Pharmacogenetic analysis of admc polymorphisms affecting the efficacy of TB treatment in tuberculosis patients from Tanzania and Uganda, and pharmalogical evaluation of potent medicinal plant extracts used in the treatment of tuberculosis

PhD (Microbiology)

Department of Biochemistry and Microbiology

Background: Despite the availability of antibiotics which can cure tuberculosis (TB), it remains the leading cause of death from a single infectious disease worldwide. Recent research has shown that the way anti-tuberculous drugs are processed by the body (pharmacokinetics) and their effects on the disease (pharmacodynamics) play a crucial role in determining the success of treatment. To find new treatments which are less harmful than current regimens, medicinal plants are being explored for their potential to produce compounds with anti-TB properties. Therefore, it was hypothesised that the effectiveness of anti-TB drugs may also depend on an individual's ADME polymorphisms. Aim: This study was aimed at evaluating the pharmacogenetics of Tanzanian and Ugandan tuberculosis patients, and pharmacological effects of medicinal plants [S. brachypetala (MMV004), R. caffra (MMV003), S. molle (MMV005), Z. mucronata (MMV002) and S. petersiana (MMV001)], used for the treatment of symptoms related to tuberculosis infection. Methodology: Saliva samples were collected from the study participants at treatment initiatiion using Genotek tissue collection kit. DNA Genotek extraction procedure was used to extact DNA from the samples and used in evaluation of custom-designed ADME targets for polymorphisms using real-time polymerase chain reaction on 96 well plates. The pharmacogenetic analysis included target genes responsible for the ADME of isoniazid, rifampin, pyrazinamide, and levofloxacin/moxifloxacin, which are currently the most widely used anti-TB drugs, including medicinal plants. For the pharmacological evaluation of the medicinal plants, plant materials were dried and ground it into fine powder and extracted with solvents of varying polarity. The phytochemical profile of each plant species was assessed using Thin Layer Chromatography (TLC) plates developed in three solvent systems - benzene/ethanol/ammonium hydroxide (BEA), chloroform/ethyl acetate/formic acid (CEF), and ethyl acetate/methanol/water (EMW) - and sprayed with vanillin sulphuric acid. Quantitative analysis of phytochemical constituents using a series of biochemical tests was conducted to determine the presence of alkaloids, cardiac glycosides, terpenoids, and tannin compounds in all plant species. Antioxidant activity was evaluated qualitatively and quantitatively using 2, 2-diphenyl-1-picrylhydrazyl (DPPH) and Ferric ion reducing power (FRAP) assays, respectively. The presence of antimycobacterial phytochemical constituents was determined using broth microdilution procedures against Mycobacterium smegmatis, Mycobacterium aurum, and Mycobacterium tuberculosis strains. To identify the phytochemical compounds responsible for the antimycobacterial activity against M. tuberculosis, ultra-performance liquid chromatography mass spectrometry (UPLC-MS) was used on R. caffra and S. molle extracts. The cytotoxicity of plant extracts was evaluated on C3A and Vero monkey kidney cell lines and the anti-inflammatory activity of the compounds against RAW 264.7 macrophages. Finally, the anticancer activity of the plant extracts was evaluated against triple-negative breast cancer MDA-MB 231 cell line and determined the type of cell death using flow cytometry. Outcomes: Through successful DNA extraction, genotyping of ADME genes was made possible. Both adult and pediatric patients from Tanzania were found to have a highly expressed slow acetylator NAT2 variant, while homogenous CC variant SLCO1B1 c.521T/C was absent in Tanzanian adults and Ugandan patients. These genotypes are highly associated with hepatotoxicity. Chromatograms showed that all plant species separated at three solvent systems had both fluorescing and vanillin reactive compounds. Biochemical tests revealed the presence of alkaloids, cardiac glycosides, terpenoids, and tannins compounds in all plant species. Antioxidant activity was observed to increase in a concentration-dependent manner, and extracts at the 0.02 mg/ml concentration showed scavenging activity lower than 50% compared to ascorbic acid. All plant extracts displayed the presence of ferric reducing antioxidant, and R. caffra absorbance was directly proportional to that of ascorbic acid. Antimycobacterial activity against M. tuberculosis was observed only in R. caffra and S. molle dichloromethane extracts, with minimum inhibitory concentration (MIC) values of 0.25 and 0.125 mg/ml. UPLCMS phytochemical profiling showed that R. caffra dichloromethane extracts had abundant alkaloids compounds, while S. molle extracts had terpenoids compounds. Cytotoxicity was higher on the C3A cell line than on the Vero cells, and toxicity was observed in a concentration-dependent manner. A high production of nitric oxide was observed with a slight change in the percentage of viable cells, suggesting potential anti-inflammatory activity of the plant species. S. molle extracts' potential anti-inflammatory activity was discovered to be due to its toxicity on the cells, as it had less than 70% viable cells at all concentrations. Antiproliferative activity on cancer cells showed that S. petersiana (D5) had percentages of early (41.97%) and late apoptosis (38.26%) which were above Cisplatin (16.15% and 30.55%). Conclusion: By understanding the ADME profile of individual TB patients or regions with a predisposition to the disease (such as East Africa) would allow personalized interventions in drug choice, dosage, and duration. This knowledge can also help inform new drug development and clinical trial planning. Additionally, the ADME profile can provide insight into the potential toxicity of phytochemical compounds and antituberculosis-phytochemical interactions. The observed antioxidant, anti-inflammatory, and anticancer activities may lead to the development of new antibiotics. This was the first study to evaluate S. molle extracts which showed antimycobacterial activity against Mycobacterium bacteria. Further evaluation of biological activities in in-vivo models is needed to facilitate communication between scientists and traditional healers.

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