Evaluating metabolism-induced toxicity using a non-hepatic cell line
- Authors: Weyers, Carli
- Date: 2018
- Subjects: Cytochrome P-450 , Drugs Metabolism , Drugs Design
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/61950 , vital:28087
- Description: The drug discovery pipeline is a complicated process taking roughly 15 years to complete, costing in excess of $1 billion per new chemical entity. It has been estimated that for every 100, 000 promising hit or lead compounds, only one will make it onto the market due to numerous drug candidates being discarded because of many complications. One such complication is metabolism-induced toxicity. Accordingly, an early understanding of the metabolism of any new chemical entity is becoming an integral part of the pipeline. In order to explore this, various methods have been developed including in silico and in vitro techniques. One such method involves performing cell viability assays on human liver cancer cell lines, which overexpress specific metabolic cytochrome P450 enzymes. If a toxic metabolite is produced it would result in reduced cell viability of the transformed cell line in comparison to a control. Since the liver is the primary site of metabolism in the human body, we were curious as to the extent to which background metabolism may play a role in the degree to which toxic metabolites would be produced in these cell lines. The aim of this project, therefore, was to establish if a non-hepatic cell-based system which overexpresses CYP3A4 could be used to detect the metabolism and any subsequent toxicity of compounds which have been reported to be substrates of the CYP450 enzyme. The HEK293 cell line was stably transfected with a plasmid vector for human CYP3A4 to create a model overexpression system for our metabolism studies. The activity of the enzyme was confirmed using the substrate, 7-benzyloxy-4-trifluoromethyl-coumarin. Subsequently, cytotoxicity testing was done on four known pharmaceuticals reported to generate toxic metabolites in hepatic cell-based assays. In silico metabolic predictions on the four known compounds were performed and compared to the results of published literature. Finally, the metabolism of one compound was studied using a combination of high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) in order to detect predicted metabolites. We observed no change in cellular toxicity nor did we detect the formation of metabolites, even though the overexpressed CYP3A4 enzyme was active. The results suggest that caution should be taken when interpreting the results of cell-based metabolism studies, and background metabolism may play a significant role in the data. , Thesis (MSc) -- Faculty of Pharmacy, Pharmacy, 2018
- Full Text:
- Date Issued: 2018
- Authors: Weyers, Carli
- Date: 2018
- Subjects: Cytochrome P-450 , Drugs Metabolism , Drugs Design
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/61950 , vital:28087
- Description: The drug discovery pipeline is a complicated process taking roughly 15 years to complete, costing in excess of $1 billion per new chemical entity. It has been estimated that for every 100, 000 promising hit or lead compounds, only one will make it onto the market due to numerous drug candidates being discarded because of many complications. One such complication is metabolism-induced toxicity. Accordingly, an early understanding of the metabolism of any new chemical entity is becoming an integral part of the pipeline. In order to explore this, various methods have been developed including in silico and in vitro techniques. One such method involves performing cell viability assays on human liver cancer cell lines, which overexpress specific metabolic cytochrome P450 enzymes. If a toxic metabolite is produced it would result in reduced cell viability of the transformed cell line in comparison to a control. Since the liver is the primary site of metabolism in the human body, we were curious as to the extent to which background metabolism may play a role in the degree to which toxic metabolites would be produced in these cell lines. The aim of this project, therefore, was to establish if a non-hepatic cell-based system which overexpresses CYP3A4 could be used to detect the metabolism and any subsequent toxicity of compounds which have been reported to be substrates of the CYP450 enzyme. The HEK293 cell line was stably transfected with a plasmid vector for human CYP3A4 to create a model overexpression system for our metabolism studies. The activity of the enzyme was confirmed using the substrate, 7-benzyloxy-4-trifluoromethyl-coumarin. Subsequently, cytotoxicity testing was done on four known pharmaceuticals reported to generate toxic metabolites in hepatic cell-based assays. In silico metabolic predictions on the four known compounds were performed and compared to the results of published literature. Finally, the metabolism of one compound was studied using a combination of high performance liquid chromatography (HPLC) and liquid chromatography-mass spectrometry (LC-MS) in order to detect predicted metabolites. We observed no change in cellular toxicity nor did we detect the formation of metabolites, even though the overexpressed CYP3A4 enzyme was active. The results suggest that caution should be taken when interpreting the results of cell-based metabolism studies, and background metabolism may play a significant role in the data. , Thesis (MSc) -- Faculty of Pharmacy, Pharmacy, 2018
- Full Text:
- Date Issued: 2018
Exploring the potential of imines as antiprotozoan agents with focus on t. Brucei and p. Falciparum
- Authors: Oluwafemi, Kola Augustus
- Date: 2018
- Subjects: Protozoa , Parasites , Imines , Nuclear magnetic resonance , HeLa cells , Plasmodium falciparum , Trypanosoma brucei , Isomerism
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/62235 , vital:28145 , DOI 10.21504/10962/62235
- Description: This work focuses on the design, synthesis and evaluation of imine-containing heterocyclic and acyclic compounds with special focus on their bioactivity against parasitic protozoans (P. falciparum and T. brucei) - given the context of drug resistance in the treatment of malaria and Human African sleeping sickness and the fact that several bioactive organic compounds have been reported to possess the imino group. Starting from 2-aminopyridine, novel #-alkylated-5-bromo-7-azabenzimidazoles and substituted 5-bromo-1-(carbamoylmethy)-7-azabenzimidazole derivatives were prepared, and their bioactivity against parasitic protozoans was assessed. NMR spectra of the substituted 5- bromo-1-(carbamoylmethy)-7-azabenzimidazole derivatives exhibited rotational isomerism, and a dynamic NMR study was used in the estimation of the rate constants and the free- energies of activation for rotation. The free-energy differences between the two rotamers were determined and the more stable conformations were predicted. Novel 2-phenyl-7-azabenzimidazoles were also synthesised from 2-aminopyridine. A convenient method for the regioselective formylation of 2,3-diaminopyridines into 2-amino- 7-(benzylimino)pyridine analogues of 2-phenyl-7-azabenzimidazole was developed, and some of the resulting imino derivatives were hydrogenated to verify the importance of the imino moiety for bioactivity. The 2-phenyl-7-azabenzimidazoles and the 2-amino-7- (benzylimino)pyridine analogues were screened for their anti-protozoal activity and their cytotoxicity level was determined against the HeLa cell line. In order to validate the importance of the pyridine moiety, novel #-(phenyl)-2- hydroxybenzylimines, #-(benzyl)-2-hydroxybenzylimines and (±)-trans-1,2-bis[2- hydroxybenzylimino]cyclohexanes were also synthesized and screened for activity against the parasitic protozoans and for cytotoxicity against the HeLa cell line. The biological assay results indicated that these compounds are not significantly cytotoxic and a good number of them show potential as lead compounds for the development of new malaria and trypanosomiasis drugs. , Thesis (PhD) -- Faculty of Science, Chemistry, 2018
- Full Text:
- Date Issued: 2018
- Authors: Oluwafemi, Kola Augustus
- Date: 2018
- Subjects: Protozoa , Parasites , Imines , Nuclear magnetic resonance , HeLa cells , Plasmodium falciparum , Trypanosoma brucei , Isomerism
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/62235 , vital:28145 , DOI 10.21504/10962/62235
- Description: This work focuses on the design, synthesis and evaluation of imine-containing heterocyclic and acyclic compounds with special focus on their bioactivity against parasitic protozoans (P. falciparum and T. brucei) - given the context of drug resistance in the treatment of malaria and Human African sleeping sickness and the fact that several bioactive organic compounds have been reported to possess the imino group. Starting from 2-aminopyridine, novel #-alkylated-5-bromo-7-azabenzimidazoles and substituted 5-bromo-1-(carbamoylmethy)-7-azabenzimidazole derivatives were prepared, and their bioactivity against parasitic protozoans was assessed. NMR spectra of the substituted 5- bromo-1-(carbamoylmethy)-7-azabenzimidazole derivatives exhibited rotational isomerism, and a dynamic NMR study was used in the estimation of the rate constants and the free- energies of activation for rotation. The free-energy differences between the two rotamers were determined and the more stable conformations were predicted. Novel 2-phenyl-7-azabenzimidazoles were also synthesised from 2-aminopyridine. A convenient method for the regioselective formylation of 2,3-diaminopyridines into 2-amino- 7-(benzylimino)pyridine analogues of 2-phenyl-7-azabenzimidazole was developed, and some of the resulting imino derivatives were hydrogenated to verify the importance of the imino moiety for bioactivity. The 2-phenyl-7-azabenzimidazoles and the 2-amino-7- (benzylimino)pyridine analogues were screened for their anti-protozoal activity and their cytotoxicity level was determined against the HeLa cell line. In order to validate the importance of the pyridine moiety, novel #-(phenyl)-2- hydroxybenzylimines, #-(benzyl)-2-hydroxybenzylimines and (±)-trans-1,2-bis[2- hydroxybenzylimino]cyclohexanes were also synthesized and screened for activity against the parasitic protozoans and for cytotoxicity against the HeLa cell line. The biological assay results indicated that these compounds are not significantly cytotoxic and a good number of them show potential as lead compounds for the development of new malaria and trypanosomiasis drugs. , Thesis (PhD) -- Faculty of Science, Chemistry, 2018
- Full Text:
- Date Issued: 2018
Synthesis and characterization of titanium dioxide nanotubes on fluorine-doped tin oxide (FTO) glass substrate using electro-anodization technique
- Zinya, Simcelile https://orcid.org/0000-0001-5864-0957
- Authors: Zinya, Simcelile https://orcid.org/0000-0001-5864-0957
- Date: 2017-12
- Subjects: Titanium dioxide , Nanotubes , Nanostructured materials
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10353/23979 , vital:62201
- Description: One-dimensional (1-D) titanium dioxide nanotubes (TNTs) have attracted much attention as a promising alternative electrode material for dye sensitized solar cell (DSSC). As compared to the randomly packed and disordered TiO2 nanoparticles (TNPs) network with numerous particle-particle interfaces, TNTs prove to have fascinating features than make them suitable candidates in DSSCs. Well-structured TNTs arrays are of great potential among the various types of 1D TiO2 nano-materials owing to their superior electron transport properties with limited grain boundaries. Vectorial transport of photon generated electrons along the TNTs has been reported to lead to higher charge mobility which is crucial for improvement of DSSC performances. In this work, highly adhesive titanium films were deposited on functional substrates (FS) using radio frequency (RF) sputtering technique at a sputtering output power of 1kW, operating pressure of 1.5 Pa and at a deposition temperature of 200 °C to obtain a thickness of 10 μm under an inert argon atmosphere. The duration period for sputter coating 10 μm thickness of titanium film layer was 122 minutes with sputter rate for titanium target of about 82 nm per minutes. Subsequently, the RF sputtered titanium films were anodized with 0.5 wt. percent ammonium fluoride + 0.35 wt. percent deionised water and 96 wt. percent glycerol electrolyte solution at room temperature at 60 V for 72 hours. The resulting TNTs on functional substrates (TNTs-FS) were subjected to thermal treatment at 350 °C, 450 °C, 550 °C and 650 °C for 3 hours under oxygen atmosphere. The effect of annealing temperature on the morphological, and structural properties have been scrutinized. The as prepared and thermally treated TNTs-FS were characterized using scanning electron microscopy (SEM), high resolution transmission electron microscopy (HRTEM), X-ray diffraction (XRD) and Confocal Raman Spectroscopy (CRS). SEM and HRTEM techniques were employed to confirm the presence of the TNTs-FS and also to study the structural-morphology of the TNTs as the annealing temperature increases. SEM revealed improvement in morphology with increase in sample annealing temperature, even at high temperatures such as 650 °C with no collapsing and sintering of the TNTs-FS occurring. SEM images revealed TNTs-FS with pore diameter sizes in the range between 85-170 nm. This is in compliance with HRTEM analysis, which revealed smooth and straight tube walls and improved surface morphology with increase in annealing temperature. In addition HRTEM images revealed pore diameter of TNTs-FS in the range between 85-165 nm. Furthermore, HRTEM revealed lattice fringes of 0.351, 0.352 and 0.353 nm between the neighbouring lattice fringes. All corresponding to (101) planes of anatase phase TNTs at different annealing temperatures (350-650 °C). The crystallographic structure of TNTs-FS was characterized by XRD measurements after thermal treatment at 350 °C, 450 °C, 550 °C and 650 °C. The XRD pattern revealed peaks in the wide angle range of 2θ (20° < 2θ > 80°) discovered at 29.43°, 45.10°, 56.52°, 63.5°, 64.92° and 74.81° corresponding to the planes (101), (112), (200), (105), (211) and (204) crystalline structures of the anatase TNTs. The intensity of the peaks increased with increasing annealing temperature. The strong sharp peaks indicate the large quantities and higher degrees of crystallinity of anatase phase of the TNTs. CRS Large Area Scan (LAS) and Depth profiling (DP) were employed to evaluate the crystallinity and phase distribution of TNTs-FS thermally treated at different temperatures. CRS LAS in the XY direction of TNTs-FS revealed the presence of differently crystallized anatase phases of TiO2 with Raman vibrational modes of 159.38 cm-1 (Eg), 208.37 cm-1 (Eg), 399.67 cm-1 (B1g), 514.25 cm-1 (A1g) and 641.58 cm-1 (Eg) for the samples annealed at 350 °C. The effect of annealing temperature on TiO2 phase evolution was meticulously evaluated using CRS for TNTs-FS for the samples annealed at 350 °C, 450 °C, 550 °C and 650 °C. The FWHM was estimated from CRS and decreases with increasing annealing temperature resulting in increasing crystallinity. Increase in anatase FWHM and anatase peak intensity implies higher degree of crystallinity and increasing crystallite sizes were also confirmed by XRD. Growing of titanium dioxide on functional substrates one novel contribution towards the fabrication of efficient electrode materials for solar cell development. Our method of characterizing TNTs-FS from a large area scan along the surface of the samples and depth profiling along the TNTs tube walls using confocal Raman spectroscopy prove to be a pivotal step in the development of efficient photoelectrode materials of the solar devices. , Thesis (MSc) -- Faculty of Science and Agriculture, 2017
- Full Text:
- Date Issued: 2017-12
- Authors: Zinya, Simcelile https://orcid.org/0000-0001-5864-0957
- Date: 2017-12
- Subjects: Titanium dioxide , Nanotubes , Nanostructured materials
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10353/23979 , vital:62201
- Description: One-dimensional (1-D) titanium dioxide nanotubes (TNTs) have attracted much attention as a promising alternative electrode material for dye sensitized solar cell (DSSC). As compared to the randomly packed and disordered TiO2 nanoparticles (TNPs) network with numerous particle-particle interfaces, TNTs prove to have fascinating features than make them suitable candidates in DSSCs. Well-structured TNTs arrays are of great potential among the various types of 1D TiO2 nano-materials owing to their superior electron transport properties with limited grain boundaries. Vectorial transport of photon generated electrons along the TNTs has been reported to lead to higher charge mobility which is crucial for improvement of DSSC performances. In this work, highly adhesive titanium films were deposited on functional substrates (FS) using radio frequency (RF) sputtering technique at a sputtering output power of 1kW, operating pressure of 1.5 Pa and at a deposition temperature of 200 °C to obtain a thickness of 10 μm under an inert argon atmosphere. The duration period for sputter coating 10 μm thickness of titanium film layer was 122 minutes with sputter rate for titanium target of about 82 nm per minutes. Subsequently, the RF sputtered titanium films were anodized with 0.5 wt. percent ammonium fluoride + 0.35 wt. percent deionised water and 96 wt. percent glycerol electrolyte solution at room temperature at 60 V for 72 hours. The resulting TNTs on functional substrates (TNTs-FS) were subjected to thermal treatment at 350 °C, 450 °C, 550 °C and 650 °C for 3 hours under oxygen atmosphere. The effect of annealing temperature on the morphological, and structural properties have been scrutinized. The as prepared and thermally treated TNTs-FS were characterized using scanning electron microscopy (SEM), high resolution transmission electron microscopy (HRTEM), X-ray diffraction (XRD) and Confocal Raman Spectroscopy (CRS). SEM and HRTEM techniques were employed to confirm the presence of the TNTs-FS and also to study the structural-morphology of the TNTs as the annealing temperature increases. SEM revealed improvement in morphology with increase in sample annealing temperature, even at high temperatures such as 650 °C with no collapsing and sintering of the TNTs-FS occurring. SEM images revealed TNTs-FS with pore diameter sizes in the range between 85-170 nm. This is in compliance with HRTEM analysis, which revealed smooth and straight tube walls and improved surface morphology with increase in annealing temperature. In addition HRTEM images revealed pore diameter of TNTs-FS in the range between 85-165 nm. Furthermore, HRTEM revealed lattice fringes of 0.351, 0.352 and 0.353 nm between the neighbouring lattice fringes. All corresponding to (101) planes of anatase phase TNTs at different annealing temperatures (350-650 °C). The crystallographic structure of TNTs-FS was characterized by XRD measurements after thermal treatment at 350 °C, 450 °C, 550 °C and 650 °C. The XRD pattern revealed peaks in the wide angle range of 2θ (20° < 2θ > 80°) discovered at 29.43°, 45.10°, 56.52°, 63.5°, 64.92° and 74.81° corresponding to the planes (101), (112), (200), (105), (211) and (204) crystalline structures of the anatase TNTs. The intensity of the peaks increased with increasing annealing temperature. The strong sharp peaks indicate the large quantities and higher degrees of crystallinity of anatase phase of the TNTs. CRS Large Area Scan (LAS) and Depth profiling (DP) were employed to evaluate the crystallinity and phase distribution of TNTs-FS thermally treated at different temperatures. CRS LAS in the XY direction of TNTs-FS revealed the presence of differently crystallized anatase phases of TiO2 with Raman vibrational modes of 159.38 cm-1 (Eg), 208.37 cm-1 (Eg), 399.67 cm-1 (B1g), 514.25 cm-1 (A1g) and 641.58 cm-1 (Eg) for the samples annealed at 350 °C. The effect of annealing temperature on TiO2 phase evolution was meticulously evaluated using CRS for TNTs-FS for the samples annealed at 350 °C, 450 °C, 550 °C and 650 °C. The FWHM was estimated from CRS and decreases with increasing annealing temperature resulting in increasing crystallinity. Increase in anatase FWHM and anatase peak intensity implies higher degree of crystallinity and increasing crystallite sizes were also confirmed by XRD. Growing of titanium dioxide on functional substrates one novel contribution towards the fabrication of efficient electrode materials for solar cell development. Our method of characterizing TNTs-FS from a large area scan along the surface of the samples and depth profiling along the TNTs tube walls using confocal Raman spectroscopy prove to be a pivotal step in the development of efficient photoelectrode materials of the solar devices. , Thesis (MSc) -- Faculty of Science and Agriculture, 2017
- Full Text:
- Date Issued: 2017-12
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