Interaction of catechol O-methyltransferase with gold and silver nanoparticles
- Authors: Usman, Aminu
- Date: 2018
- Subjects: Parkinson's disease , Methyltransferases , Catechol , Nanoparticles
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/61818 , vital:28063 , DOI https://doi.org/10.21504/10962/61818
- Description: Catechol O-methyltransferase (S-adenosyl-Z-methionine: catechol O-methyltransferase; COMT; EC 2.1.1.6) is a ubiquitous enzyme that catalyses the transfer of a methyl group from the cofactor, S-adenosyl-Z-methionine (SAM) to a hydroxyl group of endogenous and exogenous catechol-containing moieties. The physiological role of this enzyme is the methylation and thereby inactivation of the catechol-containing bio-active and bio-toxic compounds, including catechol-neurotransmitters, catechol-estrogens and catechol-containing drugs. Activity of this enzyme is implicated in the treatment of Parkinson’s disease and is associated with other diseases including breast cancer and an array neuropsychological disorders, such as schizophrenia. This thesis explores the use of gold and silver nanoparticles (NPs) (AuNPs and AgNPs) to inhibit the catalytic activity of mammalian COMT. Because of its accessibility and availability, we initially investigated bovine soluble COMT (BSCOMT) from liver tissue. Bioinformatic analyses and structural modeling revealed high (>90%) sequence similarity between BSCOMT and human soluble COMT (HSCOMT). BSCOMT was partially purified to 7.78 fold, 1.65% yield and had a specific activity of 0.052 U/mg. It had pH and temperature optima of 8.5 and 40oC, respectively. The Km, Vmax, Kcat and Kcat/Km towards esculetin methylation were respectively 1.475±0.130 pM, 0.0353±0.001 pmol/ml/min, 1.748 x 10-2±5.0x10-4 min-1 and 1.18x10-2 M-1. min-1. HSCOMT was expressed in Escherichia coli BL21(DE3) which showed optimal activity for esculetin methylation at pH and temperature of 7.0 and 30°C, respectively. It was purified to 5.62 fold, 22.6% yield with a specific activity of 3.85 U/mg. HSCOMT kinetic plots, upon incubation of the reaction mixture at 30°C for 5 min before addition of SAM was hyperbolic with Km, Vmax, Kcat and Kcat/Km values of 1.79 pM, 0.412 pmol/ml/min, 2.08 min-1 and 1.165 M-1. min-1, respectively. AuNPs and AgNPs showed a concentration dependent inhibition of HSCOMT activity upon increasing the 5 min incubation time to 1 h. Interestingly, HSCOMT kinetics, with 1 h incubation at 30°C, showed a sigmoidal curve, as well as increased activity. Incubation of the reaction mixture in the presence of 60 pM AuNPs and/or AgNPs for 1 hreversed the observed sigmoidal to a hyperbolic curve, with kinetic parameters comparable to those of 5 min incubation. SDS-PAGE analyses of HSCOMT after the kinetic experiments showed the enzyme incubated for 5 min as a monomer, while that which was incubated for 1 h migrated substantially as dimer. However, the HSCOMT incubated for 1 h in the presence of 60 pM AuNPs and/or AgNPs migrated as a monomer. This indicated that the extension of the incubation period allowed the dimerization of HSCOMT, which exhibited sigmoidal kinetics and higher activity. The presence of NPs impeded the HSCOMT dimerization which decreased the activity. Varying the concentration of SAM suggested that SAM had an allosteric modulatory effect on HSCOMT. Absorption spectroscopy indicated adsorption of HSCOMT on the gold and silver NP surfaces and the formation of NPs-HSCOMT corona. Fluorescence spectroscopy showed that the interaction of HSCOMT with both gold and silver NPs was governed by a static quenching mechanism, implying the formation of a non-fluorescent fluorophore-NP complex at the ground state. Further fluorometric analyses indicated that both gold and silver NPs had contact with Trp143; that the interactions were spontaneous and were driven by electrostatic interactions. Fourier transform infrared spectroscopic studies showed the adsorption of HSCOMT of the NPs surfaces to cause relaxation of the enzyme’s B-sheet structures. Molecular docking studies indicated involvement of largely hydrophilic amino acids, with the interacting distances of less than 3.5A. These findings signify the potential of nanotechnology in the control of COMT catalytic activity for the management of the COMT-related disorders. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Date Issued: 2018
- Authors: Usman, Aminu
- Date: 2018
- Subjects: Parkinson's disease , Methyltransferases , Catechol , Nanoparticles
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/61818 , vital:28063 , DOI https://doi.org/10.21504/10962/61818
- Description: Catechol O-methyltransferase (S-adenosyl-Z-methionine: catechol O-methyltransferase; COMT; EC 2.1.1.6) is a ubiquitous enzyme that catalyses the transfer of a methyl group from the cofactor, S-adenosyl-Z-methionine (SAM) to a hydroxyl group of endogenous and exogenous catechol-containing moieties. The physiological role of this enzyme is the methylation and thereby inactivation of the catechol-containing bio-active and bio-toxic compounds, including catechol-neurotransmitters, catechol-estrogens and catechol-containing drugs. Activity of this enzyme is implicated in the treatment of Parkinson’s disease and is associated with other diseases including breast cancer and an array neuropsychological disorders, such as schizophrenia. This thesis explores the use of gold and silver nanoparticles (NPs) (AuNPs and AgNPs) to inhibit the catalytic activity of mammalian COMT. Because of its accessibility and availability, we initially investigated bovine soluble COMT (BSCOMT) from liver tissue. Bioinformatic analyses and structural modeling revealed high (>90%) sequence similarity between BSCOMT and human soluble COMT (HSCOMT). BSCOMT was partially purified to 7.78 fold, 1.65% yield and had a specific activity of 0.052 U/mg. It had pH and temperature optima of 8.5 and 40oC, respectively. The Km, Vmax, Kcat and Kcat/Km towards esculetin methylation were respectively 1.475±0.130 pM, 0.0353±0.001 pmol/ml/min, 1.748 x 10-2±5.0x10-4 min-1 and 1.18x10-2 M-1. min-1. HSCOMT was expressed in Escherichia coli BL21(DE3) which showed optimal activity for esculetin methylation at pH and temperature of 7.0 and 30°C, respectively. It was purified to 5.62 fold, 22.6% yield with a specific activity of 3.85 U/mg. HSCOMT kinetic plots, upon incubation of the reaction mixture at 30°C for 5 min before addition of SAM was hyperbolic with Km, Vmax, Kcat and Kcat/Km values of 1.79 pM, 0.412 pmol/ml/min, 2.08 min-1 and 1.165 M-1. min-1, respectively. AuNPs and AgNPs showed a concentration dependent inhibition of HSCOMT activity upon increasing the 5 min incubation time to 1 h. Interestingly, HSCOMT kinetics, with 1 h incubation at 30°C, showed a sigmoidal curve, as well as increased activity. Incubation of the reaction mixture in the presence of 60 pM AuNPs and/or AgNPs for 1 hreversed the observed sigmoidal to a hyperbolic curve, with kinetic parameters comparable to those of 5 min incubation. SDS-PAGE analyses of HSCOMT after the kinetic experiments showed the enzyme incubated for 5 min as a monomer, while that which was incubated for 1 h migrated substantially as dimer. However, the HSCOMT incubated for 1 h in the presence of 60 pM AuNPs and/or AgNPs migrated as a monomer. This indicated that the extension of the incubation period allowed the dimerization of HSCOMT, which exhibited sigmoidal kinetics and higher activity. The presence of NPs impeded the HSCOMT dimerization which decreased the activity. Varying the concentration of SAM suggested that SAM had an allosteric modulatory effect on HSCOMT. Absorption spectroscopy indicated adsorption of HSCOMT on the gold and silver NP surfaces and the formation of NPs-HSCOMT corona. Fluorescence spectroscopy showed that the interaction of HSCOMT with both gold and silver NPs was governed by a static quenching mechanism, implying the formation of a non-fluorescent fluorophore-NP complex at the ground state. Further fluorometric analyses indicated that both gold and silver NPs had contact with Trp143; that the interactions were spontaneous and were driven by electrostatic interactions. Fourier transform infrared spectroscopic studies showed the adsorption of HSCOMT of the NPs surfaces to cause relaxation of the enzyme’s B-sheet structures. Molecular docking studies indicated involvement of largely hydrophilic amino acids, with the interacting distances of less than 3.5A. These findings signify the potential of nanotechnology in the control of COMT catalytic activity for the management of the COMT-related disorders. , Thesis (PhD) -- Faculty of Science, Biochemistry and Microbiology, 2018
- Full Text:
- Date Issued: 2018
Assessing the potential role of microorganisms in the production of seedlings for the restoration of Albany Thicket
- Authors: Mpama, Nelisa
- Date: 2017
- Subjects: Vesicular-arbuscular mycorrhizas , Rhizobacteria , Restoration ecology South Africa Albany , Microorganisms
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/64555 , vital:28558
- Description: The role of microorganisms in restoration of the Albany Thicket has not been well documented, although the benefits to plants of these various interactions has been well documented. Microorganisms are chief ecological engineers and assist in resolving environmental problems and act to restore degraded ecosystem function by forming mutual relationships with the roots of the plants. The aim of this study was to assess the potential of microorganisms for the improved biomass production of selected woody and succulent seedlings used in mesic thicket restoration. Three tree species were selected for propagation in this study namely; Mystroxylon aethiopicum Scutia myrtina and Aloe ferox. Soil samples were collected from a degraded and intact thicket site from Bathurst, South Africa. Soils were evaluated for number of arbuscular mycorrhizal (AM) spores, mycorrhizal infectivity potential and nutrient availability both before and after seedling propagation. Pasteurized soil from the degraded site was used in a pot trial. Ten replicates seedling for plant species were planted and subjected to four treatments which included inoculation with AM fungi and the rhizobacterium, Enterobacter sp., alone and in combination; the fourth treatment was an un-inouclated control. Plant growth parameters were recorded at regular intervals where appropriate and seedlings were harvested after 24 weeks for biomass measurements and AM colonisation assessments. Although generally low (< 1 spore per gram) the density of AM fungal spores was significantly higher in soils from the intact site when compared with soils from the degraded site. The mycorrhizal potential of the soils was however not significantly different. Mystroxylon aethiopicum seedling shoot height, canopy diameter and shoot biomass showed a significant increase when inoculated with AM fungi while S. myrtina seedlings showed increased shoot height when inoculated with both AM fungi and Enterobacter sp. Aloe ferox seedlings did not respond to microbial inoculation. The concentration of soil P and Na increased in treatments with Enterobacter sp. alone and in combination with AM fungi. Mystroxylon aethiopicum and S. myrtina seedlings showed a dependency on microbial inoculants indicating the importance of inoculation in the nursery before planting out into the field. Overall AM fungal inoculants applied to seedlings can be used to compensate for nutrient deficiency in soils. Although the Enterobacter isolate used was known to have various plant growth promoting capabilities. It is recommended that other rhizobacterial isolates be investigated. , Thesis (MSc) -- Faculty of Science, Environmental Science, 2017
- Full Text:
- Date Issued: 2017
- Authors: Mpama, Nelisa
- Date: 2017
- Subjects: Vesicular-arbuscular mycorrhizas , Rhizobacteria , Restoration ecology South Africa Albany , Microorganisms
- Language: English
- Type: Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/64555 , vital:28558
- Description: The role of microorganisms in restoration of the Albany Thicket has not been well documented, although the benefits to plants of these various interactions has been well documented. Microorganisms are chief ecological engineers and assist in resolving environmental problems and act to restore degraded ecosystem function by forming mutual relationships with the roots of the plants. The aim of this study was to assess the potential of microorganisms for the improved biomass production of selected woody and succulent seedlings used in mesic thicket restoration. Three tree species were selected for propagation in this study namely; Mystroxylon aethiopicum Scutia myrtina and Aloe ferox. Soil samples were collected from a degraded and intact thicket site from Bathurst, South Africa. Soils were evaluated for number of arbuscular mycorrhizal (AM) spores, mycorrhizal infectivity potential and nutrient availability both before and after seedling propagation. Pasteurized soil from the degraded site was used in a pot trial. Ten replicates seedling for plant species were planted and subjected to four treatments which included inoculation with AM fungi and the rhizobacterium, Enterobacter sp., alone and in combination; the fourth treatment was an un-inouclated control. Plant growth parameters were recorded at regular intervals where appropriate and seedlings were harvested after 24 weeks for biomass measurements and AM colonisation assessments. Although generally low (< 1 spore per gram) the density of AM fungal spores was significantly higher in soils from the intact site when compared with soils from the degraded site. The mycorrhizal potential of the soils was however not significantly different. Mystroxylon aethiopicum seedling shoot height, canopy diameter and shoot biomass showed a significant increase when inoculated with AM fungi while S. myrtina seedlings showed increased shoot height when inoculated with both AM fungi and Enterobacter sp. Aloe ferox seedlings did not respond to microbial inoculation. The concentration of soil P and Na increased in treatments with Enterobacter sp. alone and in combination with AM fungi. Mystroxylon aethiopicum and S. myrtina seedlings showed a dependency on microbial inoculants indicating the importance of inoculation in the nursery before planting out into the field. Overall AM fungal inoculants applied to seedlings can be used to compensate for nutrient deficiency in soils. Although the Enterobacter isolate used was known to have various plant growth promoting capabilities. It is recommended that other rhizobacterial isolates be investigated. , Thesis (MSc) -- Faculty of Science, Environmental Science, 2017
- Full Text:
- Date Issued: 2017
Development and assessment of gastric-retentive sustained release metronidazole microcapsules
- Authors: Makan, Anjana
- Date: 2017
- Subjects: Metronidazole , Drug delivery systems , Helicobacter pylori , High performance liquid chromatography , Gas chromatography , Drugs , Drugs Controlled release
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/59240 , vital:27491
- Description: Helicobacter pylori is one of the most common pathogenic bacterial infections and is the leading cause of gastritis, gastroduodenal ulcer disease and gastric cancers. Studies have revealed the prevalence of Helicobacter pylori is high in many countries around the globe. Although Helicobacter pylori is highly sensitive to antimicrobial agents in vitro the clinical eradication rate of the disease is still low. The instability of API at gastric pH, low concentration of API in the gastric mucosa and short gastric residence times are the main reasons for poor eradication rates. The high prevalence rate of this disease necessitates the design and development of gastric-retentive site specific oral dosage forms for the optimized delivery of existing therapeutic molecules and may be an approach to improving the eradication rate of Helicobacter pylori. Metronidazole (MTZ) is a 5-nitroimidazole derivative that exhibits antibiotic and antiprotozoal activity. MTZ is used in combination with other compounds for the treatment of Helicobacter pylori in peptic ulcer disease. MTZ is a potential candidate for inclusion in a sustained release gastric-retentive delivery system that acts in the stomach and since it is unstable in the intestinal/colonic environment enhancing gastric residence time would be a therapeutic advantage. MTZ is a cost-effective therapy that exhibits good anti-microbial activity and has a favourable pharmacokinetic profile. A sustained release gastric-retentive formulation is therefore proposed as an approach to enhance the local delivery of MTZ and improve treatment outcomes for patients infected with Helicobacter pylori. A stability indicating Reversed-Phase High Performance Liquid Chromatography (RP- HPLC) method for the quantitation of MTZ in pharmaceutical dosage forms was developed and optimised using a Central Composite Design (CCD) approach. The RP-HPLC method was found to be linear, accurate, precise, sensitive, selective, and was applied to the analysis of MTZ in commercially available medicines. Preformulation studies were conducted as preparative work prior to manufacture gastric- retentive sustained release MTZ microcapsules. The experiments conducted were tailored for the development of sustained release MTZ microcapsules using a solvent evaporation method. The particle size and shape of the microcapsules was investigated using Scanning Electron Microscopy (SEM). MTZ- excipient compatibility studies were performed using Fourier Transform Infra-red Spectroscopy (FTIR), Differential Scanning Calorimetry (DSC) and X-Ray Diffraction (XRD). The results revealed that no definite interaction between MTZ and intended excipients to be used for manufacture of MTZ formulations occurred. A solvent evaporation procedure was used for the manufacture of MTZ microcapsules. Preliminary formulations were manufactured using two different grades of Methocel® at various levels. In addition the impact of processing parameters on performance was also investigated. The formulations were assessed in terms of in vitro release, buoyancy, yield, encapsulation efficiency and microcapsule size. Formulation optimisation was undertaken using a CCD approach and numerical optimisation was used to predict an optimised formulation composition that would produce minimal initial MTZ release, maximum MTZ release at 12 hours and maximum buoyancy, encapsulation efficiency and yield. The kinetics of MTZ release from microcapsules was established by fitting in vitro release data to different mathematical models. Higuchi model and first-order model appeared to best fit the data as majority of the formulation batches had highest R2 values for these models. Short-term stability assessment of the optimised formulation was established by undertaking stability studies at 25°C/60% RH and 40°C/75%RH. No significant changes in any of the CQA were observed over 30 days of stability testing. A gas chromatographic (GC) method was developed and validated for the quantitation of residual acetone and n-hexane. The optimised formulation contained 213.60 ppm/g acetone and 25.23 ppm/g n-hexane which are well below the limits set for residual solvents. In conclusion, gastric-retentive sustained release MTZ microcapsules with potential for further development and optimisation have been successfully developed and assessed in these studies. , Thesis (MSc) -- Faculty of Pharmacy, Pharmacy, 2017
- Full Text:
- Date Issued: 2017
- Authors: Makan, Anjana
- Date: 2017
- Subjects: Metronidazole , Drug delivery systems , Helicobacter pylori , High performance liquid chromatography , Gas chromatography , Drugs , Drugs Controlled release
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/59240 , vital:27491
- Description: Helicobacter pylori is one of the most common pathogenic bacterial infections and is the leading cause of gastritis, gastroduodenal ulcer disease and gastric cancers. Studies have revealed the prevalence of Helicobacter pylori is high in many countries around the globe. Although Helicobacter pylori is highly sensitive to antimicrobial agents in vitro the clinical eradication rate of the disease is still low. The instability of API at gastric pH, low concentration of API in the gastric mucosa and short gastric residence times are the main reasons for poor eradication rates. The high prevalence rate of this disease necessitates the design and development of gastric-retentive site specific oral dosage forms for the optimized delivery of existing therapeutic molecules and may be an approach to improving the eradication rate of Helicobacter pylori. Metronidazole (MTZ) is a 5-nitroimidazole derivative that exhibits antibiotic and antiprotozoal activity. MTZ is used in combination with other compounds for the treatment of Helicobacter pylori in peptic ulcer disease. MTZ is a potential candidate for inclusion in a sustained release gastric-retentive delivery system that acts in the stomach and since it is unstable in the intestinal/colonic environment enhancing gastric residence time would be a therapeutic advantage. MTZ is a cost-effective therapy that exhibits good anti-microbial activity and has a favourable pharmacokinetic profile. A sustained release gastric-retentive formulation is therefore proposed as an approach to enhance the local delivery of MTZ and improve treatment outcomes for patients infected with Helicobacter pylori. A stability indicating Reversed-Phase High Performance Liquid Chromatography (RP- HPLC) method for the quantitation of MTZ in pharmaceutical dosage forms was developed and optimised using a Central Composite Design (CCD) approach. The RP-HPLC method was found to be linear, accurate, precise, sensitive, selective, and was applied to the analysis of MTZ in commercially available medicines. Preformulation studies were conducted as preparative work prior to manufacture gastric- retentive sustained release MTZ microcapsules. The experiments conducted were tailored for the development of sustained release MTZ microcapsules using a solvent evaporation method. The particle size and shape of the microcapsules was investigated using Scanning Electron Microscopy (SEM). MTZ- excipient compatibility studies were performed using Fourier Transform Infra-red Spectroscopy (FTIR), Differential Scanning Calorimetry (DSC) and X-Ray Diffraction (XRD). The results revealed that no definite interaction between MTZ and intended excipients to be used for manufacture of MTZ formulations occurred. A solvent evaporation procedure was used for the manufacture of MTZ microcapsules. Preliminary formulations were manufactured using two different grades of Methocel® at various levels. In addition the impact of processing parameters on performance was also investigated. The formulations were assessed in terms of in vitro release, buoyancy, yield, encapsulation efficiency and microcapsule size. Formulation optimisation was undertaken using a CCD approach and numerical optimisation was used to predict an optimised formulation composition that would produce minimal initial MTZ release, maximum MTZ release at 12 hours and maximum buoyancy, encapsulation efficiency and yield. The kinetics of MTZ release from microcapsules was established by fitting in vitro release data to different mathematical models. Higuchi model and first-order model appeared to best fit the data as majority of the formulation batches had highest R2 values for these models. Short-term stability assessment of the optimised formulation was established by undertaking stability studies at 25°C/60% RH and 40°C/75%RH. No significant changes in any of the CQA were observed over 30 days of stability testing. A gas chromatographic (GC) method was developed and validated for the quantitation of residual acetone and n-hexane. The optimised formulation contained 213.60 ppm/g acetone and 25.23 ppm/g n-hexane which are well below the limits set for residual solvents. In conclusion, gastric-retentive sustained release MTZ microcapsules with potential for further development and optimisation have been successfully developed and assessed in these studies. , Thesis (MSc) -- Faculty of Pharmacy, Pharmacy, 2017
- Full Text:
- Date Issued: 2017
Formulation development, manufacture and evaluation of hydralazine hydrochloride microspheres
- Kangausaru, Shakemore Tinashe
- Authors: Kangausaru, Shakemore Tinashe
- Date: 2017
- Subjects: Hydralazine , Microspheres , Drugs Controlled release , Drugs Design , Drug development , Hypertension Chemotherapy
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/59220 , vital:27482
- Description: Despite improvements in its detection and treatment since the 1970s, hypertension is the most common and important risk factor for cardiovascular diseases. Hypertension is a chronic condition often underdiagnosed and/or inadequately treated in Sub-Saharan Africa. Recent survey results illustrate that the condition continues to contribute significantly to mortality and morbidity in adults and that it is poorly controlled in clinical practice. Hydralazine (HYD) is used either alone or in combination for the management of chronic hypertension, chronic cardiac failure and hypertensive crises. Due to its short plasma half-life of between 2 to 4 hours, HYD is normally administered two to four times daily, therefore making it a potential candidate for inclusion in sustained release formulations. The formulation of sustained release microsphere dosage forms may be useful to improve patient adherence and to achieve predictable and optimised therapeutic plasma concentrations. A stability indicating reversed-phase high performance liquid chromatography (RP-HPLC) method for the quantitation of HYD in pharmaceutical dosage forms was developed and optimised using a Central Composite Design (CCD) approach. UV/Vis detection method was selected as HYD contains an ultraviolet light-absorbing chromophore. The method was validated with respect to linearity and range, limits of quantitation (LOQ) and detection (LOD), accuracy, precision, sensitivity, selectivity and specificity as per International Conference on Harmonisation (ICH) guidelines. The method was applied to commercially available HYD tablets. No interfering peaks were observed from excipients used in the commercially available tablets. Preformulation studies were conducted to ensure the manufacture of high quality, stable sustained release HYD microspheres. The results revealed that there was an interaction between HYD and Carbopol® 971P, therefore Carbopol® polymers were not included during formulation studies. HYD was found to be compatible with Methocel® K100LV, Eudragit® RS PO and Avicel® 101 and HYD formulations were developed and optimised using these excipients. An oil-in-oil (o/o) solvent evaporation technique was selected for the manufacture of HYD microspheres due to its simplicity and to avoid exposure of HYD to moisture that could have been encountered if a water-in-oil (w/o) manufacturing process was used. The selection of o/o solvent evaporation technique was also based on the hydrophilicity of HYD and the polymers selected. Different grades of Methocel® and Eudragit® were selected to evaluate their effect on encapsulation efficiency (EE), in vitro release and microparticle shape and morphology. The best combination of these polymers which resulted in the desired EE, in vitro release, microparticle shape and size were then selected for formulation optimisation. A numerical optimisation approach was used to predict a formulation composition that would produce minimal HYD release initially and maximum HYD release after 12 hours of dissolution testing. The release kinetics of HYD from the manufactured microspheres were established by fitting in vitro release data to several mathematical models. The in vitro release data for the optimised formulations was best described using Higuchi model. The short-term stability of the optimised formulations was established by undertaking stability studies at 4°C, 25 °C/60 % RH and 40 °C/75 % RH. The results revealed that there was no significant change in appearance and physicochemical properties of the microspheres over a period of one month. However, long-term stability studies would be required to determine the shelf-life of the formulations. In addition, a gas chromatographic (GC) method was selected for determining residual amounts of acetone and n-hexane in the optimised formulations. GC methods were developed and optimised by evaluation of process parameters. System suitability testing was performed with respect to resolution, theoretical number of plates and selectivity. Method validation was performed with respect to linearity, range, inter- and intra-day precision, retention time (Rt) precision, limit of quantitation (LOQ) and detection (LOD). A solvent extraction method was used to analyse residual solvents in the optimised formulations. The drying process was sufficient in evaporating acetone and n-hexane from the optimised formulations. Solvent evaporation technique has been successfully used in the manufacture of HYD microspheres. The microspheres have potential for further development, scale up formulation studies and long-term stability studies. , Thesis (MSc) -- Faculty of Pharmacy, Pharmacy, 2017
- Full Text:
- Date Issued: 2017
- Authors: Kangausaru, Shakemore Tinashe
- Date: 2017
- Subjects: Hydralazine , Microspheres , Drugs Controlled release , Drugs Design , Drug development , Hypertension Chemotherapy
- Language: English
- Type: Master's theses , text
- Identifier: http://hdl.handle.net/10962/59220 , vital:27482
- Description: Despite improvements in its detection and treatment since the 1970s, hypertension is the most common and important risk factor for cardiovascular diseases. Hypertension is a chronic condition often underdiagnosed and/or inadequately treated in Sub-Saharan Africa. Recent survey results illustrate that the condition continues to contribute significantly to mortality and morbidity in adults and that it is poorly controlled in clinical practice. Hydralazine (HYD) is used either alone or in combination for the management of chronic hypertension, chronic cardiac failure and hypertensive crises. Due to its short plasma half-life of between 2 to 4 hours, HYD is normally administered two to four times daily, therefore making it a potential candidate for inclusion in sustained release formulations. The formulation of sustained release microsphere dosage forms may be useful to improve patient adherence and to achieve predictable and optimised therapeutic plasma concentrations. A stability indicating reversed-phase high performance liquid chromatography (RP-HPLC) method for the quantitation of HYD in pharmaceutical dosage forms was developed and optimised using a Central Composite Design (CCD) approach. UV/Vis detection method was selected as HYD contains an ultraviolet light-absorbing chromophore. The method was validated with respect to linearity and range, limits of quantitation (LOQ) and detection (LOD), accuracy, precision, sensitivity, selectivity and specificity as per International Conference on Harmonisation (ICH) guidelines. The method was applied to commercially available HYD tablets. No interfering peaks were observed from excipients used in the commercially available tablets. Preformulation studies were conducted to ensure the manufacture of high quality, stable sustained release HYD microspheres. The results revealed that there was an interaction between HYD and Carbopol® 971P, therefore Carbopol® polymers were not included during formulation studies. HYD was found to be compatible with Methocel® K100LV, Eudragit® RS PO and Avicel® 101 and HYD formulations were developed and optimised using these excipients. An oil-in-oil (o/o) solvent evaporation technique was selected for the manufacture of HYD microspheres due to its simplicity and to avoid exposure of HYD to moisture that could have been encountered if a water-in-oil (w/o) manufacturing process was used. The selection of o/o solvent evaporation technique was also based on the hydrophilicity of HYD and the polymers selected. Different grades of Methocel® and Eudragit® were selected to evaluate their effect on encapsulation efficiency (EE), in vitro release and microparticle shape and morphology. The best combination of these polymers which resulted in the desired EE, in vitro release, microparticle shape and size were then selected for formulation optimisation. A numerical optimisation approach was used to predict a formulation composition that would produce minimal HYD release initially and maximum HYD release after 12 hours of dissolution testing. The release kinetics of HYD from the manufactured microspheres were established by fitting in vitro release data to several mathematical models. The in vitro release data for the optimised formulations was best described using Higuchi model. The short-term stability of the optimised formulations was established by undertaking stability studies at 4°C, 25 °C/60 % RH and 40 °C/75 % RH. The results revealed that there was no significant change in appearance and physicochemical properties of the microspheres over a period of one month. However, long-term stability studies would be required to determine the shelf-life of the formulations. In addition, a gas chromatographic (GC) method was selected for determining residual amounts of acetone and n-hexane in the optimised formulations. GC methods were developed and optimised by evaluation of process parameters. System suitability testing was performed with respect to resolution, theoretical number of plates and selectivity. Method validation was performed with respect to linearity, range, inter- and intra-day precision, retention time (Rt) precision, limit of quantitation (LOQ) and detection (LOD). A solvent extraction method was used to analyse residual solvents in the optimised formulations. The drying process was sufficient in evaporating acetone and n-hexane from the optimised formulations. Solvent evaporation technique has been successfully used in the manufacture of HYD microspheres. The microspheres have potential for further development, scale up formulation studies and long-term stability studies. , Thesis (MSc) -- Faculty of Pharmacy, Pharmacy, 2017
- Full Text:
- Date Issued: 2017
Prevalence of metabolic syndrome in adolescents living in Mthatha, south africa
- Sekokotla, Annah, Goswami, Nandu, Sewani-Rusike, Constance Rufaro, Iputo, Jehu Erapu, Nkeh-Chungag, Benedicta Ngwenchi
- Authors: Sekokotla, Annah , Goswami, Nandu , Sewani-Rusike, Constance Rufaro , Iputo, Jehu Erapu , Nkeh-Chungag, Benedicta Ngwenchi
- Date: 2017
- Subjects: South Africa Metabolic syndrome Journal article
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/11260/5983 , vital:45061 , xlink:href="https://doi:10.2147/TCRM.S124291"
- Description: Objective Metabolic syndrome (MetS), defined as the clustering of three of five risk factors (hypertension, obesity, triglyceridemia, dyslipidemia and hyperglycemia), is being increasingly mentioned among children and adolescents despite there being no consensus on how it should be defined in this set of population. Furthermore, very few studies have focused on MetS in children and adolescents in sub-Saharan populations. This study, therefore, aimed at determining the prevalence of the MetS and contributing risk in adolescents living in Mthatha, South Africa. Research design and methods Four hundred and ten adolescents (age range: 13–18 years, both sexes) were recruited into this cross-sectional study. In addition to a lifestyle questionnaire being administered, anthropometric measurements (weight, height and waist circumferences) were carried out for each participant. Fasting venous blood was collected for lipid profile and blood glucose assessments. Blood pressure was also measured. MetS was assessed using appropriate diagnostic criteria for children and adolescents. Results Complete data sets were collected from 371 participants. Females showed an elevation in the risk factors for MetS. Of the 371 participants, 40.2% were overweight/obese (47.5% females; 22.5% males). MetS was diagnosed in 3.1% female participants compared with 6.0% in male participants. Conclusions More risk factors for the MetS are seen among the overweight/obese as compared to lean adolescents. Given the fact that childhood overweight/obesity often continues into adulthood, it is important to address the causes of increased risk for MetS earlier in life to prevent the development of disease in adult life.
- Full Text:
- Date Issued: 2017
- Authors: Sekokotla, Annah , Goswami, Nandu , Sewani-Rusike, Constance Rufaro , Iputo, Jehu Erapu , Nkeh-Chungag, Benedicta Ngwenchi
- Date: 2017
- Subjects: South Africa Metabolic syndrome Journal article
- Language: English
- Type: text , article
- Identifier: http://hdl.handle.net/11260/5983 , vital:45061 , xlink:href="https://doi:10.2147/TCRM.S124291"
- Description: Objective Metabolic syndrome (MetS), defined as the clustering of three of five risk factors (hypertension, obesity, triglyceridemia, dyslipidemia and hyperglycemia), is being increasingly mentioned among children and adolescents despite there being no consensus on how it should be defined in this set of population. Furthermore, very few studies have focused on MetS in children and adolescents in sub-Saharan populations. This study, therefore, aimed at determining the prevalence of the MetS and contributing risk in adolescents living in Mthatha, South Africa. Research design and methods Four hundred and ten adolescents (age range: 13–18 years, both sexes) were recruited into this cross-sectional study. In addition to a lifestyle questionnaire being administered, anthropometric measurements (weight, height and waist circumferences) were carried out for each participant. Fasting venous blood was collected for lipid profile and blood glucose assessments. Blood pressure was also measured. MetS was assessed using appropriate diagnostic criteria for children and adolescents. Results Complete data sets were collected from 371 participants. Females showed an elevation in the risk factors for MetS. Of the 371 participants, 40.2% were overweight/obese (47.5% females; 22.5% males). MetS was diagnosed in 3.1% female participants compared with 6.0% in male participants. Conclusions More risk factors for the MetS are seen among the overweight/obese as compared to lean adolescents. Given the fact that childhood overweight/obesity often continues into adulthood, it is important to address the causes of increased risk for MetS earlier in life to prevent the development of disease in adult life.
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- Date Issued: 2017
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