Browsing by Author "Wiesner, Joachim"

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    Open Access
    Development of analytical techniques for probing the effects of -haematin inhibiting compounds on the malaria parasite Plasmodium falciparum
    (2024) Maepa, Makgalanoto; Wiesner, Joachim; Combrinck Jill
    Phenotypic screening for novel compounds that inhibit the growth of the malaria parasite remains one of the most effective ways to identify novel antimalarial drugs. Subsequent deconvolution of the drug target and mechanism of action (MoA) are a vital part of the drug development process. This is often a limitation since the appropriate assays can be expensive, time-consuming, or inconclusive. One of the primary MoAs of the clinically approved antimalarials against Plasmodium falciparum is via the inhibition of haem detoxification. Drugs with this MoA, including chloroquine and amodiaquine, exert their therapeutic effect by disrupting haemozoin formation, which leads to increased accumulation of cytotoxic haem. Typically, the activity of potential haemozoin inhibitors is assessed using an in vitro detergentmediated β-haematin inhibition assay. However, this is inadequate for identifying true wholecell inhibitors of haemozoin since the assay is performed extracellularly. The intracellular pyridine-based assay by Combrinck et al. is used to determine whether prioritised β-haematin inhibitors cause significant dose-dependent increases in unsequestered haem (free haem) in the parasite. However, this assay has limitations and disadvantages in terms of its low throughput and the use of toxic reagents. In addition, this method is not readily transferable to other malaria drug discovery laboratories because of the need for specific knowledge of haem chemistry, highly specialised training, and given the labour-intensive nature of the assay. Therefore, developing improved assays that are readily accessible with increased throughput will lead to substantial progress in this area. It is also notable that the processes by which haem kills the parasite are not fully understood and hence the understanding of the effects caused by the unsequestered haem levels in the parasite needs considerable advancement. Consequently, in this work, a novel 96-well plate HPLC-DAD cellular haem fractionation method was developed and validated for quantifying intracellular haem species in the P. falciparum cell. The technique has eliminated the use of toxic pyridine to complex haem for the detection of unsequestered haem. Instead, it uses non-volatile imidazole for complex formation and has also reduced parasite starting material four-fold compared with the original method. Furthermore, this 96-well plate method allows the simultaneous evaluation of four test compounds at five selected concentrations, instead of only one test compound per 24- well plate. The assay was validated against the original pyridine-based method using four known haemozoin-inhibiting drugs, including chloroquine, amodiaquine, lapatinib and lomitapide, as well as four known non-inhibiting haemozoin drugs, including mefloquine, Abstract lumefantrine, pyrimethamine and tetrahydrocannabinol (THC). This higher throughput cellular fractionation assay will significantly increase throughput while also becoming an invaluable technique for probing haemozoin inhibition as a relevant MoA for new compounds in antimalarial drug discovery pipelines. Following successful validation of the 96-well four-compound cellular haem assay, the method was modified to a 96-well 33-compound qualitative high-throughput haem fractionation assay. Applications of the high-throughput method are intended to provide a valuable technique to accommodate the standard high-throughput screening campaigns that focus on identifying haemozoin-inhibiting molecules. Thus, this qualitative high-throughput assay enabled the quantifying of intracellular haem species in P. falciparum cells for 33 compounds tested simultaneously at a single-point concentration corresponding to 2x the 50% inhibitory concentration (IC50). The compounds used to validate the assay consisted of known haemozoin inhibitors, non-haemozoin inhibitors, and novel experimental compounds sourced from different drug discovery projects at the University of Cape Town. Notably, ten of the experimental compounds were from a high-throughput screening campaign to identify βhaematin inhibiting compounds, which were invaluable for proof-of-concept application. In order to further validate the single-point results for these compounds, four (one predicted to be a non-haemozoin inhibitor and three to be haemozoin inhibitors) of this set were chosen to evaluate the method's haemozoin inhibition predictive capability using a full-dose response approach. By employing the 96-well HPLC-DAD four-compound method, we demonstrated that the single-point 33-compound high-throughput haem fractionation assay could correctly classify all four experimental compounds and the known antimalarial drugs as haemozoin or non-haemozoin inhibitors. These findings are substantial, as the validated 33-compound highthroughput assay can be used in conjunction with the extracellular β-haematin method during MoA studies for newly developed or existing compounds. This is even in cases when haemozoin inhibition is not the desired drug target, as those which cause increased unsequestered haem levels can be eliminated from further progression. After investigating a diverse series of haemozoin-inhibiting compounds using the cellular haem fractionation methods, we discovered that each compound exhibited varying levels of unsequestered haem in the parasite at their relevant IC50. Surprisingly, more active compounds (lower IC50 values) often produced lower levels of unsequestered haem than less active ones (higher IC50 values). These observations imply that the common notion that haemozoin inhibitors cause an accumulation of unsequestered haem, which directly leads to parasite death, does not hold. If this were to be the case, the unsequestered haem levels Abstract would be expected to be the same at the IC50 of each compound. This prompted an investigation of the toxicity and effect of unsequestered haem on the parasite. A flow cytometry assay was developed to investigate the killing speed and the stage-specific effects of the various haemozoin-inhibiting compounds. The findings show that compounds that produce lower levels of unsequestered haem tend to kill the parasites earlier in the life cycle. In contrast, those compounds that exhibit higher levels of unsequestered haem tend to kill later in the life cycle. Furthermore, it was also observed that the surviving parasite population postcompound incubation differed. Compounds releasing lower levels of unsequestered haem resulted in a less mature parasite population, while those releasing higher levels of unsequestered haem resulted in a more mature parasite population. These results suggest that the toxicity of haem is compound-specific. This leads to the hypothesis that the compounds present as haem–inhibitor complexes and that haemozoin inhibitors act as complexes exerting unique activities. This has important implications for the study and the design of future hemozoin-inhibiting antimalarial drugs, as well as for our understanding of parasite resistance mechanisms.
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    Open Access
    Monoclonal Antibody Therapy: The Development of a Liquid Chromatography Tandem Mass Spectrometry Method to Measure Rituximab Concentrations in Human Plasma
    (2024) Kuhar, Ana; Wiesner, Joachim
    The escalating global incidence of cancer has emphasised the urgency for innovative therapeutic strategies. Monoclonal antibodies (mAbs) have emerged as promising candidates for cancer treatment, driving the pursuit of tumour-targeted therapies. Rituximab (RTX) is one such mAb which has been of substantial interest in recent years as a therapy. The aim of this research project is to develop and optimise the extraction and quantification of RTX in human plasma using appropriate sample preparation techniques and liquid chromatography tandem mass spectrometry (LC-MS/MS) as the detection method. This was achieved by employing a bottom-up approach, culminating in the identification of a target peptide that serves as a representative of RTX. Sample preparation started with affinity binding purification using protein A bound to agarose beads. Based on the highly specific binding of protein A to the Fc region of immunoglobulins (IgG), this purification allowed for the specific extraction of IgG from plasma, including RTX, while all non-specific plasma proteins are excluded. The affinity binding process was optimised by investigating the addition of different volumes of protein A to purify the RTX from the matrix so that the available binding sites were not completely occupied by plasma IgG. The optimal volume of the protein A slurry used during the affinity binding purification procedure was found to be 200 µL. The extracted RTX molecules were subsequently digested while still bound to the protein A agarose beads, by incubation with trypsin, a protease that cleaves proteins by breaking the peptide bonds at the C-terminal side of the basic amino acids arginine and lysine. The optimal conditions for tryptic digestion were found to be in 25 mM Tris-HCl buffer at pH 8, including 10% acetonitrile and an enzyme-tosubstrate ratio of 1:20 (trypsin: RTX). Overnight incubation at room temperature was followed by a second addition of trypsin and a further 3 hours of incubation. Trypsin digestion of the bound proteins produced a large number of peptides, including the specific signature peptide (s-Pep) chosen to represent RTX. The sequence of this peptide is GLEWIGAIYPGNGDTSYNQK. The final process applied to extract the s-Pep from the total tryptic digest, was solid phase extraction (SPE), utilising Strata-X 33 µm polymeric reverse phase 30 mg / 1 mL SPE extraction cartridges, with elution using 70% acetonitrile containing 10% formic acid. Most of the method development was performed using a Sciex API-3200 triple quadrupole mass spectrometer for detection, coupled to an Agilent 1200 high performance liquid chromatography (HPLC) system used for chromatographic separation. The sensitivity of the Sciex API-3200 was however not adequate to analyse the s-Pep at the expected concentration range, and therefore, the project was concluded by transferring the analytical method to a Shimadzu Nexera X3 8050 LC-MS/MS system. Employing chromatographic separation on an Agilent Poroshell C18 column (2.1 x 50 mm 2.7-Micron) by applying a gradient mobile phase, resulted in the successful quantification of RTX at the required lower level of quantification of 12.5 µg/mL, and linearity throughout the analytical range of 12.5 – 300 µg/mL RTX in plasma
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    Open Access
    The development, validation, and evaluation of quantitative assays for determining adherence of heart failure patients to carvedilol, enalapril and perindopril
    (2023) Joubert, André; Wiesner, Joachim; Maartens Gary
    Background: Heart failure (HF) is a global pandemic with a rising prevalence rate in low- and middle-income countries (LMICs). Poor medication adherence contributes to the impact of chronic diseases such as HF. However, there are sparse adherence data on HF patients in sub-Saharan Africa (SSA). This is problematic as African HF patients have a high mortality rate, which is poorly understood. Poor medication adherence could contribute to the high mortality rate of African HF patients. Objective adherence measures are better than subjective measures (for example, patient recall) at predicting outcomes. In addition, the adherence method should be applicable to resource-scarce settings. Novel multiplex assays were developed to quantify carvedilol, enalaprilat and perindoprilat in dried blood spots (DBS) and correlated with plasma. Carvedilol, enalapril and perindopril are medications commonly used to treat HF, with enalaprilat and perindoprilat being the active metabolites of enalapril and perindopril, respectively. The developed assays were then evaluated in terms of their ability to discern between non-adherent and adherent patients and their suitability for use in resource-scarce settings. Method: The DBS and plasma assays were validated per the United States Food and Drug Administration (FDA) guidelines. The plasma assay was validated over a calibration range of 0.2–200 ng/mL for carvedilol, enalaprilat and perindoprilat. The DBS assay was validated over a range of 1.00–200 ng/mL for the three analytes. The DBS assays were correlated with plasma concentrations in a pilot intensive pharmacokinetic study of six patients. The correlation was determined using Deming regression, with Bland–Altman analysis used to establish agreement between observed and calculated plasma concentrations. Calculated plasma concentrations were obtained using the Deming regression equations describing the relationship between DBS and plasma concentrations. Results: Accuracy, precision, selectivity and sensitivity were proven with complete and reproducible extraction recovery at all concentrations tested for both assays. Stability of the analytes in the matrix and throughout sample processing was proven for both assays. The full range of plasma pharmacokinetic samples could be quantified for all analytes, with the lower limit of quantification (LLOQ) of 0.2 ng/mL proving to be sufficient. The pharmacokinetic pilot study's full range of DBS concentrations could be quantified for enalaprilat but not for carvedilol and perindoprilat. The LLOQ of 1.00 ng/mL was not sensitive enough to quantify the lowest concentrations of some patients for these two analytes. Good correlations were observed between DBS and plasma pharmacokinetic samples, with the Pearson's correlation coefficient (r) greater than 0.94 for all analytes. The difference between the observed and calculated plasma concentrations was less than 20% of their mean for > 67% of samples for all analytes, indicating good agreement between observed and calculated plasma concentrations for all analytes. Conclusions: The plasma assay is suited for evaluating patient adherence to carvedilol, enalapril and perindopril medication. The assay is robust and sensitive enough to discern between those who are adherent and non-adherent. Due to the wealth of pharmacokinetic data available for the analytes in plasma, through pharmacokinetic modelling, it is possible to determine the most appropriate dose and weight-specific adherence interpretation for that patient rather than relying on a general cut-off value. In other words, adherence interpretation can be individualised based on a patient's own dose and weight. Plasma as a matrix, however, is not very amenable to resource-scarce settings. The matrix requires strict storage and transport conditions, so creating additional logistic difficulties and expenses in resource-scarce and remote locations. These are difficulties that would have to be accommodated to use the assay. It was found that the DBS assay is more suitable as a screening assay for carvedilol and perindoprilat than as an assay to gauge adherence. The assay is suitable as an adherence determining assay for enalaprilat, however. The prolonged terminal half-life of enalaprilat allows sufficient DBS concentrations to track adherence. The DBS assay's higher LLOQ and the higher concentration of the analytes in plasma versus that of whole blood places the assay at a stark disadvantage in terms of sensitivity relative to the plasma assay. DBS samples have a significant advantage over plasma samples in their less stringent storage and transport requirements. As a matrix, DBS is far more conducive to remote and resource-scarce areas when compared to plasma. The robustness of both assays was proven with cross-validation using actual clinical samples. Good agreement between observed and calculated plasma concentrations means that DBS concentrations, once normalised, can be used interchangeably with plasma samples. DBS samples can be collected at the sampling sites, taking advantage of the DBS matrix's less stringent storage and transportation requirements. Once the samples are analysed, the concentrations can be converted to plasma concentrations, which can be interpreted more efficiently in terms of adherence. However, this would only be feasible for enalaprilat, as the DBS assay for the carvedilol and perindoprilat analytes lacked sensitivity to reflect ingestion within the last 24 hours.