl is a subtype C, CXCR4 utilising virus. Subtype C is responsible for 60 % of the HIV infections worldwide and is the prevalent subtype in SUb-Saharan Africa .. Infection enhancers were not added to the cells to improve viral infection since it was observed that the Pheroid™ in combination with DEAE-dextran or Polybrene caused cytotoxicity probably by disrupting the cell's membrane. Antioxidants were added to the Pheroid ™ formulation since it was observed that the viability of the cells incubated with the Pheroid™ decreased as the Pheroid ™ matured. The added antioxidants had no significant effect on the cells. Abacavir (ABC) was chosen as the test substance for this study since it showed low cytotoxicity in cell cultures and is water soluble and would not present solubility issues in the media. It was entrapped within the Pheroid™ and its in vitro efficacy and toxicity was tested on HIV-infected and uninfected cell cultures. One directlHIV-specific (p24 antigen ELISA assay) and one indirect (Luciferase) assays were used to asses the inhibition of HIV replication caused by ABC. The p24 antigen ELISA (Enzyme-Linked ImmunoSorbent Assay) assay required a lot of washing steps and were rather expensive to use. The Luciferase assay was only used on the M7-Luc cells; this assay was sensitive, inexpensive and easy to use. The MTT (3-(4,5-demethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) viability assay was used to measure the toxicity caused by the Pheroid ™ and/or ABC on the cells. MTT is a widely used quantitative colorimetric assay to measure the viability of cells. The vitamin E and antioxidants contained in the Pheroid ™ reduced the MTT and produced results that were misinterpreted as enhanced viability when the Pheroid™ was present during MTT analysis. To prevent this problem an additional washing step should be introduced prior to analysis to reduce the interference of the Pheroid ™ with analytical methods. In conclusion, the efficacy of ABC entrapped within the Pheroid™ is still inconclusive and further studies will have to be done. MTT should be used with care for viability analysis of cells incubated in the presence of Pheroid TM. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2009.

The aim of this in vitro study was to investigate the feasibility of the transdermal delivery of isoniazid (INH) and rifampicin (RMP) by means of the novel PheroidTM technology system. 'The application of the latter is being investigated in combination with various actives such as peptides (insulin, human growth hormone), anti-malarial drugs (chloroquine), anti-fungals (ketoconazole), local anaesthetics (lidocaine, prilocaine) as well as tuberculostatics (ethambutol, pyrazinamide etc.) via different administration routes at the North- West University. PheroidTM, a stable skin-friendly carrier, comprises of a submicron (200 nm - 2 m) emulsion type formulation for which previous studies have confirmed the ability to penetrate keratinised tissue, skin, intestinal linings, the vascular system, fungi, bacteria and even parasites. Studies involving an oral PheroidTM formulation containing the current approved regime of four anti-tuberculosis drugs showed improved efficacy results whilst an in vitro analysis of bacterial growth indicated a reduction in drug resistance in multidrug resistant tuberculosis (MDR-TB) strains. Therefore we thought it prudent to ascertain whether or not the PheroidTM system would be able to improve the transdermal delivery of a combination of INH and RMP as a possible treatment against cutaneous tuberculosis (tuberculosis involving the skin). The latter refers to pathological lesions of the skin caused by any one of the following: Mycobacterium tuberculosis, Mycobacterium bovis or the bacilli Calmette- Guerin (BCG) vaccine. Demonstration of M. tuberculosis within the infected tissues by traditional acid-fast bacilli (AFB) staining, culture or polymerase chain reaction (PCR) confirms the diagnosis. CTB lesions are associated with various degrees of one or more of the following ulceration, plaque formation, hyperkeratosis or the presence of necrotic matter. Seeing as C-TB is mostly associated with systemic involvement, current treatment comprises of the standard three/four drug regimens used for pulmonary 'TB in general. Cases of CTB usually show improvement within 1 month of therapy with anti-TB drugs, but complete resolution is only attained after 4 - 6 months. 'The major drawback to current therapy is that patients not only remain a source of infection (viable organisms can still be demonstrated in the lesions), but they also suffer from constant embarrassment due to the disfiguring nature of CTB until these lesions have healed completely. No evidence of an already existing topical formulation of this kind could be found. Therefore in vitro permeation studies were conducted using vertical Franz diffusion cells and female abdominal skin as permeation membrane over a period of 12 hours. Concentrations of 5 mg/ml and 10 mg/ml for isoniazid( INH) and rifampicin (RMP) respectively, were applied to the donor phase suspended in either phosphate buffered saline (PBS) or entrapped in PheroidTM. Permeation studies were conducted at pH 5.5. In vitro penetration of INH and RMP were assayed directly by HPLC. Particle size distribution for rifampicin and entrapment of actives within the PheroidTM carrier system was determined by polarized light and laser scanning microscopy (CLSM) respectively and revealed definite entrapment. Permeation profiles obtained for INH in PheroidTM indicated a biphasic character, whilst that obtained for RMP in PheroidTM showed a triphasic character. The PheroidTM delivery system proved more efficacious for delivery of both anti-tubercular drugs and resulted in greater percentage yield as well as flux values than that for a PBS solution. Furthermore, the PheroidTM formulation was able to deliver, the entrapped INH and RMP in concentrations sufficient to exceed their respective minimum inhibitory concentrations (MIC). / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2008.

Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2009.

Extra pulmonary tuberculosis makes up 10% of all tuberculosis cases and cutaneous tuberculosis (CTB) only a fraction of this 10%. CTB is caused by mainly Mycobacterium tuberculosis and can lead to scarring and deformities. The disease presents in different forms, from superficial granulomas to deeper ulceration and necrosis. Tissue cultures, polymerase chain reactions or purified protein derivative staining is used for the diagnosis of CTB (Barbagallo etal., 2002:320). Since the current treatment for CTB is oral anti-tubercular regimens and no topical treatment is available yet (Barbagallo et a!., 2002:320), this study aims to provide a topical preparation of isoniazide and rifampicin which will prevent the deformities and scarring caused by CTB and deliver quicker healing. This topical preparation is to be used in addition to oral treatment. Isoniazide and rifampicin are powerful first-line anti-tubercular drugs, active against both intra- and extracellular bacteria (SAMF, 2005:293). Human skin is a resistant and protective barrier against the external environment and the stratum corneum is the main barrier against diffusion of compounds through the skin (Williams, 2003:9). The physicochemical characteristics (lipophilicity and molecular size) of neither isoniazide nor rifampicin are optimal for penetration of the stratum corneum and the skin-friendly Pheroid™ delivery system was incorporated in two of the formulations to investigate the possibility of improving drug delivery. In this study the transdermal delivery of isoniazide and rifampicin was studied after formulation into four different topical preparations. The stability of these formulations were determined over a six month period under three different conditions of temperature and humidity (25°C/60% RH (relative humidity), 30°C/60% RH and 40°C/75% RH). Isoniazide and rifampicin were formulated into two Pheroid™ and two non-Pheroid™ spray formulations: lotion, Pheroid™ lotion, emulgel and Pheroid™ emulgel. Micrographs were taken with a confocal laser scanning microscope and it was seen that the formulations were homogenous and oil droplets were smaller than 10 urn, allowing permeation through skin. Vertical Franz diffusion cells were used for in vitro permeation studies, with cellulose acetate membranes, for 12 h periods at pH 7.4, to determine drug release. The donor phase was the formulation, with 5 mg/ml of isoniazide and 10 mg/ml of rifampicin. The actives were released from the formulations and small concentrations penetrated the membranes. Release for isoniazide was best from the Pheroid™ emulgel and for rifampicin from the Pheroid™ lotion. Thus it can be concluded that the Pheroid™ improved drug release. The diffusion study was repeated, substituting the membranes with female abdominal skin in order to investigate transdermal delivery. Isoniazide and rifampicin failed to permeate the skin from any of the formulations and no isoniazide or rifampicin could be found in the skin by means of tape stripping after 12 h. Stability tests performed at 4, 8, 12 and 24 weeks was the determination of drug concentrations, pH, weight loss, viscosity, particle size, physical appearance and colour change tests. In these emulsion-type formulations, rifampicin proved to be more stable than isoniazide and after 24 weeks minimal concentrations of isoniazide (20.2 ug/ml) was left. The Pheroid™ formulations were proven to be more stable than the non-Pheroid™ formulations. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2010.

The Southern parts of Africa have the highest prevalence of HIV-infected people and South Africa is the country with the highest number of infections in the world. There is still no cure for AIDS, but anti-HIV medicine can prolong and enhance the quality of life of an HIV infected person. Patient adherence with antiretroviral therapy is extremely low due to difficult dosing intervals, problematic dosage forms, instability of the antiretrovirals (ARVs) and the severe side-effects caused by these drugs; this leads to resistance of HIV to these drugs. Pheroid™ technology is a patented delivery system. Pheroid™ vesicles were used during this study. The entrapment of an active within the Pheroid™ would generally provide a safer, more effective formulation than the active alone. This could mean that the amount of drug needed for treatment of HIV can be decreased while producing fewer adverse effects and reducing the price of treatment. The main objectives of this study were to optimise and validate the cell viability and viral replication assays that can be used in an in vitro viral infection model. The MTT assay was used to asses the viability of the cells and to determine the toxicity of the antiretroviral drugs and Pheroid™ on the cells. HIV-1 assays were evaluated and used to determine the viral replication in the cells. Two different continuous cell lines were chosen for this study, an anchorage dependent GHOST cell line and suspended M7-Luc cells. Both these cell lines were best infected with the SWl virus. SWl is a subtype C, CXCR4 utilising virus. Subtype C is responsible for 60 % of the HIV infections worldwide and is the prevalent subtype in SUb-Saharan Africa .. Infection enhancers were not added to the cells to improve viral infection since it was observed that the Pheroid™ in combination with DEAE-dextran or Polybrene caused cytotoxicity probably by disrupting the cell's membrane. Antioxidants were added to the Pheroid ™ formulation since it was observed that the viability of the cells incubated with the Pheroid™ decreased as the Pheroid ™ matured. The added antioxidants had no significant effect on the cells. Abacavir (ABC) was chosen as the test substance for this study since it showed low cytotoxicity in cell cultures and is water soluble and would not present solubility issues in the media. It was entrapped within the Pheroid™ and its in vitro efficacy and toxicity was tested on HIV-infected and uninfected cell cultures. One directlHIV-specific (p24 antigen ELISA assay) and one indirect (Luciferase) assays were used to asses the inhibition of HIV replication caused by ABC. The p24 antigen ELISA (Enzyme-Linked ImmunoSorbent Assay) assay required a lot of washing steps and were rather expensive to use. The Luciferase assay was only used on the M7-Luc cells; this assay was sensitive, inexpensive and easy to use. The MTT (3-(4,5-demethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) viability assay was used to measure the toxicity caused by the Pheroid ™ and/or ABC on the cells. MTT is a widely used quantitative colorimetric assay to measure the viability of cells. The vitamin E and antioxidants contained in the Pheroid ™ reduced the MTT and produced results that were misinterpreted as enhanced viability when the Pheroid™ was present during MTT analysis. To prevent this problem an additional washing step should be introduced prior to analysis to reduce the interference of the Pheroid ™ with analytical methods. In conclusion, the efficacy of ABC entrapped within the Pheroid™ is still inconclusive and further studies will have to be done. MTT should be used with care for viability analysis of cells incubated in the presence of Pheroid TM. / Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2009.