Browsing by Author "Gibhard, Liezl"

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    Open Access
    The Artemiside-Artemisox-Artemisone-M1 Tetrad: Efficacies against Blood Stage P. falciparum Parasites, DMPK Properties, and the Case for Artemiside
    (2021-12-03) Gibhard, Liezl; Coertzen, Dina; Reader, Janette; van der Watt, Mariëtte E; Birkholtz, Lyn-Marie; Wong, Ho Ning; Batty, Kevin T; Haynes, Richard K; Wiesner, Lubbe
    Because of the need to replace the current clinical artemisinins in artemisinin combination therapies, we are evaluating fitness of amino-artemisinins for this purpose. These include the thiomorpholine derivative artemiside obtained in one scalable synthetic step from dihydroartemisinin (DHA) and the derived sulfone artemisone. We have recently shown that artemiside undergoes facile metabolism via the sulfoxide artemisox into artemisone and thence into the unsaturated metabolite M1; DHA is not a metabolite. Artemisox and M1 are now found to be approximately equipotent with artemiside and artemisone in vitro against asexual P. falciparum (Pf) blood stage parasites (IC50 1.5–2.6 nM). Against Pf NF54 blood stage gametocytes, artemisox is potently active (IC50 18.9 nM early-stage, 2.7 nM late-stage), although against the late-stage gametocytes, activity is expressed, like other amino-artemisinins, at a prolonged incubation time of 72 h. Comparative drug metabolism and pharmacokinetic (DMPK) properties were assessed via po and iv administration of artemiside, artemisox, and artemisone in a murine model. Following oral administration, the composite Cmax value of artemiside plus its metabolites artemisox and artemisone formed in vivo is some 2.6-fold higher than that attained following administration of artemisone alone. Given that efficacy of short half-life rapidly-acting antimalarial drugs such as the artemisinins is associated with Cmax, it is apparent that artemiside will be more active than artemisone in vivo, due to additive effects of the metabolites. As is evident from earlier data, artemiside indeed possesses appreciably greater efficacy in vivo against murine malaria. Overall, the higher exposure levels of active drug following administration of artemiside coupled with its synthetic accessibility indicate it is much the preferred drug for incorporation into rational new artemisinin combination therapies.
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    Efficacy and pharmacokinetic evaluation of a novel anti-malarial compound (NP046) in a mouse model
    (BioMed Central Ltd, 2015) Abay, Efrem; van der Westuizen, Jan; Swart, Kenneth; Gibhard, Liezl; Lawrence, Nina; Dambuza, Ntokozo; Wilhelm, Anke; Pravin, Kendrekar; Wiesner, Lubbe
    BACKGROUND: Even though malaria is a completely preventable and treatable disease, it remains a threat to human life and a burden to the global economy due to the emergence of multiple-drug resistant malaria parasites. According to the World Malaria Report 2013, in 2012 there were an estimated 207 million malaria cases and 627,000 deaths. Thus, the discovery and development of new, effective anti-malarial drugs are required. To achieve this goal, the Department of Chemistry at the University of the Free State has synthesized a number of novel amino-alkylated chalcones and analogues, which showed in vitro anti-malarial activity against both chloroquine-sensitive and chloroquine-resistant Plasmodium falciparum strains. The lead compound (NP046) was selected for a comprehensive pharmacokinetic (PK) and in vivo efficacy evaluation in a mouse model. METHODS: In vivo efficacy: Water solutions of NP046 were administered orally at 50 and 10mg/kg using oral gavage and IV at 5 and 1mg/kg via the dorsal penile vein to Plasmodium berghei (ANKA strain) infected male C57BL/6 mice (n=5), once a day for four days. Blood samples were collected via tail bleeding in tubes containing phosphate buffer saline (PBS) on day five to determine the % parasitaemia by flow cytometry.In vivo PK: NP046 solutions in water were administered orally (50 and 10mg/kg) and IV (5mg/kg) to male C57BL/6 mice (n=5). Blood samples were collected via tail bleeding into heparinized tubes and analysed using a validated LC-MS/MS assay. Data obtained from the concentration-time profile was evaluated using Summit PK software to determine the PK parameters of NP046. RESULTS: NP046 inhibited parasite growth for the oral and IV groups. Better parasite growth inhibition was observed for the IV group. The PK evaluation of NP046 showed low oral bioavailability (3.2% and 6% at 50mg/kg and 10mg/kg dose, respectively and a moderate mean half-life ranging from 3.1 to 4.4hours. CONCLUSION: Even though the oral bioavailability of NP046 is low, its percentage parasite growth inhibition is promising, but in order to improve the oral bioavailability, structure-activity-relationship (SAR) optimization studies are currently being conducted.
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    Optimization of a multi-well colorimetric assay to determine haem species in Plasmodium falciparum in the presence of anti-malarials
    (BioMed Central Ltd, 2015) Combrinck, Jill; Fong, Kim; Gibhard, Liezl; Smith, Peter; Wright, David; Egan, Timothy
    BACKGROUND: The activity of several well-known anti-malarials, including chloroquine (CQ), is attributed to their ability to inhibit the formation of haemozoin (Hz) in the malaria parasite. The formation of inert Hz, or malaria pigment, from toxic haem acquired from the host red blood cell of the parasite during haemoglobin digestion represents a pathway essential for parasite survival. Inhibition of this critical pathway therefore remains a desirable target for novel anti-malarials. A recent publication described the results of a haem fractionation assay used to directly determine haemoglobin, free haem and Hz in Plasmodium falciparum inoculated with CQ. CQ was shown to cause a dose-dependent increase in cellular-free haem that was correlated with decreased parasite survival. The method provided valuable information but was limited due to its low throughput and high demand on parasite starting material. Here, this haem fractionation assay has been successfully adapted to a higher throughput method in 24-well plates, significantly reducing lead times and starting material volumes. METHODS: All major haem species in P. falciparum trophozoites, isolated through a series of cellular fractionation steps were determined spectrophotometrically in aqueous pyridine (5%v/v, pH7.5) as a low spin complex with haematin. Cell counts were determined using a haemocytometer and a rapid novel fluorescent flow cytometry method. RESULTS: A higher throughput haem fractionation assay in 24-well plates, containing at most ten million trophozoites was validated against the original published method using CQ and its robustness was confirmed. It provided a minimum six-fold improvement in productivity and 24-fold reduction in starting material volume. The assay was successfully applied to amodiaquine (AQ), which was shown to inhibit Hz formation, while the antifolate pyrimethamine (PYR) and the mitochondrial electron transporter inhibitor atovaquone (Atov) demonstrated no increase in toxic cellular free haem. CONCLUSIONS: This higher throughput cellular haem fractionation assay can easily be applied to novel anti-malarials with a significantly decreased lead time, providing a valuable tool with which to probe the mechanisms of action of both new and established anti-malarials.
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    The characterization of pharmacokinetic properties and evaluation of in vitro drug combination efficacies of novel antimalarial compounds
    (2020) Laing, Lizahn; Wiesner, Lubbe Joachim; Gibhard, Liezl
    Relief of the global malaria burden relies on the management and application of effective therapies. Unfortunately, the continuous development of resistance to therapies by the deadliest parasite strain, Plasmodium falciparum, has made the treatment and control of malaria much more difficult. Derivatives of the Chinese peroxidic antimalarial drug artemisinin primarily used in first-line combination therapy for treatment of P. falciparum malaria have proved to be highly effective. However, their use also is now compromised by the development of resistance by the parasite to the artemisinin derivative in the drug combination. This event emphasizes the need for ongoing development of new and effective drug combinations. This research aimed to identify efficacious combinations selected from a group of compounds known to induce oxidative stress by redox cycling combined with an artemisinin, which as an oxidant drug also induces oxidative stress but is unable to undergo redox cycling. Combination of the artemisinin with a redox-active compound is expected to both enhance and maintain oxidative stress within the parasite's proliferative environment. These combinations should be used together with a third drug with a completely different mode of action, such as a quinolone. Selected amino artemisinins and redox active phenothiazines, phenoxazines, thiosemicarbazones, and quinolone derivatives were screened for antimalarial activity and mammalian toxicity. These were found to be potently active (11 μM) to Chinese Hamster ovarian (CHO) cells. The compounds are thus highly selective for P. falciparum, as revealed by the selectivity indices (SI) of >270. The in vitro absorption, distribution, metabolism, and elimination (ADME) properties of the compounds were also determined through the application of specific assays. In vivo pharmacokinetic (PK) profiling was also carried out by intravenous and oral administration of the individual compounds to healthy C57BL/6 mice. Biological samples were analysed via liquid chromatography-tandem mass spectrometry (LC-MS/MS) bioanalytical methods, which were validated according to the fit-for purpose recommendations by the FDA. Evaluation of the in vitro and in vivo profiles thereby facilitated the identification of suitable combination candidates. The phenoxazine and phenothiazine derivatives were identified as the best potential redox partners and were each investigated in combination with the amino-artemisinin artemisone through fixed ratio isobole analysis. A substantial synergistic interaction was observed. Overall, the investigation enabled the identification of drug combinations that are potently active in vitro. This synergistic interaction strongly supports the redox cycling rationale for identifying new antimalarial therapies and further suggests that such combinations in chemotherapy may delay the onset of resistance to the new agents. The results strongly encourage further investigation of the in vivo pharmacokinetic and pharmacodynamic (PK/PD) relationships of these combinations in the humanized murine model of P. falciparum
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    The development and validation of an LC-MS/MS method for the determination of a new anti-malarial compound (TK900D) in human whole blood and its application to pharmacokinetic studies in mice
    (2014-01-31) Abay, Efrem T; van der Westhuizen, Jan H; Swart, Kenneth J; Gibhard, Liezl; Tukulula, Matshawandile; Chibale, Kelly; Wiesner, Lubbe
    Abstract Background Malaria is one of the most lethal and life-threatening killer infectious diseases in the world, and account for the deaths of more than half a million people annually. Despite the remarkable achievement made in preventing and eradicating malaria, it still remains a threat to the public health and a burden to the global economy due to the emergence of multiple-drug resistant malaria parasites. Therefore, the need to develop new anti-malarial drugs is crucial. The chemistry department at the University of Cape Town synthesized a number of new CQ-like derivatives (TK-series), and evaluated them for in vitro activity against both CQ-sensitive and -resistant Plasmodium falciparum strains, and for general cytotoxicity against a Chinese Hamster Ovarian (CHO) mammalian cell line. The lead compounds from the TK-series were selected for a comprehensive pharmacokinetic (PK) evaluation in a mouse model. Methods A sensitive LC-MS/MS assay was developed for the quantitative determination of TK900D. Multiple reaction monitoring (MRM) in the positive ionization mode was used for detection. The analyte and the internal standard (TK900E) were isolated from blood samples by liquid-liquid extraction with ethyl acetate. Chromatographic separation was achieved with a Phenomenex® Kinetex C18 (100 × 2.0 mm id, 2.6 μm) analytical column, using a mixture of 0.1% formic acid and acetonitrile (50:50; v/v) as the mobile phase. The method was fully validated over concentrations that ranged from 3.910 to 1000 ng/ml, and used to evaluate the PK properties of the lead compounds in a mouse model. Results The assay was robust, with deviation not exceeding 11% for the intra- and inter-run precision and accuracy. Extraction recovery was consistent and more than 60%. PK evaluation showed that TK900D and TK900E have moderate oral bioavailability of 30.8% and 25.9%, respectively. The apparent half-life ranged between 4 to 6 h for TK900D and 3.6 to 4 h for TK900E. Conclusion The assay was sensitive and able to measure accurately low drug levels from a small sample volume (20 μl). PK evaluation showed that the oral bioavailability was moderate. Therefore, from a PK perspective, the compounds look promising and can be taken further in the drug development process.