Doctoral Degrees (Pharmaceutical Sciences)

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Bioavailability studies on various dosage forms of the anorectic, diethylpropion hydrochloride.
(1984) Dangor, Cassim Mahomed.; Veltman, A. M.
The stereo-chemistry, structure activity relationships and the metabolism of the anorectic drug, diethylpropion hydrochloride, have been reviewed briefly, together with the analytical methods for the determination of this drug and its metabolites in biological fluids. In addition, the physico-chemical properties, mode of action, pharmacology and uses of the metabolites have been presented. A comprehensive review on general principles of salivary excretion of drugs and their therapeutic drug monitoring in saliva with relevant published data on saliva/plasma drug concentration relationships has been outlined. Sensitive and specific assay procedures, based on gas-liquid chromatography for the identification, separation and determination of diethylpropion and its two major metabolites i.e. ethylaminopropiophenone (11) and diethylnorpseudoephedrine (IV) in aqueous and biological fluids, have been developed. These methods were used to study the urinary excreUon as well as saliva and plasma levels of the two major metabolites and, where possible, the unchanged drug, in man. Sustained release pellets with diffusion rate-controlled membranes were employed to control the rate of input into the body by oral or rectal route of administration. Urinary excretion data and plasma levels of metabolites 11 and IV in volunteers, where the urine was controlled at an acidic pH, were used for the evaluation of the bioavailabilities of different dosage forms of diethylpropion hydrochloride. The concentrations of metabolites 11 and IV were also measured in saliva and in plasma after administration of the drug in different doses and dosage forms: relationships between saliva and plasma concentrations (S/P) and between urinary excretion rates and plasma concentrations (U/P) were developed for each of the two metabolites during plateau levels after oral administration of the sustained release pellets (Lot R 7773). The potential use of salivary excretion of the metabolites as an index to monitor their plasma levels and bioavailabilities, was examined. The distinct advantage of using a subdivided controlled release system (i. .e. sustained release pellets) to a single unit sustained release tablet (erosion-core type) in relation to influence of the physical presence of food on the rate and extent of absorption has been demons t rated . It was found that the route of administration (oral or rectal) did not significantly affect the bioavailability of the sustained release pellets. The study also involved the investigation of the release of the drug from the pellets. Because the release control step was diffusion, no significant influences on dissolution rates were observed with the use of different dissolution test models and agitation intensities. The influence of the concentration and composition (presence of cations viz. Na+ and K+ i~r anions viz . phosphate and borate) of the dissolution medium on the release of the drug from sustained release pellets, was also studied. Any potential changes in the dissolution pattern on storage of the pellets under different conditions (4°C, room temperature and 37°C) ovrr, a period of at least one year, were investigated. The in vitro and in vivo correlations of two lots of sustained release pellets, each exhibiting different dissolution profiles, and administered rectally and orally, were developed: the in vitro data on the free drug were related to the sum of the urinary excretion data of metabolites II and IV. An attempt to use an empirical approach to predict urinary excretion rate profiles of metabolite II after oral administration of the sustained release pellets, was promising; the calculated profiles were reasonably comparable with those of in vivo studies. However, the complete validity of such equations needs further investigations.
A study of the relationship between the pharmacokinetics and the pharmacodynamics of atenolol in black and white subjects using an effect modelling technique.
(1991) McFadyen, Margaret Lynn.; Miller, Roger.; Miller, Roger.
Abstract is available in PDF file.
An investigation of the antidiabetic herbal remedies used by traditional healers in Northern KwaZulu-Natal and their effect on blood glucose levels.
(1998) Ziqubu-Page, Thembelihle Thandekile.; Chetty, Manoranjenni.; Makubalo, L. E.; Dangor, Cassim Mahomed.
This research study undertook to investigate and evaluate for efficacy and safety, the herbal remedies used for treating Diabetes mellitus in northern KwaZulu-Natal. In addition, it sought to gain knowledge and better understanding of traditional healing systems and the medicinal use of the natural flora. During the process of assimilating the desired information, the epidemiological and socio-economic factors which determine the form of medicine chosen by rural people in the region, were quantified. Both aspects of explanatory studies i.e. experimental and observational were used. Firstly, to evaluate the safety of the two herbal remedies, laboratory animals were given an oral dose of the herbal medicine and observed for a period of 14 days. Efficacy was assessed by treating Streptozotocin-induced diabetic rats with the herbal remedies and comparing their effect on blood glucose with that of a conventional sulphonylurea. The second part of the study was observational and it involved monitoring human subjects (patients) for twelve months, who were already taking the herbal preparations (n=56) and comparing their prognoses with that of a group taking conventional medicine (n=97). A third group using both types of medicine (n=42) was included as control measure for a possible confounding factor. Main outcome measures; Both subjective and objective measures of the perceived health of the diabetic patients were measured, as well as the determinants of using traditional medicine versus conventional medicine. The battery of toxicity tests which utilises behavioural and functional observations of the laboratory animals, yielded no signs of toxicity or abnormal behaviour. The histopathological examination results of the sample organs from the treated rats also revealed no signs of abnormality that could be attributed to the herbal remedies tested. There was no sex variation recorded in the response. The first HP tested (HP-1) demonstrated minimal hypoglycaemic effect whereas HP-2 significantly lowered the blood glucose of the streptozotocin-induced diabetic rats by an average of 59%. This was comparable to the conventional medicine (Glibenclamide) used in the experiment. After 12 months of follow-up, 93 % of traditional medicine users (n=56) were convinced that their blood sugar was controlled because of the traditional remedy they were using. The proportion of diabetic cases who used conventional medicine were no better off than those who used traditional medicine or vice versa. Health status and the financial situation (income) of the respondents greatly influenced their choice for diabetic treatment. The herbal remedies that were investigated were non-toxic and safe for use and internal consumption. One preparation demonstrated a significant hypoglycaemic effect, which was comparable to the conventional allopathic medicine used in treating Diabetes mellitus. This study should serve as a springboard to encourage more pharmacological evaluation of herbal medicines.
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.
Vasoactive peptides in acute renal allograft rejection and renal diseases: the role of endothelins, atrial natriuretic peptide and kinins.
(2001) Naicker, Saraladevi.; Bhoola, Keshavlal Daya Narotam.
Abstract available in PDF.
The role of kinins and cytokines in rheumatoid arthritis.
(2001) Cassim, Bilkish.; Bhoola, Keshavlal Daya Narotam.
Introduction: Rheumatoid arthritis (RA) is a systemic inflammatory disease characterized by inflammatory synovitis. The histopathological features include synovial hyperplasia, an inflammatory cell infiltration, angiogenesis and an inflammatory exudate into the synovial joint with progression to bone and joint destruction. While the exact aetiology of RA is unknown, a number of inflammatory cells and mediators have been implicated in the pathogenesis. Kinins are vasoactive peptides that have the capacity to induce the cardinal features of inflammation and considerable evidence exists for a role for the kallikrein-kinin cascade in inflammatory arthritis. The proinflarnmatory cytokines are also important mediators in rheumatoid arthritis and there is evidence for a functional relationship between the kallikrein-kinin and cytokine cascades in rheumatoid arthritis. Methods: Following approval from the Ethics Committee of the University of Natal, synovial tissue samples were obtained at arthroscopy from patients with RA and at autopsy (for controls). The tissue samples were processed for light microscopy and immunostained by the immunoperoxidase method to detect tissue kallikrein and the kinin B 1 and B2 receptors. The intensity of the immunostaining was quantified by image analysis. Blood and synovial fluid samples were obtained from patients with RA and blood from age and sex matched healthy volunteers. The RA patients were assessed clinically to determine the degree of disease activity and the presence or concomitant diseases. Disease activity was determined by the duration of morning stiffness, the twenty eight tender and swollen joint counts, pain on a visual analogue scale, patient's and physician's global assessment of disease activity (Likert scale), a local activity index, the modified Health Assessment Questionnaire (HAQ), disease activity score (DAS) and the erythrocyte sedimentation rate (ESR) and C reactive protein (CRP). In the synovial fluid (SF) samples, the functional activity of tissue kallikrein (TK) was demonstrated using an amidolytic assay and the total amount of TK was measured in a sandwich enzyme linked immunosorbent assay (ELISA). The Pearson's correlation coefficient was used to correlate the TK levels with measures of disease activity. Further, basal and generated kinins were measured in the SF by competitive ELISA, and the levels correlated with measures of disease activity. In the cytokine study, interleukin lP (IL IP) and tumour necrosis factor p (TNFP) were measured in the synovial fluid samples by ELISA, and the relationship between the cytokine levels and disease activity as well as TK, basal and generated kinins determined. Neutrophils were isolated from the blood and synovial fluid samples from the rheumatoid arthritis patients and from the blood samples from healthy volunteers. The circulating and synovial fluid neutrophils were immunostained to detect tissue kallkrein, the kinin moiety in the kininogen molecule and kinin BI and B2 receptors, and the immunofluorescence visualized by confocal microscopy. The images were digitally analysed using the Analysis 2.1 Pro system. The Kruskal Wallis and one-way ANOVA tests were used to compare the mean intensity of imrnunostaining in the control neutrophils with that present on the circulating and SF neutrophils harvested from RA patients. The intensity of labeling for these antigens was correlated with measures of disease activity. Results: 1. Synovial tissue samples: Labeling for tissue kallikrein was observed in the synovial lining and endothelial cells in control and rheumatoid tissue. There was a significant increase in the intensity of TK labeling in the endothelial cells of the rheumatoid tissue (p < 0.05). The kinin Bl and B2 receptor were visualized in the synovial lining cells, endothelial cells and the subintimal fibroblasts and macrophages in the control and rheumatoid synovial samples, with a significant increase in B 1 receptor labeling in the synovial lining cells in rhewnatoid synovial tissue (p < 0.01). 2. Tissue kallikrein activity and the total TK concentration was measured in the synovial fluid obtained from 20 patients with RA. There was no direct correlation between the between the enzymic and antigenic tissue kallikrein. There w as a significant negative correlation between the enzymic TK and the twenty eight swollen joint count (r = -0.464; p <0.05). 3. There was a significant negative correlation between the basal kinin and generated kinin levels (r= -0.454; p < 0.05). In addition, there was a negative correlation between the basal kinin levels and the C RP (r = -0.537; p < 0.05) and the disease activity score (r = -0.458; p < 0.05). In contrast, there was a positive correlation between the generated kinin levels and the twenty-eight tender and swollen joint counts (r = 0.536; p < 0.05 and r = 0.509; p < 0.05 respectively), the ESR (r = 0.598; p < 0.01), CRP (r = 0.725; p < 0.01) and the disease activity score (r = 0.676; p < 0.01). There was a significant correlation between the SF levels of IL IP and pain (r = 0.462; p < 0.05), physician's global assessment of disease activity (r = 0.549; p < 0.05), 28 tender joint count (r = 0.4 72; p < 0.05) and CRP (r = 0.530; p < 0.05). Although there appeared to be a correlation between the IL 1 p and disease activity score, this was not significant (r = 0.412; p = 0.07). In addition, the levels of synovial fluid lNF a correlated with the 28 tender joint (r = 0.458; p < 0.05) count and CRP (r = 0.653; p < 0.01). 4. There appeared to be a trend towards a negative correlation between the SF amidase TK levels and IL 1 p, however this was not significant. While there was no direct relationship between the SF levels of IL 1 P and the generated kinins, there was a positive correlation between low to moderate levels of IL 1 p and the generated kinins (r = 0.51, p < 0.05). In contrast, there was a negative correlation with higher levels of IL Ip (r = -0.5, p < 0.05). 5. Immunoreactive TK, kinin moiety and the Bl and B2 receptors were visualized on the circulating neutrophils from the healthy volunteers and the circulating and SF neutrophils from the RA patients. There was no statistically significant difference in the mean intensity of TK labeling in the circulating neutrophils from healthy volunteers (n=8) and the circulating and synovial fluid neutrophils of the RA patients n=8). However, when the intensity of labeling of the SF neutrophils (n=80) from the RA patients was compared to the circulating neutrophils (n=80) of healthy volunteers, there was a significant loss of TK labeling in the SF neutrophils of the RA patients (1-Way ANOVA, p < 0.01). In the RA patients, there was a loss of the kinin moiety from both the SF and circulating neutrophils compared to controls (Kruskal-Wallis: p < 0.05 and < 0.01 respectively). Although there was a clear increase in the intensity of labeling of the kinin BI receptor on the SF neutrophils from RA patients (n=8), when compared to the circulating neutrophils from healthy volunteers (n=8), the mean values did not reach significance (Kruskal Wallis; p > 0.05). However, a significant increase in Bl receptor labeling was observed on both the circulating and SF neutrophils of the RA patients (n=80) when compared to circulating neutrophils of the healthy volunteers (n=80) (1 Way ANOVA, p < 0.01 and < 0.05 respectively). In addition, there was a positive correlation between the immunoreactivity for the BI kinin receptor on the circulating neutrophils from the RA patients and the local activity index (r = 0.783; p < 0.05). Although there was a clear increase of the kinin B2 receptor on the circulating and SF neutrophils from the RA patients compared to circulating neutrophils from healthy volunteers, this was only significant for the circulating neutrophils from the RA patients (Kruskal-Wallis, p = 0.05). There was no correlation between the intensity of labeling of TK, the kinin moiety and the B2 receptor and measures of disease activity. Discussion and conclusions 1. This study provides the first evidence for the localization of TK and the kinin receptors in control and rheumatoid synovial tissue using antibodies specific for each protein and standard immunolabelling techniques. Synovial fibroblasts, macrophages and endothelial cells through the release of enzymes and cytokines have the ability to mediate the inflammatory changes and cartilage and bone destruction in RA. The presence of TK and the kinin receptors in these cells therefore provides evidence for a pathogenetic role for the kallikrein-kinin cascade in RA. 2. In addition, in RA there is an exudation of fluid into the joint space. Tissue kallikrein has been previously reported in the synovial fluid obtained from RA patients, however the correlation of TK levels and disease activity has not been previously studied. The negative correlation between enzymic TK and the twenty-eight swollen joint count, an indicator of disease activity suggests that there is a consumption of TK in inflammation, presumably due to increased kininogenase activity. Similarly, the kinin generating capacity of synovial fluid obtained from RA patients has been previously reported, however, this is the first study demonstrating a link between kinin generating capacity and validated markers of disease activity. The kinin generating capacity is a complex and dynamic cascade involving the bioregulation of all the components of the kallikrein-kinin system and is therefore more likely to accurately define the role of kinins in inflammatory arthritis than are individual components of the kallikrein-kinin cascade. Measurement of tissue kallikrein and basal kinins is affected by the presence of natural inhibitors and their short half-life in biological fluids. In addition to the synovial fluid study a decrease in the urinary TK activity and an increase in urine kinin generated kinins was demonstrated, suggesting that there is a systemic activation of the kallikrein kinin cascade in RA. 3. Although there is evidence for an interaction between the kallikrein-kinin and cytokine cascades in inflammation, in this study a direct correlation between the levels of interleukin 1 J3 and tumour necrosis factor J3 in the synovial fluid and TK and generated kinin levels was not found. This may be due to the wide variations in the levels of cytokines, the presence of inhibitors and anti-inflammatory cytokines, or a complex and dynamic relationship between the two cascades. However, the correlation of both generated kinins and interleukin l J3 and tumour necrosis factor J3 with disease activity provides circumstantial evidence for a synergistic role for these mediators in inflammatory arthritis. 4. In the neutrophil study, loss of immunoreactive tissue kallkrein and kinin moiety from the neutrophils obtained from RA patients was demonstrated. This finding supports the hypothesis that kinins are released from the neutrophils by the enzymatic action of tissue kallkrein and suggests that the kallkrein-kinin system is activated both locally and systemically in patients with RA. Further, there was upregulation of the both the kinin B 1 and B2 receptors on the neutrophils from the RA patients. While the B2 receptor is thought mediate most of the actions of kinins, the correlation of the intensity of B 1 receptors and the local activity index implies that the B 1 receptor may be important in inflammation. 5. These findings provide convincing evidence for the role of the kallikrein-kinin cascade in the pathogenesis of inflammation in RA. Further development of kinin receptor antagonists may provide a novel therapeutic modality.
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.
Some pharmacological properties of Harpagophytum procumbens DC [Pedaliaceae] secondary root extract
(2004) Mahomed, Ismail Mall.; Ojewole, John Akanni Oluwole.
Abstract available on PDF file.
Beta-lactamase mediated resistance in Escherichia Coli isolated from state hospitals in KwaZulu-Natal.
(2008) Mocktar, Chunderika.; Essack, Sabiha Yusuf.; Sturm, Adriaan Willem.
Escherichia coli, one of the most common pathogens causing urinary tract infections, has shown increased resistance to commonly used antibiotics. In this study we analyzed the β-lactamase profiles of 38 inhibitor-resistant E. coli isolates obtained from public hospitals at three different levels of healthcare in KwaZulu-Natal, selected on the basis of their resistance profiles to the three antibiotic/inhibitor combinations, viz., amoxicillin/clavulanate, ampicillin/ sulbactam and piperacillin/ tazobactam. The isolates were subjected to MIC determinations, IEF analysis, plasmid profile analysis, PCR of the different β-lactamase genes and sequencing thereof to detect the possible mechanism/s of resistance. A range of β-lactamases including two novel inhibitor-resistant TEM β-lactamases, TEM-145 and TEM-146 were detected in two isolates whilst a novel plasmid-mediated AmpC-type β-lactamase, CMY-20 was detected in three isolates. Other β-lactamases included OXA-1, TEM-55, SHV-2, CTX-M-l and TEM-1. Changes were detected in the chromosomal AmpC promoter/attenuator regions in one isolate. Diverse β-lactamase genes and plasmid profiles inferred extensive mobilization of β-lactamase genes causing the concern of limited therapeutic options in the face of increasing resistance.
Synthesis and biological activities of natural homoisoflavanones.
(2011) Shaikh, Mahidansha Mahiboob.; Du Toit, Karen.; Kruger, Hendrik Gerhardus.
Plants have formed the foundation of traditional medicine systems throughout the world for thousands of years and continue to provide mankind with new remedies for various ailments. A large portion of the black South African population still depends on medicinal plants as primary health care due to its affordability, accessibility and cultural importance. These medicinal plants need to be investigated since new lead compounds are often found in nature. Homoisoflavanones isolated from South African and Indian plants were found to exhibit anti inflammatory activities although the mechanism of action has not yet been determined. A few reports on the anti fungal activities of these compounds were also found. Four new and three known homoisoflavanones of the 3-benzylidene-4-chromanone type were synthesized and tested for anti-inflammatory and antifungal activities. Two novel intermediates were also synthesised. Enantiomers of a homoisoflavanone of the 3-benzyl-4- chromanone types were also synthesized from the corresponding 3,5-dimethoxy phenol via 4- chromanone in six steps. This is the first report of the synthesis of an enantiomerically pure homoisoflavanone compound together with its opposite isomer. The enantiomers and racemate were tested for anti-inflammatory activity. All the synthesized homoisoflavanones were screened for cytotoxicity. The structures of these homoisoflavanones were elucidated by NMR spectroscopy along with HRMS data. The crystal structure of a homoisoflavanone with anti-inflammatory and antifungal activity is reported. The anti-inflammatory activity of the homoisoflavanones was determined in an acute croton oil-induced auricular dermatitis mouse model. The antifungal activity was performed in vitro against a Candida albicans strain. Compounds were tested for cytotoxicity against a Chinese Hamster Ovarian (CHO) cell line using the 3-(4,5-dimethylthiazol-2-yl)-3,5- diphenyltetrazoliumbromide (MTT) assay. In conclusion, the synthetic homoisoflavanones showed anti-inflammatory as well as antifungal activity. Some of the compounds showed anti-inflammatory activity comparable to that of the commercially available diclofenac.
Design, synthesis and screening of novel PCU-peptide/peptoid derived HIV protease inhibitors.
(2011) Makatini, Maya Mellisa.; Govender, Patrick.; Kruger, Hendrik Gerhardus.; Maguire, Glenn Eamonn Mitchel.; Govender, Thavendran.
The AIDS epidemic in Africa has reached dramatic proportions. Of the 42 million people infected with HIV worldwide, 30 million are in Africa. Current available therapies have begun to transform this fatal disease into a chronic condition but there are still major obstacles that have resulted in a great demand for new and better drugs. The aim of this study was to synthesize novel and effective HIV protease inhibitors. This work describes the first account of pentacycloundecane (PCU)-peptide and peptoid based protease inhibitors. These inhibitors are proposed to bind the wild type C-South African HIV protease (C-SA) catalytic site via the norstatine or dihydroxylethelene type functional group of the PCU. The desired compounds were synthesized by the coupling of the peptides and peptoids to the PCU cage which resulted in a series of promising and structurally diverse HIV-1 protease inhibitors. The inhibitors were characterized by Nuclear Magnetic Resonance (NMR) and evaluated against the wild type C-SA enzyme for its ability to inhibit 50 % of the enzyme’s activity (IC50). Two of the compounds reported herein, inhibited the enzyme activity at concentrations less than 80 nM. NMR investigations indicated that the activity was related to the chirality of the PCU moiety and its ability to induce conformations of the coupled peptide side chain. Employing the new Efficient Adiabatic Symmetrized Rotating Overhauser Effect Spectroscopy (EASY-ROESY) technique enabled us to obtain vital information about the 3D structure of these small linear peptides and peptoids in solution. This technique is the first example describing the successful through space correlations of such small peptides. Furthermore, docking and a combined quantum mechanics/molecular mechanics (QM/MM) molecular dynamics MD simulation at the AM1 semi empirical level mirrored the observed NMR results and the experimental IC50 activity profile of the considered inhibitors. The combination of these experimental and theoretical methods provided a powerful insight into the interaction mode of these cage peptide and peptoid inhibitors with the enzyme.
Studies on some pharmacological properties of Capsicum frutescens-driven capsaicin in experimental animal models.
(2012) Jolayemi, Adebayo Taiwo Ezekiel.; Ojewole, John Akanni Oluwole.
The present study investigated pharmacological properties of Capsicum frutescens-derived capsaicin, including its analgesic, anti-inflammatory and coagulatory properties. The effects of capsaicin on gastrointestinal and myocardial muscles, as well as on myocardial ischaemic-reperfusion, were also investigated. Capsaicin pre-treatment in neonatal rats has been found to abolish the development of thermal hyperalgesia produced in a model of neuropathic pain in rats (Toth-Kasa et al., 1986). In addition, capsaicin sensitivity has been found to be dependent on continued presence of nerve growth factor (NGF), whose concentration increases in inflamed tissues (Bevan and Winter, 1995). By stimulating the release of excitatory amino acids (EAA); such as glutamate and neuropeptides [(CGRP, neurokinin A (NKA) and Substance P (SP)] from both the peripheral and central terminals of sensory neurones by two mechanisms (Kroll et al., 1990; Del Bianco et al., 1991; Lou et al., 1992; 1994; Woolf et al., 1994); capsaicin has been shown to produce a longer-term inhibitory effect. This is one likely mechanism for capsaicin analgesic and anti-inflammatory actions (Bleakman et al., 1990). Within the gastro-intestinal tract, SP and NKA are involved in the physiological control of several digestive functions, such as motility, fluid and electrolyte secretion, blood flow, and tissue homeostasis (Otsuka, 1993; Holzer et al., 1997). Consistent with this finding, upsurge of SP in irritable bowel syndrome (IBD) was confirmed by Mantyh et al, (1988). Pre-treatment of rats with either capsaicin or NK-1R antagonists dramatically reduced fluid secretion, mucosal permeability, and intestinal inflammation in animal models of acute and chronic inflammation (McCafferty et al, 1994; Pothoulakis et al., 1994). Capsaicin can modulate endocrine and paracrine activities, immune responses, as well as gastro-intestinal and cardiovascular functions. Moreover, up-regulation of Substance P receptors was found to be associated with chronic inflammatory conditions (De et al., 1990). Stimulation of transient receptor potential vanilloid 1 also results in the activation of nociceptive and neurogenic inflammatory responses (Rigoni et al., 2003). vi The pharmacodynamic effects of capsaicin on the cardiovascular system remain elusive. Some actions of capsaicin on the heart were attributed to an interaction at K+ channels (Castle, 1992), or liberation of neuropeptides, most notably calcitonin-gene-related-peptide (CGRP) from the vanilloid-sensitive innervation of the heart (Franco-Cereceda et al., 1988; 1991). The possibility of a direct effect of capsaicin on the heart via a cardiac vanilloid receptor (VR), or through interaction of vanilloid receptors with purinergic receptors, and subsequent release of nitric oxide (NO), leading to vasodilatation were considered. Evidence abound in the literature that Ca2+ ions are released through 1, 4, 5 inositol phosphatase by the release of phospholipase C, or through interaction of the vanilloid receptors with cannabinoids. In an earlier study, Jaiarj et al. (1998) found that capsaicin acting on the heat-sensitive vanilloid receptors, had thrombolytic effects. Though weak evidence, Jaiarj et al. (1998) observed that individuals who consume large amounts of Capsicum have lower incidence of thromboembolism. Following ethical approval, the study reported in this thesis was conducted in phases. Identification of Capsicum frutescens (facilitated by a botanist in the Department of Botany, Westville campus of the University of KwaZulu Natal). Chromatographic extraction of capsaicin from Capsicum frutescens was followed by Nuclear Magnetic Resonance (NMR) analysis of the extract. Animal studies were conducted using capsaicin extract (CFE) and/or a reference capsaicin (CPF), using „hot plate. and „acetic acid. test methods to investigate the role of capsaicin on analgesia. Fresh egg albumin-induced inflammation was used to investigate the role of capsaicin in inflammation, following pre-treatment with CFE and CPF. Concentraton-response curves of increasing concentrations of capsaicin, acetylcholine and other agonist drugs with specific antagonists on strips of chick oesophagus, guinea-pig ileum, and rabbit duodenum were constructed following investigations on gastrointestinal (GIT) smooth muscles. The effect of capsaicin on coagulation was assessed by measuring international normalized ratio (INR) of animals that were exposed to different concentrations of capsaicin (CFE and CPF). Furthermore, parallel control studies were conducted in each of these investigations using distilled water or saline as placebo-control or specific-prototype agonists. negative-control. Cardiovascular investigations included studies on the effects of capsaicin on the heart rate, inotropy, vii coronary perfusion pressure, and ischaemic-reperfusion injury, using Langendorf.s rat heart models. Collated data were triangulated by manual hand-written and PowerLab data acquisition, or computerised capture. Statistical analysis were performed by either one or two of the following: Student.s t-test, ANOVA (repeated or single–use modes), facilitated and confirmed by Graph Pad Prism, Microsoft Excel or CPSS software(s). Reproducibility and relevance to the stated objectives of the various studies were confirmed by assessing which of the Null or Alternative hypothesis is validated by the results from the test. Treatment with CFE or CPF at all doses significantly (p<0.01) increased MRT. By comparison with control, writhing responses to acetic acid were significantly reduced following pre-treatment with various doses of CFE or CPF. The results in both parallel groups of CFE and CPF in the hot plate and acetic acid tests had Pearson correlation of one (1). Compared to the diclofenac (DIC) group, the degree of inhibition of paw oedema by CFE and CPF was statistically significant (P<0.05-0.001), best in the first 4 hours of treatment. The results of the in vitro laboratory animal study indicate that relatively low concentration of CPF (20 or 40 .g) produced significant (p.0.05), concentration-related inhibitions of acetylcholine (0.1-5 .g)-induced contractions of the chick isolated oesophagus, guinea-pig isolated ileum and rabbit isolated duodenum. Biphasic effects, which were noticed at low concentrations, consisted of initial brief contractions, followed by longer-lasting relaxations and reductions of the contractile amplitudes of the muscle preparations. Percentage inhibitions of the smooth muscle contractions by CFE or CPF were concentration-dependent, ranging from 20-70% (p<0.02).
Grapefruit juice ameliorates nephropathy in streptozotocin induced diabetes.
(2012) Hayangah, Julia Achieng'.; Owira, Peter Mark Oroma.
Background Diabetic Nephropathy (DN) is the leading cause of end stage renal disease and mortality in diabetic patients. Over the years, medicinal plants have been used to manage diabetes and its complications. Metformin, the synthetic analogue of galegine is used as first line therapy for Diabetes Mellitus (DM) but its use is contraindicated in patients with kidney dysfunction. The management of DN currently is limited to the use of anti-hypertensive agents; ACE inhibitors and angiotensin receptor blockers. Grapefruit juice (GFJ) has shown potential as an anti-diabetogenic agent because of its ability to ameliorate hyperglycaemia and dyslipidemia but its effect on fluid and electrolyte disturbance is not known. This study was designed to investigate the effect of GFJ on renal dysfunction in streptozotocin (STZ) induced male wistar rats. Materials and Methods Male wistar rats weighing between 250-300g were divided into 7 groups (n=7) and kept in cages for the treatment period of 8 weeks. Laboratory conditions of 12 hour light/dark cycle, temperature 25±20C and humidity 50-55 % were maintained throughout the study period. Non-diabetic animals group 1 (Control) were treated orally with 1.0 ml /Kg BW of distilled water, while group 2 (ND-GFJ) were treated orally with 3.0 ml /kg BW of GFJ. The diabetic groups 3, 4, 5, 6 and 7 were starved overnight in preparation for the STZ injection. Fasting blood glucose concentration was obtained via tail prick before 45 mg or 60 mg of STZ was administered via a single injection in the peritoneal cavity. STZ was prepared by dissolving it in 0.2 ml of 0.1 M Citrate buffer at pH 4.5. Groups 3, 4 and 7 received 60.0 mg/ Kg BW of STZ while group 5 and 6 received 45 mg/kg BW of STZ. Three days following STZ induction, the diabetic state was confirmed by measuring fasting blood glucose and animals with glucose concentration greater than 6 mmol/L were included in the study. Group 4 (INS- D60) and group 5 (INS- D45) were additionally treated with 4.0 U/kg BW of insulin via subcutaneous injection (S.C) twice a day while Group 6 (GFJ-D45) and group 7 (GFJ-D60) were treated orally with 3.0ml/Kg BW of GFJ. Group 3 (D-60) were similarly treated with 1.0 ml /Kg BW of distilled water. Fasting blood glucose (FBG) and glucose tolerance tests (GTT) were done on days 1 and 58 respectively in all the treatment groups. Urine was collected for a period of 24 hours on day 59 and on the last day animals were sacrificed by halothane overdose. Blood samples were obtained via cardiac puncture; kidney tissues were removed and preserved in formalin while the liver was snap frozen with liquid nitrogen and stored in a freezer (-800C) before analysis. Results Reduced plasma insulin was accompanied by decrease in body weight and an increase in FBG accompanied by polyuria, polydipsia and glucose intolerance in the non-treated diabetic animals compared to the control. Fasting blood glucose was significantly (p<0.0001) increased in the diabetic groups and treatment with GFJ or insulin lowered FBG in groups (GFJ-D45, GFJ-D60 & INS-D60) compared to the diabetic control (D60). GFJ significantly (p=0.0034) improved glucose intolerance in diabetic animals (GFJ-D60 & GFJ-D45) when compared to diabetic control groups D-60 & D-45 respectively. Hepatic glycogen content was reduced in diabetic animals (P=0.024) and treatment with GFJ significantly (P=0.00016) increased the glycogen concentration. In the non-diabetic group (GFJ-ND) treatment with GFJ significantly (P=0.0013) increased the glycogen concentration when compared to the control group. In the diabetic animals, decreased GFR was accompanied by Na+ retention accompanied by low urinary K+ and Cl- concentration. Treatment with GFJ significantly (p<0.05) increased urinary Na+ and K+ and Cl- in the diabetic group (GFJ-D60) but did not increase urinary Cl- in non-diabetic group and consequently improved GFR in the diabetic group. Renal pathology showed structural changes in the glomerulus and treatment with GFJ had some reno-protective effect. Conclusion GFJ lowered the fasting blood glucose and improved glucose tolerance in the STZ-induced diabetic rats in a comparable manner to insulin treated diabetic rats. GFJ decreased Na+ retention and increased GFR in the diabetic animals. This study suggests that GFJ could ameliorate nephropathy associated with diabetes mellitus. These results are the first to show that GFJ has renoprotective effects in STZ-Induced diabetic rats.
Synthesis and evaluation of novel tetrahydroisoquinoline organocatalysts in asymmetric catalysis.
(2012) Naicker, Tricia.; Govender, Thavendran.; Kruger, Hendrik Gerhardus.; Maguire, Glenn Eamonn Mitchel.; Arvidsson, Per Ingemar.
Organocatalysis has rapidly expanded in the last decade to encompass a wide variety of small organic molecules that are capable of either activating substrates or transforming them into more reactive forms. The aim of this study was to develop novel chiral organocatalysts based on the tetrahydroisoquinoline backbone and evaluate them on asymmetric reactions. Three organocatalytic modes of activation have been investigated for C-C bond forming asymmetric reactions. In chapter 2, for the first time organocatalysts bearing a secondary nitrogen within a cyclohexane ring were evaluated in the asymmetric Diels–Alder reaction. These catalysts were tested over a range of dienes and dienophiles and displayed promising chemical conversions of up to 100 % with up to 64 % ee when triflic acid was employed as the cocatalyst. Density functional theory computational studies and 2D NMR spectroscopy were used to determine the structure of the intermediate iminium ion formed between the most efficient catalyst and cinnamaldehyde. Chapter 3 includes a series of novel tetrahydroisoquinoline chiral N-oxide organocatalysts and their evaluation in the asymmetric allylation reaction of aromatic and α-β-unsaturated aldehydes with allyltrichlorosilane. The chiral homoallyl products were obtained with good chemical efficiency (up to 93 % yield) and high enantioselectivity (up to 91 % ee) under mild reaction conditions (23 °C). Chapter 4 is the simple and practical microwave-assisted synthesis of new tetrahydroisquinoline guanidine organocatalysts and their evaluation in the asymmetric Michael addition reaction of malonates and β-ketoesters with nitro-olefins. In addition, a novel microwave assisted procedure of introducing the guanidine unit onto amino amide derivatives is reported. The chiral products were obtained with quantitative chemical efficiency (up to 99 % yield) and excellent enantioselectivity (up to 97 % ee). Chapter 5 is a collection of all X-ray crystal structures that were published from novel compounds synthesized pertaining to Chapters 2-4, it contains 15 published crystal structures while Chapters 3-4 contain 3 other X-ray crystal structures. It should be noted that with the exception of the introduction and Chapter 4 (submitted for publication), the remaining chapters of this thesis have been published in international peer reviewed journals. In the next section (DECLARATION 2 – PUBLICATIONS) a precise description of my contribution to each of the publications/chapters is provided.
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.
Investigating plasmepsin flexibility as a function of the flap region : a unique structural and dynamic feature of aspartic protease.
(2015) McGillewie, Lara.; Mahmoud, E Soliman.
Malaria is one of the most deadly infectious protozoan diseases known to man. It is spread by the Plasmodium parasite through the bite of the female Anopheles mosquito. Increasing resistance to currently available antimalarial drugs is a growing concern. Plasmepsins are malarial aspartic proteases, due to their characteristic mechanism of action, the fact that they are found in all Plasmodium species and are essential to parasitic survival they represent novel targets in the design of antimalarials. A unique structural feature of aspartic proteases and plasmepsins is the flap region lying perpendicular to the catalytic aspartic acid active, partially covering the active site. The flap region plays an important structural (and kinetic) role in regulating access to the active site, thereby regulating ligand binding. The present study focused on the flap dynamics of Plm I – V, proposing and validating parameters to accurately quantify the dynamic behaviour of the flap region. The catalytic aspartic acids is highly conserved in the plasmepsin family; sequence analysis revealed that although all plasmepsins are similar in structure, they differ greatly in the residues in the flap region. The heterogeneity in this region gives each plasmepsin unique substrate specificity and response to inhibitors. The parameters proposed in the present study gives a detailed account for the twisting of the flaps which move away from the active site in the absence of an inhibitor. Upon inhibitor binding, residues in the flap region form hydrogen bonds with the inhibitor pulling it inward towards the active site rendering the enzyme inactive. The parameters proposed in the present study will be of great value in the design of novel plasmepsin inhibitors, with increased efficacy and potency.
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.
Meeting South Africa's pharmacovigilance challenges in the face of rapidly increasing public health treatment programmes.
(2016) Dheda, Mukesh.; Oosthuizen, Frasia.
Among South Africa‘s (SA‘s) many public health challenges, having the largest treatment programmes globally is a significant challenge. The latter requires a robust pharmacovigilance (PV) programme. The purpose of this study was to conduct a review of the PV landscape in SA to meet the PV challenge of these treatment programmes. Aims and Objectives The aim of this study was to explore the decentralised PV approach to support key public health programmes. The specific objectives were firstly, to trace and reconstruct the history of PV activities to date. Secondly, to benchmark the current PV activities through an appropriate baseline assessment. Thirdly, to determine the impact of a PV training intervention. And finally, to evaluate the effectiveness of a low cost SAspecific PV strategy implemented in response to major public health challenges, namely HIV/AIDS and tuberculosis, through analysis of the data collected. Methodology This thesis followed a mixed methods approach including a literature survey, a structured questionnaire-based evaluation (baseline and before- and after-training assessments) and finally a retrospective review of ADR reports. Results The study reviewed published and grey literature to reconstruct the evolution of pharmacovigilance in SA. Through a baseline assessment in Eastern Cape Province, it also demonstrated areas that need strengthening and provided recommendations of simple, cost-effective interventions to close these gaps in that province, as well as generally in SA. Training, a key intervention recommended, was also tested and the study found a positive shift in knowledge gained by healthcare professionals (HCPs) from a one-day pharmacovigilance training intervention (p<0.002). Finally, a retrospective analysis of ADR data collected was conducted. Among others, this revealed the effectiveness of this low cost PV programme in detecting top causative agents, most common ADRs and their incidence across gender. Conclusions This study provided a review of the PV landscape in SA. The findings have the potential to inform treatment guidelines. Scaling up the methods used herein has the potential to detect trends that can be acted upon to reduce morbidity and mortality from large public health treatment programmes, especially in low-income settings.
Novel series of dehydrozingerone inspired potential antimycobacterial agents: design, synthesis, spectral studies and in vitro biological evaluation.
(2016) Hampannavar, Girish Appasaheb.; Karpoormath, Rajshekhar.
Tuberculosis (TB) is a key health burden globally. With the emergence of resistance issue, the antitubercular research has been challenging. Novel effective drugs are immediately required to treat this serious epidemic disease. Innovative potential antitubercular drug candidates are momentously required to combat the disadvantages linked with existing drugs or line of treatments. Synthetic manipulations of natural sources are being extensively investigated worldwide for developing potent and efficient drugs. Besides, these manipulations also offer effective leads for further optimization. Therefore, this project is an effort in identifying a novel and effective antitubercular leads based on natural product model dehydrozingerone (DZG), a curcumin degradant. In this project we have performed an extensive literature survey of DZG for its known biological activities. And further, we have synthesized some novel series of DZG fused heterocyclic compounds with three different 5 membered heterocyclic scaffolds namely, thiazole, thiazolidon- 4-one and pyrazole. A total of 53 compounds comprising of styryl hydrazine thiazole hybrids (6ao, Chapter 3), styryl hydrazine thiazolidin-4-one hybrids (7a-d, 10a-l and 13a-b, Chapter 4) and lastly styryl fused pyrazole derivatives of acid hydrazides, semicarbazone and thiosemicarbazones (8a-i, 11a-h and 14a-c, Chapter 5) have been synthesized by versatile synthetic routes as outlined in schemes of respective chapters. The completion of reaction and the purity of synthesized compounds were established by chromatographic analysis. All the newly synthesized compounds displayed acceptable analysis for their anticipated structures, which were established based on physicochemical and spectral data (IR, 1H NMR, 13C NMR and HRMS). These newly synthesized compounds were primarily evaluated for their in vitro antimycobacterial activities at Infectious Disease Research Institute (IDRI) within the National Institute of Allergy and Infectious Diseases (NIAID) screening program, Bethesda, USA or Department of Microbiology, Inkosi Albert Luthuli Hospital, Durban, South Africa. From the systematic analysis of antimycobacterial activity results obtained following key observations were made. i. Degradants of curcumin have been looked upon for molecular variations in developing diverse scaffolds. DZG is an imperative scaffold and its numerous analogs have emerged as a promising leads in the design and development of some novel medicinally active compounds with improved metabolic, pharmacokinetic and pharmacological profiles, indicating that there is much scope for considering DZG as a structural framework for developing effective leads. ii. Chapter 3: Of the fifteen novel styryl hydrazine thiazole derivatives synthesized and tested, compound 6o exhibited significant antimycobacterial activity (H37Rv; MIC = 1.5 μM; IC50 = 0.48 μM) along with bactericidal (MBC = 12 μM) and intracellular antimycobacterial activities (IC50 = < 0.098 μM). Furthermore, 6o displayed prominent antimycobacterial activity under hypoxic (MIC = 46 μM) and normal oxygen (MIC = 0.28 μM) conditions along with anti-mycobacterial efficiency against isoniazid (MIC = 3.2 μM for INH-R1; 1.5 μM for INH-R2) and rifampicin (MIC = 2.2 μM for RIF-R1; 6.3 μM for RIF-R2) resistant strains of Mycobacterium tuberculosis. Presence of electron donating groups on the phenyl ring of thiazole moiety had positive correlation for antimycobacterial activity. iii. Chapter 4: From the eighteen novel styryl hydrazine thiazolidin-4-one hybrids derivatives synthesized and tested, Compounds 7a (MIC = 110 μM; IC50 = 67 μM), 7c (MIC = 120 μM; IC50 = 66 μM) and 10g (MIC = 100 μM; IC50 = 100 μM) exhibited noteworthy antimycobacterial activity. Further, the title compounds displayed least cytotoxic effects against a mammalian Vero cell determined using MTT assay. iv. Chapter 5: Among the twenty novel styryl pyrazolo carbazone derivatives synthesized and tested, Compounds 8a, 8c, 8d, 8g, 8h, 8i and 11f showed reasonable antibacterial activity (MIC = 50 μg/mL) against B. subtilis, compound 11a demonstrated noteworthy activity towards P. aeruginosa (MIC = 25 μg/mL). Further, compounds 8a, 8d, 8e, 8f, 8i, and 11h showed good to moderate antifungal activity ranging from 25 to 50 μg/mL towards C. neoformans (MIC = 25 μg/mL) and C. albicans (MIC = 50 μg/mL). Besides, compound 8a, comprising of isonicotinoyl hydrazide portion displayed remarkable antitubercular activity (MIC = 0.78 μg/mL) against H37Rv. Substituted urea derivatives, 14a-c and 11d also exhibited encouraging activity (MIC = 12.5 and 25 μg/mL, respectively) whereas, derivative with carbothioamide portion 11a, (MIC = 0.78 μg/mL) illustrated significant activity against H37Rv. Moreover, some of the tested compounds showed reasonable activity against MDR (multi drug resistant) and MOTT (mycobacteria other that tuberculosis) strains.
A computational perspective of influenza a virus targets : neuraminidase and endonuclease.
(2016) Singh, Ashona.; Soliman, Mahmoud Elsayed Soliman.
Through the ages the viruses have plagued mankind claiming the lives of millions, pre-dating any advancements in the medicinal sciences. One such pathogenic virus is influenza A, which has been implicated in the 1918-Spanish flu, the 2006-avian flu outbreak and the 2009-swine flu pandemic. It is a highly sophisticated species, alluding efforts to thwart the spread of disease and infection. One of the main reasons influenza has survived this long is simple evolution. Natural mutation within the genome of virions expressed in proteins, enzymes or molecular structure render us unable to predict or take preventative measures against possible infection. Thus, research efforts toward the competitive inhibition of biological pathways that lead to the spread of disease, have become attractive targets. The influenza A virus has a number of chemotherapeutic targets, such as: 1) The surface antigens, hemagglutinin and neuraminidase, 2) RNA-dependent RNA polymerase, and 3) The M2 proton channel. Influenza RNA polymerase is composed of three large segments encoding polymerase acidic protein (PA), polymerase basic protein 1 (PB1) and polymerase basic protein 2 (PB2). The PA protein is an N-terminal domain subunit which contains the endonuclease activity. The influenza virus is incapable of synthesizing a 5’-mRNA cap, so it has adapted a cap-snatching mechanism whereby the PB2 subunit binds to the 5’-end of host mRNA, after which 10-14 nucleotides downstream the PA-subunit (aka PAN) cleaves the strand forming a primer for viral mRNA synthesis which is catalysed by the PB1 subunit. Influenza target identification is based primarily on evidence suggesting sequence conservation of each entity and its selective expression in the virus and not the host. In this thesis two enzymatic targets were investigated, the PA protein of RNA polymerase and neuraminidase. The studies focussed on using computational tools to: 1) provide insight into the mechanism of drug-resistance, 2) describe the conformational structure of the protein in the presence of point mutations and in complex with an inhibitor, 3) determine the essential binding pharmacophoric features to aid the design of new drug therapies. An array of computational techniques were employed in the studies, such as: molecular dynamics (MD) simulation, structure-based and ligand-based in silico screening, principal component analysis, radius of gyration analysis, binding free energy calculations and solventaccessible surface area analysis. The first study (Chapter 5) determined the mechanism of drug-resistance in influenza A neuraminidase as a consequence of antigenic variations. Two distinct mutations in the enzyme sequence that were investigated are H274Y and I222K. The active site residues of neuraminidase are conserved among the subtypes of influenza A. However, it was discovered that the occurrence of resistance to the drug oseltamivir, in the H1N1 species was different to the H5N1 virus. Although both systems shared a loss in hydrophobicity of the active site, the conformational distortion of the active site pocket distinguished the enzyme of the two viral entities, from one another. The discoveries made in the first study laid the foundation for the second study (Chapter 6), which was based on the in silico design and screen of potential neuraminidase inhibitors. As a result 10 characteristic molecular scaffolds were suggested as potential inhibitors. The pharmacophore design was constructed with consideration to the new conformational structure of the active site pocket. Chapter 7 is the third study of this thesis. The active site pocket enclosing the endonuclease activity of the PA subunit was investigated. Using molecular dynamics simulations and postdynamic analyses, a description of the protein conformation was offered. Subsequently, a pharmacophore was proposed as a potential scaffold to which endonuclease inhibitors may be modelled upon. It is my belief that the impact of the results derived from the above mentioned studies would greatly contribute to the development of new and effective anti-influenza drugs.

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