Browsing by Author "Botha, Julia Hilary."

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Effects of a prostaglandin precursor, gamma-linolenic acid (GLA), on malignant cells in vitro and in vivo.
(1985) Ramchurren, Nirasha.; Botha, Julia Hilary.; Robinson, K. M.
Recent studies have shown that the proliferation of various human and murine tumour lines can be inhibited by the addition of gamma-linolenic acid (GLA) to the culture medium. These findings are consistent with the proposal put forward by Horrobin (1980) that malignant cells lack the enzyme/ A 6 desaturase; which is responsible for the conversion of linoleic acid (LA) to GLA. Since GLA is a prostaglandin (PG) precursor/ inadequate conversion of LA to GLA would result in decreased production of PGs/ particularly PGEi/ which has been shown to have an inhibitory effect on cell growth. Provision of GLA to enzyme deficient malignant cells should therefore bypass this blockade/ increase PGET synthesis and thus "normalise malignant cells". This study was performed to examine further the effects of exogenous GLA on growth of malignant cells in vitro and in vivo. Cells of the continuous murine sarcoma (M52B) line and primary cultures of non malignant fibroblasts were used to investigate effects of GLA in vitro. Cultures were exposed to either single or multiple doses of a range of concentrations of GLA. Radioimmunoassay (RIA) was performed to compare the amounts of PGE and PGF released into the medium by GLA treated and control M52B cultures and thus determine whether the addition of GLA in vitro significantly affected production of these PGs. Athymic BALB/c mice and immunocompetent BALB/c and Biozze mice as well as mice of the "Onderstepoort Strain" were used in various in vivo studies. Tumours were induced by the subcutaneous inoculation of approximately 1 x 106 cells of either the M52B line (into immunocompetent and athymic mice) or human breast carcinoma (NUB 1) line (into athymic mice). Take rates and latent periods were recorded. GLA treatment was initiated after tumours were established. In one study the fatty acid in hydrogenated coconut oil (HCO), which contains no PG precursors/ was administered parenterally (100 ug/ml/day) to Biozze mice. Control mice were either untreated or injected with HCO only. In another study, BALB/c mice and mice of the "Onderstepoort Strain" had their diet supplemented with GLA (in the form of EPO) to an extent of 3.5%. Control mice consumed either standard laboratory chow only or, chow supplemented with either 35% sunflower seed oil (SSO) or 35% HCO/ neither of which contain GLA. All diets were supplied ad libitum. Tumour sizes were measured every 48 hours and at the end of each experiment at which time tumours were excised and examined histologically. GLA was found to produce inhibitory and toxic effects on growth of both M52B cells and non malignant fibroblasts in vitro/ although the effect in the latter was observed only with high concentrations of the fatty acid. The inhibition of malignant cell growth was concentration dependant and was positively related to the duration of exposure to the fatty acid. Prior to death/ cells treated with GLA accumulated vii paranuclear granules which were shown histochemically to be lipid in nature. Electron microscopy confirmed the presence of large lipid deposits. Cultured M52B cells treated with GLA also released more PGE and PGF into the medium than did cells not exposed to the fatty acid. However, analysis of results using the Mann Whitney U test showed these differences to be statistically non significant for both PGE and PGF on two tailed tests. In contrast to the inhibition of M52B cell growth observed in vitro, growth of solid M52B sarcomas and NUB 1 carcinoma xenografts in athymic mice was apparently unaffected by administration of dietary GLA. Analysis of data using an unpaired student's t-test showed that the differences in tumour volumes between control and treated groups were not statistically significant either before or at the end of the experiment. While the inhibition of malignant cell growth caused by GLA in vitro was consistent with Horrobin's proposal that malignant cells may lack this PG precursor, whether or not these actions are mediated by the PGs remains obscure. Although an increase in PGE production by M52B cells was observed following GLA treatment, besides this increase being statistically non significant, it was not possible to determine whether this was due to PGE, (as suggested by Horrobin) or PGE2. It is possible that the effect produced in vitro was due to some factor other than raised PGE production, for example a non-specific fat-overload effect or a change in cell membrane fluidity. The lack of effect of GLA on tumour growth in vivo may have been due to inadequate delivery of the fatty acid to the tumour site. However, whatever the mechanism of action of GLA in vitro/ since oral GLA was supplemented to the maximum tolerated extent and produced no effect in immunodeficient mice inyiyo, it would seem that in a similar clinical situation oral doses which would be practical may be ineffective.
Effects of Z-venusol and other pure compounds from medicinal plants on prostate, cervical and breast cancer cells.
(2017) Mathibe, Lehlohonolo John.; Naidoo, Strinivasen.; Botha, Julia Hilary.
Introduction According to recent World Health Organisation (WHO) estimates, cancer causes more deaths than coronary heart diseases globally (GLOBOCAN, 2012). While communicable diseases such as HIV/AIDS continue to burden African populations, cancer is increasingly recognised as a critical public and private health problem in Africa (Igene, 2008). It is estimated that by 2030, about 112 921 new cases of cancer will be diagnosed in South Africa (Singh et al., 2015). This would represent a 50% increase of new cancer cases as compared to 2012’s estimates by the WHO. Although there is little doubt about the incidences of cancer, there are, unfortunately, divergent theories in as far as tumourigenesis and the aetiology of cancer. Some researchers hold the view that cancer originates from malignant transformation of normal tissue progenitor and stem cells (Reya et al., 2001). Others believe that cancer is as a result of mature cells that have undergone de-differentiation (Sell, 2004). Notably, latest research has shown that there is a strong association between tissue-specific cancer risk and the lifetime cumulative number of cell divisions of tissue or organ-specific stem cells (Tomasetti & Vogelstein, 2015). Although there are still differing views on the origins of cancer, it is widely accepted that this devastating disease occurs as a result of abnormal cell development and is characterised by uncontrollable cell proliferation. The majority of currently-available cancer treatments target cell proliferation. However, the effectiveness of many cytotoxic drugs, including those that were discovered from plants, is limited by their serious side-effects and cost (Abratt, 2016). Chemotherapeutic agents that were originally discovered from medicinal plants include vinblastine (isolated from Catharanthus roseus), etoposide (isolated from Podophyllum peltatum), paclitaxel (isolated from Taxus brevifolia) and topotecan and camptothecin (isolated from Camptotheca acumenata). Thus, medicinal plants continue to play a critical role in the management of diseases in the world. In Africa, decoctions, which contain extracts from various medicinal plants (Bruneton, 1995; Balunas & Kinghorn, 2005), are widely used for traditional management of many diseases including cancer. However, apart from subjective oral evidence regarding the effectiveness of extracts from various plants, the identity of ingredients, as well as the science and pharmacology of active compounds found in numerous popular concoctions and decoctions are not known. Objectives The main objectives of this study were:  To assess anti-proliferative potential of three plant-derived-compounds, i.e. hypoxoside, ent-Beyer-15-en-19-ol and Z-venusol on human cancer cells, namely DU-145 (prostate), HeLa (cervical) and MCF-7 (breast) in vitro.  To determine the type of cell death, i.e. whether a compound with potential causes apoptotic or necrotic cell death on both human cancer and normal cell lines (such as MCF-12, HMECs and dMVECs).  To investigate how a potential compound exerts its cytotoxicity. Materials and Methods Initially dimethylthiazol-diphenyltetrazolium bromide (MTT) assays were conducted to find the concentrations which may inhibit proliferation in prostate (DU-145), cervical (HeLa) and breast (MCF-7) cancer cells. Normal human cell lines, which were used for control purposes, were the primary human mammary epithelial cells (HMECs), MCF-12 and the dermal microvascular endothelial cells (dMVECs). Initially, cells were exposed for 48 hr to hypoxoside, ent-Beyer-15-en-19-ol and Z-venusol, which were isolated from Hypoxis hemerocallidea, Helichrysum tenax, and Gunnera perpensa, respectively. The concentrations ranged from 2.34 μg/mL to 2400 μg/mL, dissolved in cell specific media. In subsequent experiments, the more sensitive sulforhodamine B (SRB) methodology was used, and cells were exposed to Z-venusol for 24 hr, 48 hr and 72 hr, to much lower concentrations, which ranged from 1.9 μg/mL to 240 μg/mL dissolved in dimethyl sulphoxide (DMSO). To investigate possible pathways of observed cell death, two assays were conducted. These were the fluorescein isothiocyanate (FITC) Annexin V apoptosis detection assay (using the FACS Calibur “JO” E5637 flow cytometer for analysis), and the lactate dehydrogenase (LDH) assay. To explore possible mechanism(s) of action, the activities of interleukin-6 (IL-6) and cyclic adenosine monophosphate (cAMP) were assessed. To investigate the activity of IL-6, cells were exposed for 48 hr to various working concentrations of Z-venusol; that is, 37.5 μg/mL and 75 μg/mL. To investigate the activity of direct cAMP, cells were exposed for 48 hr to various working concentrations of Z-venusol; that is, 37.5 μg/mL, 75 μg/mL, and 150 μg/mL. Absorbance, which is inversely proportional to the concentration of cAMP in both the samples and the standards, was measured using a BioRad (Model 3550) microplate reader. Epinephrine (10 μM) and propranolol (10 μM), were used separately and in combination, added to the highest concentration of Z-venusol for comparison. Main Results & Discussion Hypoxoside resulted in a statistically significant (p < 0.001) 38% and 77% increases in proliferation in MCF-7s at concentrations of hypoxoside 1200 μg/mL and 2400 μg/mL, respectively, after 48 hr exposure. In support of the current findings, Xulu (2013) also reported that hypoxoside, and its active derivative known as rooperol, significantly increases cell proliferation of both cancer and normal mammary cells in vitro (Xulu, 2013). This was considered an undesirable finding with regards to the aim of finding a cure for cancer. Therefore, no further test were carried out on this compound beyond the initial screening stages. The highest concentration (i.e., 2400 μg/mL) of the second compound, that is ent-Beyer-15- en-19-ol, decreased proliferation in prostate cancer cells (DU-145) and in breast cancer cells (MCF-7) by 6% and 19%, respectively. Interestingly, much lower concentrations, i.e. 4.7 μg/mL and 9.4 μg/mL, of ent-beyer-15-en-19-ol significantly (p < 0.05) decreased cell proliferation in cervical cancer cells (HeLa) by 37% and 41%, respectively. The differences in expression of vimentin gene, which is over-expressed in HeLa cells and suppressed in MCF- 7s and DU-145s may explain why this compound showed significant activity only in the cervical cancer cells (Oshima, 2002; Satelli & Li, 2011). More importantly, the ability of this compound to significantly inhibit cell proliferation in the HeLa cell line by almost 50% at lower concentrations offers an opportunity for further studies. The findings with regards to the third compound, i.e. Z-venusol, were the most exciting. Hence investigations on it were developed beyond the screening stages. This compound demonstrated a statistically significant, concentration-dependent, apoptotic inhibitory effect on the proliferation of MCF-7 cells, with an IC50 of 53.7 μg/mL after 72 hr exposure, while the highest concentration (250 μg/mL) resulted in 69% inhibition. Both the FITC Annexin V and LDH results suggested that apoptosis contributed to most of the effects observed. Further, there was non-significant inhibition (20%) of HMEC proliferation observed when the concentration of Z-venusol was increased beyond 16.6 μg/mL. The highest concentration of Z-venusol used in this study resulted in a statistically significant (p < 0.001) 51% inhibition of IL-6 activity in the MCF-7 after 48 hr exposure. None of the Z-venusol concentrations, either alone or in combination with epinephrine, an agonist of the adrenergic receptors, showed any statistically significant effect on the levels of cAMP in the MCF-7s. Surprisingly, there was a significant (p ≤ 0.028) 34% elevation of cAMP levels in cells which were exposed to a combination of Zvenusol and propranolol. If Z-venusol was ever able to be used clinically, there might be a need to increase the dose high enough for the attainment of desired therapeutic effects with minimal cytotoxicity on normal cells, because its potency is much lower than that of cisplatin. Increasing Z-venusol to a therapeutically-effective concentration would be possible as there was no plateauing-off of inhibition of proliferation in MCF-7s. It was only in primary normal human mammary epithelial cells (HMECs) that formation of “plateaus” was observed. Favourably, this selective plateauing-effect might allow the ‘gold-standard’ attainment of the desired cytotoxic effect on cancer cells while preserving normal cells at higher concentrations. There are no studies with which to directly compare the findings of this study. However, reports on effects of the extracts of G. perpensa on various other cancer cell lines provide an opportunity for comparison. For instance, the results of this research support the findings of Simelane and colleagues. They recently reported that G. perpensa extracts caused an inhibition of proliferation of hepatocellular carcinoma cells (HepG2) with an IC50 of 222.33 μg/mL and human embryonic kidney 293 (HEK293) cells, with an IC50 of 279.43 μg/mL both after 48 hr of treatment (Simelane et al., 2012). Conclusion Z-venusol, unlike other compounds studied, has a firm potential to play a role in the treatment of cancer in the future. Its mechanism of action involves IL-6 signaling, which may trigger other downstream mediators and may also involve cAMP “cross-talk”. Recommendations More basic science investigations using other hormone-dependent and highly invasive breast cancer cell lines such as the triple-negative MB-231 cells are needed. In vivo studies, such as using the nude mice model, are needed to confirm the in vitro results and to provide an insight into the benefits of Z-venusol in living systems.
The impact of therapeutics tutorials on the reasoning of fourth year medical students with regard to the prescribing process.
(2005) Harries, Catherine Sara.; Mbali, Valerie Charlotte.; Botha, Julia Hilary.
This research was initiated as a response to a request for assistance from a group of students at the Nelson R Mandela School of Medicine who had reported feeling unprepared to prescribe medicines. This led to an interest in the level of competence shown by students in making prescribing decisions and the extent to which they were confident of their prescribing judgments. Student prescribing competence and confidence were assessed using quantitative and qualitative methods. The quantitative assessment comprised a test where students were asked to rate their confidence in some of their responses. A stratified sample of 10 of these student interviewed, where they were asked to choose treatment for four paper cases. Prescribing skills were found to be lacking, with test results averaging 47%. appropriate treatment selected for only 4 of the total of 40 paper cases. Upon reviewing the literature, it became apparent that poor prescribing skills, leading to the problem of irrational prescribing was a worldwide phenomenon The study aimed to address areas of weak prescribing skill using a short intense intervention comprising of several different learning strategies. Student change in confidence following the course was assessed using an evaluation form where students rated their perceived changes in key competences. Students showed improved confidence for each of the prescribing abilities measured. These findings have been compiled into 3 research publications, the texts of which are bound together as they were submitted together to comply with the research requirement of an M.Ed. The findings are reported in a paper titled Building successful therapeutics into a problembased medical curriculum in Africa in the South African Journal of Higher Education (see Appendices). I was also interested in how prescribing ability builds as students develop new prescribing skills. The student interviews provided an opportunity to explore the variation shown between the students relating to the quality of the treatment they prescribed for a given paper case. A sample of two sets of paper cases were assessed using a phenomenographic method, yielding two different perspectives of student experience. The research outlined above is the focus of the dissertation, which also includes an exploration of the teaching and learning issues which guided the design of the intervention and which I believe led to the positive finding of improved student prescribing confidence. Also included in the dissertation is an analysis of the quantitative assessment according to the cognitive categories of Bloom's Taxonomy, as well as qualitative data gathered from student interviews which revealed an understanding about prescribing abilities which predominated at differing Bloom cognitive levels for different students. In the second paper titled Undergraduate medical students' reasoning with regard to the prescribing process which has been submitted to Medical Teacher, (see Appendices) the range of student cognition associated with prescribing is explored. Each question from the quantitative assessment of prescribing abilities were grouped according to the Bloom Category it had been assigned, student scores according to each Bloom category were calculated. Students scored highest for the lowest cognitive category ('knowledge') and lowest for the highest ranked cognitive categories( 'evaluation' and 'synthesis'). These findings along with the qualitative findings and the phenomenographic assessment were reported here.
The influence of tuberculosis treatment on efavirenz clearance in patients co-infected with HIV and tuberculosis.
(Springer-Verlag., 2011) Gengiah, Tanuja Narayansamy.; Holford, Nicholas H. G.; Botha, Julia Hilary.; Gray, Andrew Lofts.; Naidoo, Kogieleum.; Abdool Karim, Salim Safurdeen.
Purpose: Drug interactions are of concern when treating patients co-infected with human immunodeficiency virus (HIV) and tuberculosis. Concomitant use of efavirenz (EFV) with the enzyme inducer rifampicin might be expected to increase EFV clearance. We investigated the influence of concomitant tuberculosis treatment on the plasma clearance of EFV. Methods: Fifty-eight patients were randomized to receive their EFV-containing antiretroviral therapy either during or after tuberculosis treatment. Steady-state EFV plasma concentrations (n = 209 samples) were measured, 83 in the presence of rifampicin. Data were analyzed using a non-linear mixed effects model, and the model was evaluated using non-parametric bootstrap and visual predictive checks. Results: The patients had a median age of 32 (range 19–55) years and 43.1% were women. There was a bimodal distribution of apparent clearance, with slow EFV metabolizers accounting for 23.6% of the population and having a metabolic capacity 36.4% of that of the faster metabolizers. Apparent EFV clearance after oral administration in fast metabolizers was 12.9 L/h/70 kg whilst off tuberculosis treatment and 9.1 L/h/70 kg when on tuberculosis treatment. In slow metabolizers, the clearance estimates were 3.3 and 4.7 L/h/70 kg in the presence and absence of TB treatment, respectively. Overall there was a 29.5% reduction in EFV clearance during tuberculosis treatment. Conclusion: Unexpectedly, concomitant rifampicin-containing tuberculosis treatment reduced apparent EFV clearance with a corresponding increase in EFV exposure. While the reasons for this interaction require further investigation, cytochrome P450 2B6 polymorphisms in the population studied may provide some explanation.
The injectable contraceptive : user, social and pharmacological perspectives.
(2003) Smit, Jennifer Ann Bodley.; McFadyen, Margaret Lynn.; Botha, Julia Hilary.; Preston-Whyte, Eleanor.
Despite its widespread use, little research has been undertaken on the use of progestogen-only injectable contraceptives by South African women. This thesis is comprised of two sections. Section 1 provides the first comprehensive description of injectable contraceptive use among rural South African women. It includes an analysis of the contraceptive method mix, prevalence of injectable contraceptive use, discontinuation patterns and reported side effects. A comparison of depot medroxyprogesterone acetate (DMPA) versus norethisterone oenanthate (NET-EN) focuses on utilization patterns and costs. The second section gives an account of the pharmacokinetics of DMPA including the first ever population analysis. A cross-sectional, community-based household survey was undertaken in the Hlabisa sub-district of KwaZulu-Natal, South Africa. Interviews were held during 1998 and 1999, with 848 randomly selected women (aged 15-49 years) and with 14 focus groups. There was a heavy reliance on injectable contraceptives which were used by 74% of women practising contraception. By contrast, the condom was the current method of only 4%. The injectable method was the most commonly used method among teenagers. However, in most cases, contraceptive use appeared to commence only after the first pregnancy. Slightly more NET-EN (54%) than DMPA (46%) was used, with younger women more likely to use NET-EN than DMPA (p=0.001). No significant differences in self-reported side effects were found between current users of the two injectables. Health workers played an important role in women's decisions to use the injectable, and in product selection, with NET-EN being recommended for younger women on the basis of concerns about method reversibility. While some women used injectables for long periods of time, discontinuation rates at two years were high, most commonly due to menstrual disturbances. Many side effects were reported by users of both DMPA and NET-EN, with amenorrhoea the most common, experienced by 63% of current injectable users. Heavy bleeding was most commonly reported by previous users (38%). Vaginal wetness was also common, mentioned by 18% and 29% of current and previous users respectively. Utilisation patterns of the two injectable products (DMPA and NET-EN) were analysed by means of a Pareto analysis of injectables issued from four South African provincial pharmaceutical depots over three financial years (1997/8, 1998/9 and 1999/2000). Injectables accounted for a substantial share of total state expenditure on drugs. While more DMPA than NET-EN was issued, NET-EN distribution from two depots increased over the period of analysis, even though DMPA was the cheaper option. The pharmacokinetic analysis was undertaken amongst DMPA users routinely attending family planning services in three Durban clinics in 1996. Medroxyprogesterone acetate levels at the end of the dosing interval were analysed for 94 women. In addition a population pharmacokinetic analysis of 291 serum levels from 111 DMPA users was undertaken. This involved the use of Non Linear Mixed Effect Modelling (NONMEM) to fit the data and determine the pharmacokinetic parameters, apparent clearance (CLIP) and apparent volume of distribution (VIP), and to estimate the influence of covariates on CLIP and VIP (where P is the bioavailability). The final model estimates for CLIP and VIP were 1080 (95% confidence interval: 994, 1166) litres/day and 86200 litres (95% confidence interval: 68246, 104154) respectively. No significant relationships were found between the covariates tested and CLIP and VIP. Concerns raised in the literature about the influence of weight or ethnicity on the pharmacokinetics of DMPA were shown to be unfounded. In the context of South Africa's HIV epidemic, the heavy reliance on injectable contraceptives, which offer no protection against HIV, should be addressed by expanding the contraceptive method mix to include barrier methods such as the female condom. Health providers are influential in contraceptive decision-making and should be encouraged and supported to redress the dependence on the injectable method alone, taking into account the need of many for dual protection against HIV and unwanted pregnancy. Provider counseling should also focus on adherence to dosing regimens, improving continuation rates, and should provide appropriate advice for women complaining about vaginal wetness with injectable use. Promotion of one injectable product over another to younger women is not appropriate. Since DMPA is the cheaper product, provider training about the rational use of injectable contraceptives should include cost considerations.
Integrating human immunodeficiency virus and tuberculosis drug treatment.
(2014) Gengiah, Tanuja Narayansamy.; Botha, Julia Hilary.
The human immunodeficiency virus (HIV) and tuberculosis (TB) epidemics are major global public health challenges. Worldwide, approximately 42% of TB patients are also co-infected with HIV, and sub-Saharan Africa (SSA) is home to the majority of the world’s infections of both HIV and TB. Dual infection has been shown to be associated with a higher risk of death. Integrating drug treatment for both diseases is therefore essential to improve survival. However, drug interactions between antiretroviral therapy (ART) and anti-TB medication remain a challenge to effective treatment integration. Although several drug interactions have been identified, only some are clinically relevant. The impact of significant interactions on public health outcomes is expected to be greatest when large numbers of patients are prescribed interacting drugs. Efavirenz (EFV) is the most commonly prescribed nucleoside reverse transcriptase inhibitor (NNRTI) component of first line ART in sub-Saharan Africa, particularly when rifampicin (RIF) based TB treatment is co-administered. RIF is known to up-regulate cytochrome P450 (CYP450) drug metabolizing enzymes resulting in decreased exposure to concomitantly administered drugs that utilize similar metabolic pathways. Therefore, the concomitant use of EFV with RIF would be expected to increase EFV clearance while absorption of TB drugs may also be compromised by advanced HIV disease. The efficacy of both TB and HIV treatment may thus be compromised by pharmacokinetic interactions, while more recent evidence also implicates genetic variation in drug metabolism as a predictor of drug exposure. To understand the significance of the EFV-RIF interaction better in a South African population, the pharmacokinetics of EFV during and after RIF-based TB treatment were investigated as an ancillary study of the ‘Starting Tuberculosis and Antiretroviral Therapy’ (START) trial (CAPRISA 001: NCT00091936). Participants were randomized to receive both ART and TB treatment simultaneously (integrated arm) or to initiate ART only on completion of TB treatment (sequential arm). In both arms, the ART regimen included once daily enteric-coated didanosine (400 mg for participants >60 kg; 250 mg for participants <60 kg), lamivudine 300mg and efavirenz. Based on the expected drug interactions, when EFV was administered in the presence of TB treatment, participants weighing less than 50kg received 600mg and those weighing 50kg or more received 800mg daily. After TB treatment was successfully completed, all patients received EFV 600mg. Blood samples for trough EFV plasma concentrations were obtained at the end of months 1, 2 and 3 during TB treatment and at the same time points after TB treatment was successfully completed. Additionally, approximated peak RIF concentrations were measured 2.5 hours post-dose at the end of months 1, 2 and 3 of TB treatment. The influence of single nucleotide polymorphisms, in CYP2B6, CYP2A6, and UGT2B7 on EFV concentrations, and in drug transporter genes (SLCO1B1) on RIF concentrations, was assessed post-trial from stored peripheral blood mononuclear cell (PBMC) samples. EFV concentration-time data were analyzed using a population pharmacokinetic nonlinear mixed effects model (NONMEM) to quantify the impact of RIF-based TB treatment on EFV clearance. Unexpectedly, there was an overall 29.5% reduction in EFV clearance during TB treatment. A bimodal distribution of EFV apparent clearance (CL/F) was evident and indicated that slow EFV metabolisers accounted for 21.9% of the population. EFV clearance after oral administration in fast metabolisers was 11.5 L/h/70kg off TB treatment and 7.6 L/h/70kg when on TB treatment. In slow metabolisers, however, the clearance estimates were 2.9 and 4.3 L/h/70kg in the presence and absence of TB treatment respectively. Building on the findings of the NONMEM analysis and in response to the US FDA prescribing change in 2012, that approved an EFV dose increase from 600mg to 800mg in patients weighing 50kg and more when on concomitant RIF, the presence and influence of pharmacogenetic polymorphisms of the CYP450 enzyme system on NNRTI plasma exposure during and after TB co-treatment and the effect of increasing the EFV dose was investigated. During TB treatment, median (IQR) EFV Cmin was 3.2 (2.6-6.3) μg/mL and 3.3 (2.4-9.5) μg/mL in the EFV 800mg and 600mg groups respectively, while off TB treatment Cmin was 2.0 (1.4 - 3.5) μg/mL. The frequency of the CYP2B6 *1, *6 and *18 haplotypes was 18.5%, 38.9% and 25.9% respectively. Polymorphisms in all three CYP2B6 genes studied (516T-785G-983C) were present in 11.1% of patients. Median (IQR) EFV concentrations in patients with the three mutations were 19.2 (9.5-20) μg/mL and 4.7 (3.5-5.6) μg/mL when on and off TB treatment. TB treatment, composite genotypes CYP2B6 516 GT/TT, CYP2B6 983 TC/CC or being a CYP2A6*9B carrier predicted median EFV Cmin > 4 μg/mL. Therefore, increasing the EFV dose to 800mg during TB treatment is unnecessary in African patients with these polymorphisms. As a critical component of first line TB treatment concerns about sub-optimal TB drug bioavailability were examined for RIF. The influence of drug transporter gene polymorphisms on RIF concentrations was also assessed. Median RIF (IQR) C2.5hr was found to be 3.6 (2.8-5.0) μg/mL while polymorphism frequency of the SLCO1B1 (rs4149032) drug transporter gene was high (0.76) and was associated with low RIF concentrations as was male gender and having a low haemoglobin. Increased RIF dosage warrants urgent consideration in African TB-HIV co-infected patients. In conclusion, concomitant RIF-containing TB treatment unexpectedly reduced EFV CL/F with a corresponding increase in EFV exposure. Polymorphisms of EFV metabolizing enzymes were frequent in this population and contribute to this outcome. While in South Africa where TB-HIV co-treatment is associated with elevated EFV concentrations, peak RIF concentrations were alarmingly low and well below the recommended target range of 8 to 24 μg/mL. Increased RIF dosage may be warranted in African TB-HIV co-infected patients whilst the need for EFV dose increase is not supported by these data. Recommendations for public health benefit, in this generalized epidemic in South Africa, include the consideration of an EFV dose reduction as a cost saving to improve life-long treatment sustainability, and a RIF dose increase to curb TB treatment failure and future development of multiple-drug resistant (MDR) TB.
Investigation into drug dose practices and proportional reasoning in mathematics among medical students.
(2019) Harries, Catherine Sara.; Botha, Julia Hilary.
Multilingual preclinical South African medical students, receiving a developing quantitative literacy program, were studied. Their preparedness for drug dosage practices, aspects they found difficult, the underlying reasons, and factors or interventio ns that helped them were investigated A mixed methods action -research-type study involved three cycles, each testing a different consenting student cohort. In Cycle 1 dosage calculation ability, when using different concentration formats, was assessed progressively over two years. Individual teaching observations explored struggling students' experience. In Cycle II, the focus broadened to include retrieving dosage information from guidelines and preparing a dose in a syringe. In Cycle ill, assessment in eluded drug selection, requiring interpretation of statistics to compare treatments. In all cycles, the percentage of successful students was calculated, error frequencies were determined, and associations were sought. Observations were coded for themes of student difficulties, supportive strategies and improvement, responses were mapped to stage progress toward proportional reasoning and assessments were analysed for linguist ic and mathematical difficulty. In Cycle I (n = 364), competence rose from 23% to 66% after extended exposure to training and assessment, peer learning and individual tuition. Observed tuition themes produced curriculum change. In Cycle II, 239 students were randomised to four groups. The two groups given standard numerical information fared best (46% success with the addition of equipment and 43% without). Where information was extracted from guidelines, 25% were successful when provided with equipment and only 10% without. Groups with equipment calculated fewer implausible doses and u sed fewer incorrect units. In Cycle ill, after training, only 26% of 231 students understood relative risk, but a mere 6% understood the concepts of absolute risk and number needed to treat. Sixty students (26%) made a rational drug choice. Proportional re asoning ability was not associated with interpreting risk statistics successfully or making a rational treatment selection. In all cycles, English as home language predicted success, although in Cycle I, this effect was lost after peer tuition. To prepare medical students for the complexities of drug dose practices, early training and repeated assessment should include extraction of information from guidelines, use of the relevant equipment as well as training in interpreting risk statistics for rational drug selection.
An investigation into the use of complementary and alternative medicine for atopic eczema.
(2016) Thandar, Yasmeen.; Botha, Julia Hilary.; Mosam, Anisa.
Atopic eczema (AE) is one of the most common skin diseases that patients frequently present with to dermatological practices in South Africa (SA). It has shown to impact negatively on the quality of life of many patients suffering from it. Epidemiological studies have shown high rates of AE prevalence, ranging from 2-7% in adults and 7-20% in children. Over the last decade, the lifetime prevalence of physician-diagnosed AE has almost doubled in SA. This rise continues despite accessible effective treatments. Due to AE’s chronic and relapsing nature and the unattainability of complete clinical cure, patients are progressively exploring complementary and alternative medicines (CAM) in search of a solution. Although the global popularity of CAM for AE is on the rise, a review of the literature demonstrated contradictory evidence with regards to their efficacy with shortcomings in many of the published data thus making it difficult for clinicians to assess their role, if any, in the management of AE. Objective One To objectively evaluate the information on the efficacy and safety of CAM in light of the most recent findings, the study entitled “Complementary Therapy in Atopic Eczema: The Latest Systematic Reviews” in Chapter Two of this thesis collectively evaluated all published systematic reviews (SRs) to date on the most popular CAM modalities for AE. These SRs included those of Chinese herbal medicines(CHM), homeopathy, oral herbal remedies (including evening primrose oil and borage oil), probiotics and certain dietary supplements. The study concluded that none of the alternative therapies evaluated demonstrated obvious and indisputable evidence of efficacy due to many limitations in study design, poor methodologies, patient numbers etc. Further studies may be warranted with some therapies (CHM, different probiotic strains and fish oil), whereas homoeopathy failed to show any treatment effect and further studies with evening primrose oil and borage oil may be difficult to justify. This overview was able to provide objective information to enable dermatologists and general practitioners to advise and manage their patients holistically in the light of the most recent findings. Objective Two Topical corticosteroids remain the mainstay of treatment for AE. However, many patients are concerned about their long-term safety and thus seek evidence-based safer alternatives. Many published papers have made reference to the wide use of topical herbal creams for AE and many of these been tested, but few in controlled clinical trials. No SRs of these trials could be found, although SRs of topical herbal extracts have been published for other chronic skin conditions. The study entitled “Topical Herbal Medicines for Atopic Eczema: A Systematic Review of Randomised Controlled Trials” in Chapter Three of this thesis was the first SR to be conducted for topical herbal preparations for AE. Using Cochrane SR methodology, numerous databases were searched from inception until June 2014. All controlled clinical trials of topical herbal medicines for AE in humans of any age and published in English were included regardless of the control intervention or randomisation. Of eight studies that met the inclusion criteria, seven investigated extracts of single plants and one an extract from multiple plants. The study concluded that there is currently insufficient evidence of efficacy for any topical herbal extract in AE with many studies having methodological flaws. Even studiesthat did show efficacy over placebo were single trials with small patient cohorts. Together with providing clarity to both prescribers and patients, the study was able to identify opportunities for future research in better designed trials with topical extracts that showed a promising effect and had a low risk of bias across all domains. These were randomised controlled trials (RCTs) of licorice gel and Hypericum perforatum. Objective Three The literature has thus far reported on numerous international studies on the widespread use of CAM for AE. These studies not only investigated the prevalence of CAM use but also the modalities used, motivations for use and demographic variables that influence their use. All these factors potentially impact on the treatment of AE. No such studies conducted anywhere in Africa could be found. Given the lack of literature in SA, the study entitled “Complementary and Alternative Medicine Use amongst patients with Atopic Eczema - a South African Perspective” in Chapter Four of this thesis was a cross-sectional study that was conducted amongst AE patients in Durban, KwaZulu-Natal to bridge this gap in knowledge. This study found a 66% current or previous CAM use, which was moderately higher than those reported in other countries. Frequently used CAM were vitamins, aromatherapy oils, herbal creams, traditional African medicines and homeopathy. Non-disclosure to the dermatologist was high and almost half of the patients interviewed said they were not questioned about CAM use. More Indian patients used CAM and Muslims were the most frequent CAM users. Duration of AE was also a predictor of use. Although not statistically significant, the more educated and higher income bracket used CAM more. The study was able to provide detailed trends of CAM use by South Africans for AE which is an important addition to the literature. This information is able to highlight to dermatologists and healthcare professionals treating AE patients, the need to be more conversant with CAM that patients explore, as this could impact overall clinical outcome. Objective Four Although evident from the literature that patients have embraced CAM, it is uncertain whether mainstream healthcare professionals are as embracing. Their attitude and knowledge of CAM will influence their pro-activeness in enquiring about CAM and confidently discussing proven/unproven remedies with their patients, thereby influencing an overall positive clinical experience and disease course. Several international studies have explored the knowledge, attitudes and practices amongst general practitioners (GPs), physicians, pharmacists, paediatricians, academic doctors and other healthcare workers towards CAM, but none within the context of a specific disease. No published studies conducted in SA or elsewhere investigating HCPs’ knowledge, attitudes and norms of practice with regards to CAM for AE could be found. As a result, and given the extensive use among SA patients with AE as per the study’s previous findings, a cross-sectional study entitled “Knowledge, Attitude and Practices of South African Healthcare Professionals towards Complementary and Alternative Medicine Use for Atopic Eczema - A Descriptive Survey” was conducted. Results amongst GPs, dermatologists, paediatricians and pharmacists are reported in Chapter Five of this thesis. GPs and pharmacists were significantly more embracing of CAM compared to dermatologists and paediatricians. The study revealed poor CAM knowledge and communication between HCPs and patients, however there was a strong interest to learn more. It was also found that there is an urgent need for continuing education programmes on CAM and inclusion into undergraduate curriculums as most HCPs were interested in learning more about CAM. Conclusion Overall, this thesis was able to fill a gap in the knowledge of CAM use for AE both globally and within the context of SA. The study provided clarity and objective conclusions from the many SRs previously published for popular oral CAM therapies. Furthermore, the study conducted and published the first SR on topical herbal therapies for AE. This SR identified therapies that have demonstrated positive results for AE with low risk of bias and is thus able to provide direction for future research in this regard. Within the SA context, the study described the perspectives and practices of both patients and mainstream healthcare professionals on CAM use for AE, which was lacking in Africa. With this information we were able to ascertain the popular CAM that SA patients are using, the extent of their use as well as establish CAM education needs for local healthcare professionals.
Low rifampicin concentrations in tuberculosis patients with HIV infection.
(Open Learning on Enteric Pathogens., 2014) Gengiah, Tanuja Narayansamy.; Botha, Julia Hilary.; Soowamber, Deepak.; Naidoo, Kogieleum.; Abdool Karim, Salim Safurdeen.
Abstract available in pdf.
The pharmacokinetics/pharmacodynamics of theophylline in premature neonates during the first few days after birth.
(2000) Du Preez, Marie J.; Botha, Julia Hilary.; McFadyen, Margaret Lynn.
Theophylline is one of the few preparations available for the treatment of apnoea of prematurity. Currently little data is available on the pharmacokinetics and the pharmacokinetic/pharmacodynamic relationships of theophylline for premature neonates during the first few days of life, a time when neonates undergo profound physiological changes and when the drug is most often used. Furthermore, the influence of theophylline on hypoxaemic episodes has not yet been quantified. The study aimed to investigate optimal theophylline dosing in this group by establishing pharmacokinetic parameters, assessing the effectiveness of the drug in abolishing apnoea and hypoxaemic episodes and investigating the concentration/effect relationship. The project was conducted in the neonatal wards of King Edward VIII Hospital, Durban, South Africa. The study group comprised a total of 105 Black, apnoeic, premature neonates, with respiratory distress syndrome, who were receiving intravenous theophylline. Serum samples (263), collected from patients during routine care, were analysed for theophylline. Forty-six patients were monitored before and after theophylline therapy with a neonatal capnograph linked to a data acquisition. Apnoea incidents were classified into total (all apnoea <_5 seconds) and pathologic (all apnoea >_20 seconds) and a hypoxaemic episode was defined as a >_10% fall for >10 seconds in peripheral oxygen saturation. Within each of these groups patients were assessed as responders (>_50% reduction in the clinical effect from baseline to the last recording) and non-responders. Patient characteristics were identified as possible markers of non-response to theophylline therapy. The Nonlinear Mixed Effects Model (NONMEM) was used to derive population pharmacokinetic models and parameters for theophylline as well as to assess the concentration-effect relationship. The pharmacokinetic analysis estimated a low clearance and volume of distribution, with oxygen support enhancing clearance. Relatively high inter-individual and residual variability values were obtained prompting testing for inter-occasion variability. This resulted in a decrease of inter-individual variability for clearance and volume of distribution as well as in residual variability. In the theophylline doses used, a significant reduction in total and pathologic apnoea but not in hypoxaemic episodes occurred over the first three days after birth. The most positive improvement was seen on the first day of treatment after the loading dose. A statistically significant increase in the average pulse rate and a decrease in episodes of bradycardia from baseline to all three days of monitoring were recorded. Most patients responded at serum theophylline concentrations of 3 to 9 mg/L. Most serum theophylline concentration measurements were also in this range and it was not possible to clearly define a concentration-effect relationship. The cumulative percentage of non-responders was relatively high for total apnoea (48%) and hypoxaemic episodes (45%), but low for pathological apnoea (13%). Being one of a set of twins was identified as a marker of poor response for both total apnoea and hypoxaemic episodes. Other possible markers for poor response, in terms of total hypoxaemic episodes, were being born by caesarean section and having more than the 75th percentile pathologic apnoea per hour at baseline. It was interesting to note that, with regard to total apnoea, there were some features that seemed to predict a favourable response to theophylline. These were birth weight and 5 minute Apgar score below the 25th percentile, and patients with baseline total apnoea counts above the 75th percentile. The cumulative graphs of the responders and non-responders resembled the fixed effect model, which is the simplest model to explain drug-effect relationships. More sophisticated analysis of the concentration-effect relationship, using NONMEM and the count model proved difficult. None of the models tested were found to be satisfactory, but that which included the influence of a hypothetical respiratory depressant factor gave the most realistic value of EC50. It is suggested that further even more complex modelling may be required to accurately define the concentration-effect relationship (and hence the therapeutic range) for theophylline in neonatal apnoea.
Regulation of tumour-angiogenesis by protease inhibitors and receptor antagonists.
(2012) Naidu, Naressa.; Naidoo, Strinivasen.; Botha, Julia Hilary.
Introduction Angiogenesis, the growth of new blood vessels from the pre-existing vasculature, is a pre-requisite for tumour growth and metastasis. Tumour-angiogenesis is regulated by various pro- and anti-angiogenic factors released by both endothelial and tumour cells, as well as by the micro-environment. Numerous studies have implicated various systems in the acquisition of the angiogenic phenotype. The present study sought to investigate the role of the kallikrein-kinin system (KKS) in tumour-angiogenesis. The kallikreins consist of two serine proteases, plasma and tissue kallikrein (TK), involved in the release of kinin peptides by enzymatic cleavage of kininogens. Stimulation of the cognate bradykinin receptors (BKR), B1R and B2R, mediates the mitogenic and vasoactive properties of kinins. In addition, TK activates matrix metallo-proteinases (MMPs) involved in extracellular matrix (ECM) degradation. The expression profiles of TK and kinins have been found to be dys-regulated in numerous human cancers, and several studies have demonstrated the involvement of the KKS in growth and metastasis of prostate tumours. Further, previous in vitro models in our laboratory have established an association between the KKS and prostate tumour-angiogenesis. In those studies it was postulated that the up-regulated TK (produced by endothelial and tumour cells) stimulated endothelial cell proliferation. Thus, the aim of the present study was to define the effects of the KKS and seek a direct correlation with angiogenesis using in vitro models with tumour conditioned medium (CM), kinin receptor agonists and antagonists. Methods Ethical approval for this project was granted by the Biomedical Research Ethics Committee, University of KwaZulu-Natal (reference number BE152/08). Micro-vascular endothelial cells represent a suitable in vitro angiogenic model and dermal micro-vascular endothelial cells (dMVECs) were obtained commercially for this purpose. The tumour model used in this study was an immortalised prostate cancer (DU145) cell line. The CM model involves the treatment of one cell line with the metabolites of another. In the angiogenic model, dMVECs were exposed to increasing concentrations of DU145 CM. Stimulation was further augmented with BKR agonists. Specific BKR antagonists were used to test the specificity of stimulation. In addition, vascular endothelial growth factor (VEGF) was tested as a positive proliferation control. The potential of these agents to induce proliferation and migration was determined using the 3-[4,5 dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide (MTT) assay and a modified Boyden chamber assay, respectively. Previous studies investigating the pro-angiogenic effects of CM differed, in many respects, in terms of their models and methodologies. In an attempt to fully explore the pro-mitogenic effects of CM on endothelial cells, various modifications, as well as alternate endothelial and tumour cell types, were employed in the present study. The mitogenic and migratory effect of BKR agonists and antagonists on DU145 cells was also assessed. Further, the tumour model was expanded to investigate the autocrine potential of the KKS, by investigating the effect of DU145 CM on DU145 migration. Results In the angiogenic model, although the addition of DU145 CM elicited a statistically significant increase in micro-vascular endothelial cell proliferation, this increase was very small (<10%) and not dose-dependent. Pre-incubation of dMVECs with a B1R or B2R antagonist did not influence this small effect of CM on proliferation. In addition, neither B1R nor B2R agonists, at any concentration, produced any significant proliferative effect on endothelial cells. In contrast to these findings VEGF, a well-known mitogen, was able to stimulate proliferation of dMVECs. Migration assays revealed that DU145 CM failed to stimulate endothelial cell motility. Further, neither BKR agonist displayed any chemo-attractant potential in those assays. The most important finding was in the tumour model, where stimulation with a B1R agonist significantly enhanced proliferation and especially migration of DU145 cells. In addition, pre-treatment with a B1R antagonist abolished both these effects. B2R agonists could not produce the same positive effect as the B1R agonist on growth and migration of prostate tumour cells. DU145 CM did not prove to be a migratory stimulus for DU145 cells at any concentration. Discussion Previous studies in our laboratory have shown prostate-tumour CM to promote proliferation of endothelial cells and have postulated that TK up-regulation may be the reason for this. However, the present study could not reproduce this effect of CM. Further, BKR antagonists had no notable or consistent effect on the minimal promotion of proliferation that had been produced by DU145 CM. In addition, selective BKR agonists failed to induce proliferation or migration of endothelial cells, key events in the angiogenic cascade. Although in contrast to some studies, the present study was unable to implicate the KKS in angiogenesis, tumour neo-vascularisation is a consequence of several angiogenic factors functioning together as opposed to a single, isolated factor. For example, we were able to demonstrate a positive mitogenic effect of VEGF on endothelial cells and it may be this as well as other factors in the CM that are responsible for the small proliferation we observed. Up-regulation of kallikreins and kinins in tumours may enhance fundamental events in tumourigenesis in an autocrine manner, and bradykinin (BK) has previously been shown to promote tumour growth in mouse models. Our study supported the involvement of the KKS in tumourigenesis. Although CM from DU145 cells did not self-stimulate the migration of these cells, a B1R agonist enhanced both proliferation and migration, an effect that was also abrogated by the relevant antagonist, indicating a role for kinins. In contrast to the findings of another study, stimulation of the B2R failed to significantly promote tumour growth or motility. However, this is not an unexpected finding because it is thought that the ubiquitous B2R mediates physiological effects in the prostate while the inducible B1R plays a role in prostate cancer pathology. In summary, this study lends support to the ongoing exploration of BKR antagonists as possible candidates in the development of alternate approaches to cancer therapy. This may be particularly beneficial to hormone-independent tumours, such as those of the prostate, for which there exists few effective treatment options.
A retrospective analysis of prevention of mother to child transmission (PMTCT) outcomes in a group of infants attending paediatric practices in central Durban.
(2009) Cassim, Shakira Mahomed.; Botha, Julia Hilary.
The vast majority of paediatric HIV occurs in sub-Saharan Africa and could be averted through implementation of effective Prevention of Mother to Child Transmission (PMTCT) strategies. At the United Nations General Assembly Special Session on HIV/AIDS in 2001, members committed themselves to the goal of reducing paediatric HIV by 20% by 2005 and by 50% by 2010. In South Africa, rates of HIV infection range between 28% in KwaZulu-Natal and 16% in the Western Cape. The South African National Department of Health has, over the past few years, phased in a comprehensive package for PMTCT of HIV. KwaZulu-Natal implemented its programme in 2002. The South African private healthcare sector follows guidelines of those of developed countries for PMTCT. Not much data is available of the outcome of infants born to HIV positive mothers managed in private practice. In view of this, the present study aimed to assess success or otherwise of PMTCT in private paediatric practice in South Africa. Eight of the 20 private paediatricians, in the central region of Ethekweni Metro of KwaZulu-Natal (Durban Central Area), agreed to participate in a retrospective study. Data for all their HIV exposed infants between January 2004 and June 2005 were reviewed. One hundred and one Black African infants were born to 100 HIV positive women aged 29.85 years (SD 5.38; range 18-44 years). The median CD4 count was 426 (IQR 244-613). The median viral load at first presentation was 3.97 logs (IQR 1.6-5.8) or 11 391 copies/ml (IQR 2 013-41 502). Eighty six women had HAART, nine had other antiretroviral therapy and five had no prophylaxis. Treatment started before 34 weeks in 72 women. There were 93 caesarean sections. There were 20 low birth weight neonates, 18 were preterm and all had been formula fed and received AZT for six weeks. Of the 92 tested, two (one preterm) were positive. Although caesarean deliveries, both these mothers had not adhered to the optimal treatment protocol. Of the rest, eight did not return for HIV testing and one died (the only neonatal death). This death was unlikely to have been HIV related. The transmission rate of less than one percent in those women who followed the protocol optimally is much better than that in the SA public sector, and is consistent with transmission rates in the developed world.
The role of the kallikrein-kinin system in prostate and breast tumourigenesis and tumour-associated angiogenesis..
(2007) Wright, Jaclyn.; Naidoo, Strinivasen.; Botha, Julia Hilary.
This thesis consists of three main parts. An introduction to diode-pumped solid-state lasers, thermal modelling of solid-state lasers and rate-equation modelling of solid-state lasers. The first part explains the basic components and operation principles of a typical diode-end-pumped solid-state laser. The stimulated emission process, solid-state laser gain media, various pump geometries and a basic end-pumped laser resonator configuration are among the topics that are explained. Since thermal effects are one of the main limiting factors in the power-scaling of diode-pumped solid-state lasers, the second part of this thesis describes numerical and analytical thermal models that determine the thermal lens and thermally induced stresses in a laser crystal. As a first step, a time-independent numerical thermal model which calculates the three-dimensional temperature distribution in the laser crystal is implemented. In order to calculate the time dependent thermally induced stresses in a laser crystal, a coupled thermal-stress finite element analysis model was implemented. Even though some steady-state analytical solutions for simple crystal geometries do exist, the finite element analysis approach was taken so that the time dependent thermally induced stresses could be calculated for birefringent crystals of various geometries. In order to validate the numerical results, they are compared to experimental data and analytical solutions where possible. In the last part, the population dynamics inside the laser gain medium are described and modelled with a quasi-three-level rate-equation model. A comprehensive spatially resolved rate-equation model is developed and discussed. In order to simplify the implementation of the rate-equation model as a computer simulation, the spatial dependence of the laser parameters is ignored so that the model reduces to a singleelement plane-wave model. The simplified rate-equation model is implemented and solved numerically. The model is applied to a four-level CW and Q-switched Nd:YLF laser as well as a quasi-three-level QCW Tm:GdV04 laser. The models' predictions are thoroughly verified with experimental results and also with analytical solutions where possible.