Masters Degrees (Clinical Pharmacology)
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- ItemThe role of estrogen receptors in fibrosis: in search of therapeutics(Stellenbosch : Stellenbosch University, 2024-02) Alberts, Janneke; Smith, Carine; Ollewagen, Tracey; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine. Division of Clinical Pharmacology.ENGLISH ABSTRACT: Background: Autoimmune rheumatic diseases, affecting 5-8% of people globally, exhibit a 2:1 female prevalence bias, influencing severity and prognosis. Chronic inflammation leads to immune complex formation, which causes vessel occlusion, and resulting in fibrosis, contributing to cardiovascular diseases, particularly myocarditis. Fibrosis - driven by activated fibroblasts - results from extracellular matrix dysregulation, causing tissue scarring and organ dysfunction. Therefore, investigating antifibrotic interventions is crucial for global healthcare. 17β-estradiol (E2), the predominant female hormone, has the potential to modulate fibrosis, but literature in this niche is contradictory. Zebrafish have been identified as an ideal model for studying sex bias in autoimmune rheumatic-related myocarditis, exploring the role of E2 in chronic inflammation and fibrosis. Methods: To elucidate underlying mechanisms, a multidisciplinary approach was imperative. Employing diverse model systems, the thesis incorporated two pilot studies. The initial pilot study utilised male human dermal fibroblasts (HDFs) in vitro to assess the impact of E2 on cell function linked to fibrotic outcome. Cell viability and fibronectin production were assessed after 24 hours (h) E2- exspoure relative to that of untreated cells. Subsequently, a second pilot study evaluated behavioural outcomes in larval zebrafish, in response to exposure of a range of E2 dosages for 60 h, informing the selection of an appropriate, potentially beneficial, E2 concentration for the main experiment. A significant innovation was the establishment of a novel larval zebrafish myocarditis model, by means of microinjection with a sclerosing agent (bleomycin). Using this model, the effect of added E2 and modulation of estrogen receptor (ER) signalling on fibrosis was determined by staining for collagen (Masson-Goldner Trichrome) and vimentin (immunofluorescence staining). Results: Results generated in HDFs elucidated that treatment with E2 did not significantly alter the fibroblast viability or fibronectin production. Microinjected zebrafish larvae exhibited lower basal activity levels than controls (p
- ItemThe role of estrogen receptors in anxiety disorders: an investigation in zebrafish(Stellenbosch : Stellenbosch University, 2023-11) Balshaw, Aidan Glenn; Smith, Carine; Pretorius, Lesha; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Dept. of Medicine. Division of Clinical PharmacologyENGLISH ABSTRACT: Background & Aim Anxiety disorders are the most prevalent psychiatric disorders with approximately twice the prevalence in females compared to males. Clinical studies on the exacerbation of anxiety symptoms in the premenstrual phase of the menstrual cycle implicate the role of declining estradiol levels. Preclinical evidence most consistently indicating the anxiolytic effect of increased estrogen receptor beta-signalling exacerbations in anxiety symptoms have been demonstrated in clinical studies. This thesis aimed to simulate estrogen-linked anxiety behaviour in larval zebrafish to assess the role of estrogen receptors (ER) in this phenomenon. Methods Using an estradiol-treatment-withdrawal-model, behaviour of zebrafish larvae was determined in the LDTT on 7 days post-fertilisation (dpf) in a series of experiments. Firstly, the estradiol treatment withdrawal model was optimised by increasing the duration of treatment withdrawal and reducing the number of estradiol concentrations used. Further optimisation included comparing behaviour after estradiol washout with behaviour after prolonged tamoxifen, an ER modulator, administration. Secondly, ER modulators (WAY-200070, PHTPP, and tamoxifen) were administered for 45 minutes, in the presence of absence of exogenous estradiol exposure, before behavioural analysis. Thirdly, ERβ expression was determined after estradiol treatment. Lastly, redox status (hydrogen peroxide levels, antioxidant capacity, and lipid peroxidation) and behaviour of estradiol-treated larvae were evaluated. Results Estradiol withdrawal decreased basal and anxiety-like behaviour. The accuracy of the estradiol washout model was not improved by longer durations of treatment withdrawal or refinement of estradiol concentrations used. Prolonged tamoxifen administration reduced basal and anxiety-like behaviour. ER modulation did not alter anxiety-like behaviour, while basal activity slightly altered by supraphysiological concentrations of WAY-200070 in the absence of estradiol. WAY-200070 and tamoxifen altered basal activity when administered in the presence of exogenous estradiol exposure. ERβ expression was not upregulated in larvae exposed to low concentrations of estradiol. Longer exposure to low concentrations of estradiol increased antioxidant capacity and slightly decreased lipid peroxidation while hydrogen peroxide levels were unaltered. In addition, acute exposure to low concentrations of estradiol increased basal activity in the LDTT. Conclusion Current data suggest that developing zebrafish larvae are likely not suitable for modelling exacerbations in anxiety symptoms associated with fluctuating estrogen levels. Rather, current investigations demonstrated that increased activity levels were not linked to anxiety but better redox status, in the context of free estrogen availability. Considering the effect of redox status on larval behaviour, it is recommended that behavioural analysis be conducted in parallel with mechanistic studies in the context of ER signalling.
- ItemThe development and validation of an LC-MS/MS method for the quantification of metformin in human plasma(Stellenbosch : Stellenbosch University, 2023-08) du Plessis, Christiena Hendriena; Kellermann, Tracy; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Dept. of Medicine. Division of Clinical Pharmacology.ENGLISH ABSTRACT: Background: The rising incidence of type 2 diabetes mellitus among people living with HIV residing in lower-and middle-income countries such as South Africa, leads to a greater proportion of people being prescribed both the first-line anti-diabetic medication, metformin, as well as the first-line antiretroviral drug, dolutegravir (DTG). DTG has been found to interact with metformin by increasing its plasma concentration. Drug interactions can result in supratherapeutic drug concentrations, and for this reason it is essential to develop and validate a selective, sensitive, rapid, and affordable bioanalytical method for the quantification of metformin in human plasma. This carries true clinical value, as it will not only enhance our understanding of the drug itself, but also inform on the magnitude of possible drug interactions. Methods: A Shimadzu 8040 instrument was used for analysis, to monitor the transition of metformin and metformin-d6 hydrochloride (ISTD), in the positive ion mode, [M+H]+ : m/z 129.9 → 60.1 and 136.2 → 60.1, respectively. An Agilent Zorbax Eclipse XDB-C8 column was used with gradient chromatography and mobile phases of 0.1% formic acid in water (A) and 100% acetonitrile (B) at a flow rate of 0.5 mL/min, and a retention time of ~2.50 min. A protein precipitation method was used where 200 μL of acetonitrile (ACN) was added to 50 μL plasma and 200 μL of the supernatant was transferred to 96-well plates, before injection for analysis. The % cross-talk, concomitant medication effects, whole blood stability (2 h), matrix effects, % recovery, process efficiency and the effect of 2% hemolysis were determined. Intra- and inter-batch validations were performed together with bench-top stability for ~4 h, freeze-thaw stability (3 cycles), autosampler stability (48 h). Results: The calibration curve had a quadratic regression with a weighting of 1/C2 and a concentration range of 15.6 ng/mL – 4 000 ng/mL in plasma. Metformin was stable in stock and working solutions at 4 °C, -20 °C, and -80 °C for 24 h and on bench at room temperature for ~4 h. The % cross-talk was negligible, and the presence of concomitant had no effect on the method’s performance. The analyte was stable in whole blood for 2 h. No matrix effects were observed after evaluating plasma from 6 different sources. The average percentage recovery was 69.9%, and process efficiency was 106.4%. Hemolysis at 2% did not have an effect on the quantification of metformin. Intra- and inter-batch validation results met the FDA (2018) and EMA (2011) acceptance criteria. The calibration standards had a % accuracy ranging from 98.1% - 102.1% (% CV of 5.1% - 10.2%). The quality controls had a % accuracy ranging from 91.6% – 101.5% (% CV of 4.9% - 10.5%). The analyte was stable in plasma through 3 freeze/thaw cycles, and on bench for ~4 h. Metformin was stable in the autosampler at 15 °C for ~48 h. This method was successfully applied to clinical samples. Conclusions: The developed LC-MS/MS method, using a simple protein precipitation extraction protocol, was successfully validated according to FDA and EMA guidelines. Subsequently, the robust method was applied to clinical samples to quantify metformin in human plasma to better inform patient care.
- ItemUnderstanding the effect of protocol variations in the zebrafish light/dark transition test(Stellenbosch : Stellenbosch University, 2023-03) Gelderblom, Michelle; Smith, Carine; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Dept. of Medicine. Division of Clinical Pharmacology.ENGLISH ABSTRACT: Anxiety disorders have devastating individual and societal costs, and are a major contributor to years lost to disability worldwide. They appear to be increasing in prevalence, both in South Africa and the world at large. Although there are medications to treat anxiety, there is a need for more treatment options. Anxiety is often not treated effectively due to treatment resistance or non-compliance to medication due to side effects. One of the best options for identifying novel anxiolytics is to use animal models. Zebrafish are useful for screening of potential anxiolytic treatments because they are the most well-studied vertebrate model that shares the size, cost and fecundity benefits of invertebrate models. The light/dark transition test (LDTT) is the most widely used zebrafish larvae behavioural test. It has many applications in pharmacology, particularly in toxicology and screening for potential pharmaceuticals, including treatments for anxiety disorders. It is likely to be one of the first tests used when screening for neuroactivity in zebrafish larvae. During the test, zebrafish larvae are exposed to a period of light followed by an abrupt transition to darkness which produces a hyperlocomotion response that responds to anxiolytics and anxiogenics. The design of the LDTT varies between studies, but it is unclear how common protocol variations affect the comparison of results and contextualisation of data generated using slightly varied protocols. Through both prospective experiments and retrospective data analysis, the effect of age (from 2 dpf to 5 dpf), lighting conditions during rearing (standard or continuous darkness), capture order, repeated light/dark cycles, repeated light/dark transition tests, duration of the light period (1 minute or 10 minutes), light intensity during the light period, and breeding stocks on the response to the light/dark transition test was measured. All experiments consisted of an acclimation period, and at least one cycle consisting of a light period and a dark period. Experiments were recorded using the DanioVision system and activity was measured automatically using the EthoVision XT software. Variations in age, time of day, light period and breeding stock had a significant impact on the response to the light/dark transition test and should therefore be carefully controlled. Light conditions during rearing did not have a statistically significant effect, but more research is needed to confirm that variations in light-rearing do not affect response to the light/dark transition test. Finally, capture order, repeated cycles, repeated light/dark transition tests and light/dark transition intensity did not have a significant effect, suggesting that they can vary according to logistical requirements without affecting results. This opens up the use of repeated measurements that facilitate identifying neuroactivity when the amount of time it will take for the onset of action is unknown. This informs both experimental design, and which studies are comparable. It will also facilitate the use of the light/dark transition test to screen for potential anxiolytics.
- ItemNeurological risk of prolonged low dose exposure to imidacloprid in zebrafish(Stellenbosch : Stellenbosch University, 2022-11) McCulloch, Megan; Kellermann, Tracy; Smith, Carine; Faculty of Medicine and Health Sciences. Dept. of Medicine. Division of Clinical Pharmacology.ENGLISH ABSTRACT: Imidacloprid (IMI) is a systemic neonicotinoid insecticide intended to replace the organophosphate pesticides in agriculture. Extensive use of these pesticides increases the risk to the environment and non-target organisms such as humans due to their potential bioaccumulation and toxicity. Researchers studying the effect of IMI on human nicotinic receptors (α4β2) have reported that IMI may have more substantial side effects on humans than originally anticipated. This research project aimed to assess the possibility of long-term neurological risks following prolonged, low dose IMI exposure within an in vivo zebrafish model. Methods A protein precipitation extraction from zebrafish brain, liver and gill homogenate was applied followed by LC-MS/MS detection of neurotransmitters and IMI and its primary metabolites. Protein precipitation was conducted using methanol:acetonitrile (1:1 v/v) as the precipitating solvent. Phenethylamine-d4 and IMI-d4 were used as internal standards for the neurotransmitter and IMI LC-MS/MS methods, respectively. For the neurotransmitters, chromatographic separation was achieved using a Poroshell column (3.0 x 100 mm, 2.7 μm) using a gradient elution mode at a flow rate of 0.45 mL/min and an analysis time of 7 min. Mobile phase A and B consisted of water with 0.1% formic acid and acetonitrile, respectively. For IMI and its metabolites, chromatographic separation was achieved using a biphenyl column (2.1 x 100 mm, 2.7 μm) with gradient elution at a flow rate of 0.4 mL/min. The total analysis time was 8.5 min. Mobile phase A and B consisted of water and methanol respectively, both with 5 mM ammonium formate and 0.1% formic acid. The two developed methods underwent a partial validation to certify that both methods were precise, accurate and reliable. Zebrafish larvae were exposed to IMI at four and five days post fertilisation to determine the no observed adverse effect level (NOAEL) of IMI. After this, adult zebrafish were exposed to the NOAEL concentration for 21 days. Key endpoints included behaviour indicative of neurocognitive decline and possible bioaccumulation in the adult zebrafish brain, liver and gills. Neurotransmitter concentrations were measured in the adult zebrafish brain tissue at the end of the treatment period to evaluate changes in neurotransmitter signalling and potential neurological risks using the developed LC-MS/MS method. Bioaccumulation of IMI and its metabolites in zebrafish brain, liver and gills was evaluated using LC-MS/MS. Results The calibration curve fits a quadratic (weighted 1/C) regression over the concentration range of 31.3 - 1000 ng/mL for acetylcholine, gamma-aminobutyric acid, serotonin and dopamine. The calibration curve for IMI and its metabolites fits a quadratic (weighted 1/C) regression over the concentration range of 1.95 - 125 ng/Ml for imidacloprid-urea and IMI, 0.244 - 125 ng/mL for desnitro-imidacloprid and 3.91 - 125 ng/mL for 5-hydro imidacloprid. The NOAEL of IMI in zebrafish larvae was determined to be 2.5 μg/L. No significant morphological changes were observed in the adult zebrafish during the treatment period. Behavioural changes observed during the pesticide exposure period included decrease in appetite of the treatment group. The treatment group was also observed swimming at the bottom of the tank in comparison to the control group. Although IMI, IMI-urea and desnitro-IMI could not be detected in any of the tissue specimens, 5-hydro IMI was detected at relatively high concentrations in the liver (0.793 ng/mg tissue) and gill epithelial tissue (117 ng/mg tissue). Only concentrations of gamma-aminobutyric acid and acetylcholine were detected and quantified in both the treated and control group. The treated group showed a 1.4-fold decrease and 1.9-fold increase in acetylcholine and gamma-aminobutyric acid, respectively in comparison to the control. Serotonin and dopamine could not be detected due to their levels being below the limit of quantitation of this method. Conclusion Robust LC-MS/MS methods were developed for the detection and quantitation of IMI, desnitro-imidacloprid, imidacloprid-urea and 5-hydro-imidacloprid, as well as serotonin, dopamine, acetylcholine and gamma-aminobutyric acid neurotransmitters in 200 μL zebrafish brain, liver and gill epithelial tissue homogenate. The behavioural changes observed in the adult zebrafish could be an indication of one of two things, anxiety or sedative effect. This is verified by the increase in gamma-aminobutyric acid neurotransmitter levels. Together with the evaluation of bioaccumulation of imidacloprid and its metabolites within the brain, liver and gill epithelial tissue of zebrafish, this study provides an indication of the potential risk to human health following chronic neonicotinoid exposure. Overall, our findings further contribute to existing literature and suggest that IMI does pose a threat to more than just insects and therefore requires further investigation.