Clinical Pharmacology

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This division was known as Pharmacology until 27 June 2013.

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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.
Determination of relationship between dolutegravir and trace amine profile, using advanced liquid chromatography tandem mass spectrometry analysis of various tissues
(Stellenbosch : Stellenbosch University, 2023-12) Henning, Natasha; Smith, Carine; Kellermann, Tracy; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences. Dept. of Medicine. Division of Clinical Pharmacology.
ENGLISH ABSTRACT: The immunopathogenic mechanisms of human immunodeficiency virus (HIV) are complex and require a multidimensional approach to pharmacological management. While it is known that antiretroviral therapy (ART) comprising of multi-drug treatment regimens often lead to the presentation of adverse effects, mechanisms leading to adverse effects require more elucidation. This is especially true for dolutegravir (DTG) – an integrase strand inhibitor (INSTI) – currently part of the first line treatment for HIV. Recent literature has elucidated that the neurological and gastrointestinal adverse events could be related to accumulation of DTG in these tissue compartments because of its physiochemical properties. However, few reports are available for methodology to assess DTG accumulation at tissue levels. Trace amines are biogenic amines which are endogenously produced in trace amounts in the brain, as well as in larger amounts by the gut microbiome and are known to differentially regulate inflammatory outcome. We proposed that a dysregulated trace amine profile may exacerbate the persistent inflammation associated with HIV in both neuronal and gastrointestinal tissue in response to DTG treatment. To elucidate the potential impact of DTG administration on the trace aminergic system, a multidisciplinary approach was required. Therefore, a wistar rat model and novel liquid chromatography tandem mass spectrometry methodology was combined to accurately determine both tissue DTG and trace amine concentrations. Following this approach, data generated illustrated that DTG indeed accumulated in tissue following a chronic DTG dosing regimen which mimics human monotherapy. In addition, DTG altered the urinary and gastrointestinal trace amine profile of wistar rats. In line with higher adverse event reporting by female patients, DTG quantification in plasma and various tissue matrices illustrated significantly higher DTG concentration in female rats when compared to males. In addition, there was a direct relationship between concentrations of DTG in plasma vs concentrations observed in muscle and liver, but not adipose tissue. Since DTG accumulated in tissue, further analytical assessments were conducted to determine potential dysregulation of the trace amine profile by DTG. Data suggest modulation of the trace amine profile by DTG administration, with confounding effect of sex. In conclusion, this dissertation contributes to available literature aimed at elucidating potential mechanisms causing inflammation-centred adverse events following DTG treatment. Data presented illustrate the importance of including both males and females in experimental pharmacology studies. From a clinical perspective, the data presented highlight the importance of more accurate dosage adjustment for body size and/or sex, to minimize risk of over-dosing individuals with relatively smaller body size.
The 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.
Development of a LC-MS/MS method for the detection of snake venom toxins in human plasma
(Stellenbosch : Stellenbosch University, 2022-12) Lermer, Anne; Kellermann, Tracy; Faculty of Medicine and Health Sciences. Dept. of Medicine. Division of Clinical Pharmacology.
ENGLISH ABSTRACT: Background: Snakebite envenomation in sub-Saharan Africa significantly threatens human health. Species that was responsible for the envenomation are rarely positively identified. There are no rapid methods/diagnostics available for conclusive identification of the organism/species responsible. Venomous species can be categorised based on the composition and primary effect caused by their venom components as neurotoxic, cytotoxic and/or hemotoxic. Aim: To develop an LC-MS/MS method for the species-specific detection of snake venom toxins from human plasma. Methods: An epidemiological study on the incidence of snakebite in South Africa as reported to Poisons Information Helpline of Western Cape (PIHWC) was performed to provide rationale for the analytical study. Analytical methodology includes identification of species-specific venom toxins by high resolution (HR) LC-MS/MS, fractionation of Naja nivea venom by size exclusion chromatography and compositional analysis of the fractions by HR-LC-MS/MS. Venom fractions were screened for toxicity using a zebrafish model, utilising DanioVision for phenotypic screening and behavioural tracking of the larvae. Thereafter, a triple quadrupole LC-MS/MS method was developed containing species-specific toxins. Results: Analysis of PIHWC call data revealed that in 43.73% of the recorded snakebite cases the causative snake species were unidentified. The snake species Naja nivea, Bitis arietans and Bitis atropos species were identified as most medically significant in South Africa and consequently included in the study. Venom from N. nivea was chosen as the species for fractionation after common peptides from inter-region venom were identified as unique to the species. Behavioural changes in larvae were observed after exposure to sub-lethal concentrations of N. nivea venom fractions. Significant (p<0.0001) changes in larval behaviour were observed in two treatment groups compared to the control. Using transitions that was generated during HR-LC-MS/MS analysis, FASTA files were generated and converted into MRM’s onto the triple quadrupole LC-MS. Toxins were positively identified from human plasma by LC-MS/MS. Discussion: This study identified the species predominantly responsible for snakebites from PIHWC call data, and highlighted a need for a diagnostic to identify the species responsible for envenomation. Analysis of the venom proteomes by HR-LC-MS/MS revealed similarities and differences in the venom profiles of Naja nivea, Bitis arietans and Bitis atropos species. In-solution, HILIC assisted tryptic digestion produced the identification of more proteins from the N. nivea crude venom compared to in-gel digestion and while fractionation by size exclusion chromatography prior to MS analysis increased the detection of low molecular weight toxins. It was also shown that using a combination of conventional HR-MS (with database/library searches) and triple quadrupole MS, a method could be created that identified species specific venom peptides from human plasma to aid diagnosis. Conclusion: This is a proof of concept for future work that will include the development of a lateral flow assay to detect venom from envenomed plasma that is cost-effective to produce, aids in defining diagnosis and importantly serves victims of snakebite envenomation in rural communities.
Development of LC-MS/MS toxicology screening methods for common poisoning agents in South Africa
(Stellenbosch : Stellenbosch University, 2020-12) Tiya, Luthando Lukhanyo; Stander, Marietjie; Kellermann, Tracy; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Medicine: Clinical Pharmacology.
ENGLISH ABSTRACT: Background: Hospitals admit many patients suffering from acute poisoning on a regular basis. Over a single year period the Tygerberg Poison Information Centre (TPIC) dealt with 4 771 consultations related to human exposure to various poisonous substances. Of these consultations, exposures to medicines and pesticides accounted for the majority of cases. As a response to this medical burden, this project aims to develop effective screening tools for both medicines and pesticides in human plasma. It is believed that such a tool could enhance how medical practitioners accurately treat and respond to serious poisonings. There are many toxicological screening methods employed today. In the past immunoassays (IAs) and gas chromatography coupled to mass spectrometry (GC-MS) were seen to be the gold standard for the identification of compounds. Due to false positive results associated with immunoassays and the labour intensive sample preparation associated with GC-MS, it is clear that there is a need for faster, more robust and more sensitive bioanalytical methods. This study’s objective therefore is to develop a cost effective, fast and robust liquid chromatography tandem mass spectrometry (LC-MS/MS) screening tools for the identification of drugs and pesticides. Relevant drug and pesticide selection was done by consulting with the TPIC, a centre which deals with poisoning cases on a daily basis, as well as clinicians in the Division of Clinical Pharmacology who deal with patients admitted at Tygerberg Hospital daily and CropLife SA, an organisation that has the knowledge of which pesticides are currently in use in South Africa. A total of 37 drugs and 10 pesticides were identified as those often suspected to be involved in poisoning/overdose cases. Two methods were developed: An untargeted liquid chromatography quadrupole time-of-flight mass spectrometry (LC-TOFMS) method for the determination of therapeutic drugs; and a targeted LC-MS/MS method for the determination of pesticides. Method: Analytes were extracted from CPD plasma using protein precipitation (PP). For the untargeted method, a Waters Synapt G2 Quadrupole Time-of-flight (QTOF) mass spectrometer (MS) fitted with an ultra-high performance liquid chromatography (UHPLC) was used. The separation was done using a Waters HSS T3 (1.8 μm, 2.1 ×150 mm) column. A gradient consisting of a mobile phase of 0.1% formic acid in both water (A) and in acetonitrile (B) at a flow rate of 0.25 mL/min. Ionisation mode was set for both positive and negative (ESI +/-); Nitrogen as the desolvation gas was set at a flow rate of 650L/h; Desolvation temperature was set at 275˚C. The instrument was operated in the MSE mode. The total run time was 37 min. Four concentration levels (10, 50, 100, 1000 ng/mL) were determined to cover the pharmacokinetic peak concentrations of all drugs. This range was determined according to sub-therapeutic, therapeutic and overdose type concentrations. Furthermore, the data generated from the untargeted method development process was used to accurately identify analytes using online search libraries. For the targeted method, two liquid chromatographic separation methods were developed on a Shimadzu 8040 LC-MS/MS instrument. One gradient method using the Agilent Poroshell 120 EC-C18 (2.7 μm, 4.6 ×100 mm) column was developed for atrazine, MCPA, fipronil, methomyl and aldicarb, while a second isocratic method utilizing of the Restek Raptor Biphenyl (2.7 μm, 2.1 x 100 mm) column was also developed for imidacloprid. Both methods used mobile phase consisting of 0.1% formic acid in water (A) and in acetonitrile (B) with a flow rate of 0.5 mL/min. The LC program on the Poroshell method starts at 30% (B) for 2 min followed by a 0.5 minute ramp to 90% (B); 2.5 min at 90% (B); 0.5 min to 30% (B), equilibrate at 30% (B) for 2.5 min. The total run time is 9 min. The biphenyl method isocratic run comprises of (65%:35%, v/v) for a run time of 1.75 min. A semi-quantitative method with a quadratic curve fit with 1/x regression over the range of 20 to 600 ng/mL was chosen. The two targeted methods were subjected to a partial validation to ensure that the methods were accurate, robust and reliable. Results: The untargeted method was able to detect and identify 92% of the therapeutic drugs included in the screen. Due to matrix effects and differences in compound ionisation some could not be detected at lower concentrations. Polarity and thus lack of retention on the columns, difficulty in ionisation, and the inability to produce stable fragments made the detection of valproate, levetiracetam and metformin not possible on the method. A screening tool for simultaneous determination of pesticides was optimized. Due to several challenges associated with the LC-MS/MS determination of glyphosate, paraquat and deltamethrin, the study resorted to excluding these three pesticides from the current methods. The targeted method was developed for the determination of seven pesticides. The USA Food and Drug Administration (FDA) and the European Medicines Agency (EMA) guidelines were followed for a partial validation of the semi-quantitative method. Three independent runs were assessed to measure accuracy and precision. The calibration curve was linear over the ranges 20 to 600 ng/mL. Matrix effects, recovery, process efficiency, on-bench stability, freeze/thaw stability, storage stability, and whole blood stability for the methods was successfully determined for the seven pesticides. Conclusion: To respond to the medical burden of acute poisoning cases faced by our hospitals, this study has developed efficient screening tools for both relevant medicines and relevant pesticides in human plasma. The latter method was subjected to a partial validation, ensuring accuracy and reliability of results that will be applied to the clinical management of poisoning patients.
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.
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.
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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