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
Evaluation of the pharmaceutical availability of erythromycin from topical formulations
- Authors: Mandimika, Nyaradzo
- Date: 2008
- Subjects: Pharmacy -- Research Chromatographic analysis Gel permeation chromatography Gels (Pharmacy) Chemistry, analytic Acne -- Treatment Sebaceous glands -- Diseases -- Treatment Drugs -- Testing Erythromycin -- Bioavailability
- Language: English
- Type: Thesis , Masters , MPharm
- Identifier: vital:3771 , http://hdl.handle.net/10962/d1003249
- Description: Erythromycin (ERY) is a macrolide antibiotic which is used in the treatment of acne vulgaris.Acne is a common skin condition that occurs when the sebaceous glands and hair shafts become infected by the bacteria Propionibacterium acnes. Acne is a chronic condition that may last for years and the severity of the effects of the disease on patients is often undermined especially in third world countries where more emphasis is placed on other more life-threatening diseases. It may cause considerable physical and emotional distress to sufferers along with the possibility of permanent scarring. Although use of topical ERY formulations is not the first line of treatment it has proven to be effective in treating inflammation of skin and skin structures cause by the responsible bacteria. To-date there are a variety of vehicles which are used in preparing topical ERY formulations namely ointment and gel bases, alcoholic solutions and pledgets. All the gel formulations on the market contain hydroxypropyl cellulose, alcohol and water along with the active ingredient(s). However, some gel formulations contain propylene glycol in addition to these excipients an example being Emgel®. Propylene glycol has been shown to affect the penetration of topically applied drugs through the skin suggesting that it would be highly likely that those formulations which contain propylene glycol may release more ERY into the skin following application. With this in mind, two ERY gel formulations were produced which contained different percentages of propylene glycol. According to the FDA guidelines, pharmacokinetic measurements in blood, plasma and/or urine of topical dermatological drug products are not feasible to document bioequivalence since the active ingredient(s) in topical formulations is/are not intended to be absorbed into the systemic circulation and in addition, concentrations in extracutaneous biological tissues would generally not be measurable. This limits determination of bioavailability and assessment of bioequivalence of such products to pharmacodynamic measurements, clinical trials and dermatopharmacokinetic (DPK) measurements such as tape stripping (TS) and microdialysis (MD).TS is a sampling technique which involves sequential removal of layers of the stratum corneum using strips of adhesive tape. This technique has found increasing use in DPK studies for investigation of drug kinetics in the skin following the application of a topical formulation. The technique has also been used as a diagnostic tool in assessing the quality of the stratum corneum in diseased skin. In the current research study, the tape stripping technique was used to investigate the pharmaceutical/biological availability of topical gel formulations containing ERY. MD is another DPK sampling technique which has been used to determine the amount of a topically applied drug that penetrates through the stratum corneum to reach deeper tissues of the skin. The in vivo sampling technique involves the insertion of microdialysis probes beneath the skin surface in the dermal tissue and allows for real-time sampling of the analyte at its target site. Recently in vitro MD has also been successfully used to assess the pharmaceutical availability of a topical corticosteroid, mometesone furoate, from topical formulations. Based on this work, microdialysis was used to determine the pharmaceutical availability of ERY from gel formulations which were developed for use in this research. The results of the pharmaceutical availability of ERY from in vivo tape stripping studies and the in vitro microdialysis studies were compared to establish correlation between the data. Pharmaceutical equivalence and bioequivalence data obtained from the respective studies on the gel formulations were investigated by statistical analysis of the data generated from both the in vitro and in vivo experiments. In summary the objectives of this research were: 1. To develop and validate a high performance liquid chromatography method suitable to analyse ERY concentrations obtained from in vitro microdialysis studies and in vivo tape stripping studies. 2. To prepare two different ERY gel formulations with different percentage content of propylene glycol. 3. To determine the pharmaceutical availability of ERY from two different gel formulations using in vitro microdialysis. 4. To develop and validate a tape stripping technique which could be used to determine percutaneous penetration and bioequivalence of the gel formulations. 5. To compare in vitro microdialysis and in vivo tape stripping data and attempt to establish a correlation between the two different approaches.
- Full Text:
- Date Issued: 2008
- Authors: Mandimika, Nyaradzo
- Date: 2008
- Subjects: Pharmacy -- Research Chromatographic analysis Gel permeation chromatography Gels (Pharmacy) Chemistry, analytic Acne -- Treatment Sebaceous glands -- Diseases -- Treatment Drugs -- Testing Erythromycin -- Bioavailability
- Language: English
- Type: Thesis , Masters , MPharm
- Identifier: vital:3771 , http://hdl.handle.net/10962/d1003249
- Description: Erythromycin (ERY) is a macrolide antibiotic which is used in the treatment of acne vulgaris.Acne is a common skin condition that occurs when the sebaceous glands and hair shafts become infected by the bacteria Propionibacterium acnes. Acne is a chronic condition that may last for years and the severity of the effects of the disease on patients is often undermined especially in third world countries where more emphasis is placed on other more life-threatening diseases. It may cause considerable physical and emotional distress to sufferers along with the possibility of permanent scarring. Although use of topical ERY formulations is not the first line of treatment it has proven to be effective in treating inflammation of skin and skin structures cause by the responsible bacteria. To-date there are a variety of vehicles which are used in preparing topical ERY formulations namely ointment and gel bases, alcoholic solutions and pledgets. All the gel formulations on the market contain hydroxypropyl cellulose, alcohol and water along with the active ingredient(s). However, some gel formulations contain propylene glycol in addition to these excipients an example being Emgel®. Propylene glycol has been shown to affect the penetration of topically applied drugs through the skin suggesting that it would be highly likely that those formulations which contain propylene glycol may release more ERY into the skin following application. With this in mind, two ERY gel formulations were produced which contained different percentages of propylene glycol. According to the FDA guidelines, pharmacokinetic measurements in blood, plasma and/or urine of topical dermatological drug products are not feasible to document bioequivalence since the active ingredient(s) in topical formulations is/are not intended to be absorbed into the systemic circulation and in addition, concentrations in extracutaneous biological tissues would generally not be measurable. This limits determination of bioavailability and assessment of bioequivalence of such products to pharmacodynamic measurements, clinical trials and dermatopharmacokinetic (DPK) measurements such as tape stripping (TS) and microdialysis (MD).TS is a sampling technique which involves sequential removal of layers of the stratum corneum using strips of adhesive tape. This technique has found increasing use in DPK studies for investigation of drug kinetics in the skin following the application of a topical formulation. The technique has also been used as a diagnostic tool in assessing the quality of the stratum corneum in diseased skin. In the current research study, the tape stripping technique was used to investigate the pharmaceutical/biological availability of topical gel formulations containing ERY. MD is another DPK sampling technique which has been used to determine the amount of a topically applied drug that penetrates through the stratum corneum to reach deeper tissues of the skin. The in vivo sampling technique involves the insertion of microdialysis probes beneath the skin surface in the dermal tissue and allows for real-time sampling of the analyte at its target site. Recently in vitro MD has also been successfully used to assess the pharmaceutical availability of a topical corticosteroid, mometesone furoate, from topical formulations. Based on this work, microdialysis was used to determine the pharmaceutical availability of ERY from gel formulations which were developed for use in this research. The results of the pharmaceutical availability of ERY from in vivo tape stripping studies and the in vitro microdialysis studies were compared to establish correlation between the data. Pharmaceutical equivalence and bioequivalence data obtained from the respective studies on the gel formulations were investigated by statistical analysis of the data generated from both the in vitro and in vivo experiments. In summary the objectives of this research were: 1. To develop and validate a high performance liquid chromatography method suitable to analyse ERY concentrations obtained from in vitro microdialysis studies and in vivo tape stripping studies. 2. To prepare two different ERY gel formulations with different percentage content of propylene glycol. 3. To determine the pharmaceutical availability of ERY from two different gel formulations using in vitro microdialysis. 4. To develop and validate a tape stripping technique which could be used to determine percutaneous penetration and bioequivalence of the gel formulations. 5. To compare in vitro microdialysis and in vivo tape stripping data and attempt to establish a correlation between the two different approaches.
- 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
Neuroprotective mechanisms of nevirapine and efavirenz in a model of neurodegeneration
- Authors: Zheve, Georgina Teurai
- Date: 2008
- Subjects: HIV infections -- Treatment AIDS (Disease) -- Treatment AIDS dementia complex -- Treatment Nervous system -- Degeneration -- Treatment Melatonin Neurotoxic agents Quinolinic acid
- Language: English
- Type: Thesis , Masters , MPharm
- Identifier: vital:3807 , http://hdl.handle.net/10962/d1003285
- Description: AIDS Dementia Complex (ADC) is a neurodegenerative disorder implicated in HIV-1 infection that is associated with elevated levels of the neurotoxin, quinolinic acid (QA) which causes a cascade of events to occur, leading to the production of reactive oxygen species (ROS), these being ultimately responsible for oxidative neurotoxicity. In clinical studies, Non-nucleoside reverse transcriptase inhibitors (NNRTIs), efavirenz (EFV) and nevirapine (NVP) have been shown to potentially delay the progressive degeneration of neurons, thus reducing the frequency and neurological deficits associated with ADC. Despite these neuroprotective implications, there is still no biochemical data to demonstrate the mechanisms through which these agents offer neuroprotection. The present study aims to elucidate and further characterize the possible antioxidant and neuroprotective mechanisms of NVP and EFV in vitro and in vivo, using QA-induced neurotoxicity as a model. Research has demonstrated that antioxidants and metal chelators have the ability to offer neuroprotection against free radical induced injury and may be beneficial in the prevention or treatment of neurodegeneration. Hence the antioxidant and metal binding properties of these agents were investigated respectively. Inorganic studies, including the 1, 1-diphenyl-2 picrylhydrazyl (DPPH) assay, show that these agents readily scavenge free radicals in vitro, thus postulating the antioxidant property of these agents. The enhancement of superoxide radical generation and iron mediated Fenton reaction by QA is related to lipid peroxidation in biological systems, the extent of which was assayed using the nitroblue tetrazolium and thiobarbituric acid method respectively. Both agents significantly curtail QA-induced lipid peroxidation and potentially scavenge superoxide anions generated by cyanide in vitro. Furthermore, in vivo results demonstrate the ability of NVP and EFV to protect hippocampal neurons against lipid peroxidation induced by QA and superoxide radicals generated as a consequence thereof. The alleviation of QA-induced oxidative stress in vitro possibly occurs through the binding of iron (II) and / or iron (III), and this argument is further strengthened by the ability of EFV and not NVP to reduce iron (II)-induced lipid peroxidation in vitro directly. In addition the ferrozine and electrochemistry assay were used to measure the extent of iron (II) Fe[superscript 2+] and iron (III) Fe[superscript 3+] chelation activity. Both assays demonstrate that these agents bind iron (II) and iron (III), and prevent redox recycling of iron and subsequent complexation of Fe[superscript 2+] with QA which enhances neuronal damage. Both NNRTIs inhibit the endogenous biosynthesis of QA by inhibiting liver tryptophan 2, 3-dioxygenase activity in vivo and subsequently increasing hippocampal serotonin levels. Furthermore, these agents reduce the turnover of hippocampal serotonin to 5-hydroxyindole acetic acid. NVP and not EFV increase 5-hydroxyindole acetic acid and norepinephrine levels in the hippocampus. The results of the pineal indole metabolism study show that NVP increases the synthesis of melatonin, but decreases N-acetylserotonin, 5-hydroxyindole acetic acid and 5-hydroxytryptophol levels. Furthermore, it shows that EFV decreases 5-hydroxyindole acetic acid and melatonin synthesis. Behavioural studies using a Morris water maze show that the post-treatment of rats with NVP and EFV significantly improves QA-induced spatial memory deficits in the hippocampus. This study therefore provides novel information regarding the neuroprotective mechanisms of NVP and EFV. These findings strengthen the argument that these NNRTIs not only have antiviral effects but possess potential neuroprotective properties, which may contribute to the effectiveness of these drugs in the treatment of ADC.
- Full Text:
- Date Issued: 2008
- Authors: Zheve, Georgina Teurai
- Date: 2008
- Subjects: HIV infections -- Treatment AIDS (Disease) -- Treatment AIDS dementia complex -- Treatment Nervous system -- Degeneration -- Treatment Melatonin Neurotoxic agents Quinolinic acid
- Language: English
- Type: Thesis , Masters , MPharm
- Identifier: vital:3807 , http://hdl.handle.net/10962/d1003285
- Description: AIDS Dementia Complex (ADC) is a neurodegenerative disorder implicated in HIV-1 infection that is associated with elevated levels of the neurotoxin, quinolinic acid (QA) which causes a cascade of events to occur, leading to the production of reactive oxygen species (ROS), these being ultimately responsible for oxidative neurotoxicity. In clinical studies, Non-nucleoside reverse transcriptase inhibitors (NNRTIs), efavirenz (EFV) and nevirapine (NVP) have been shown to potentially delay the progressive degeneration of neurons, thus reducing the frequency and neurological deficits associated with ADC. Despite these neuroprotective implications, there is still no biochemical data to demonstrate the mechanisms through which these agents offer neuroprotection. The present study aims to elucidate and further characterize the possible antioxidant and neuroprotective mechanisms of NVP and EFV in vitro and in vivo, using QA-induced neurotoxicity as a model. Research has demonstrated that antioxidants and metal chelators have the ability to offer neuroprotection against free radical induced injury and may be beneficial in the prevention or treatment of neurodegeneration. Hence the antioxidant and metal binding properties of these agents were investigated respectively. Inorganic studies, including the 1, 1-diphenyl-2 picrylhydrazyl (DPPH) assay, show that these agents readily scavenge free radicals in vitro, thus postulating the antioxidant property of these agents. The enhancement of superoxide radical generation and iron mediated Fenton reaction by QA is related to lipid peroxidation in biological systems, the extent of which was assayed using the nitroblue tetrazolium and thiobarbituric acid method respectively. Both agents significantly curtail QA-induced lipid peroxidation and potentially scavenge superoxide anions generated by cyanide in vitro. Furthermore, in vivo results demonstrate the ability of NVP and EFV to protect hippocampal neurons against lipid peroxidation induced by QA and superoxide radicals generated as a consequence thereof. The alleviation of QA-induced oxidative stress in vitro possibly occurs through the binding of iron (II) and / or iron (III), and this argument is further strengthened by the ability of EFV and not NVP to reduce iron (II)-induced lipid peroxidation in vitro directly. In addition the ferrozine and electrochemistry assay were used to measure the extent of iron (II) Fe[superscript 2+] and iron (III) Fe[superscript 3+] chelation activity. Both assays demonstrate that these agents bind iron (II) and iron (III), and prevent redox recycling of iron and subsequent complexation of Fe[superscript 2+] with QA which enhances neuronal damage. Both NNRTIs inhibit the endogenous biosynthesis of QA by inhibiting liver tryptophan 2, 3-dioxygenase activity in vivo and subsequently increasing hippocampal serotonin levels. Furthermore, these agents reduce the turnover of hippocampal serotonin to 5-hydroxyindole acetic acid. NVP and not EFV increase 5-hydroxyindole acetic acid and norepinephrine levels in the hippocampus. The results of the pineal indole metabolism study show that NVP increases the synthesis of melatonin, but decreases N-acetylserotonin, 5-hydroxyindole acetic acid and 5-hydroxytryptophol levels. Furthermore, it shows that EFV decreases 5-hydroxyindole acetic acid and melatonin synthesis. Behavioural studies using a Morris water maze show that the post-treatment of rats with NVP and EFV significantly improves QA-induced spatial memory deficits in the hippocampus. This study therefore provides novel information regarding the neuroprotective mechanisms of NVP and EFV. These findings strengthen the argument that these NNRTIs not only have antiviral effects but possess potential neuroprotective properties, which may contribute to the effectiveness of these drugs in the treatment of ADC.
- Full Text:
- Date Issued: 2008
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