Masters Degrees (Clinical Pharmacology)

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The effect of dolutegravir on adipose tissue health
(Stellenbosch : Stellenbosch University, 2022-10) Coetzee, Johanna Adriana; Smith, Carine; Ross, Kelly Shirley; Faculty of Medicine and Health Sciences. Dept. of Medicine. Division of Clinical Pharmacology.
ENGLISH ABSTRACT: Introduction: Dolutegravir, an integrase strand transfer inhibitor (INSTI), forms part of the first-line antiretroviral (ARV) therapy. Despite low cost, fewer drug-drug interactions and minimal reported side effects, dolutegravir use has clinically been associated with weight gain. The significance of this in terms of adipose- and overall patient health remains to be fully elucidated. Methods: The effects of dolutegravir on the adipose tissue health were assessed in a 12-week treatment intervention study in Wistar rats, allowing an assessment of risk profile for dolutegravir in the absence of potential confounding effects of retroviral infection or other ARV agents commonly found in combination therapy. Dolutegravir was administered as a human equivalent dose set in jelly blocks, once daily for 12 weeks, starting at 8 weeks of age. Visceral adipose tissue morphology and fibrosis profile, adipokine and inflammatory cytokine levels were assessed. Results: Dolutegravir did not change the rate of body mass increase in the rats (when compared to a placebo group). Females presented to have smaller adipocyte size and increased adiponectin secretion – more pronounced in the treated group compared to the controls. While pro-inflammatory cytokines (TNF-α and MCP-1) were also elevated in both the female groups compared to the males. Conclusion: Current data illustrates that in terms of potential effects of dolutegravir on AT health, females seem more vulnerable to undesired longer term outcome. The overall small effect sizes seen is in line with a chronic, low-grade dysregulation which may predispose dolutegravir-treated patients to development of co-morbidities with a chronic inflammatory character.
The effect of Dolutegravir on redox and vascular integrity
(Stellenbosch : Stellenbosch University, 2022-10) Conradie, Janica; Smith, Carine; Ollewagen, Tracey; Faculty of Medicine and Health Sciences. Dept. of Medicine. Division of Clinical Pharmacology.
ENGLISH ABSTRACT: Background: Of the estimated 7.2 million people living with HIV (PLWH) in South Africa, more than 50% are on antiretroviral (ARV) medication. Dolutegravir (DTG), an integrase strand transfer inhibitor (INSTI), is increasingly used in South Africa (and Africa) as one of the first line treatments for HIV. Both in vitro and clinical studies indicate that DTG-based therapies have been associated with adverse effects (such as abnormal weight gain, hyperglycemia, and neurological dysfunction) alongside increased oxidative stress. Another disease state common in the South African context is obesity. DTG is administered to an individual regardless of their weight. As obesity is also associated with oxidative stress and inflammation, this might be a potential confounding factor in the efficacy and safety of DTG treatment. Endothelial dysfunction is one of the most common risk factors associated with non-communicable disease (such as obesity and CVD) and would therefore be an important phenomenon to investigate in the context of ARVs and obesity. Aim: This study aimed to elucidate potential effects of chronic DTG on redox profile and endothelial integrity in the presence or absence of overfeeding as a confounder. Methods: Adult zebrafish were subjected to chronic administration of DTG in the absence and presence of obesity (overfeeding model) over a period of 2 weeks. The lean zebrafish received Hikari dry pellets twice per day, whereas the overfed fish received the same food, six times per day over 4 weeks. A parallel 12-week DTG administration study was also executed in lean male and female Wistar rats. Body mass, reactive oxygen species (ROS) levels (hydrogen peroxide (H2O2)) and vascular endothelial tight junctions (claudin-5 and ZO-1) and adherens junction (VE-Cadherin) protein profile were evaluated. Results: DTG did not have a significant effect on the body mass of adult zebrafish or rats. DTG administration showed no detrimental effect on ROS levels in either zebrafish or rats. When considering the effect of DTG on the expression of tight and adherens junction proteins, DTG showed no effect on the expression of ZO-1 and Claudin-5 in both zebrafish and rats. In terms of obesity, the overfed zebrafish had significantly higher body mass when compared to lean fish confirming successful execution of the overfeeding protocol. Overfeeding did not impact ROS, however there was a decrease in VE-cadherin expression in the zebrafish dorsal aorta, pointing to a cumulative effect of DTG and obesity (overfeeding). Conclusion: This study suggests that in the absence of HIV, DTG does not have an effect on redox status and endothelial dysfunction in terms of tight and adherens junction integrity. However, the addition of overfeeding highlighted the cumulative effect of DTG and obesity on endothelial function, an effect that can potentially impact the human population.
Neurological 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.
The 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 Pharmacology
ENGLISH 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.
The 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
Understanding 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.
A zebrafish larval model for drug-induced hepatic Injury due to first-line antituberculosis drugs and possible prevention/treatment with N-acetyl-cysteine
(Stellenbosch : Stellenbosch University, 2022-11) Motha, Khetiwe; Kellermann, Tracy; Smith, Carine; Faculty of Medicine and Health Sciences. Dept. of Medicine. Division of Clinical Pharmacology.
ENGLISH ABSTRACT: Background Tuberculosis (TB) is ranked as the second most deadly infectious disease worldwide. First-line TB medication is associated with the development of drug-induced liver injury (DILI). The hepatotoxicity from this combination therapy is mostly due to pyrazinamide (PZA), isoniazid (INH) and rifampicin (RIF), however, no information has been reported on the hepatotoxicity potential of ethambutol (EMB). DILI typically occurs within the initial few weeks of the intensive phase of therapy. The only way to treat DILI is by stopping the medication and considering a liver transplant in the case of liver failure. Halting treatment can lead to the development of multi-drug resistant TB (MDR-TB). Therefore, close monitoring during therapy is required. Moreover, a preventative intervention needs to be put in place in order to prevent TB-DILI from developing in the first place. Acetaminophen (APAP) has been shown to cause DILI due to the accumulation of the drug’s toxic metabolites in the liver following overdose of the drug. N- acetylcysteine (NAC) is an effective treatment and works by replenishing cellular glutathione in hepatocytes, thereby preventing liver injury from progressing to liver failure. It is therefore hypothesized that NAC may be able to reverse liver injury due to first line TB medication since it has been shown to be highly effective in the treatment of DILI associated with APAP, and has in fact become the standard of care. Previous studies have reported the potential of NAC in treating TB-dug associated DILI. However, this needs to be confirmed through further studies. Therefore, new models are needed for predicting which therapeutic compounds could cause DILI in humans, and new markers and mediators of DILI need to be identified. The physiological and metabolic processes in zebrafish (Danio rerio) are similar to that of humans and the transparency of the zebrafish larvae makes this vertebrate a suitable model for studying TB-DILI mechanism and treatment. This study therefore aims to develop a zebrafish larval model (<5dpf) for DILI due to TB drugs, using APAP as a positive control that is known to cause DILI and to investigate the potential of NAC in preventing liver injury. Methods High-performance liquid chromatography (HPLC) analysis of INH, PZA and RIF was performed using previously developed methods, while liquid chromatography tandem mass spectrometry (LC-MS/MS) using a previously developed method was used for the evaluation of EMB. An HPLC method for the quantitation of APAP was developed and partially validated. The method used a Shimadzu HPLC system coupled with a PDA detector. Successful separation was achieved by isocratic elution on a reverse-phase Venusil XBP C18 (4.6 X 100 mm, 5 μm) column using a mobile phase consisting of 0.1% formic acid in water and acetonitrile (82:18; A:B, v:v) at 0.650 ml/min flow rate, detection wavelength of 247 nm, column oven temperature of 28°C and injection volume of 10 μl. The chromatographic retention time was consistent at 3.26 min. The calibration curve covered a range of 3.13 to 200 μg/ml with a quadratic regression weighted 1/c (c: concentration). These analytical methods were used to determine the solubility and stability of these drugs in E3 medium at 28°C for 3 days. Thereafter, dose response experiments were performed for all drugs to obtain the dose (s) that resulted in liver steatosis as an indicator of DILI using Oil red O positive liver stains and EthoVision movement tracking software (for NAC) as endpoints. Optimisation of the doses and the evaluation of the ability of NAC to prevent or reverse TB drug-associated DILI was attempted in zebrafish larvae. For NAC, APAP, INH and PZA, previously reported zebrafish larval doses for each drug were used a as reference for the study. However, for rifampicin (RIF) and EMB, the doses were adjusted according to the human dose. Results Solubility and stability data showed that APAP, INH, PZA and EMB were soluble in E3 embryo water and stable at 28˚C for 3 days. However, RIF remained insoluble and unstable in E3 medium at 28°C after 24 hours, even with the addition of ascorbic acid at 20 μg/ml. Doses of 1 mM, 7 mM, 10 μM, 1.2 mM and 0.5 mM were selected as the doses associated with DILI for INH, PZA, RIF, EMB and APAP, respectively. EthoVision data showed that 12 μM and 16 μM NAC resulted in irritation and toxicity, respectively. However, 8 μM NAC was shown to be consistent with the negative control and was therefore selected as the safe dose for NAC. Due to time constraints and difficulties in breeding, NAC was not evaluated for its ability to prevent or reverse DILI due to first-line TB drugs and this will be elucidated in future. Conclusion A time efficient, economical zebrafish larval model for DILI was successfully developed. Current data illustrates that this model is able to accurately simulate known toxicity effects of first-line antituberculosis drugs on the liver. Furthermore, this model can be used to evaluate potential treatment interventions and prophylaxis for the reversal and/or prevention of DILI.

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Browsing Masters Degrees (Clinical Pharmacology) by browse.metadata.advisor "Smith, Carine"

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