Masters Degrees (Clinical Pharmacology)

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    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.
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    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.
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    An analytical investigation of the impact of crushing of first-line antituberculosis medication and administration via a nasogastric tube
    (Stellenbosch : Stellenbosch University, 2022-11) Phogole, Cassius; Kellermann, Tracy; Faculty of Medicine and Health Sciences. Dept. of Medicine. Division of Clinical Pharmacology.
    ENGLISH ABSTRACT: Background Currently, the treatment of TB patients admitted to an intensive care unit (ICU) in South African Hospitals is performed by the off-label practice of crushing the first-line antituberculosis drugs isoniazid (INH), rifampicin (RIF), pyrazinamide (PZA) and ethambutol (EMB) and administration to the patients through a nasogastric (NG) tube as the majority of these patients are often sedated or intubated and therefore cannot swallow the whole tablets. This has, however, been associated with low drug exposure insufficient to effectively treat the Mycobacterial infection. Additionally, there is a paucity of alternative intravenous (IV) formulations of first-line antituberculosis drugs, especially in developing countries. The stability and solubility of these crushed drugs dissolved in water are questionable. Furthermore, the impact of the removal of the protective outer tablet coating and its effect on absorption and subsequent bioavailability has not been elucidated in the literature. Moreover, drug loss of crushed first-line antituberculosis drugs by adsorption to the surface materials used during medication preparation and administration via NG tube has also not been documented. Therefore, the present study aimed to investigate the root cause of the poor plasma drug exposure observed when crushing the first-line antituberculosis drugs and administration through an NG tube using laboratory-based techniques with possible translational interventions that can be applied in clinical settings to ameliorate their bioavailability. Methods The aqueous solubility of crushed drugs under the inversion mixing method was evaluated against easily implementable mixing methods (sonication and vortexing) with/without ascorbic acid (Asc). Moreover, the aqueous stability assessment of these crushed first-line antituberculosis drugs in mono-suspensions with/without Asc and co-suspensions at room and low temperatures was executed as well. The stability of the whole/crushed tablets in fasted-state simulated gastrointestinal fluids (FSSGIFs) was evaluated with/without Asc. Lastly, the drug loss by adsorption to the surface materials used during medication preparation and in vitro administration through an NG tube was quantitatively determined. Results Rifampicin (RIF) was the only drug showing poor aqueous solubility and instability in the simulated-gastric fluid. However, the addition of Asc has been shown to significantly (P<0.001) improve RIF solubility in both water and FSSGIFs with no detrimental effects. A minimum recommended volume of water (10 ml) to rinse the NG tube after administration of medication was shown to be inadequate to clear off all the residues of crushed antituberculosis medication. However, when an additional rinsing step with another 10 mL of water (total volume of 20 mL) was employed in the current study the amount of APIs of crushed first-line antituberculosis drugs adsorbed to these surface materials was significantly (p<0.001) reduced. Conclusion Co-administration of first-line antituberculosis medication with Asc when off-label crushing practice is used may improve RIF bioavailability. Furthermore, rinsing the NG tube with 20 mL of water after administering TB medication was shown to ensure adequate drug delivery, which may improve the bioavailability of nonpolar drugs that adhere to the surface of an NG tube.
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    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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    LC-MS/MS methods for the quantification of sulfasalazine and sulfapyridine in various matrices: application to a pharmacokinetic study
    (Stellenbosch : Stellenbosch University, 2022-11) Louw, Vanessa; Kellermann, Tracy; Faculty of Medicine and Health Sciences. Dept. of Medicine. Division of Clinical Pharmacology.
    ENGLISH ABSTRACT: Introduction: An early phase clinical trial which took place at The Mercy Hospital for Women in Australia assessed the use of sulfasalazine as a treatment for preterm pre-eclampsia. This project consisted of the development and validation of a Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) method according to the Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines to simultaneously quantitate sulfasalazine and its metabolite, sulfapyridine, in placenta for pharmacokinetic analysis. Methods: A Shimadzu 8040 mass spectrometer was operated in multiple reaction monitoring (MRM) mode to monitor the mass-to-charge (m/z) transition of the protonated precursor ions m/z 398.90 and m/z 250.07 to the product ions m/z 381.05 and m/z 156.00 for sulfasalazine and sulfapyridine, respectively. Sulfasalazine-d4 and sulfapyridine-d4 were used as internal standards. 100 μL of placental tissue homogenate was extracted using acetonitrile:methanol (90:10, v/v) and the supernatant was eluted through hydrophilic-lipophilic balanced cartridges. The extraction procedure was followed by liquid chromatographic separation using a Poroshell C18 column. Gradient elution using a mobile phase combination of water + 0.1% formic acid (A) and acetonitrile:methanol (90:10, v/v) + 0.1% formic acid (B) was used. Accuracy and precision were assessed over three consecutive, independent runs. The ratios of analyte peak area to internal standard peak area were plotted against the nominal concentrations to generate a calibration curve which fits a quadratic regression (weighted by 1/x, x= concentration) over the range 30-30 000 ng/mL for both sulfasalazine and sulfapyridine. Results and Discussion: The average accuracy of calibration standards during inter-day validations ranged from 94.2-103.2% (%CV= 1.4-10.8) for sulfasalazine and 96.6-103.4% (%CV= 1.4-8.3) for sulfapyridine. The accuracy of quality controls ranged from 101.6-112.7% (%CV= 4.4-6.7) and 97.4-108.4% (%CV= 3.7-10.0) for sulfasalazine and sulfapyridine, respectively. Endogenous matrix components were shown to have no impact on the reproducibility of the method when placental tissue from six different sources were analysed. The average recovery of sulfasalazine and sulfapyridine from placental tissue homogenate was 121.5% and 119.6%, respectively. Autosampler stability experiments indicated that placental tissue homogenate extracts were stable on instrument for up to 48-hours at the method-defined temperature. Re-injection reproducibility experiments illustrated that the method remained accurate and precise for analysis of both analytes following a re-injection of a batch for up to 48 hours after the initial injection. Furthermore, sulfasalazine and sulfapyridine were found to be stable in placental tissue homogenate for 10 days when stored at -80 °C, for six hours when left on bench at room temperature, and when subjected to three-freeze thaw cycles. Upon analysis of patient samples (n= 9), the concentrations ranged from 491-4201 ng/g tissue for sulfasalazine and 637-26756 ng/g tissue for sulfapyridine, with two patient samples below the limit of quantitation (BLQ) of the assay for both analytes. Conclusion: An LC-MS/MS method for the quantification of sulfasalazine and sulfapyridine in human placenta was successfully validated and applied to a clinical study to evaluate the efficacy of sulfasalazine as an intervention for pre-eclampsia.