Synthesis and structure-activity relationship studies of 1,4-naphthoquinone derivatives as potential anti-trypanosomal agents
- Authors: Chakaingesu, Chikomborero
- Date: 2014
- Subjects: African trypanosomiasis , Trypanosoma brucei , Naphthoquinone , Protozoan diseases , Drugs -- Structure-activity relationships , Millennium Development Goals
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3873 , http://hdl.handle.net/10962/d1020959
- Description: Human African Trypanosomiasis (HAT) is an infectious, vector-borne protozoal disease which is amongst the so-called neglected diseases. In 2000, at a summit of the United Nations, eight Millennium Development Goals (MDGs) were set, to be achieved by 2015. MDG 6 states “to combat HIV/AIDS, malaria & other diseases”. With just under 2 years to go before the end of 2015, HAT is still thriving in developing countries. The drugs currently used for the treatment of HAT are in short supply, have severe side effects and those used to treat late stages of the disease are very difficult to administer. The aforementioned challenges call for research into this neglected disease in order to develop new, safe and easy-to-use medicines. Naphthoquinones are a class of compounds shown to possess anti-parasitic activity, amongst a variety of other biological activities, and therefore this pharmacophore was selected for this study. The purpose of this study was to synthesise derivatives of 2,3-dichloro-1,4- naphthoquinone to be tested for anti-trypanosomal activity and thereafter conduct structureactivity relationship studies. A series of reactions were carried out using thiophenol, phenol and aniline nucleophiles to synthesise thioether (-S-), ether (-O-) and amino (-NH-) derivatives of 2,3-dichloro-1,4-naphthoquinone with various halogen or methyl substituents. Purification of the products was carried out by recrystallisation. Nuclear magnetic resonance (NMR), infra-red (IR) and high pressure liquid chromatography coupled to an electro-spray ionisation mass spectrometer (HPLC-ESI-MS) were the analytical methods used for structural confirmation of the products. There were eighteen 1,4-naphthoquinone derivatives that were successfully synthesised using ethanolic solutions. Unfortunately, attempts to synthesise 1,4-naphthoquinones in reactions involving 2-(trifluoro-methyl)aniline and 2-isopropyl-5-methylphenol were unsuccessful, presumably due to steric hindrance by the bulky ortho-substituents. Although the aims of the synthetic procedures were to obtain both mono- and disubstituted products by nucleophilic displacement of the chlorine atom(s) of 2,3-dichloro-1,4- naphthoquinone, only monosubstituted products were obtained from substitution with aniline and phenol nucleophiles. Thiol nucleophiles, however, selectively yielded disubstituted products only. Synthesised naphthoquinone derivatives were tested against Trypanosoma brucei and calculation of the EC₅₀ values from the obtained dose-response curves was carried out using the four parametric equation. All the 1,4-naphthoquinones showed a degree of potency, except compounds 1b, 3c and 3e, which had little or lack of potency. Structure-activity relationship studies (SARs and QSARs) were carried out to determine which structural features or functional group substituents of the naphthoquinone derivatives contribute or take away from the desired anti-trypanosomal activity. It was found that compounds with the best in vitro anti-trypanosomal potencies in the series of analogous 1,4-naphthoquinone derivatives had EC₅₀ values in the range 2.137 to 2.884 μM. The most potent compound in the series was 2-chloro-3-(4-(trifluoromethyl)phenylamino)-1,4- naphthoquinone 1e; but it was 142-fold less potent than the reference standard of melarsoprol.
- Full Text:
- Date Issued: 2014
- Authors: Chakaingesu, Chikomborero
- Date: 2014
- Subjects: African trypanosomiasis , Trypanosoma brucei , Naphthoquinone , Protozoan diseases , Drugs -- Structure-activity relationships , Millennium Development Goals
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3873 , http://hdl.handle.net/10962/d1020959
- Description: Human African Trypanosomiasis (HAT) is an infectious, vector-borne protozoal disease which is amongst the so-called neglected diseases. In 2000, at a summit of the United Nations, eight Millennium Development Goals (MDGs) were set, to be achieved by 2015. MDG 6 states “to combat HIV/AIDS, malaria & other diseases”. With just under 2 years to go before the end of 2015, HAT is still thriving in developing countries. The drugs currently used for the treatment of HAT are in short supply, have severe side effects and those used to treat late stages of the disease are very difficult to administer. The aforementioned challenges call for research into this neglected disease in order to develop new, safe and easy-to-use medicines. Naphthoquinones are a class of compounds shown to possess anti-parasitic activity, amongst a variety of other biological activities, and therefore this pharmacophore was selected for this study. The purpose of this study was to synthesise derivatives of 2,3-dichloro-1,4- naphthoquinone to be tested for anti-trypanosomal activity and thereafter conduct structureactivity relationship studies. A series of reactions were carried out using thiophenol, phenol and aniline nucleophiles to synthesise thioether (-S-), ether (-O-) and amino (-NH-) derivatives of 2,3-dichloro-1,4-naphthoquinone with various halogen or methyl substituents. Purification of the products was carried out by recrystallisation. Nuclear magnetic resonance (NMR), infra-red (IR) and high pressure liquid chromatography coupled to an electro-spray ionisation mass spectrometer (HPLC-ESI-MS) were the analytical methods used for structural confirmation of the products. There were eighteen 1,4-naphthoquinone derivatives that were successfully synthesised using ethanolic solutions. Unfortunately, attempts to synthesise 1,4-naphthoquinones in reactions involving 2-(trifluoro-methyl)aniline and 2-isopropyl-5-methylphenol were unsuccessful, presumably due to steric hindrance by the bulky ortho-substituents. Although the aims of the synthetic procedures were to obtain both mono- and disubstituted products by nucleophilic displacement of the chlorine atom(s) of 2,3-dichloro-1,4- naphthoquinone, only monosubstituted products were obtained from substitution with aniline and phenol nucleophiles. Thiol nucleophiles, however, selectively yielded disubstituted products only. Synthesised naphthoquinone derivatives were tested against Trypanosoma brucei and calculation of the EC₅₀ values from the obtained dose-response curves was carried out using the four parametric equation. All the 1,4-naphthoquinones showed a degree of potency, except compounds 1b, 3c and 3e, which had little or lack of potency. Structure-activity relationship studies (SARs and QSARs) were carried out to determine which structural features or functional group substituents of the naphthoquinone derivatives contribute or take away from the desired anti-trypanosomal activity. It was found that compounds with the best in vitro anti-trypanosomal potencies in the series of analogous 1,4-naphthoquinone derivatives had EC₅₀ values in the range 2.137 to 2.884 μM. The most potent compound in the series was 2-chloro-3-(4-(trifluoromethyl)phenylamino)-1,4- naphthoquinone 1e; but it was 142-fold less potent than the reference standard of melarsoprol.
- Full Text:
- Date Issued: 2014
Aspects of the bioavailability of topical corticosteroid formulations
- Authors: Magnus, Ashley Denis
- Date: 2013-02-12
- Subjects: Adrenocortical hormones -- Therapeutic use , Dermatologic agents , Transdermal medication -- Evaluation , Dermatopharmacology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3843 , http://hdl.handle.net/10962/d1009516 , Adrenocortical hormones -- Therapeutic use , Dermatologic agents , Transdermal medication -- Evaluation , Dermatopharmacology
- Description: Two possible variables of the McKenzie/Stoughton blanching assay, namely amount applied to the test site and occlusion time have been investigated. Subsequently, two topical steroid preparations, Synalar cream (0,025% fluocinolone acetonide) and Betnovate cream (0,1% betamethasone 17-valer ate) were extemporaneously diluted with five and six placebo bases respectively. Taking cognizance of the two possible variables, these diluted preparations were assessed in vivo using a modified version of the McKenzie/Stoughton blanching assay for blanching activity over a 14 month period. It was found that the base E45, which is slightly alkali, had the greatest effect on both preparations. In the case of betamethasone 17-valerate this base c aused the conversion to the less active isomer, betamethasone 21-valerate whereas at the end of the 14 month test period it was found that the Synalar/E45 dilution contained no fluocinolone acetonide. Quantitative analysis of all the diluted preparations by high performance liquid chromatography using a reverse-phase system was performed. The data obtained f r om the systematic stUdies of the effects of varying concentrations and occlusion times were presented at the Eleventh National Congress of the South African Pharmacological Society. , KMBT_363 , Adobe Acrobat 9.53 Paper Capture Plug-in
- Full Text:
- Authors: Magnus, Ashley Denis
- Date: 2013-02-12
- Subjects: Adrenocortical hormones -- Therapeutic use , Dermatologic agents , Transdermal medication -- Evaluation , Dermatopharmacology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3843 , http://hdl.handle.net/10962/d1009516 , Adrenocortical hormones -- Therapeutic use , Dermatologic agents , Transdermal medication -- Evaluation , Dermatopharmacology
- Description: Two possible variables of the McKenzie/Stoughton blanching assay, namely amount applied to the test site and occlusion time have been investigated. Subsequently, two topical steroid preparations, Synalar cream (0,025% fluocinolone acetonide) and Betnovate cream (0,1% betamethasone 17-valer ate) were extemporaneously diluted with five and six placebo bases respectively. Taking cognizance of the two possible variables, these diluted preparations were assessed in vivo using a modified version of the McKenzie/Stoughton blanching assay for blanching activity over a 14 month period. It was found that the base E45, which is slightly alkali, had the greatest effect on both preparations. In the case of betamethasone 17-valerate this base c aused the conversion to the less active isomer, betamethasone 21-valerate whereas at the end of the 14 month test period it was found that the Synalar/E45 dilution contained no fluocinolone acetonide. Quantitative analysis of all the diluted preparations by high performance liquid chromatography using a reverse-phase system was performed. The data obtained f r om the systematic stUdies of the effects of varying concentrations and occlusion times were presented at the Eleventh National Congress of the South African Pharmacological Society. , KMBT_363 , Adobe Acrobat 9.53 Paper Capture Plug-in
- Full Text:
The isolation and characterisation of secondary metabolites from selected South African marine red algae (Rhodophyta)
- Authors: Fakee, Jameel
- Date: 2013
- Subjects: Metabolites Marine algae -- South Africa Marine algae -- Therapeutic use Metabolites -- Therapeutic use Marine metabolites Plocamocera Red algae Laurencia Delisea flaccida
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3733 , http://hdl.handle.net/10962/d1001472
- Description: Secondary metabolites from natural sources are fast growing as popular drug leads. The structural novelty and favourable biological activity that these compounds display contribute to their popularity as drugs of the future. Examples of such compounds include the potent anticancer drug paclitaxel isolated from the bark of a yew tree as well as the more commonly known analgesic aspirin which stems from the bark of the willow tree. The biological activities exhibited by these secondary metabolites are vast and range from antimicrobial to anticancer activity to mention but a few. As a result, the isolation of novel compounds from natural sources is on the rise. The South African seaboard is home to a wealth of various marine algal species which produce fascinating secondary metabolites. For example, Portierria hornemanii was shown to produce halomon, a halogenated monoterpene which has displayed promising cytotoxic activity. This study thus focused primarily on pursuing novel compounds from three endemic South African marine algal species which have never been analysed previously from a chemical perspective. These are Plocamium rigidum (Bory de Saint-Vincent), Laurencia natalensis (Kylin) and Delisea flaccida (Suhr) Papenfuss. Four known compounds and one new halogenated monoterpene, (2E,5E,7Z)-8-chloro- 7-(dichloromethyl)-4-hydroxy-3-methylocta-2,5,7-trienal, were isolated from Plocamium rigidum. The breast cancer (MCF-7 cell line) inhibitory activity for these compounds was assessed and it was observed that an increase in the lipophilic nature of the compounds produced more favourable IC50 values. A pre-cursor to bromofucin type compounds, cis-laurencenyne, was isolated from Laurencia natalensis, as well as a new acetoxy chamigrane type compound, 4-bromo- 3,10-dichloro-7-hydroxy-3,7,11,11-tetramethylspiro [6.6] undec-1-yl acetate. Delisea flaccida was seen to contain two known bromofuranone type compounds isolated as an isomeric mixture, 1-[(5Z)-4-bromo-5-(bromomethylidene)-2-oxo-2,5- dihydrofuran-3-yl] butyl acetate and 1-[(5E)-4-bromo-5-(bromomethylidene)-2- oxo-2,5-dihydrofuran-3-yl]butyl acetate. These compounds are famous for their ability to inhibit bacterial biofilm production and they have been isolated before from an Australian Delisea spp , Adobe Acrobat 9.53 Paper Capture Plug-in
- Full Text:
- Date Issued: 2013
- Authors: Fakee, Jameel
- Date: 2013
- Subjects: Metabolites Marine algae -- South Africa Marine algae -- Therapeutic use Metabolites -- Therapeutic use Marine metabolites Plocamocera Red algae Laurencia Delisea flaccida
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3733 , http://hdl.handle.net/10962/d1001472
- Description: Secondary metabolites from natural sources are fast growing as popular drug leads. The structural novelty and favourable biological activity that these compounds display contribute to their popularity as drugs of the future. Examples of such compounds include the potent anticancer drug paclitaxel isolated from the bark of a yew tree as well as the more commonly known analgesic aspirin which stems from the bark of the willow tree. The biological activities exhibited by these secondary metabolites are vast and range from antimicrobial to anticancer activity to mention but a few. As a result, the isolation of novel compounds from natural sources is on the rise. The South African seaboard is home to a wealth of various marine algal species which produce fascinating secondary metabolites. For example, Portierria hornemanii was shown to produce halomon, a halogenated monoterpene which has displayed promising cytotoxic activity. This study thus focused primarily on pursuing novel compounds from three endemic South African marine algal species which have never been analysed previously from a chemical perspective. These are Plocamium rigidum (Bory de Saint-Vincent), Laurencia natalensis (Kylin) and Delisea flaccida (Suhr) Papenfuss. Four known compounds and one new halogenated monoterpene, (2E,5E,7Z)-8-chloro- 7-(dichloromethyl)-4-hydroxy-3-methylocta-2,5,7-trienal, were isolated from Plocamium rigidum. The breast cancer (MCF-7 cell line) inhibitory activity for these compounds was assessed and it was observed that an increase in the lipophilic nature of the compounds produced more favourable IC50 values. A pre-cursor to bromofucin type compounds, cis-laurencenyne, was isolated from Laurencia natalensis, as well as a new acetoxy chamigrane type compound, 4-bromo- 3,10-dichloro-7-hydroxy-3,7,11,11-tetramethylspiro [6.6] undec-1-yl acetate. Delisea flaccida was seen to contain two known bromofuranone type compounds isolated as an isomeric mixture, 1-[(5Z)-4-bromo-5-(bromomethylidene)-2-oxo-2,5- dihydrofuran-3-yl] butyl acetate and 1-[(5E)-4-bromo-5-(bromomethylidene)-2- oxo-2,5-dihydrofuran-3-yl]butyl acetate. These compounds are famous for their ability to inhibit bacterial biofilm production and they have been isolated before from an Australian Delisea spp , Adobe Acrobat 9.53 Paper Capture Plug-in
- Full Text:
- Date Issued: 2013
Design and evaluation of illustrated information leaflets as an educational tool for low-literate asthma patients
- Authors: Wrench, Wendy Merle
- Date: 2012 , 2012-10-08
- Subjects: Asthma -- South Africa -- Study and teaching , Asthmatics -- South Africa -- Education
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3867 , http://hdl.handle.net/10962/d1016236
- Description: Asthma is a chronic non-communicable disease associated with an increase in morbidity, mortality and economic burden. Globally 300 million people have asthma and it is estimated that one in every 250 deaths worldwide are due to asthma. South Africa has the highest asthma prevalence (8.1%) in Africa and the disease is 18th in the top 20 causes of death. Inadequate home management, poor availability of health care, and poor transport and emergency services are recognised as important contributing factors. Patients with a low level of education and limited literacy skills may be unable to understand instructions on frequency and use of asthma medicines, which could result in unintentional non-adherence leading to serious complications and increased health care costs. The aim of this study was to investigate the impact of a tailored educational intervention on low-literate patients with asthma. Objectives to achieve this aim included designing patient information leaflets (PILs) containing information on asthma, management of asthma and asthma therapy, and using the PILs to educate low-literate asthma patients. A before-andafter intervention type design evaluated self-reported selected health-related quality of life measures, self-reported self-efficacy, knowledge of asthma and asthma management, knowledge of the use of metered dose inhalers (MDIs) and MDI technique. The acceptability and understanding of the tailored PILs was also investigated. Two simple, readable PILs containing pictograms were developed in English and then translated into isiXhosa, the home language of the majority of the target population. Various guidelines on the design of health-related information for people with low-literacy were consulted and input on the design was received from health care providers, patients and graphic artists. A pilot study was conducted at a local primary health care (PHC) clinic to evaluate the PILs and final modifications to the PILs were made based on feedback received. For the main study, patients were recruited from the KwaNonqubela PHC clinic in Alexandria in the Eastern Cape, South Africa. Patients were 18 years or older, dependent on public sector health care facilities, diagnosed with asthma, prescribed a MDI (beclomethasone and/or salbutamol) for at least one month and English or isiXhosa-speaking. The exclusion criterion for patients in this study was involvement in any other asthma educational intervention during the period of study. Interviewer-led structured questionnaires were administered to 55 patients at the baseline and follow-up. Data collected include demographics, brief medical history and current asthma medications. Self-efficacy and iii health-related quality of life were assessed. Knowledge of asthma and asthma management was evaluated, and the use of beclomethasone and/or salbutamol metered dose inhalers was assessed. The PIL ‘Understanding asthma and trigger factors of asthma’ formed part of the educational intervention to explain asthma and aspects related to its management. Inhaler technique was evaluated and corrected using the PIL ‘How to use your pump’ together with a demonstration of correct technique by the investigator. Follow-up interviews were conducted approximately four weeks after baseline. PIL acceptability, readability and understanding of each pictogram were investigated at follow-up only. The educational intervention resulted in a significant increase in mean knowledge of asthma from 52.7% at baseline to 75.5% at follow-up. Gender was not associated with knowledge, but there was a significant age effect at baseline only, with the younger patients achieving better knowledge results. In both phases, patients with higher education had improved scores. A significant increase (2.4% to 38.6%) in the number of patients taking the minimum recommended adult dose of beclomethasone was noted but it is a matter of concern that the majority of patients were taking less than this. Patient self-reports suggested a significant increase in adherence, with the number of patients taking beclomethasone daily increasing from 33.3% to 61.3%. Self-reported management and control of asthma improved and this was reflected by the enhanced HRQOL results. MDI technique also improved significantly with an increase in the mean number of correct steps from 4.6 ± 2.2 to 7.9 ± 2.7. Education had a significant effect on MDI technique with more errors associated with lower educational status. There were no significant age or gender effects on the total number of correct steps in either phase. The illustrated PILs were received favourably with the majority of literate patients reporting that they were easy to read. Patients commented positively on the inclusion of pictograms and stated that the pictograms had served as aids in the understanding of asthma, trigger factors of asthma and correct MDI technique. The results of this study show that specially designed illustrated PILs can be an effective tool in educating low-literate patients with asthma. , Adobe Acrobat Pro 11.0.0 Paper Capture Plug-in
- Full Text:
- Date Issued: 2012
- Authors: Wrench, Wendy Merle
- Date: 2012 , 2012-10-08
- Subjects: Asthma -- South Africa -- Study and teaching , Asthmatics -- South Africa -- Education
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3867 , http://hdl.handle.net/10962/d1016236
- Description: Asthma is a chronic non-communicable disease associated with an increase in morbidity, mortality and economic burden. Globally 300 million people have asthma and it is estimated that one in every 250 deaths worldwide are due to asthma. South Africa has the highest asthma prevalence (8.1%) in Africa and the disease is 18th in the top 20 causes of death. Inadequate home management, poor availability of health care, and poor transport and emergency services are recognised as important contributing factors. Patients with a low level of education and limited literacy skills may be unable to understand instructions on frequency and use of asthma medicines, which could result in unintentional non-adherence leading to serious complications and increased health care costs. The aim of this study was to investigate the impact of a tailored educational intervention on low-literate patients with asthma. Objectives to achieve this aim included designing patient information leaflets (PILs) containing information on asthma, management of asthma and asthma therapy, and using the PILs to educate low-literate asthma patients. A before-andafter intervention type design evaluated self-reported selected health-related quality of life measures, self-reported self-efficacy, knowledge of asthma and asthma management, knowledge of the use of metered dose inhalers (MDIs) and MDI technique. The acceptability and understanding of the tailored PILs was also investigated. Two simple, readable PILs containing pictograms were developed in English and then translated into isiXhosa, the home language of the majority of the target population. Various guidelines on the design of health-related information for people with low-literacy were consulted and input on the design was received from health care providers, patients and graphic artists. A pilot study was conducted at a local primary health care (PHC) clinic to evaluate the PILs and final modifications to the PILs were made based on feedback received. For the main study, patients were recruited from the KwaNonqubela PHC clinic in Alexandria in the Eastern Cape, South Africa. Patients were 18 years or older, dependent on public sector health care facilities, diagnosed with asthma, prescribed a MDI (beclomethasone and/or salbutamol) for at least one month and English or isiXhosa-speaking. The exclusion criterion for patients in this study was involvement in any other asthma educational intervention during the period of study. Interviewer-led structured questionnaires were administered to 55 patients at the baseline and follow-up. Data collected include demographics, brief medical history and current asthma medications. Self-efficacy and iii health-related quality of life were assessed. Knowledge of asthma and asthma management was evaluated, and the use of beclomethasone and/or salbutamol metered dose inhalers was assessed. The PIL ‘Understanding asthma and trigger factors of asthma’ formed part of the educational intervention to explain asthma and aspects related to its management. Inhaler technique was evaluated and corrected using the PIL ‘How to use your pump’ together with a demonstration of correct technique by the investigator. Follow-up interviews were conducted approximately four weeks after baseline. PIL acceptability, readability and understanding of each pictogram were investigated at follow-up only. The educational intervention resulted in a significant increase in mean knowledge of asthma from 52.7% at baseline to 75.5% at follow-up. Gender was not associated with knowledge, but there was a significant age effect at baseline only, with the younger patients achieving better knowledge results. In both phases, patients with higher education had improved scores. A significant increase (2.4% to 38.6%) in the number of patients taking the minimum recommended adult dose of beclomethasone was noted but it is a matter of concern that the majority of patients were taking less than this. Patient self-reports suggested a significant increase in adherence, with the number of patients taking beclomethasone daily increasing from 33.3% to 61.3%. Self-reported management and control of asthma improved and this was reflected by the enhanced HRQOL results. MDI technique also improved significantly with an increase in the mean number of correct steps from 4.6 ± 2.2 to 7.9 ± 2.7. Education had a significant effect on MDI technique with more errors associated with lower educational status. There were no significant age or gender effects on the total number of correct steps in either phase. The illustrated PILs were received favourably with the majority of literate patients reporting that they were easy to read. Patients commented positively on the inclusion of pictograms and stated that the pictograms had served as aids in the understanding of asthma, trigger factors of asthma and correct MDI technique. The results of this study show that specially designed illustrated PILs can be an effective tool in educating low-literate patients with asthma. , Adobe Acrobat Pro 11.0.0 Paper Capture Plug-in
- Full Text:
- Date Issued: 2012
Development, assessment and optimisation of oral famciclovir formulations for paediatric use
- Authors: Magnus, Laura
- Date: 2012
- Subjects: Drugs -- Dosage forms , Drugs -- Analysis , Capsules (Pharmacy) , Antiviral agents , Pediatrics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3870 , http://hdl.handle.net/10962/d1018244
- Description: Many Active Pharmaceutical Ingredients (API) such as the antiviral agent famciclovir (FCV) are required for paediatric treatment but are not commercially available in age-appropriate dosage forms. It is common practice to prepare oral liquid dosage forms using commercially available tablets, capsules or powdered API and then dispersing or dissolving the crushed and/or powdered materials in a vehicle that the patient can swallow. Vehicles that are commonly used for this purpose include methylcellulose, syrup or combinations of these carriers where possible or commercially available suspending agents such as Ora-Sweet®, if available, can be used. However, several critical factors are overlooked when manufacturing extemporaneous formulations including, but not limited to, physical and chemical properties of the API, excipients, compatibility, stability and bioavailability issues. A stability-indicating High Performance Liquid Chromatography (HPLC) method for the analysis of FCV was developed and validated according to the International Conference on Harmonization (ICH) guidelines. The method is sensitive, selective, precise, accurate and linear over the concentration range 2-120 μg/ml. The stability of 25 mg/ml FCV formulations was assessed in vehicles manufactured from syrup simplex, hydroxypropyl methylcellulose (HPMC), Ora-Sweet® and an aqueous buffer (pH 6) following storage at 25 °C/60% RH and 40 °C/75% RH over six (6) to eight (8) weeks. The shelf life of the products was calculated as the longest period of storage for approximately 90% of the added FCV to be recovered. Formulations were manufactured using syrup simplex or HPMC with methylparaben and propylparaben individually or in combination and with sodium metabisulphite, ascorbic acid or citric acid as antioxidants. The resultant products were subject to quality control analysis for API content, viscosity, pH and appearance and the resultant data were subject to statistical analysis. The degradation rates were calculated for each product and a degradation profile plotted. The degradation rates of FCV in extemporaneous formulations were compared to those of FCV manufactured using a commercially available suspending agent and a buffered vehicle. FCV undergoes major degradation in the presence of sucrose, as observed for formulations in which the vehicle was syrup and Ora-Sweet®. FCV was found to be most stable when dissolved/dispersed in an HPMC vehicle incorporating sodium metabisulphite and a combination of parabens. The formulation that exhibited the maximum stability was manufactured using an aqueous solution buffered to pH 6. Due to the enhanced stability of FCV when added to a buffered vehicle a formulation in which an HPMC vehicle buffered to pH 6 with sodium metabisulphite, methylparaben and propylparaben was selected for optimisation using a Central Composite Design approach (CCD). In this way it was possible to establish a relationship between input variables such as pH, % w/v HPMC, % w/v antioxidant and % w/v preservative and the responses selected for monitoring by means of response surface modelling. A quadratic model was found to be the most appropriate to describe the relationship between input and output variables. Thirty batches of product were randomly manufactured according to the CCD and analysed to establish the stability in respect of viscosity, pH and the amount of FCV remaining following storage and the data were fitted to models using Design-Expert® software. A correlation between input variables and the responses was best described by a quadratic polynomial model. Analysis of Variance indicated that the response surface models were significant (P-value < 0.0001). The pH to which a FCV formulation was buffered was the most significant factor to effect the % drug content and the ultimate pH of the formulation, while the % w/v HPMC had the most significant effect on the viscosity of the product. The optimum composition for the manufacture of an oral liquid FCV formulation was predicted using the optimisation function of the Design-Expert® software. A low % error of prediction was established, indicating that the model is robust and that RSM is an appropriate formulation optimisation tool as it has a high prognostic ability. A liquid FCV formulation was developed, optimised and found to be suitable for its intended purpose. However further optimisation is required in respect of colourants, sweeteners and/or flavourants. The approach followed is useful in ensuring the development of quality products and can be applied in future.
- Full Text:
- Date Issued: 2012
- Authors: Magnus, Laura
- Date: 2012
- Subjects: Drugs -- Dosage forms , Drugs -- Analysis , Capsules (Pharmacy) , Antiviral agents , Pediatrics
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3870 , http://hdl.handle.net/10962/d1018244
- Description: Many Active Pharmaceutical Ingredients (API) such as the antiviral agent famciclovir (FCV) are required for paediatric treatment but are not commercially available in age-appropriate dosage forms. It is common practice to prepare oral liquid dosage forms using commercially available tablets, capsules or powdered API and then dispersing or dissolving the crushed and/or powdered materials in a vehicle that the patient can swallow. Vehicles that are commonly used for this purpose include methylcellulose, syrup or combinations of these carriers where possible or commercially available suspending agents such as Ora-Sweet®, if available, can be used. However, several critical factors are overlooked when manufacturing extemporaneous formulations including, but not limited to, physical and chemical properties of the API, excipients, compatibility, stability and bioavailability issues. A stability-indicating High Performance Liquid Chromatography (HPLC) method for the analysis of FCV was developed and validated according to the International Conference on Harmonization (ICH) guidelines. The method is sensitive, selective, precise, accurate and linear over the concentration range 2-120 μg/ml. The stability of 25 mg/ml FCV formulations was assessed in vehicles manufactured from syrup simplex, hydroxypropyl methylcellulose (HPMC), Ora-Sweet® and an aqueous buffer (pH 6) following storage at 25 °C/60% RH and 40 °C/75% RH over six (6) to eight (8) weeks. The shelf life of the products was calculated as the longest period of storage for approximately 90% of the added FCV to be recovered. Formulations were manufactured using syrup simplex or HPMC with methylparaben and propylparaben individually or in combination and with sodium metabisulphite, ascorbic acid or citric acid as antioxidants. The resultant products were subject to quality control analysis for API content, viscosity, pH and appearance and the resultant data were subject to statistical analysis. The degradation rates were calculated for each product and a degradation profile plotted. The degradation rates of FCV in extemporaneous formulations were compared to those of FCV manufactured using a commercially available suspending agent and a buffered vehicle. FCV undergoes major degradation in the presence of sucrose, as observed for formulations in which the vehicle was syrup and Ora-Sweet®. FCV was found to be most stable when dissolved/dispersed in an HPMC vehicle incorporating sodium metabisulphite and a combination of parabens. The formulation that exhibited the maximum stability was manufactured using an aqueous solution buffered to pH 6. Due to the enhanced stability of FCV when added to a buffered vehicle a formulation in which an HPMC vehicle buffered to pH 6 with sodium metabisulphite, methylparaben and propylparaben was selected for optimisation using a Central Composite Design approach (CCD). In this way it was possible to establish a relationship between input variables such as pH, % w/v HPMC, % w/v antioxidant and % w/v preservative and the responses selected for monitoring by means of response surface modelling. A quadratic model was found to be the most appropriate to describe the relationship between input and output variables. Thirty batches of product were randomly manufactured according to the CCD and analysed to establish the stability in respect of viscosity, pH and the amount of FCV remaining following storage and the data were fitted to models using Design-Expert® software. A correlation between input variables and the responses was best described by a quadratic polynomial model. Analysis of Variance indicated that the response surface models were significant (P-value < 0.0001). The pH to which a FCV formulation was buffered was the most significant factor to effect the % drug content and the ultimate pH of the formulation, while the % w/v HPMC had the most significant effect on the viscosity of the product. The optimum composition for the manufacture of an oral liquid FCV formulation was predicted using the optimisation function of the Design-Expert® software. A low % error of prediction was established, indicating that the model is robust and that RSM is an appropriate formulation optimisation tool as it has a high prognostic ability. A liquid FCV formulation was developed, optimised and found to be suitable for its intended purpose. However further optimisation is required in respect of colourants, sweeteners and/or flavourants. The approach followed is useful in ensuring the development of quality products and can be applied in future.
- Full Text:
- Date Issued: 2012
The development of an orodispersible sildenafil citrate tablet intended for paediatric use
- Authors: Dagnolo, Bianca
- Date: 2012
- Subjects: Drug development -- Children -- Research -- South Africa , Pulmonary hypertension -- Children -- Research , Tablets (Medicine) -- Development , Pharmaceutical chemistry -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3751 , http://hdl.handle.net/10962/d1003229 , Drug development -- Children -- Research -- South Africa , Pulmonary hypertension -- Children -- Research , Tablets (Medicine) -- Development , Pharmaceutical chemistry -- Research
- Description: Sildenafil citrate (SC) is a phosphodiesterase-5 inhibitor that is used to treat pulmonary hypertension (PH) in paediatric patients. The purpose of these studies was to develop a formulation and manufacture an orodispersible tablet (ODT) that can be easily administered to neonates and children with PH. The advantages of ODT dosage forms include ease of administration, rapid dissolution of the API, SC. Furthermore the dosage form can be taken without water which is beneficial to patients without immediate access to potable fluids. A simple, rapid, accurate, precise and selective reversed-phase HPLC method was developed and validated in accordance with International Conference on Harmonization (ICH) guidelines and was successfully used for the analysis of SC as raw material and in SC containing pharmaceutical dosage forms. Preformulation studies were performed on SC, alone and in combination with potential excipients that could be used to make tablets. Investigations into potential interactions between SC and the excipients were performed using Differential Scanning Calorimetry (DSC) and Infrared Spectroscopy (IR). DSC results revealed that SC was compatible with all potential excipients except mannitol and magnesium stearate. However these interactions were not observed with IR and therefore it was concluded that the interactions were induced by the high temperatures that DSC operates at. Particle size and shape was also established by use of Scanning Electron Microscopy (SEM) and flow properties were monitored by calculating Carr’s Index (CI) and the Hausner Ratio (HR). Direct compression was used as the method of manufacture for SC tablets as this approach is simple and the most economic production approach. The powder blends were assessed for bulk and tapped density and the CI and HR were used to determine the flowability of the blends. The quality attributes of the resultant tablets that were monitored included uniformity of weight, friability, crushing strength, tensile strength, disintegration, wetting and in vitro dispersion times. Design of Experiments is an efficient statistical approach that has become a popular tool used in the pharmaceutical industry to optimize formulation compositions, as it allows for the investigation of several input factors at the same time whilst not using the tedious and traditional “ modification of one variable at a time” approach. A Central composite experimental design was chosen as the most appropriate means to optimize the formulation as it produces more accurate results as opposed to other experimental designs approaches as input factors are investigated at five different levels. Through the use of mathematical modelling, optimum concentrations of disintegrant(s) and an appropriate blending time were established. Analysis of the data from the experimental design and mathematical modelling studies reveal that no changes in disintegrant concentration or blending time altered the disintegration time of the formulation to any significant extent. This result is most likely due to the fact that the critical disintegrant concentration has been reached and increasing the disintegrant concentration further has no effect on disintegration time. It was also established that a change in the concentration of CMS and CRP altered the wetting time of the tablet significantly. Finally it was noted that there was a linear relationship between blending time and the uniformity of content of the tablets produced in these studies. The optimized product was a white tablet with a diameter of 7.31 mm with a thickness of 2.80mm.The dosage form had no visible cracks or evidence of picking or sticking. The tablet exhibits suitable friability and tensile strength while exhibiting a disintegration time of only 8s. Therefore an orodispersible tablet containing SC intended for paediatric use has been successfully developed, manufactured and optimized through the use of preformulation studies, appropriate quality control monitoring and mathematical modelling. These formulations require further optimization in respect of addition of flavours and or additional sweetening agents.
- Full Text:
- Date Issued: 2012
- Authors: Dagnolo, Bianca
- Date: 2012
- Subjects: Drug development -- Children -- Research -- South Africa , Pulmonary hypertension -- Children -- Research , Tablets (Medicine) -- Development , Pharmaceutical chemistry -- Research
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3751 , http://hdl.handle.net/10962/d1003229 , Drug development -- Children -- Research -- South Africa , Pulmonary hypertension -- Children -- Research , Tablets (Medicine) -- Development , Pharmaceutical chemistry -- Research
- Description: Sildenafil citrate (SC) is a phosphodiesterase-5 inhibitor that is used to treat pulmonary hypertension (PH) in paediatric patients. The purpose of these studies was to develop a formulation and manufacture an orodispersible tablet (ODT) that can be easily administered to neonates and children with PH. The advantages of ODT dosage forms include ease of administration, rapid dissolution of the API, SC. Furthermore the dosage form can be taken without water which is beneficial to patients without immediate access to potable fluids. A simple, rapid, accurate, precise and selective reversed-phase HPLC method was developed and validated in accordance with International Conference on Harmonization (ICH) guidelines and was successfully used for the analysis of SC as raw material and in SC containing pharmaceutical dosage forms. Preformulation studies were performed on SC, alone and in combination with potential excipients that could be used to make tablets. Investigations into potential interactions between SC and the excipients were performed using Differential Scanning Calorimetry (DSC) and Infrared Spectroscopy (IR). DSC results revealed that SC was compatible with all potential excipients except mannitol and magnesium stearate. However these interactions were not observed with IR and therefore it was concluded that the interactions were induced by the high temperatures that DSC operates at. Particle size and shape was also established by use of Scanning Electron Microscopy (SEM) and flow properties were monitored by calculating Carr’s Index (CI) and the Hausner Ratio (HR). Direct compression was used as the method of manufacture for SC tablets as this approach is simple and the most economic production approach. The powder blends were assessed for bulk and tapped density and the CI and HR were used to determine the flowability of the blends. The quality attributes of the resultant tablets that were monitored included uniformity of weight, friability, crushing strength, tensile strength, disintegration, wetting and in vitro dispersion times. Design of Experiments is an efficient statistical approach that has become a popular tool used in the pharmaceutical industry to optimize formulation compositions, as it allows for the investigation of several input factors at the same time whilst not using the tedious and traditional “ modification of one variable at a time” approach. A Central composite experimental design was chosen as the most appropriate means to optimize the formulation as it produces more accurate results as opposed to other experimental designs approaches as input factors are investigated at five different levels. Through the use of mathematical modelling, optimum concentrations of disintegrant(s) and an appropriate blending time were established. Analysis of the data from the experimental design and mathematical modelling studies reveal that no changes in disintegrant concentration or blending time altered the disintegration time of the formulation to any significant extent. This result is most likely due to the fact that the critical disintegrant concentration has been reached and increasing the disintegrant concentration further has no effect on disintegration time. It was also established that a change in the concentration of CMS and CRP altered the wetting time of the tablet significantly. Finally it was noted that there was a linear relationship between blending time and the uniformity of content of the tablets produced in these studies. The optimized product was a white tablet with a diameter of 7.31 mm with a thickness of 2.80mm.The dosage form had no visible cracks or evidence of picking or sticking. The tablet exhibits suitable friability and tensile strength while exhibiting a disintegration time of only 8s. Therefore an orodispersible tablet containing SC intended for paediatric use has been successfully developed, manufactured and optimized through the use of preformulation studies, appropriate quality control monitoring and mathematical modelling. These formulations require further optimization in respect of addition of flavours and or additional sweetening agents.
- Full Text:
- Date Issued: 2012
The isolation, quantification and synthetic modification of antiplasmodial natural products from sargassum heterophyllum
- Authors: Munedzimwe, Tatenda Carol
- Date: 2012
- Subjects: Malaria -- Developing countries -- Prevention , Antimalarials
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3871 , http://hdl.handle.net/10962/d1018252
- Description: Malaria is one of the most deadly parasitic diseases known to man. Although the number of malaria cases reported each year is decreasing, this disease continues to pose health and economic problems mainly in developing countries. Significant progress has been made in the fight against this disease. This includes the discovery and development of potent antimalarial agents. However, the development of resistance to most of these potent antimalarials has made the development of new antiplasmodial agents of paramount importance. Several promising antiplasmodial agents have been found from the marine environment. Amongst these are the tetraprenylated toluquinols from the brown alga: Sargassum heterophyllum. These metabolites have been reported to exhibit a range of antiplasmodial activity; however, the mechanisms by which these compounds bring about their antiplasmodial activity and the pharmacophoric groups responsible for such activity are unknown. Two species of Sargassum algae were encountered during the course of this project. From the investigation of the geographical and seasonal variation of metabolites of S. heterophyllum and S. elegans we established that there were no significant intra and inter site variations amongst metabolite profiles of both species both within and between the sampled seasons. These results enabled us to establish that the collection of both species from three different sites on the eastern coast of South Africa namely; Kenton on Sea, Port Alfred and Noordhoek in autumn, winter or spring would qualitatively yield the same metabolites. A comparison of metabolite profiles of both species also revealed no qualitative differences between metabolites of S. heterophyllum and S. elegans. The quantities of selected prenylated metabolites extracted from S. heterophyllum using four different extraction techniques was also assessed using qNMR as the method of quantification. This led to the identification of optimal extraction techniques and conditions for the extraction of sargahydroquinoic acid (1.38), sargaquinoic aid (1.39) and sargachromenol (2.10) from S. heterophyllum. From this study, the extraction of algae by soxhlet extraction using EtOH as the extraction solvent led to the extraction of the highest quantities of sargahydroquinoic acid. The potential of other extraction techniques such as microwave assisted extraction, to yield high quantities of the selected metabolites were also identified. With gram quantities of sargahydroquinoic acid (1.38) in hand, this compound was modified by oxidation, reduction, acetylation, methylation and cyclization reactions to yield nine derivatives. The derivatives and four naturally occurring prenylated toluquinols were assessed for antiplasmodial and cytotoxic activity against the FCR-3 Gambian Chloroquine resistant strain of P. falciparum and the MDA-MB-231 breast carcinoma cell line respectively. Comparison of antiplasmodial data for all twelve compounds showed that the hydroquinone moeity of sargahydroquinoic acid (1.38) is important for antiplasmodial activity while esterification of the carboxylic acid group in 1.38 resulted in more potent antiplasmodial compounds. Of all twelve compounds, compound 5.2, the hydroquinone methyl ester of 1.38 was found to be the most potent antiplasmodial compound with an IC₅₀ value of 1.94 μM and a selectivity index of 22.68.
- Full Text:
- Date Issued: 2012
- Authors: Munedzimwe, Tatenda Carol
- Date: 2012
- Subjects: Malaria -- Developing countries -- Prevention , Antimalarials
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3871 , http://hdl.handle.net/10962/d1018252
- Description: Malaria is one of the most deadly parasitic diseases known to man. Although the number of malaria cases reported each year is decreasing, this disease continues to pose health and economic problems mainly in developing countries. Significant progress has been made in the fight against this disease. This includes the discovery and development of potent antimalarial agents. However, the development of resistance to most of these potent antimalarials has made the development of new antiplasmodial agents of paramount importance. Several promising antiplasmodial agents have been found from the marine environment. Amongst these are the tetraprenylated toluquinols from the brown alga: Sargassum heterophyllum. These metabolites have been reported to exhibit a range of antiplasmodial activity; however, the mechanisms by which these compounds bring about their antiplasmodial activity and the pharmacophoric groups responsible for such activity are unknown. Two species of Sargassum algae were encountered during the course of this project. From the investigation of the geographical and seasonal variation of metabolites of S. heterophyllum and S. elegans we established that there were no significant intra and inter site variations amongst metabolite profiles of both species both within and between the sampled seasons. These results enabled us to establish that the collection of both species from three different sites on the eastern coast of South Africa namely; Kenton on Sea, Port Alfred and Noordhoek in autumn, winter or spring would qualitatively yield the same metabolites. A comparison of metabolite profiles of both species also revealed no qualitative differences between metabolites of S. heterophyllum and S. elegans. The quantities of selected prenylated metabolites extracted from S. heterophyllum using four different extraction techniques was also assessed using qNMR as the method of quantification. This led to the identification of optimal extraction techniques and conditions for the extraction of sargahydroquinoic acid (1.38), sargaquinoic aid (1.39) and sargachromenol (2.10) from S. heterophyllum. From this study, the extraction of algae by soxhlet extraction using EtOH as the extraction solvent led to the extraction of the highest quantities of sargahydroquinoic acid. The potential of other extraction techniques such as microwave assisted extraction, to yield high quantities of the selected metabolites were also identified. With gram quantities of sargahydroquinoic acid (1.38) in hand, this compound was modified by oxidation, reduction, acetylation, methylation and cyclization reactions to yield nine derivatives. The derivatives and four naturally occurring prenylated toluquinols were assessed for antiplasmodial and cytotoxic activity against the FCR-3 Gambian Chloroquine resistant strain of P. falciparum and the MDA-MB-231 breast carcinoma cell line respectively. Comparison of antiplasmodial data for all twelve compounds showed that the hydroquinone moeity of sargahydroquinoic acid (1.38) is important for antiplasmodial activity while esterification of the carboxylic acid group in 1.38 resulted in more potent antiplasmodial compounds. Of all twelve compounds, compound 5.2, the hydroquinone methyl ester of 1.38 was found to be the most potent antiplasmodial compound with an IC₅₀ value of 1.94 μM and a selectivity index of 22.68.
- Full Text:
- Date Issued: 2012
The synthesis and breast cancer inhibitory activity of cinnamic acid analogues based on the halogenated monoterpene pharmacophore
- Authors: Chiwakata, Maynard Tendai
- Date: 2012
- Subjects: Halocarbons , Cancer -- Treatment , Breast -- Cancer -- Treatment
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3866 , http://hdl.handle.net/10962/d1016129
- Description: Breast cancer is one of the leading causes of death, with mortality rate estimates of 465 000 deaths per annum. It is estimated that 1.3 million women are diagnosed with the disease each year especially in the developing countries. Current chemotherapy relies on the use of high doses of non-specific toxic agents that possess adverse side effects and compromise patient’s compliance and adherence to treatment. Paclitaxel, one of the common drugs used in breast cancer chemotherapy results in sensory and motor neuropathy, whilst hormonal therapy e.g. Herceptin causes severe cardiovascular, gastrointestinal and cutaneous side effects. There has been a demand in developing newer cancer agents that demonstrate selective cytoxicity with minimal effect on normal body tissue. Numerous studies have shown that marine organisms produce a wide range of halogenated compounds that possess cytotoxic properties, and hence can be a source of new drug hits or leads for cancer therapy. Halomon, a polyhalogenated monoterpene from Portieria hornemannii, displayed interesting activity against brain, renal and lung cancer tumours with selective/differential cytotoxicity. This inspired us to focus our project on halogenated monoterpenes isolated from the same Rhodophyta class as P. hornemannii but with particular attention to Plocamium species. Several metabolites have been isolated from P. cornutum, P. corallorhiza and P. suhrii that possess interesting cytotoxicities against a breast cancer cell line (MCF7) and an oesophageal cancer line (WHCO1). The aim of the project was therefore centred at isolating target compounds for preliminary structure-activity studies against a breast cancer cell line, and use this information to synthesize a series of analogues that are more stable than the natural products and yet as active using a fragment-based type approach to map out pharmacophoric elements. Five metabolites were isolated from P. cornutum and five from P. corallorhiza. Cell-based assays were conducted using an MTT assay kit against MCF7 and MDA-MB-231 breast cancer cell lines and (1E,3E,5S,6R)-1,5,6-trichloro-2-(dichloromethyl)-6-methylocta-1,3,7-triene, isolated from P. cornutum was the most active with IC50 values of 3.0 μM and 6.15 μM respectively. Introduction of a terminal aromatic ring to enhance stability, together with varying substituents (H, CH3, CF3, Br, CN, CHO, CHCl2) on position 7 of the molecule, gave rise to a series of cinnamate ester derivatives inspired by (1E,3E,5S,6R)-1,5,6-trichloro-2-(dichloromethyl)-6-methylocta-1,3,7-triene. The analogues were synthesized from their benzaldehyde precursors via Aldol condensation, esterification and Wittig reactions. Their carboxylic acid counterparts were synthesized by hydrolysis of the parent esters in an attempt to promote water solubilities of the analogues. Biological activity assays were then conducted with the cinnamate analogues against the MDA-MB-231 breast cancer cell line using an MTT assay kit. Ester derivatives with -CHO and -CHCl2 functionalities had IC50 values of 43.45 μM and 100.01 μM respectively whilst the other ester derivatives were inactive. It was concluded that either an aldehyde (-CHO) or gem-dichlorides (-CHCl2) is specifically required for cytotoxic activity to be observed. None of the carboxylic acids were active which could have been due to failure of the compounds to enter the breast cancer cells and reach the target site because of their polar nature. Compounds with -CHO and -CHCl2 functionalities were therefore selected for future SARs studies.
- Full Text:
- Date Issued: 2012
- Authors: Chiwakata, Maynard Tendai
- Date: 2012
- Subjects: Halocarbons , Cancer -- Treatment , Breast -- Cancer -- Treatment
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3866 , http://hdl.handle.net/10962/d1016129
- Description: Breast cancer is one of the leading causes of death, with mortality rate estimates of 465 000 deaths per annum. It is estimated that 1.3 million women are diagnosed with the disease each year especially in the developing countries. Current chemotherapy relies on the use of high doses of non-specific toxic agents that possess adverse side effects and compromise patient’s compliance and adherence to treatment. Paclitaxel, one of the common drugs used in breast cancer chemotherapy results in sensory and motor neuropathy, whilst hormonal therapy e.g. Herceptin causes severe cardiovascular, gastrointestinal and cutaneous side effects. There has been a demand in developing newer cancer agents that demonstrate selective cytoxicity with minimal effect on normal body tissue. Numerous studies have shown that marine organisms produce a wide range of halogenated compounds that possess cytotoxic properties, and hence can be a source of new drug hits or leads for cancer therapy. Halomon, a polyhalogenated monoterpene from Portieria hornemannii, displayed interesting activity against brain, renal and lung cancer tumours with selective/differential cytotoxicity. This inspired us to focus our project on halogenated monoterpenes isolated from the same Rhodophyta class as P. hornemannii but with particular attention to Plocamium species. Several metabolites have been isolated from P. cornutum, P. corallorhiza and P. suhrii that possess interesting cytotoxicities against a breast cancer cell line (MCF7) and an oesophageal cancer line (WHCO1). The aim of the project was therefore centred at isolating target compounds for preliminary structure-activity studies against a breast cancer cell line, and use this information to synthesize a series of analogues that are more stable than the natural products and yet as active using a fragment-based type approach to map out pharmacophoric elements. Five metabolites were isolated from P. cornutum and five from P. corallorhiza. Cell-based assays were conducted using an MTT assay kit against MCF7 and MDA-MB-231 breast cancer cell lines and (1E,3E,5S,6R)-1,5,6-trichloro-2-(dichloromethyl)-6-methylocta-1,3,7-triene, isolated from P. cornutum was the most active with IC50 values of 3.0 μM and 6.15 μM respectively. Introduction of a terminal aromatic ring to enhance stability, together with varying substituents (H, CH3, CF3, Br, CN, CHO, CHCl2) on position 7 of the molecule, gave rise to a series of cinnamate ester derivatives inspired by (1E,3E,5S,6R)-1,5,6-trichloro-2-(dichloromethyl)-6-methylocta-1,3,7-triene. The analogues were synthesized from their benzaldehyde precursors via Aldol condensation, esterification and Wittig reactions. Their carboxylic acid counterparts were synthesized by hydrolysis of the parent esters in an attempt to promote water solubilities of the analogues. Biological activity assays were then conducted with the cinnamate analogues against the MDA-MB-231 breast cancer cell line using an MTT assay kit. Ester derivatives with -CHO and -CHCl2 functionalities had IC50 values of 43.45 μM and 100.01 μM respectively whilst the other ester derivatives were inactive. It was concluded that either an aldehyde (-CHO) or gem-dichlorides (-CHCl2) is specifically required for cytotoxic activity to be observed. None of the carboxylic acids were active which could have been due to failure of the compounds to enter the breast cancer cells and reach the target site because of their polar nature. Compounds with -CHO and -CHCl2 functionalities were therefore selected for future SARs studies.
- Full Text:
- Date Issued: 2012
Development, manufacture and assessment of Clobetasol 17-propionate cream formulations
- Fauzee, Ayeshah Fateemah Beebee
- Authors: Fauzee, Ayeshah Fateemah Beebee
- Date: 2011
- Subjects: Adrenocortical hormones , Adrenocortical hormones -- Physiological effect , Adrenocortical hormones -- Testing , Drugs -- Testing , Drugs -- Development , Dermatopharmacology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3856 , http://hdl.handle.net/10962/d1013324
- Description: Eczema or dermatitis is the most common dermatological condition accounting for one-third of all diagnoses in the total population surveyed in South Africa. The prevalence of seborrhoeic dermatitis, extreme photodermatitis and severe psoriasis has increased markedly over the last decade and this increase may be ascribed to the HIV epidemic, first diagnosed in South Africa in 1982. Potent innovator corticosteroids, such as clobetasol 17-propionate (CP) that are used to treat skin disorders, are expensive and there is therefore a need for the production of generic topical corticosteroid products. Formulation and manufacturing processes can be challenging aspects for formulation scientists to produce a robust product that will elicit an appropriate and desirable pharmacokinetic-pharmacodynamic profile. Laboratory scale CP creams were manufactured using different concentrations of Gelot® 64 and propylene glycol in order to establish a composition that would produce a formulation, with similar physical and chemical characteristics and in vitro release profile as an innovator product, Dermovate®. These formulations were assessed in terms of their viscosity, spreadability, pH, content uniformity and in vitro release characteristics using a Franz diffusion cell apparatus. A formulation containing 3% w/w Gelot® 64 and 46% v/v propylene glycol (CPLS-02) was found to exhibit similar viscosity and spreadability characteristics and released CP in a manner similar to Dermovate®. The mechanism of drug release was evaluated using mathematical models such as zero order, first order and Higuchi models. In addition, the in vitro release profiles were characterised by use of difference (f1) and similarity (f2 and Sd) factors. A scale-up formulation with the same % w/w composition as the laboratory scale was also investigated following manufacture using a Wintech® cream/ointment mixer. A Central Composite Design approach was used to investigate the effect of process variables on the performance of the scale-up cream formulations. The homogenisation speed, anchor speed, homogenisation time and cooling time were the process variables investigated. Thirty scale-up batches were manufactured and analysed in terms of their viscosity, spreadability, pH, % drug content and cumulative % drug released per unit area over 72 hours. Model fitting using Design-Expert® software was undertaken and revealed that a correlation between the process variables and the cream responses was most suitably described by quadratic polynomial relationships. The homogenisation speed had the most significant effect on the quality of the scale-up formulations, whereas the anchor speed had a secondary effect on the measured responses, for the formulations investigated. The qualitative interpretation and statistical analysis of the in vitro release data from the scale-up formulations using ANOVA and the f1, f2 and Sd factors revealed that one scale-up batch (CPSU-04), for which the process variables were a homogenisation speed of 1900 rpm, an anchor speed of 35 rpm, a homogenisation time of 100 minutes and a cooling time of 100 minutes, released CP at a similar rate and extent to Dermovate®. A diffusion-controlled mechanism appeared to be predominant in these formulations. A human skin blanching study, using both visual and chromameter assessments, was performed to establish whether batch CPSU-04 was bioequivalent to Dermovate®. The bioequivalence of the selected scale-up formulation to Dermovate® was confirmed, following the calculation of a 90% CI.
- Full Text:
- Date Issued: 2011
- Authors: Fauzee, Ayeshah Fateemah Beebee
- Date: 2011
- Subjects: Adrenocortical hormones , Adrenocortical hormones -- Physiological effect , Adrenocortical hormones -- Testing , Drugs -- Testing , Drugs -- Development , Dermatopharmacology
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3856 , http://hdl.handle.net/10962/d1013324
- Description: Eczema or dermatitis is the most common dermatological condition accounting for one-third of all diagnoses in the total population surveyed in South Africa. The prevalence of seborrhoeic dermatitis, extreme photodermatitis and severe psoriasis has increased markedly over the last decade and this increase may be ascribed to the HIV epidemic, first diagnosed in South Africa in 1982. Potent innovator corticosteroids, such as clobetasol 17-propionate (CP) that are used to treat skin disorders, are expensive and there is therefore a need for the production of generic topical corticosteroid products. Formulation and manufacturing processes can be challenging aspects for formulation scientists to produce a robust product that will elicit an appropriate and desirable pharmacokinetic-pharmacodynamic profile. Laboratory scale CP creams were manufactured using different concentrations of Gelot® 64 and propylene glycol in order to establish a composition that would produce a formulation, with similar physical and chemical characteristics and in vitro release profile as an innovator product, Dermovate®. These formulations were assessed in terms of their viscosity, spreadability, pH, content uniformity and in vitro release characteristics using a Franz diffusion cell apparatus. A formulation containing 3% w/w Gelot® 64 and 46% v/v propylene glycol (CPLS-02) was found to exhibit similar viscosity and spreadability characteristics and released CP in a manner similar to Dermovate®. The mechanism of drug release was evaluated using mathematical models such as zero order, first order and Higuchi models. In addition, the in vitro release profiles were characterised by use of difference (f1) and similarity (f2 and Sd) factors. A scale-up formulation with the same % w/w composition as the laboratory scale was also investigated following manufacture using a Wintech® cream/ointment mixer. A Central Composite Design approach was used to investigate the effect of process variables on the performance of the scale-up cream formulations. The homogenisation speed, anchor speed, homogenisation time and cooling time were the process variables investigated. Thirty scale-up batches were manufactured and analysed in terms of their viscosity, spreadability, pH, % drug content and cumulative % drug released per unit area over 72 hours. Model fitting using Design-Expert® software was undertaken and revealed that a correlation between the process variables and the cream responses was most suitably described by quadratic polynomial relationships. The homogenisation speed had the most significant effect on the quality of the scale-up formulations, whereas the anchor speed had a secondary effect on the measured responses, for the formulations investigated. The qualitative interpretation and statistical analysis of the in vitro release data from the scale-up formulations using ANOVA and the f1, f2 and Sd factors revealed that one scale-up batch (CPSU-04), for which the process variables were a homogenisation speed of 1900 rpm, an anchor speed of 35 rpm, a homogenisation time of 100 minutes and a cooling time of 100 minutes, released CP at a similar rate and extent to Dermovate®. A diffusion-controlled mechanism appeared to be predominant in these formulations. A human skin blanching study, using both visual and chromameter assessments, was performed to establish whether batch CPSU-04 was bioequivalent to Dermovate®. The bioequivalence of the selected scale-up formulation to Dermovate® was confirmed, following the calculation of a 90% CI.
- Full Text:
- Date Issued: 2011
Investigations of the assessment of bioequivalence of topical clotrimazole products using a dermatopharmacokinetic approach
- Authors: Parfitt, Natalie Rae
- Date: 2011 , 2010-07-05
- Subjects: Dermatopharmacology , Drugs -- Therapeutic equivalency , Antifungal agents , Therapeutics, Cutaneous and external
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3840 , http://hdl.handle.net/10962/d1007659 , Dermatopharmacology , Drugs -- Therapeutic equivalency , Antifungal agents , Therapeutics, Cutaneous and external
- Description: The specialised nature of the stratum corneum makes it an efficient barrier to foreign substances, including drug molecules. Therefore, cutaneous drug absorption is a slow and complex process of which stratum corneum penetration is the rate limiting step. The rate and extent of stratum corneum penetration by a drug compound depends greatly on the presence of penetration enhancing/retarding excipients and therefore the clinical outcomes of a product rely greatly on the components and quality of the formulation. Hence, establishing bioequivalence between topical products is crucial to ensure that patients receiving multisource drug products are assured of the same efficacy and safety as the brand product. Since locally acting topical formulations do not target the systemic circulation, conventional methods of assessing bioequivalence using plasma levels are not appropriate. Consequently, the current regulatory guidelines require comparative clinical trials to be carried out to show bioequivalence between topical products. As these studies are very expensive and time consuming, the development of a more direct and relatively rapid and inexpensive method for determining bioequivalence between topical products is required. Clotrimazole is an anti-fungal agent where the target site of action is in the stratum corneum. In this work, tape stripping, which involves the sampling of stratum corneum, was investigated as a tool for the determination of bioequivalence between topical clotrimazole products. The tape stripping method involved the analysis of each tape strip individually and standardization of stratum corneum thickness between subjects was carried out using TEWL measurements. This approach provided detailed information regarding the amount of clotrimazole present in the stratum corneum as well as the extent of drug penetration. Prior to the tape stripping studies an HPLC method was developed for the quantitative analysis of clotrimazole from the tape strip samples. This method was shown to be accurate and reproducible across the required range. It was also shown to be selective for clotrimazole in the presence of possible interfering substances such as those present in the tape adhesive and also skin components. The bioequivalence studies were conducted using a single “uptake” time point. In order to determine an appropriate dose duration for these studies a novel approach was employed, involving a preliminary dose duration study. For the bioequivalence investigations, Canesten® Topical cream was used as both test and reference products to determine if the method was capable of showing bioequivalence. Subsequently, Canesten® Topical cream was also compared to a 1% gel formulation to determine if the method could detect formulation differences. The conventional BE limits of 0.8 – 1.25 were used for the assessment of BE, however, the clinical relevance of using these limits for dermal studies is debatable since they are derived from oral pharmacokinetic studies. Therefore, the data from the tape stripping investigations were also assessed using more realistic limits of 0.75 – 1.33 and even 0.7 – 1.44. In addition to the tape stripping studies a novel method of determining the amount of drug present in the stratum corneum, the “Residual Method”, was investigated. This method involved assaying the amount of clotrimazole found in the residual formulation after a specified dose duration had elapsed and subtracting that amount from the amount of clotrimazole initially applied. The results of tape stripping investigations showed that, if the study is sufficiently powered, tape stripping may be used to determine bioequivalence according to the conventional limits, as well as possibly detect formulation differences between different clotrimazole products. Bioequivalence assessment using the widened intervals showed that fewer subjects were required to achieve a sufficient statistical power. The variability associated with this method was acceptable and tape stripping may therefore have the potential to be used as a BE tool in a regulatory setting for clotrimazole or other antifungal topical formulations. The “Residual Method” also showed promising results as a bioequivalence tool, but further investigation and extensive validation of this method is required before it can be suggested as a regulatory method. The results of these studies have clearly indicated that tape stripping has the potential to be used as an alternative to comparative clinical trails for the assessment of bioequivalence between clotrimazole formulations and also to assess bioequivalence between other antifungal products.
- Full Text:
- Date Issued: 2011
- Authors: Parfitt, Natalie Rae
- Date: 2011 , 2010-07-05
- Subjects: Dermatopharmacology , Drugs -- Therapeutic equivalency , Antifungal agents , Therapeutics, Cutaneous and external
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3840 , http://hdl.handle.net/10962/d1007659 , Dermatopharmacology , Drugs -- Therapeutic equivalency , Antifungal agents , Therapeutics, Cutaneous and external
- Description: The specialised nature of the stratum corneum makes it an efficient barrier to foreign substances, including drug molecules. Therefore, cutaneous drug absorption is a slow and complex process of which stratum corneum penetration is the rate limiting step. The rate and extent of stratum corneum penetration by a drug compound depends greatly on the presence of penetration enhancing/retarding excipients and therefore the clinical outcomes of a product rely greatly on the components and quality of the formulation. Hence, establishing bioequivalence between topical products is crucial to ensure that patients receiving multisource drug products are assured of the same efficacy and safety as the brand product. Since locally acting topical formulations do not target the systemic circulation, conventional methods of assessing bioequivalence using plasma levels are not appropriate. Consequently, the current regulatory guidelines require comparative clinical trials to be carried out to show bioequivalence between topical products. As these studies are very expensive and time consuming, the development of a more direct and relatively rapid and inexpensive method for determining bioequivalence between topical products is required. Clotrimazole is an anti-fungal agent where the target site of action is in the stratum corneum. In this work, tape stripping, which involves the sampling of stratum corneum, was investigated as a tool for the determination of bioequivalence between topical clotrimazole products. The tape stripping method involved the analysis of each tape strip individually and standardization of stratum corneum thickness between subjects was carried out using TEWL measurements. This approach provided detailed information regarding the amount of clotrimazole present in the stratum corneum as well as the extent of drug penetration. Prior to the tape stripping studies an HPLC method was developed for the quantitative analysis of clotrimazole from the tape strip samples. This method was shown to be accurate and reproducible across the required range. It was also shown to be selective for clotrimazole in the presence of possible interfering substances such as those present in the tape adhesive and also skin components. The bioequivalence studies were conducted using a single “uptake” time point. In order to determine an appropriate dose duration for these studies a novel approach was employed, involving a preliminary dose duration study. For the bioequivalence investigations, Canesten® Topical cream was used as both test and reference products to determine if the method was capable of showing bioequivalence. Subsequently, Canesten® Topical cream was also compared to a 1% gel formulation to determine if the method could detect formulation differences. The conventional BE limits of 0.8 – 1.25 were used for the assessment of BE, however, the clinical relevance of using these limits for dermal studies is debatable since they are derived from oral pharmacokinetic studies. Therefore, the data from the tape stripping investigations were also assessed using more realistic limits of 0.75 – 1.33 and even 0.7 – 1.44. In addition to the tape stripping studies a novel method of determining the amount of drug present in the stratum corneum, the “Residual Method”, was investigated. This method involved assaying the amount of clotrimazole found in the residual formulation after a specified dose duration had elapsed and subtracting that amount from the amount of clotrimazole initially applied. The results of tape stripping investigations showed that, if the study is sufficiently powered, tape stripping may be used to determine bioequivalence according to the conventional limits, as well as possibly detect formulation differences between different clotrimazole products. Bioequivalence assessment using the widened intervals showed that fewer subjects were required to achieve a sufficient statistical power. The variability associated with this method was acceptable and tape stripping may therefore have the potential to be used as a BE tool in a regulatory setting for clotrimazole or other antifungal topical formulations. The “Residual Method” also showed promising results as a bioequivalence tool, but further investigation and extensive validation of this method is required before it can be suggested as a regulatory method. The results of these studies have clearly indicated that tape stripping has the potential to be used as an alternative to comparative clinical trails for the assessment of bioequivalence between clotrimazole formulations and also to assess bioequivalence between other antifungal products.
- Full Text:
- Date Issued: 2011
The development and assessment of a fixed dose combination tablet of Ranitidine and Metronidazole
- Authors: King'ori, Loti David
- Date: 2011 , 2011-04-07
- Subjects: Ulcers -- Treatment , Ranitidine -- Evaluation , Metronidazole -- Evaluation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3858 , http://hdl.handle.net/10962/d1013359
- Description: The oral route of drug administration is convenient since it is acceptable to most patients and the manufacturing processes used to produce tablets and capsules are relatively simple when compared to those used to manufacture other types of dosage forms. Metronidazole (MTZ) and Ranitidine (RTD) have been used in combination, as part of triple therapy for the treatment of ulcers. However the use of large numbers of tablets and long duration of therapy makes adherence to drug treatment challenging for patients. Therefore the formulation of a fixed dose combination (FDC) of MTZ and RTD may improve patient adherence to therapy and consequently may reduce morbidity and mortality due to ulcers. A stability indicating HPLC method for the simultaneous analysis of MTZ and RTD was developed and validated according to the International Conference on Harmonization (ICH) guidelines. The method was sensitive, selective, precise, accurate and linear.Preformulation studies were performed on the active pharmaceutical ingredients (API) alone and in combination with potential excipients. Differential scanning calorimetry (DSC) studies revealed a potential interaction between MTZ and RTD, however the interaction was not apparent following IR analysis of the same samples. DSC analyses of the API in combination with potential excipients revealed that the compounds were compatible with most materials with the exception of a binary mixture of RTD and Dibasic calcium phosphate (DCP) that exhibited a potential interaction. Thermal gravimetric analysis (TGA) of MTZ and RTD revealed that both compounds exhibited thermal stability. The Carrs Index (CI) and Hausner Ratio (HR) values of MTZ and RTD indicated that both compounds exhibited poor flow and compressibility properties, whereas the CI and HR values for (Microcrystalline cellulose) MCC and DCP indicated better flowability and compressibility characteristics.Direct compression and wet granulation processes were assessed to identify a suitable method of manufacture of FDC tablets of MTZ and RTD. The blends were evaluated using bulk and tapped density and the resultant tablets were evaluated for weight uniformity, crushing strength, tensile strength and disintegration time. The wet granulation method of manufacture produced tablets that showed acceptable pharmacotechnical properties: this approach was therefore used as the method of manufacture of FDC tablets of MTZ and RTD. Tablet formulations comprised of API, viz. MTZ and RTD and different compositions of MCC, DCP, Sodium starch glycolate (SSG) and Croscarmellose sodium (CCS), were manufactured in order to screen for an appropriate diluent and disintegrant composition for use in response surface studies. Assays of tablet content and in vitro drug release were undertaken using the validated HPLC method. Tablets in which MCC and CCS were used appeared to produce better assay and dissolution results as compared to those manufactured using DCP and SSG. Consequently a formulation comprised of MCC and CCS was selected and used in studies in which the effect(s) of level two formulation and composition changes as described in the Scale and Post Approval Changes for Immediate Release (SUPAC-IR) Guidelines on tablet disintegration and in vitro release were assessed. A Box-Behnken statistical design was used for the investigation of the effect of input factors, viz. CCS, (Polyvinyl pyrollidone K30) PVP-K30 and magnesium stearate on measured responses, viz. disintegration time and percent drug release in 10 minutes (Q10). CCS appeared to have an inverse linear relationship on disintegration time and a linear relationship with the Q10 for MTZ and RTD, whereas PVP-K30 and magnesium stearate appeared to have an antagonistic effect on the measured responses. Furthermore CCS and magnesium stearate exhibited an interaction that had an agonistic effect on the Q10 value for RTD. A numerical optimization approach was used to predict a formulation composition that would produce tablets that exhibited a disintegration time and Q10 values for MTZ and RTD that fell within the constraints set in our laboratory. The resultant model was found to be accurate and had a percent prediction error of < 5% for all measured response variables.FDC tablets of MTZ and RTD have been successfully produced. The disintegration of the tablet and dissolution of the API were within compendial specifications and the tablets are of suitable quality and have the potential to be further investigated to reduce the pill burden in the treatment of ulcers.
- Full Text:
- Date Issued: 2011
- Authors: King'ori, Loti David
- Date: 2011 , 2011-04-07
- Subjects: Ulcers -- Treatment , Ranitidine -- Evaluation , Metronidazole -- Evaluation
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3858 , http://hdl.handle.net/10962/d1013359
- Description: The oral route of drug administration is convenient since it is acceptable to most patients and the manufacturing processes used to produce tablets and capsules are relatively simple when compared to those used to manufacture other types of dosage forms. Metronidazole (MTZ) and Ranitidine (RTD) have been used in combination, as part of triple therapy for the treatment of ulcers. However the use of large numbers of tablets and long duration of therapy makes adherence to drug treatment challenging for patients. Therefore the formulation of a fixed dose combination (FDC) of MTZ and RTD may improve patient adherence to therapy and consequently may reduce morbidity and mortality due to ulcers. A stability indicating HPLC method for the simultaneous analysis of MTZ and RTD was developed and validated according to the International Conference on Harmonization (ICH) guidelines. The method was sensitive, selective, precise, accurate and linear.Preformulation studies were performed on the active pharmaceutical ingredients (API) alone and in combination with potential excipients. Differential scanning calorimetry (DSC) studies revealed a potential interaction between MTZ and RTD, however the interaction was not apparent following IR analysis of the same samples. DSC analyses of the API in combination with potential excipients revealed that the compounds were compatible with most materials with the exception of a binary mixture of RTD and Dibasic calcium phosphate (DCP) that exhibited a potential interaction. Thermal gravimetric analysis (TGA) of MTZ and RTD revealed that both compounds exhibited thermal stability. The Carrs Index (CI) and Hausner Ratio (HR) values of MTZ and RTD indicated that both compounds exhibited poor flow and compressibility properties, whereas the CI and HR values for (Microcrystalline cellulose) MCC and DCP indicated better flowability and compressibility characteristics.Direct compression and wet granulation processes were assessed to identify a suitable method of manufacture of FDC tablets of MTZ and RTD. The blends were evaluated using bulk and tapped density and the resultant tablets were evaluated for weight uniformity, crushing strength, tensile strength and disintegration time. The wet granulation method of manufacture produced tablets that showed acceptable pharmacotechnical properties: this approach was therefore used as the method of manufacture of FDC tablets of MTZ and RTD. Tablet formulations comprised of API, viz. MTZ and RTD and different compositions of MCC, DCP, Sodium starch glycolate (SSG) and Croscarmellose sodium (CCS), were manufactured in order to screen for an appropriate diluent and disintegrant composition for use in response surface studies. Assays of tablet content and in vitro drug release were undertaken using the validated HPLC method. Tablets in which MCC and CCS were used appeared to produce better assay and dissolution results as compared to those manufactured using DCP and SSG. Consequently a formulation comprised of MCC and CCS was selected and used in studies in which the effect(s) of level two formulation and composition changes as described in the Scale and Post Approval Changes for Immediate Release (SUPAC-IR) Guidelines on tablet disintegration and in vitro release were assessed. A Box-Behnken statistical design was used for the investigation of the effect of input factors, viz. CCS, (Polyvinyl pyrollidone K30) PVP-K30 and magnesium stearate on measured responses, viz. disintegration time and percent drug release in 10 minutes (Q10). CCS appeared to have an inverse linear relationship on disintegration time and a linear relationship with the Q10 for MTZ and RTD, whereas PVP-K30 and magnesium stearate appeared to have an antagonistic effect on the measured responses. Furthermore CCS and magnesium stearate exhibited an interaction that had an agonistic effect on the Q10 value for RTD. A numerical optimization approach was used to predict a formulation composition that would produce tablets that exhibited a disintegration time and Q10 values for MTZ and RTD that fell within the constraints set in our laboratory. The resultant model was found to be accurate and had a percent prediction error of < 5% for all measured response variables.FDC tablets of MTZ and RTD have been successfully produced. The disintegration of the tablet and dissolution of the API were within compendial specifications and the tablets are of suitable quality and have the potential to be further investigated to reduce the pill burden in the treatment of ulcers.
- Full Text:
- Date Issued: 2011
Bioethanol production from waste paper through fungal biotechnology
- Authors: Voigt, Paul George
- Date: 2010
- Subjects: Biomass energy , Cellulose -- Biodegradation , Waste paper -- Recycling , Biomass chemicals -- Economic aspects , Renewable energy sources , Fungi -- Biotechnology , Enzymes -- Biotechnology
- Language: English
- Type: Thesis , MSc , Masters
- Identifier: vital:3861 , http://hdl.handle.net/10962/d1013447
- Description: Bioethanol is likely to be a large contributor to the fuel sector of industry in the near future. Current research trends are geared towards utilizing food crops as substrate for bioethanol fermentation; however, this is the source of much controversy. Utilizing food crops for fuel purposes is anticipated to cause massive food shortages worldwide. Cellulose is the most abundant renewable resource on earth and is subject to a wide array of scientific study in order to utilize the glucose contained within it. Waste paper has a high degree of cellulose associated with it, which makes it an ideal target for cellulose biotechnology with the ultimate end goal of bioethanol production. This study focussed on producing the necessary enzymes to hydrolyse the cellulose found in waste paper and using the sugars produced to produce ethanol. The effects of various printing inks had on the production of sugars and the total envirorunental impact of the effluents produced during the production line were also examined. It was found that the fungus Trichoderma longibrachiatum DSM 769 grown in Mandel's medium with waste newspaper as the sole carbon source at 28 °C for 6 days produced extracellular cellulase enzymes with an activity of 0.203 ± 0.009 FPU.ml⁻¹, significantly higher activity as compared to other paper sources. This extracellular cellulase was used to hydrolyse waste newspaper and office paper, with office paper yielding the highest degree of sugar production with an end concentration of 5.80 ± 0.19 g/1 at 40 °C. Analysis by HPLC showed that although glucose was the major product at 4.35 ± 0.12 g/1, cellobiose was also produced in appreciable amounts (1.97 ± 0.71 g/1). The sugar solution was used as a substrate for Saccharomyces cerevisiae DSM 1333 and ethanol was produced at a level of 1.79 ± 0.26 g/1, the presence of which was confirmed by a 600 MHz NMR spectrum. It was found that cellobiose was not fermented by this strain of S. cerevisiae. Certain components of inks (the PAHs phenanthrene and naphthalene) were found to have a slight inhibitory effect (approximately 15% decrease) on the cellulase enzymes at very high concentrations (approximately 600 μg/1 in aqueous medium), while anthracene had no effect. Whole newsprint ink was shown not to sorb glucose. The environmental analysis of the effluents produced showed that in order for the effluents to be discharged into an aqueous ecosystem they would have to be diluted up to 200 times. They were also shown to have the potential to cause severe machinery damage if reused without proper treatment.
- Full Text:
- Date Issued: 2010
- Authors: Voigt, Paul George
- Date: 2010
- Subjects: Biomass energy , Cellulose -- Biodegradation , Waste paper -- Recycling , Biomass chemicals -- Economic aspects , Renewable energy sources , Fungi -- Biotechnology , Enzymes -- Biotechnology
- Language: English
- Type: Thesis , MSc , Masters
- Identifier: vital:3861 , http://hdl.handle.net/10962/d1013447
- Description: Bioethanol is likely to be a large contributor to the fuel sector of industry in the near future. Current research trends are geared towards utilizing food crops as substrate for bioethanol fermentation; however, this is the source of much controversy. Utilizing food crops for fuel purposes is anticipated to cause massive food shortages worldwide. Cellulose is the most abundant renewable resource on earth and is subject to a wide array of scientific study in order to utilize the glucose contained within it. Waste paper has a high degree of cellulose associated with it, which makes it an ideal target for cellulose biotechnology with the ultimate end goal of bioethanol production. This study focussed on producing the necessary enzymes to hydrolyse the cellulose found in waste paper and using the sugars produced to produce ethanol. The effects of various printing inks had on the production of sugars and the total envirorunental impact of the effluents produced during the production line were also examined. It was found that the fungus Trichoderma longibrachiatum DSM 769 grown in Mandel's medium with waste newspaper as the sole carbon source at 28 °C for 6 days produced extracellular cellulase enzymes with an activity of 0.203 ± 0.009 FPU.ml⁻¹, significantly higher activity as compared to other paper sources. This extracellular cellulase was used to hydrolyse waste newspaper and office paper, with office paper yielding the highest degree of sugar production with an end concentration of 5.80 ± 0.19 g/1 at 40 °C. Analysis by HPLC showed that although glucose was the major product at 4.35 ± 0.12 g/1, cellobiose was also produced in appreciable amounts (1.97 ± 0.71 g/1). The sugar solution was used as a substrate for Saccharomyces cerevisiae DSM 1333 and ethanol was produced at a level of 1.79 ± 0.26 g/1, the presence of which was confirmed by a 600 MHz NMR spectrum. It was found that cellobiose was not fermented by this strain of S. cerevisiae. Certain components of inks (the PAHs phenanthrene and naphthalene) were found to have a slight inhibitory effect (approximately 15% decrease) on the cellulase enzymes at very high concentrations (approximately 600 μg/1 in aqueous medium), while anthracene had no effect. Whole newsprint ink was shown not to sorb glucose. The environmental analysis of the effluents produced showed that in order for the effluents to be discharged into an aqueous ecosystem they would have to be diluted up to 200 times. They were also shown to have the potential to cause severe machinery damage if reused without proper treatment.
- Full Text:
- Date Issued: 2010
Comparison of the neuroprotective potential of theanine and minocycline
- Authors: Mpofu, Tariro Ann-Maureen
- Date: 2010 , 2010-09-20
- Subjects: Nervous system -- Degeneration -- Treatment , Tetracyclines , Antibiotics -- Side effects , Theanine -- Evaluation , Drugs -- Administration , Cerebrovascular disease -- Prevention
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3775 , http://hdl.handle.net/10962/d1003253 , Nervous system -- Degeneration -- Treatment , Tetracyclines , Antibiotics -- Side effects , Theanine -- Evaluation , Drugs -- Administration , Cerebrovascular disease -- Prevention
- Description: Stroke is one of the most common causes of disability and death worldwide. The most commonly experienced stroke in the clinical setting is focal ischaemia in which the middle cerebral artery (MCA) is occluded and leads to a complex series of various pathophysiological pathways that ultimately lead to neuronal cell death. Several studies have been conducted on various therapeutic agents in the search for a neuroprotective drug and various animal models have been used to carry out this research. While theanine, a component of green tea and minocycline, a tetracycline antibiotic, have been shown to possess some neuroprotective properties, the mechanisms by which these two agents carry out these effects still remains unclear. The objectives of this study were to investigate the mechanisms by which these drugs carry out these neuroprotective effects and their neuroprotective ability in a MCA occlusion model of focal ischaemia. Ischaemia leads to oxidative stress due to the imbalance of free radicals and the endogenous antioxidant defence system. An antioxidant assay using the stable 2, 2-diphenyl-1-picrylhydrazyl (DPPH●) radical was used to assess the antiradical properties of each drug. It was found that minocycline showed superior antioxidant activity in vitro when compared to theanine. Further studies on the drugs‟ ability to attenuate the Fenton reaction (in which iron catalyses the formation of reactive species) were elucidated using electrochemical analysis, UV/VIS studies, ferrozine and ferritin assays. It was found that minocycline, in contrast to theanine, was able to bind to iron ions and thus potentially prevent the participation of iron in metal catalysed radical reaction. The antioxidant activity of both drugs was further investigated by assessing their effect on cyanide-induced superoxide generation and quinolinic acid (QA)-induced lipid peroxidation (LP). Experimental evidence shows that both drugs had no significant effect on the generation of superoxide in vitro and that there was a significant decrease in LP for minocycline in vitro and theanine in vivo. The metal binding and antioxidant properties were postulated to be a possible mechanism through which these agents reduced lipid peroxidation. A study was conducted to determine the effects of the drugs on the biosynthesis of the neurotoxin, QA and it was found that minocycline increases the levels of holoenzyme activity of tryptophan-2, 3-dioxygenase (TDO) in vitro and that theanine reduces the levels of the same enzyme in vivo after treatment for 10 days. TDO is the enzyme that converts tryptophan to other products that enable enzymatic activity to change it to QA. Minocycline was thought to bring about this effect as it has been shown from preceding experimental studies that it is an effective reducing agent. Theanine on the other hand is hypothesised to bring about a reduction in holoenzyme activity by changing the binding of tryptophan to the enzyme or affecting the radicals that participate in the enzymatic degradation of tryptophan. A focal ischaemic model of stroke was induced by occluding the MCA. Histological examination of the hippocampus post -ischaemia shows a reduction in the size of the infarct after pre-treatment with minocycline only. A further study into the effects of the drugs on the generation of superoxide and on the levels of the endogenous glutathione after a stroke was carried out. Pre-treatment of the animals with either theanine or minocycline showed no significant effects on the generation of the radical species or of the endogenous antioxidant which ruled out these as a mechanism of neuroprotection of both drugs, post-ischaemia.The findings of this study provide novel information on the possible mechanisms by which both theanine and minocycline act to bring about neuroprotection. In particular in this study, pre-treatment with minocycline has shown promise in the focal ischaemic model of stroke.
- Full Text:
- Date Issued: 2010
- Authors: Mpofu, Tariro Ann-Maureen
- Date: 2010 , 2010-09-20
- Subjects: Nervous system -- Degeneration -- Treatment , Tetracyclines , Antibiotics -- Side effects , Theanine -- Evaluation , Drugs -- Administration , Cerebrovascular disease -- Prevention
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3775 , http://hdl.handle.net/10962/d1003253 , Nervous system -- Degeneration -- Treatment , Tetracyclines , Antibiotics -- Side effects , Theanine -- Evaluation , Drugs -- Administration , Cerebrovascular disease -- Prevention
- Description: Stroke is one of the most common causes of disability and death worldwide. The most commonly experienced stroke in the clinical setting is focal ischaemia in which the middle cerebral artery (MCA) is occluded and leads to a complex series of various pathophysiological pathways that ultimately lead to neuronal cell death. Several studies have been conducted on various therapeutic agents in the search for a neuroprotective drug and various animal models have been used to carry out this research. While theanine, a component of green tea and minocycline, a tetracycline antibiotic, have been shown to possess some neuroprotective properties, the mechanisms by which these two agents carry out these effects still remains unclear. The objectives of this study were to investigate the mechanisms by which these drugs carry out these neuroprotective effects and their neuroprotective ability in a MCA occlusion model of focal ischaemia. Ischaemia leads to oxidative stress due to the imbalance of free radicals and the endogenous antioxidant defence system. An antioxidant assay using the stable 2, 2-diphenyl-1-picrylhydrazyl (DPPH●) radical was used to assess the antiradical properties of each drug. It was found that minocycline showed superior antioxidant activity in vitro when compared to theanine. Further studies on the drugs‟ ability to attenuate the Fenton reaction (in which iron catalyses the formation of reactive species) were elucidated using electrochemical analysis, UV/VIS studies, ferrozine and ferritin assays. It was found that minocycline, in contrast to theanine, was able to bind to iron ions and thus potentially prevent the participation of iron in metal catalysed radical reaction. The antioxidant activity of both drugs was further investigated by assessing their effect on cyanide-induced superoxide generation and quinolinic acid (QA)-induced lipid peroxidation (LP). Experimental evidence shows that both drugs had no significant effect on the generation of superoxide in vitro and that there was a significant decrease in LP for minocycline in vitro and theanine in vivo. The metal binding and antioxidant properties were postulated to be a possible mechanism through which these agents reduced lipid peroxidation. A study was conducted to determine the effects of the drugs on the biosynthesis of the neurotoxin, QA and it was found that minocycline increases the levels of holoenzyme activity of tryptophan-2, 3-dioxygenase (TDO) in vitro and that theanine reduces the levels of the same enzyme in vivo after treatment for 10 days. TDO is the enzyme that converts tryptophan to other products that enable enzymatic activity to change it to QA. Minocycline was thought to bring about this effect as it has been shown from preceding experimental studies that it is an effective reducing agent. Theanine on the other hand is hypothesised to bring about a reduction in holoenzyme activity by changing the binding of tryptophan to the enzyme or affecting the radicals that participate in the enzymatic degradation of tryptophan. A focal ischaemic model of stroke was induced by occluding the MCA. Histological examination of the hippocampus post -ischaemia shows a reduction in the size of the infarct after pre-treatment with minocycline only. A further study into the effects of the drugs on the generation of superoxide and on the levels of the endogenous glutathione after a stroke was carried out. Pre-treatment of the animals with either theanine or minocycline showed no significant effects on the generation of the radical species or of the endogenous antioxidant which ruled out these as a mechanism of neuroprotection of both drugs, post-ischaemia.The findings of this study provide novel information on the possible mechanisms by which both theanine and minocycline act to bring about neuroprotection. In particular in this study, pre-treatment with minocycline has shown promise in the focal ischaemic model of stroke.
- Full Text:
- Date Issued: 2010
Development and assessment of minocycline sustained release capsule formulations
- Sachikonye, Tinotenda Chipo Victoria
- Authors: Sachikonye, Tinotenda Chipo Victoria
- Date: 2010
- Subjects: Drugs -- Controlled release , Drugs -- Dosage forms , Capsules (Pharmacy) , Drugs -- Administration , Acne -- Treatment , Tetracyclines , Antibiotics -- Side effects
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3854 , http://hdl.handle.net/10962/d1013127
- Description: The use of minocycline for the treatment of a broad range of systemic infections and for severe acne has been associated with vestibular side effects. The severity of side effects may lead to poor adherence to therapy by patients. The use of sustained release formulations of minocycline that display slow dissolution of minocycline following administration may be beneficial in reducing the incidence and severity of side effects. Therefore, sustained release capsule dosage forms containing 100 mg minocycline (base) were manufactured and assessed for use as sustained release oral dosage forms of minocycline. Minocycline sustained release capsules were manufactured based on matrix technologies using hydroxypropylmethyl cellulose (HPMC) and Compritol® as release retarding polymers. The rate and extent of minocycline release from the capsules was evaluated using USP Apparatus 1 and samples were analysed using a validated High Performance Liquid Chromatographic (HPLC) method with ultraviolet (UV) detection. Differences in the rate and extent of minocycline release from formulations manufactured using HPMC or Compritol® were influenced by the concentration of polymer used in the formulations. The rate and extent of minocycline release was faster and greater when low concentrations of polymer were used in formulations. The effect of different excipients on the release pattern(s) of minocycline and particularly their potential to optimise minocycline release from experimental formulations was investigated. The use of diluents such as lactose and microcrystalline cellulose (MCC) revealed that lactose facilitated minocycline release when HPMC was used as the polymer matrix. In contrast, the use of lactose as diluent resulted in slower release of minocycline from Compritol® based formulations. The addition of sodium starch glycolate to HPMC based formulations resulted in slower release of minocycline than when no sodium starch glycolate was used. Compritol® based formulations were observed to release minocycline faster following addition of sodium starch glycolate and Poloxamer 188 to experimental formulations. In vitro dissolution profiles were compared to a target or reference profile using the difference and similarity factors, ƒ1 and ƒ2 , and a one way analysis of variance (ANOVA). In addition, the mechanism of minocycline release was elucidated following fitting of dissolution data to the Korsmeyer-Peppas, Higuchi and Zero order models. Minocycline release kinetics were best described by the Korsmeyer-Peppas model and the values of the release exponent, n (italics), revealed that drug release was a result of the combined effects of minocycline diffusion through matrices and erosion of the matrices. These in vitro dissolution profiles were better fit to the Higuchi model than to the Zero order model. Two formulations that displayed a fit to the Zero order model were identified for further studies as potential dosage forms for sustained release minocycline.
- Full Text:
- Date Issued: 2010
- Authors: Sachikonye, Tinotenda Chipo Victoria
- Date: 2010
- Subjects: Drugs -- Controlled release , Drugs -- Dosage forms , Capsules (Pharmacy) , Drugs -- Administration , Acne -- Treatment , Tetracyclines , Antibiotics -- Side effects
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3854 , http://hdl.handle.net/10962/d1013127
- Description: The use of minocycline for the treatment of a broad range of systemic infections and for severe acne has been associated with vestibular side effects. The severity of side effects may lead to poor adherence to therapy by patients. The use of sustained release formulations of minocycline that display slow dissolution of minocycline following administration may be beneficial in reducing the incidence and severity of side effects. Therefore, sustained release capsule dosage forms containing 100 mg minocycline (base) were manufactured and assessed for use as sustained release oral dosage forms of minocycline. Minocycline sustained release capsules were manufactured based on matrix technologies using hydroxypropylmethyl cellulose (HPMC) and Compritol® as release retarding polymers. The rate and extent of minocycline release from the capsules was evaluated using USP Apparatus 1 and samples were analysed using a validated High Performance Liquid Chromatographic (HPLC) method with ultraviolet (UV) detection. Differences in the rate and extent of minocycline release from formulations manufactured using HPMC or Compritol® were influenced by the concentration of polymer used in the formulations. The rate and extent of minocycline release was faster and greater when low concentrations of polymer were used in formulations. The effect of different excipients on the release pattern(s) of minocycline and particularly their potential to optimise minocycline release from experimental formulations was investigated. The use of diluents such as lactose and microcrystalline cellulose (MCC) revealed that lactose facilitated minocycline release when HPMC was used as the polymer matrix. In contrast, the use of lactose as diluent resulted in slower release of minocycline from Compritol® based formulations. The addition of sodium starch glycolate to HPMC based formulations resulted in slower release of minocycline than when no sodium starch glycolate was used. Compritol® based formulations were observed to release minocycline faster following addition of sodium starch glycolate and Poloxamer 188 to experimental formulations. In vitro dissolution profiles were compared to a target or reference profile using the difference and similarity factors, ƒ1 and ƒ2 , and a one way analysis of variance (ANOVA). In addition, the mechanism of minocycline release was elucidated following fitting of dissolution data to the Korsmeyer-Peppas, Higuchi and Zero order models. Minocycline release kinetics were best described by the Korsmeyer-Peppas model and the values of the release exponent, n (italics), revealed that drug release was a result of the combined effects of minocycline diffusion through matrices and erosion of the matrices. These in vitro dissolution profiles were better fit to the Higuchi model than to the Zero order model. Two formulations that displayed a fit to the Zero order model were identified for further studies as potential dosage forms for sustained release minocycline.
- Full Text:
- Date Issued: 2010
African traditional medicines-antiretroviral drug interactions: the effect of African potato (Hypoxis hemerocallidea) on the pharmacokinetics of efavirenz in humans
- Authors: Mogatle, Seloi
- Date: 2009
- Subjects: Potatoes -- Africa Potatoes -- Therapeutic use Medicinal plants Traditional medicine AIDS (Disease) -- Treatment HIV infections -- Drug therapy Drug interactions Antiretroviral agents Pharmacokinetics Hypoxidaceae -- Therapeutic use High performance liquid chromatography
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3773 , http://hdl.handle.net/10962/d1003251
- Description: African Potato (Hypoxis hemerocallidea), (AP) is an African traditional medicine (TM) that is commonly used for various nutritional/medicinal purposes and also by people infected with the human immuno deficiency virus HIV and AIDS patients as an immune booster. The use of AP has also been recommended by the former Minister of Health of South Africa for use by HIV positive people. The main phytochemical component of AP is a norlignan glucoside, hypoxoside, and other relatively minor components have also been reported. A recent in vitro study reported the effects of AP extracts, hypoxoside and rooperol (the metabolite of hypoxoside) on human metabolic enzymes such as the cytochrome P450 (CYP450) group of enzymes and also on the transporter protein, p-glycoprotein (P-gp). This research focussed on investigating the clinical significance of those in vitro effects on the pharmacokinetics of efavirenz (EFV) in humans. EFV was chosen as the substrate drug because it is in first-line regimen of treatment of HIV/AIDS in South Africa, and also has been reported to be a substrate for the specific CYP isozymes, 3A4 and 2B6, in common with APs metabolic involvement with 3A4. A high performance liquid chromatography method with ultra-violet detection (HPLC-UV) for the quantitative determination of EFV in plasma was developed and successfully validated according to international standards with good reproducibility, accuracy, recovery, linear response and requisite sensitivity. The preparation of the plasma samples for analysis was effected by using a simple and rapid precipitation method, and the mobile phase consisted of readily available solvents. EFV in plasma samples was found to be stable under the relevant storage conditions studied. The oral dose of AP, administered as a freshly prepared traditional decoction, was standardised based on the hypoxoside content, and the quality of all the AP decoctions was analysed immediately prior to administration, using a validated HPLC-UV method. A single dose, two-phase sequential study was conducted over a period of 31 days in 10 healthy volunteers. The clinical study was approved by the Rhodes University Ethical Standards Committee, and all the participants agreed to the conditions of the study by giving their informed consent. On day 1 of the study, human subjects were administered a 600 mg EFV tablet and blood samples were collected before dosing and at various intervals over a period of 48 hr post dosing. From day 16, a traditionally prepared AP decoction was administered daily at a standardized dose of 15 mg/kg/day per subject until day 30. On day 29, volunteers were administered a single 600 mg dose of EFV as was done on day 1. Plasma samples were harvested immediately after blood sample collection and frozen at -80 ºC until assayed. Geometric mean ratios of relevant pharmacokinetic parameters, Cmax (maximum plasma concentration achieved following dosing) and AUC0-48 (area under the curve of a plot of drug plasma concentrations versus time representing the extent of absorption) of EFV before and after co-administration of 14 successive daily doses of AP were compared and evaluated to determine whether an interaction had occurred. All subjects completed the study and the geometric mean ratios of Cmax and AUC0-48 were 97.30 and 102.82 with corresponding 90% confidence intervals (CIs) of 78.81-120.14% and 89.04-118.80%, respectively. Whereas the acceptance criteria for the ratios of the AUCs fell within the preset 90% CIs indicating no interaction, the Cmax ratios fell outside the limits. Although the protocol was developed in accordance with the United States of America Food & Drug Administration’s Guidance for Drug Interactions, a priori stating that both criteria need to fall within the acceptance limits to indicate no interaction, an argument is presented to waive the Cmax requirement for the declaration of an interaction. As a result, the pharmacokinetic data generated during this study indicated that the effect of AP on the pharmacokinetics of EFV is not clinically significant. Hence, co-administration of AP is unlikely to affect the clinical use of EFV. In summary the objectives of this project were: 1. To develop and validate a suitable HPLC-UV method for the quantitative determination of EFV in plasma. 2. To perform a mini-validation of the determination of hypoxoside for use as a marker in the quality control and standardisation of AP decoctions. 3. To conduct a clinical interaction study in order to determine whether AP affects the pharmacokinetics of EFV following concurrent administration. 4. To apply the validated HPLC-UV method to determine plasma concentrations of EFV in plasma of human subjects. 5. To use appropriate statistical methods and treatments such as a non-compartmental pharmacokinetic analysis to determine the occurrence of an interaction.
- Full Text:
- Date Issued: 2009
- Authors: Mogatle, Seloi
- Date: 2009
- Subjects: Potatoes -- Africa Potatoes -- Therapeutic use Medicinal plants Traditional medicine AIDS (Disease) -- Treatment HIV infections -- Drug therapy Drug interactions Antiretroviral agents Pharmacokinetics Hypoxidaceae -- Therapeutic use High performance liquid chromatography
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3773 , http://hdl.handle.net/10962/d1003251
- Description: African Potato (Hypoxis hemerocallidea), (AP) is an African traditional medicine (TM) that is commonly used for various nutritional/medicinal purposes and also by people infected with the human immuno deficiency virus HIV and AIDS patients as an immune booster. The use of AP has also been recommended by the former Minister of Health of South Africa for use by HIV positive people. The main phytochemical component of AP is a norlignan glucoside, hypoxoside, and other relatively minor components have also been reported. A recent in vitro study reported the effects of AP extracts, hypoxoside and rooperol (the metabolite of hypoxoside) on human metabolic enzymes such as the cytochrome P450 (CYP450) group of enzymes and also on the transporter protein, p-glycoprotein (P-gp). This research focussed on investigating the clinical significance of those in vitro effects on the pharmacokinetics of efavirenz (EFV) in humans. EFV was chosen as the substrate drug because it is in first-line regimen of treatment of HIV/AIDS in South Africa, and also has been reported to be a substrate for the specific CYP isozymes, 3A4 and 2B6, in common with APs metabolic involvement with 3A4. A high performance liquid chromatography method with ultra-violet detection (HPLC-UV) for the quantitative determination of EFV in plasma was developed and successfully validated according to international standards with good reproducibility, accuracy, recovery, linear response and requisite sensitivity. The preparation of the plasma samples for analysis was effected by using a simple and rapid precipitation method, and the mobile phase consisted of readily available solvents. EFV in plasma samples was found to be stable under the relevant storage conditions studied. The oral dose of AP, administered as a freshly prepared traditional decoction, was standardised based on the hypoxoside content, and the quality of all the AP decoctions was analysed immediately prior to administration, using a validated HPLC-UV method. A single dose, two-phase sequential study was conducted over a period of 31 days in 10 healthy volunteers. The clinical study was approved by the Rhodes University Ethical Standards Committee, and all the participants agreed to the conditions of the study by giving their informed consent. On day 1 of the study, human subjects were administered a 600 mg EFV tablet and blood samples were collected before dosing and at various intervals over a period of 48 hr post dosing. From day 16, a traditionally prepared AP decoction was administered daily at a standardized dose of 15 mg/kg/day per subject until day 30. On day 29, volunteers were administered a single 600 mg dose of EFV as was done on day 1. Plasma samples were harvested immediately after blood sample collection and frozen at -80 ºC until assayed. Geometric mean ratios of relevant pharmacokinetic parameters, Cmax (maximum plasma concentration achieved following dosing) and AUC0-48 (area under the curve of a plot of drug plasma concentrations versus time representing the extent of absorption) of EFV before and after co-administration of 14 successive daily doses of AP were compared and evaluated to determine whether an interaction had occurred. All subjects completed the study and the geometric mean ratios of Cmax and AUC0-48 were 97.30 and 102.82 with corresponding 90% confidence intervals (CIs) of 78.81-120.14% and 89.04-118.80%, respectively. Whereas the acceptance criteria for the ratios of the AUCs fell within the preset 90% CIs indicating no interaction, the Cmax ratios fell outside the limits. Although the protocol was developed in accordance with the United States of America Food & Drug Administration’s Guidance for Drug Interactions, a priori stating that both criteria need to fall within the acceptance limits to indicate no interaction, an argument is presented to waive the Cmax requirement for the declaration of an interaction. As a result, the pharmacokinetic data generated during this study indicated that the effect of AP on the pharmacokinetics of EFV is not clinically significant. Hence, co-administration of AP is unlikely to affect the clinical use of EFV. In summary the objectives of this project were: 1. To develop and validate a suitable HPLC-UV method for the quantitative determination of EFV in plasma. 2. To perform a mini-validation of the determination of hypoxoside for use as a marker in the quality control and standardisation of AP decoctions. 3. To conduct a clinical interaction study in order to determine whether AP affects the pharmacokinetics of EFV following concurrent administration. 4. To apply the validated HPLC-UV method to determine plasma concentrations of EFV in plasma of human subjects. 5. To use appropriate statistical methods and treatments such as a non-compartmental pharmacokinetic analysis to determine the occurrence of an interaction.
- Full Text:
- Date Issued: 2009
The investigation of novel marine microorganisms for the production of biologically active metabolites
- Authors: Sunkel, Vanessa Ann
- Date: 2009 , 2013-07-15
- Subjects: Antibiotics , Drugs -- Research , Metabolites , Marine biotechnology , Marine metabolites -- Therapeutic use , Microorganisms -- Effect of drugs on , Penicillium
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3812 , http://hdl.handle.net/10962/d1004579 , Antibiotics , Drugs -- Research , Metabolites , Marine biotechnology , Marine metabolites -- Therapeutic use , Microorganisms -- Effect of drugs on , Penicillium
- Description: New drugs, particularly antibiotics, are urgently required to combat the increasing problem of antibiotic resistant human pathogens. Due to the scarcity of products available today, the pharmaceutical industry is now under pressure to reassess compounds derived from plants, soil and marine organisms. Pharmaceutical companies are showing renewed interest in marine biotechnology as the oceans represent a rich source of both biological and chemical diversity of novel molecular structures with anti-cancer, anti-inflammatory and antibiotic properties. Formerly unexplored locations, such as deep ocean sediments, show great potential as a source of genetically novel microorganisms producing structurally unique secondary metabolites. In this research, a metabolite producing marine Pseudoalteromonas strain, known as AP5, was initially used to develop methods for the detection, optimisation of production and extraction of bioactive metabolites from other potentially novel marine isolates. Two hundred and seventy six (276) marine isolates from water and sediment samples from the Antarctic Ocean and Marion Island were isolated. Ten visually different isolates were screened for bioactivity against Gram-positive and -negative bacteria, fungi and yeast. Three out of the 10 isolates, WL61 , WL 114 and WL 136, appeared to be novel Streptomyces spp. showing activity against different test organisms. Many of these marine microorganisms are difficult to culture in the laboratory, particularly when they are cultivated continuously in shake flasks as they can stop producing bioactive compounds. The cultivation of marine isolates in bioreactors may be a more beneficial process for the optimisation of metabolite production compared to conventional liquid fermentation techniques whereby the solid-liquid-air interface of membrane bioreactors can imitate the natural environment of microbes. The membrane bioreactor system is a stable growth environment with low shear that supports steady-state biofilm growth consisting of a high cell density due to a high mass transfer of nutrients and oxygen to the cells. This approach was employed and isolates WL61, WL114 and WL136 were immobilised onto ceramic membranes using Quorus single fibre bioreactors (SFR). The SFRs were used to establish the most suitable growth medium for continuous secondary metabolite production. The best growth conditions were applied to the Quorus multifibre bioreactor (MFR) for scale up of biologically active metabolites, highlighting the potential of bioreactor technology for use in bioprospecting for isolating and screening novel and known organisms for new and interesting natural products. Furthermore, the Quorus MFR was shown to be suitable for the production of high yields of antimicrobial metabolites and is an efficient new fermentation production system. Purification by HPLC fractionation was used to characterise four major compounds from isolate WL 114 extracts. NMR structure elucidation identified one of the two primary compounds as Bisphenol A. The complete chemical structure for the second potent bioactive compound could not be determined due to the low concentration and volume of material. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 2009
- Authors: Sunkel, Vanessa Ann
- Date: 2009 , 2013-07-15
- Subjects: Antibiotics , Drugs -- Research , Metabolites , Marine biotechnology , Marine metabolites -- Therapeutic use , Microorganisms -- Effect of drugs on , Penicillium
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3812 , http://hdl.handle.net/10962/d1004579 , Antibiotics , Drugs -- Research , Metabolites , Marine biotechnology , Marine metabolites -- Therapeutic use , Microorganisms -- Effect of drugs on , Penicillium
- Description: New drugs, particularly antibiotics, are urgently required to combat the increasing problem of antibiotic resistant human pathogens. Due to the scarcity of products available today, the pharmaceutical industry is now under pressure to reassess compounds derived from plants, soil and marine organisms. Pharmaceutical companies are showing renewed interest in marine biotechnology as the oceans represent a rich source of both biological and chemical diversity of novel molecular structures with anti-cancer, anti-inflammatory and antibiotic properties. Formerly unexplored locations, such as deep ocean sediments, show great potential as a source of genetically novel microorganisms producing structurally unique secondary metabolites. In this research, a metabolite producing marine Pseudoalteromonas strain, known as AP5, was initially used to develop methods for the detection, optimisation of production and extraction of bioactive metabolites from other potentially novel marine isolates. Two hundred and seventy six (276) marine isolates from water and sediment samples from the Antarctic Ocean and Marion Island were isolated. Ten visually different isolates were screened for bioactivity against Gram-positive and -negative bacteria, fungi and yeast. Three out of the 10 isolates, WL61 , WL 114 and WL 136, appeared to be novel Streptomyces spp. showing activity against different test organisms. Many of these marine microorganisms are difficult to culture in the laboratory, particularly when they are cultivated continuously in shake flasks as they can stop producing bioactive compounds. The cultivation of marine isolates in bioreactors may be a more beneficial process for the optimisation of metabolite production compared to conventional liquid fermentation techniques whereby the solid-liquid-air interface of membrane bioreactors can imitate the natural environment of microbes. The membrane bioreactor system is a stable growth environment with low shear that supports steady-state biofilm growth consisting of a high cell density due to a high mass transfer of nutrients and oxygen to the cells. This approach was employed and isolates WL61, WL114 and WL136 were immobilised onto ceramic membranes using Quorus single fibre bioreactors (SFR). The SFRs were used to establish the most suitable growth medium for continuous secondary metabolite production. The best growth conditions were applied to the Quorus multifibre bioreactor (MFR) for scale up of biologically active metabolites, highlighting the potential of bioreactor technology for use in bioprospecting for isolating and screening novel and known organisms for new and interesting natural products. Furthermore, the Quorus MFR was shown to be suitable for the production of high yields of antimicrobial metabolites and is an efficient new fermentation production system. Purification by HPLC fractionation was used to characterise four major compounds from isolate WL 114 extracts. NMR structure elucidation identified one of the two primary compounds as Bisphenol A. The complete chemical structure for the second potent bioactive compound could not be determined due to the low concentration and volume of material. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 2009
An investigation into the neuroprotective and neurotoxic properties of levodopa, dopamine and selegiline
- Authors: Scheepers, Mark Wesley
- Date: 2008
- Subjects: Parkinson's disease , Nervous system -- Degeneration -- Treatment , Neurotoxic agents , Neuroanatomy , Oxidative stress , Pharmacology , Dopamine , Selegiline , Dopaminergic neurons
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3789 , http://hdl.handle.net/10962/d1003267 , Parkinson's disease , Nervous system -- Degeneration -- Treatment , Neurotoxic agents , Neuroanatomy , Oxidative stress , Pharmacology , Dopamine , Selegiline , Dopaminergic neurons
- Description: Parkinson’s disease (PD) is a neurodegenerative disorder characterized by a profound loss of dopaminergic neurons from the substantia nigra (SN). Among the many pathogenic mechanisms thought to be responsible for the demise of these cells, dopamine (DA)-dependent oxidative stress and oxidative damage has taken center stage due to extensive experimental evidence showing that DA-derived reactive oxygen species (ROS) and oxidized DA metabolites are toxic to SN neurons. Despite its being the most efficacious drug for symptom reversal in PD, there is concern that levodopa (LD) may contribute to the neuronal degeneration and progression of PD by enhancing DA concentrations and turnover in surviving dopaminergic neurons. The present study investigates the potential neurotoxic and neuroprotective effects of DA in vitro. These effects are compared to the toxicity and neuroprotective effects observed in the rat striatum after the administration of LD and selegiline (SEL), both of which increase striatal DA levels. The effects of exogenous LD and/or SEL administration on both the oxidative stress caused by increased striatal iron (II) levels and its consequences have also been investigated. 6-Hydroxydopamine (6-OHDA) is a potent neurotoxin used to mimic dopaminergic degeneration in animal models of PD. The formation of 6-OHDA in vivo could destroy central dopaminergic nerve terminals and enhance the progression of PD. Inorganic studies using high performance liquid chromatography with electrochemical detection (HPLC-ECD) show that hydroxyl radicals can react with DA to form 6-OHDA in vitro. SEL results in a significant decrease in the formation of 6-OHDA in vitro, probably as a result of its antioxidant properties. However, the exogenous administration of LD, with or without SEL, either does not lead to the formation of striatal 6-OHDA in vivo or produces concentrations below the detection limit of the assay. This is despite the fact that striatal DA levels in these rats are significantly elevated (two-fold) compared to the control group. The auto-oxidation and monoamine oxidase (MAO)-mediated metabolism of DA causes an increase in the production of superoxide anions in whole rat brain homogenate in vitro. In addition to this, DA is able to enhance the production of hydroxyl radicals by Fenton chemistry (Fe(III)-EDTA/H2O2) in a cell free environment. Treatment with systemic LD elevates the production of striatal superoxide anions, but does not lead to a detectable increase in striatal hydroxyl radical production in vivo. The co-adminstration of SEL with LD is able to prevent the LD induced rise in striatal superoxide levels. It has been found that the presence of DA or 6-OHDA is able to reduce lipid peroxidation in whole rat brain homogenate induced by Fe(II)-EDTA/H2O2 and ascorbate (Fenton system). However, DA and 6-OHDA increase protein oxidation in rat brain homogenate, which is further increased in the presence of the Fenton system. In addition to this, the incubation of rat brain homogenate with DA or 6-OHDA is also accompanied by a significant reduction in the total GSH content of the homogenate. The exogenous administration of LD and/or SEL was found to have no detrimental effects on striatal lipids, proteins or total GSH levels. Systemic LD administration actually had a neuroprotective effect in the striatum by inhibiting iron (II) induced lipid peroxidation. Inorganic studies, including electrochemistry and the ferrozine assay show that DA and 6-OHDA are able to release iron from ferritin, as iron (II), and that DA can bind iron (III), a fact that may easily impede the availability of this metal ion for participation in the Fenton reaction. The binding of iron (III) by DA appears to discard the involvement of the Fenton reaction in the increased production of hydroxyl radicals induced by the addition of DA to mixtures containing Fe(II)-EDTA and hydrogen peroxide. 6-OHDA did not form a metal-ligand complex with iron (II) or iron (III). In addition to the antioxidant activity and MAO-B inhibitory activity of SEL, the iron binding studies show that SEL has weak iron (II) chelating activity and that it can also form complexes with iron (III). This may therefore be another mechanism involved in the neuroprotective action of SEL. The results of the pineal indole metabolism study show that the systemic administration of SEL increases the production of N-acetylserotonin (NAS) by the pineal gland. NAS has been demonstrated to be a potent antioxidant in the brain and protects against 6-OHDA induced toxicity. The results of this study show that DA displays antioxidant properties in relation to lipid eroxidation and exhibits pro-oxidant properties by causing an increase in the production of hydroxyl radicals and superoxide anions, as well as protein oxidation and a loss of total GSH content. Despite the toxic effects of DA in vitro, the treatment of rats with exogenous LD does not cause oxidative stress or oxidative damage. The results also show that LD and SEL have some neuroprotective properties which make these agents useful in the treatment of PD.
- Full Text:
- Date Issued: 2008
- Authors: Scheepers, Mark Wesley
- Date: 2008
- Subjects: Parkinson's disease , Nervous system -- Degeneration -- Treatment , Neurotoxic agents , Neuroanatomy , Oxidative stress , Pharmacology , Dopamine , Selegiline , Dopaminergic neurons
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3789 , http://hdl.handle.net/10962/d1003267 , Parkinson's disease , Nervous system -- Degeneration -- Treatment , Neurotoxic agents , Neuroanatomy , Oxidative stress , Pharmacology , Dopamine , Selegiline , Dopaminergic neurons
- Description: Parkinson’s disease (PD) is a neurodegenerative disorder characterized by a profound loss of dopaminergic neurons from the substantia nigra (SN). Among the many pathogenic mechanisms thought to be responsible for the demise of these cells, dopamine (DA)-dependent oxidative stress and oxidative damage has taken center stage due to extensive experimental evidence showing that DA-derived reactive oxygen species (ROS) and oxidized DA metabolites are toxic to SN neurons. Despite its being the most efficacious drug for symptom reversal in PD, there is concern that levodopa (LD) may contribute to the neuronal degeneration and progression of PD by enhancing DA concentrations and turnover in surviving dopaminergic neurons. The present study investigates the potential neurotoxic and neuroprotective effects of DA in vitro. These effects are compared to the toxicity and neuroprotective effects observed in the rat striatum after the administration of LD and selegiline (SEL), both of which increase striatal DA levels. The effects of exogenous LD and/or SEL administration on both the oxidative stress caused by increased striatal iron (II) levels and its consequences have also been investigated. 6-Hydroxydopamine (6-OHDA) is a potent neurotoxin used to mimic dopaminergic degeneration in animal models of PD. The formation of 6-OHDA in vivo could destroy central dopaminergic nerve terminals and enhance the progression of PD. Inorganic studies using high performance liquid chromatography with electrochemical detection (HPLC-ECD) show that hydroxyl radicals can react with DA to form 6-OHDA in vitro. SEL results in a significant decrease in the formation of 6-OHDA in vitro, probably as a result of its antioxidant properties. However, the exogenous administration of LD, with or without SEL, either does not lead to the formation of striatal 6-OHDA in vivo or produces concentrations below the detection limit of the assay. This is despite the fact that striatal DA levels in these rats are significantly elevated (two-fold) compared to the control group. The auto-oxidation and monoamine oxidase (MAO)-mediated metabolism of DA causes an increase in the production of superoxide anions in whole rat brain homogenate in vitro. In addition to this, DA is able to enhance the production of hydroxyl radicals by Fenton chemistry (Fe(III)-EDTA/H2O2) in a cell free environment. Treatment with systemic LD elevates the production of striatal superoxide anions, but does not lead to a detectable increase in striatal hydroxyl radical production in vivo. The co-adminstration of SEL with LD is able to prevent the LD induced rise in striatal superoxide levels. It has been found that the presence of DA or 6-OHDA is able to reduce lipid peroxidation in whole rat brain homogenate induced by Fe(II)-EDTA/H2O2 and ascorbate (Fenton system). However, DA and 6-OHDA increase protein oxidation in rat brain homogenate, which is further increased in the presence of the Fenton system. In addition to this, the incubation of rat brain homogenate with DA or 6-OHDA is also accompanied by a significant reduction in the total GSH content of the homogenate. The exogenous administration of LD and/or SEL was found to have no detrimental effects on striatal lipids, proteins or total GSH levels. Systemic LD administration actually had a neuroprotective effect in the striatum by inhibiting iron (II) induced lipid peroxidation. Inorganic studies, including electrochemistry and the ferrozine assay show that DA and 6-OHDA are able to release iron from ferritin, as iron (II), and that DA can bind iron (III), a fact that may easily impede the availability of this metal ion for participation in the Fenton reaction. The binding of iron (III) by DA appears to discard the involvement of the Fenton reaction in the increased production of hydroxyl radicals induced by the addition of DA to mixtures containing Fe(II)-EDTA and hydrogen peroxide. 6-OHDA did not form a metal-ligand complex with iron (II) or iron (III). In addition to the antioxidant activity and MAO-B inhibitory activity of SEL, the iron binding studies show that SEL has weak iron (II) chelating activity and that it can also form complexes with iron (III). This may therefore be another mechanism involved in the neuroprotective action of SEL. The results of the pineal indole metabolism study show that the systemic administration of SEL increases the production of N-acetylserotonin (NAS) by the pineal gland. NAS has been demonstrated to be a potent antioxidant in the brain and protects against 6-OHDA induced toxicity. The results of this study show that DA displays antioxidant properties in relation to lipid eroxidation and exhibits pro-oxidant properties by causing an increase in the production of hydroxyl radicals and superoxide anions, as well as protein oxidation and a loss of total GSH content. Despite the toxic effects of DA in vitro, the treatment of rats with exogenous LD does not cause oxidative stress or oxidative damage. The results also show that LD and SEL have some neuroprotective properties which make these agents useful in the treatment of PD.
- Full Text:
- Date Issued: 2008
An investigation of the antimicrobial and antifouling properties of marine algal metabolites
- Authors: Mann, Maryssa Gudrun Ailsa
- Date: 2008 , 2013-07-11
- Subjects: Anti-infective agents , Marine metabolites -- Therapeutic use , Marine algae , Pharmacognosy , Fouling , Marine fouling organisms
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3831 , http://hdl.handle.net/10962/d1007465 , Anti-infective agents , Marine metabolites -- Therapeutic use , Marine algae , Pharmacognosy , Fouling , Marine fouling organisms
- Description: Prevention of the accumulation of undesirable biological material i.e. biofouling upon a solid surface requires the use of antifouling systems. The solid surface may be a contact lens, an off shore oil rig or a living organism. When chemicals are employed as a mechanism of defense against biofouling, the agents involved are known as antifouling agents. Marine algae must protect themselves from fouling organisms and it is thought that one of the mechanisms used by these organisms is the production of secondary metabolites with an array of biological activities. In vitro studies have shown numerous compounds isolated from marine algae to possess antibacterial, antifungal and antimacrofouling activity. The aim of this study was to evaluate the secondary metabolite extracts of selected Southern African marine macro-algae as a potential source of compounds that inhibit biofilm formation and that could be used as antifouling agents. In this project, marine macro-algae were collected from various sites along the South African coastline. Their extracts were screened for antimicrobial activity against four ubiquitous microorganisms, Staphylococcus aureus, Klebsiella pneumoniae, Mycobacterium aurm and Candida albicans. Results of screening assays guided the fractionation of two Rhodophyta, Plocamium corallorhiza and Laurencia flexuosa. The algae were fractionated using silica gel column chromatography and compounds were isolated by semi-preparative normal phase HPLC. Compound characterization was performed using UV, IR and advanced one- and two-dimensional NMR (¹H, ¹³C NMR, COSY, HSQC, HMBC and NOESY) spectroscopy and mass spectrometry. Ten halogenated monoterpenes including four members of the small class of halogenated monoterpene aldehydes were isolated from extracts of P. corallorhiza. The compounds isolated included the known compounds 3,4,6,7-tetrachloro-3,7-dimethyl-1-octene; 4,6-dibromo-1, 1-dichloro-3,7 -dimethyl-2E,7 octadiene; 4,8-d ibromo-1,1,7 -trichloro-3, 7-dimethyl-2,5Eoctadiene;1 ,4,8-tribromo-3, 7 -dichloro-3,7-dimethyl-1 E,5E-octadiene; 8-bremo-6, 7-dichloro-3,7-dimethyl-octa-2E,4E-dienal; 4-Bromo-8-chloro-3,7-dimethyl-octa-2E,6E-dienal; 4,6- Dibromo-3,7-dimethyl-octa-2E,7-dienal; 2,4-dichloro-1-(2-chlorovinyl)-1-methyl-5-methylidene-cyclohexane and two new metabolites 4,8-chloro-3,7-dimethyl-2Z,4,6Z-octatrien-1-al and Compound 3.47. Methodology was developed for the chemical derivatization and mass spectrometric analysis of the aldehydic compounds, The aldehyde trapping reagent 0-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride was used to derivatize the molecules, stabilizing them and allowing for their complete characterization. From Laurencia flexuosa a new cuparene sesquiterpene 4-bremo-2-(5-hydroxy-1,2,2- trimethylcyclopent-3-enyl)-5-methylphenol was isolated along with two geometric isomers of the vinyl acetylene bromofucin , An halogenated monoterpene 3S*,4R*-1-bromo-3,4,8-trichloro-9-dichloromethyl-1-E,5-E,7-Z-octatriene was also isolated but was suspected to be a contaminant and an investigation into its biological source revealed that it originated from Plocamium suhrii. A third alga, Martensia elegans was extracted based on published reports of antimicrobial compounds in related species. A new a-alkyl malate derivative was isolated and characterized. Selected compounds isolated during the course of the study were employed in preliminary assays that tested their ability to inhibit biofilm formation by Pseudomonas aeruginosa. The halogenated monoterpenes isolated from the Plocamium species were the only active compounds. 3S*,4R*-1-bromo-3,4,S-trichloro-g-dichloromethyl-1-E,5-E,7-octatriene from P. suhrii inhibited biofilm formation through antibacterial activity on planktonic cells but could not prevent biofilm formation when employed as a film on the surface of microtitre plate wells. 1,4,8-tribromo-3,7-dichloro-3,7-dimethyl-1E,5E-octadiene and 4,6-dibromo-1,1-dichloro-3,7-dimethyl-2E,7-octadiene inhibited biofilm formation when applied as a film to the microtitre plate wells but had no significant antibacterial activity. No potential antifouling agents were identified in this project but the antimicrobial activity exhibited by the crude algal extracts was highly encouraging and a number of new research areas have been identified. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 2008
- Authors: Mann, Maryssa Gudrun Ailsa
- Date: 2008 , 2013-07-11
- Subjects: Anti-infective agents , Marine metabolites -- Therapeutic use , Marine algae , Pharmacognosy , Fouling , Marine fouling organisms
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3831 , http://hdl.handle.net/10962/d1007465 , Anti-infective agents , Marine metabolites -- Therapeutic use , Marine algae , Pharmacognosy , Fouling , Marine fouling organisms
- Description: Prevention of the accumulation of undesirable biological material i.e. biofouling upon a solid surface requires the use of antifouling systems. The solid surface may be a contact lens, an off shore oil rig or a living organism. When chemicals are employed as a mechanism of defense against biofouling, the agents involved are known as antifouling agents. Marine algae must protect themselves from fouling organisms and it is thought that one of the mechanisms used by these organisms is the production of secondary metabolites with an array of biological activities. In vitro studies have shown numerous compounds isolated from marine algae to possess antibacterial, antifungal and antimacrofouling activity. The aim of this study was to evaluate the secondary metabolite extracts of selected Southern African marine macro-algae as a potential source of compounds that inhibit biofilm formation and that could be used as antifouling agents. In this project, marine macro-algae were collected from various sites along the South African coastline. Their extracts were screened for antimicrobial activity against four ubiquitous microorganisms, Staphylococcus aureus, Klebsiella pneumoniae, Mycobacterium aurm and Candida albicans. Results of screening assays guided the fractionation of two Rhodophyta, Plocamium corallorhiza and Laurencia flexuosa. The algae were fractionated using silica gel column chromatography and compounds were isolated by semi-preparative normal phase HPLC. Compound characterization was performed using UV, IR and advanced one- and two-dimensional NMR (¹H, ¹³C NMR, COSY, HSQC, HMBC and NOESY) spectroscopy and mass spectrometry. Ten halogenated monoterpenes including four members of the small class of halogenated monoterpene aldehydes were isolated from extracts of P. corallorhiza. The compounds isolated included the known compounds 3,4,6,7-tetrachloro-3,7-dimethyl-1-octene; 4,6-dibromo-1, 1-dichloro-3,7 -dimethyl-2E,7 octadiene; 4,8-d ibromo-1,1,7 -trichloro-3, 7-dimethyl-2,5Eoctadiene;1 ,4,8-tribromo-3, 7 -dichloro-3,7-dimethyl-1 E,5E-octadiene; 8-bremo-6, 7-dichloro-3,7-dimethyl-octa-2E,4E-dienal; 4-Bromo-8-chloro-3,7-dimethyl-octa-2E,6E-dienal; 4,6- Dibromo-3,7-dimethyl-octa-2E,7-dienal; 2,4-dichloro-1-(2-chlorovinyl)-1-methyl-5-methylidene-cyclohexane and two new metabolites 4,8-chloro-3,7-dimethyl-2Z,4,6Z-octatrien-1-al and Compound 3.47. Methodology was developed for the chemical derivatization and mass spectrometric analysis of the aldehydic compounds, The aldehyde trapping reagent 0-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride was used to derivatize the molecules, stabilizing them and allowing for their complete characterization. From Laurencia flexuosa a new cuparene sesquiterpene 4-bremo-2-(5-hydroxy-1,2,2- trimethylcyclopent-3-enyl)-5-methylphenol was isolated along with two geometric isomers of the vinyl acetylene bromofucin , An halogenated monoterpene 3S*,4R*-1-bromo-3,4,8-trichloro-9-dichloromethyl-1-E,5-E,7-Z-octatriene was also isolated but was suspected to be a contaminant and an investigation into its biological source revealed that it originated from Plocamium suhrii. A third alga, Martensia elegans was extracted based on published reports of antimicrobial compounds in related species. A new a-alkyl malate derivative was isolated and characterized. Selected compounds isolated during the course of the study were employed in preliminary assays that tested their ability to inhibit biofilm formation by Pseudomonas aeruginosa. The halogenated monoterpenes isolated from the Plocamium species were the only active compounds. 3S*,4R*-1-bromo-3,4,S-trichloro-g-dichloromethyl-1-E,5-E,7-octatriene from P. suhrii inhibited biofilm formation through antibacterial activity on planktonic cells but could not prevent biofilm formation when employed as a film on the surface of microtitre plate wells. 1,4,8-tribromo-3,7-dichloro-3,7-dimethyl-1E,5E-octadiene and 4,6-dibromo-1,1-dichloro-3,7-dimethyl-2E,7-octadiene inhibited biofilm formation when applied as a film to the microtitre plate wells but had no significant antibacterial activity. No potential antifouling agents were identified in this project but the antimicrobial activity exhibited by the crude algal extracts was highly encouraging and a number of new research areas have been identified. , KMBT_363 , Adobe Acrobat 9.54 Paper Capture Plug-in
- Full Text:
- Date Issued: 2008
Isolation and characterization of antiplasmodial metabolites from South African marine alga
- Authors: Afolayan, Anthonia Folake
- Date: 2008
- Subjects: Malaria -- Africa Antimalarials -- Therapeutic use Malaria -- Prevention Malaria -- Drug therapy Marine algae -- Therapeutic use Natural products -- Therapeutic use Plasmodium
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3739 , http://hdl.handle.net/10962/d1003063
- Description: Malaria is one of the three most deadly diseases in Africa. Although there are available treatments, their efficacy has been greatly reduced over the past two decades due to the development of resistance to currently available drugs. This has necessitated the search for new and effective antimalarial agents. This project approached the search for new antimalarial compounds in two ways: (i) by screening natural products isolated from marine algae against the Plasmodium parasite and (ii) by modification of selected isolated active compounds to target 1-deoxY-đ-xylulose 5-phosphate reductoisomerase (DXR), an enzyme found in the nonmevalonate isoprenoid biosynthetic pathway of Plasmodium Jalciparum. It was envisaged that such a compound would exhibit dual action on the Plasmodium parasite. Extracts obtained from 22 marine algae were prefractionated by solvent partitioning and were screened for anti plasmodial activity against the chloroquine sensitive (CQS) P. Jalciparum D 10 strain. Overall, 50% of the algae screened produced at least one crude fraction with activity against P. Jalciparum. Extracts of the algae Sargassum heterophyllum, Plocamium cornutum, Amphiroa ephedrea and Pterosiphonia cloiophylla gave the most promising results. Fractionation of S. heterophyllum afforded three tetraprenyltoluquinols (3.1, 3.2 and 3.5) and an all-trans-fucoxanthin (3.6). Three new compounds (4.5, 4.6 and 4.7) and two known halogenated monoterpenes (4.1 and 4.4) were isolated from P. cornutum. Each of the isolated compounds from both S. heterophyllum and P. cornutum showed antiplasmodial activity with IC₅₀ values ranging from 2.0 - 15.3 μM for S. heterophyllum and 13 - 230 μM for P. cornutum. Attempts to synthetically modify halogenated monoterpene 4.4 by dihydroxylation and phosphorylation in order to inhibit the DXR enzyme was unsuccessful. However, the hemiterpene analogue (5.42) of the halogenated monoterpenes was successfully phosphorylated and dihydroxylated to give compound 5.45 which showed promising activity against DXR. The result obtained indicated that the proposed phosphorylation and dihydroxylation of the halogenated monoterpene 4.4 would result in the synthesis of a potent DXR inhibitor and therefore a potential antimalarial agent with dual mode of action on the Plasmodium parasite.
- Full Text:
- Date Issued: 2008
- Authors: Afolayan, Anthonia Folake
- Date: 2008
- Subjects: Malaria -- Africa Antimalarials -- Therapeutic use Malaria -- Prevention Malaria -- Drug therapy Marine algae -- Therapeutic use Natural products -- Therapeutic use Plasmodium
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3739 , http://hdl.handle.net/10962/d1003063
- Description: Malaria is one of the three most deadly diseases in Africa. Although there are available treatments, their efficacy has been greatly reduced over the past two decades due to the development of resistance to currently available drugs. This has necessitated the search for new and effective antimalarial agents. This project approached the search for new antimalarial compounds in two ways: (i) by screening natural products isolated from marine algae against the Plasmodium parasite and (ii) by modification of selected isolated active compounds to target 1-deoxY-đ-xylulose 5-phosphate reductoisomerase (DXR), an enzyme found in the nonmevalonate isoprenoid biosynthetic pathway of Plasmodium Jalciparum. It was envisaged that such a compound would exhibit dual action on the Plasmodium parasite. Extracts obtained from 22 marine algae were prefractionated by solvent partitioning and were screened for anti plasmodial activity against the chloroquine sensitive (CQS) P. Jalciparum D 10 strain. Overall, 50% of the algae screened produced at least one crude fraction with activity against P. Jalciparum. Extracts of the algae Sargassum heterophyllum, Plocamium cornutum, Amphiroa ephedrea and Pterosiphonia cloiophylla gave the most promising results. Fractionation of S. heterophyllum afforded three tetraprenyltoluquinols (3.1, 3.2 and 3.5) and an all-trans-fucoxanthin (3.6). Three new compounds (4.5, 4.6 and 4.7) and two known halogenated monoterpenes (4.1 and 4.4) were isolated from P. cornutum. Each of the isolated compounds from both S. heterophyllum and P. cornutum showed antiplasmodial activity with IC₅₀ values ranging from 2.0 - 15.3 μM for S. heterophyllum and 13 - 230 μM for P. cornutum. Attempts to synthetically modify halogenated monoterpene 4.4 by dihydroxylation and phosphorylation in order to inhibit the DXR enzyme was unsuccessful. However, the hemiterpene analogue (5.42) of the halogenated monoterpenes was successfully phosphorylated and dihydroxylated to give compound 5.45 which showed promising activity against DXR. The result obtained indicated that the proposed phosphorylation and dihydroxylation of the halogenated monoterpene 4.4 would result in the synthesis of a potent DXR inhibitor and therefore a potential antimalarial agent with dual mode of action on the Plasmodium parasite.
- Full Text:
- Date Issued: 2008
Development and assessment of an oxytocin parenteral dosage form prepared using pluronic ® F127
- Authors: Chaibva, Faith Anesu
- Date: 2007
- Subjects: Oxytocin -- Therapeutic use , Drugs -- Dosage forms , Pregnancy -- Complications -- Management
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3747 , http://hdl.handle.net/10962/d1003225 , Oxytocin -- Therapeutic use , Drugs -- Dosage forms , Pregnancy -- Complications -- Management
- Full Text:
- Date Issued: 2007
- Authors: Chaibva, Faith Anesu
- Date: 2007
- Subjects: Oxytocin -- Therapeutic use , Drugs -- Dosage forms , Pregnancy -- Complications -- Management
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:3747 , http://hdl.handle.net/10962/d1003225 , Oxytocin -- Therapeutic use , Drugs -- Dosage forms , Pregnancy -- Complications -- Management
- Full Text:
- Date Issued: 2007