Lipid nanocarriers : a novel approach to delivering ophthalmic clarithromycin
- Authors: Makoni, Pedzisai Anotida
- Date: 2021
- Subjects: Clarithromycin , Nanomedicine , Nanostructures , Antibiotics , Eye -- Diseases -- Treatment , Ocular pharmacology , Ophthalmic drugs , Karatitis -- Chemotherapy
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/171678 , vital:42109 , 10.21504/10962/171678
- Description: The feasibility of incorporating clarithromycin (CLA) into innovative solid lipid nanoparticles (SLN) and nanostructured lipi d carriers (NLC) using hot emulsification ultrasonication (HEUS) was investigated. This approach was investigated in an attempt to address the shortcomings associated with the use of lyophilized parenteral formulations administered via the ocular route suc h as toxic reactions, intolerance and patient discomfort due to frequent insti llation of topical solutions of CLA. In particular, sustained release approaches to delivery may enhance precorneal retention, increase ocular availability and permit dose reduction or use of a longer dosing frequency when treating ocular non - tuberculous m ycobacterial (NTM) keratitis infections. This approach may potentially improve the delivery of CLA to the eye, thereby addressing some or all of the unmet clinical needs described vide infra . Prior to initiating pre - formulation, formulation development a nd optimization studies of CLA - loaded SLN and/or NLC, Design of Experiments (DoE), specifically a Central Composite Design (CCD) was used in conjunction with Response Surface Methodology (RSM) to develop and optimize a suitable method for the quantitative determination of CLA in pharmaceutical formulations and for monitoring CLA release from SLN and/or NLC in vitro . A simple, accurate, precise, sensitive and stability - indicating reversed phase - high performance liquid chromatography (RP - HPLC) method with ele ctrochemical (EC) detection was developed, validated and optimized for the in vitro analysis of CLA loaded SLN and/or NLC formulations. Pre - formulation studies were undertaken to investigate the thermal stability of CLA and bulk lipids to facilitate the s election of lipid excipients for the manufacture of nanocarriers in addition to establishing compatibility of CLA with the excipients. It was established that CLA was thermostable up to a temperature of approximately 300 °C thereby indicating that HEUS cou ld be used for the manufacture of CLA - loaded SLN and/or NLC. Lipid screening revealed that CLA i s, in general, poorly soluble in solid and liquid lipids however a combination of stearic acid (SA) and Transcutol ® HP (THP) exhibited the best dissolution pote ntial for CLA of all lipids tested . Stearic acid appears to exist as polymorphic form B prior to exposure to heat however occurs as the form C polymorph following heating at 85 °C for one hour. The best ratio for the mixture of SA and THP for the manufactu re of CLA - NLC ii was an 80:20 ( w/w ) ratio of SA: THP as the two lipids are miscible in this ratio and exhibited the greatest dissolution potential for CLA. Furthermore, an investigation of binary mixtures of CLA/SA and SA/Transcutol ® HP, in addition to eutect ic mixtures of CLA, SA and Transcutol ® HP, revealed no obvious interaction between CLA and the lipids selected for the production of the nanocarriers. Due to the relatively high solubility of CLA in THP in comparison to SA, NLC are likely to exhibit a hig her loading capacity (LC) and encapsulation efficiency (EE) for CLA than SLN. Consequently the feasibility of incorporating CLA (10% w/w ) into NLC was investigated and evaluation of the production of SLN was not undertaken as the production of these might not result in the manufacture of a delivery technology with a high EE and LC for CLA. Tween ® 20 was used as the surfactant as it is readily available, exhibits little or no cytotoxicity and is relatively cheap. Polyethylene glycol (PEG) was used as a coati ng polymer to impart muco - adhesive properties the formulated CLA - NLC. Response surface methodology (RSM) in conjunction with DoE, specifically a Box - Behnken Design (BBD) used as a screening design was used to identify a formulation composition which would produce a product that would meet the pre - defined target critical quality attributes (CQA) for the nanoparticles viz. particle size (PS) in the nano - range, polydispersity index (PDI) < 0.5, Zeta Potential (ZP) ≥ ± 30 mV, and EE > 80%. The formulation composition identified was subsequently used for the optimization of the manufacturing parameters viz. sonication time and amplitude, using a Central Composite Design (CCD) . The LC and EE, in vitro CLA release, cytotoxicity, osmolarity, pH, degree of crystallinity and lipid modification, elemental analysis and surface morphology of the optimized batch was investigated and mon itored to ensure that CLA - loaded NLC, of the desirable quality, had been produced. On the day of manufacture the mean PS and PDI of the optimized CLA - loaded NLC formulation adjusted to physiological osmolarity (250 – 450 mOsm/kg) was 461.9 ± 40.16 nm and 0. 523 ± 0.104, respectively. The ZP for the optimized NLC generated on the day of manufacture using HPLC grade water as the dispersion medium was - 20.5 ± 4.82 mV. The pH and osmolarity of the optimized CLA - loaded NLC formulation was 7.76 ± 0.01 and 316 ± iii 2 m Osm/Kg, respectively and the EE was 88.62 ± 0.23 %. The optimized NLC exhibited a decreased crystallinity in comparison to the bulk lipid materials. DSC, WAXS and FT - IR revealed that CLA was molecularly dispersed in the nanocarriers. The optimized CLA - load ed NLC exhibited muco - adhesive properties, when tested under stationary conditions using laser doppler anemometry (LDA). The optimized formulation also exhibited sustained release of CLA over 24 hours during in vitro release testing and CLA release was bes t described using the Baker - Lonsdale model . The cumulative % CLA released over 24 hours was 56.13 ± 0.23% and mass balance analysis revealed 41.38 ± 0.02% CLA had been retained in the NLC. In vitro cytotoxicity testing revealed that the optimized CLA - NLC w ere less cytotoxic to HeLa cells when compared to CLA alone and further confirmed that the lipids and excipients used in these studies were of GRAS status . Stability studies revealed that the EE reduced over 28 days by 14.42% and 5.14% when stored at 4 °C and 22 °C , respectively. In addition, the particle size increased from the nm to μm range for samples stored at 22 °C. The findings are a good starting point but require further optimization to ensure prolongation of stability. In addition , the technology requires additional developmental studies and a powder for reconstitution for use as a single - dose considered as single dose packaging may be a solution to the compromised formulation stability observed in these studies. The CLA - NLC produced in these stu dies exhibit sound product attributes which serve as a useful foundation for the novel delivery of antibiotics to the eye. The results suggest that the optimized NLC have the potential to enhance precorneal retention and increase ocular availability of CLA , which in turn may be useful to reduce the required dose and dosing frequency when administering CLA as a reconstituted solution to treat susceptible organisms that infect ocular tissues.
- Full Text:
- Date Issued: 2021
- Authors: Makoni, Pedzisai Anotida
- Date: 2021
- Subjects: Clarithromycin , Nanomedicine , Nanostructures , Antibiotics , Eye -- Diseases -- Treatment , Ocular pharmacology , Ophthalmic drugs , Karatitis -- Chemotherapy
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/171678 , vital:42109 , 10.21504/10962/171678
- Description: The feasibility of incorporating clarithromycin (CLA) into innovative solid lipid nanoparticles (SLN) and nanostructured lipi d carriers (NLC) using hot emulsification ultrasonication (HEUS) was investigated. This approach was investigated in an attempt to address the shortcomings associated with the use of lyophilized parenteral formulations administered via the ocular route suc h as toxic reactions, intolerance and patient discomfort due to frequent insti llation of topical solutions of CLA. In particular, sustained release approaches to delivery may enhance precorneal retention, increase ocular availability and permit dose reduction or use of a longer dosing frequency when treating ocular non - tuberculous m ycobacterial (NTM) keratitis infections. This approach may potentially improve the delivery of CLA to the eye, thereby addressing some or all of the unmet clinical needs described vide infra . Prior to initiating pre - formulation, formulation development a nd optimization studies of CLA - loaded SLN and/or NLC, Design of Experiments (DoE), specifically a Central Composite Design (CCD) was used in conjunction with Response Surface Methodology (RSM) to develop and optimize a suitable method for the quantitative determination of CLA in pharmaceutical formulations and for monitoring CLA release from SLN and/or NLC in vitro . A simple, accurate, precise, sensitive and stability - indicating reversed phase - high performance liquid chromatography (RP - HPLC) method with ele ctrochemical (EC) detection was developed, validated and optimized for the in vitro analysis of CLA loaded SLN and/or NLC formulations. Pre - formulation studies were undertaken to investigate the thermal stability of CLA and bulk lipids to facilitate the s election of lipid excipients for the manufacture of nanocarriers in addition to establishing compatibility of CLA with the excipients. It was established that CLA was thermostable up to a temperature of approximately 300 °C thereby indicating that HEUS cou ld be used for the manufacture of CLA - loaded SLN and/or NLC. Lipid screening revealed that CLA i s, in general, poorly soluble in solid and liquid lipids however a combination of stearic acid (SA) and Transcutol ® HP (THP) exhibited the best dissolution pote ntial for CLA of all lipids tested . Stearic acid appears to exist as polymorphic form B prior to exposure to heat however occurs as the form C polymorph following heating at 85 °C for one hour. The best ratio for the mixture of SA and THP for the manufactu re of CLA - NLC ii was an 80:20 ( w/w ) ratio of SA: THP as the two lipids are miscible in this ratio and exhibited the greatest dissolution potential for CLA. Furthermore, an investigation of binary mixtures of CLA/SA and SA/Transcutol ® HP, in addition to eutect ic mixtures of CLA, SA and Transcutol ® HP, revealed no obvious interaction between CLA and the lipids selected for the production of the nanocarriers. Due to the relatively high solubility of CLA in THP in comparison to SA, NLC are likely to exhibit a hig her loading capacity (LC) and encapsulation efficiency (EE) for CLA than SLN. Consequently the feasibility of incorporating CLA (10% w/w ) into NLC was investigated and evaluation of the production of SLN was not undertaken as the production of these might not result in the manufacture of a delivery technology with a high EE and LC for CLA. Tween ® 20 was used as the surfactant as it is readily available, exhibits little or no cytotoxicity and is relatively cheap. Polyethylene glycol (PEG) was used as a coati ng polymer to impart muco - adhesive properties the formulated CLA - NLC. Response surface methodology (RSM) in conjunction with DoE, specifically a Box - Behnken Design (BBD) used as a screening design was used to identify a formulation composition which would produce a product that would meet the pre - defined target critical quality attributes (CQA) for the nanoparticles viz. particle size (PS) in the nano - range, polydispersity index (PDI) < 0.5, Zeta Potential (ZP) ≥ ± 30 mV, and EE > 80%. The formulation composition identified was subsequently used for the optimization of the manufacturing parameters viz. sonication time and amplitude, using a Central Composite Design (CCD) . The LC and EE, in vitro CLA release, cytotoxicity, osmolarity, pH, degree of crystallinity and lipid modification, elemental analysis and surface morphology of the optimized batch was investigated and mon itored to ensure that CLA - loaded NLC, of the desirable quality, had been produced. On the day of manufacture the mean PS and PDI of the optimized CLA - loaded NLC formulation adjusted to physiological osmolarity (250 – 450 mOsm/kg) was 461.9 ± 40.16 nm and 0. 523 ± 0.104, respectively. The ZP for the optimized NLC generated on the day of manufacture using HPLC grade water as the dispersion medium was - 20.5 ± 4.82 mV. The pH and osmolarity of the optimized CLA - loaded NLC formulation was 7.76 ± 0.01 and 316 ± iii 2 m Osm/Kg, respectively and the EE was 88.62 ± 0.23 %. The optimized NLC exhibited a decreased crystallinity in comparison to the bulk lipid materials. DSC, WAXS and FT - IR revealed that CLA was molecularly dispersed in the nanocarriers. The optimized CLA - load ed NLC exhibited muco - adhesive properties, when tested under stationary conditions using laser doppler anemometry (LDA). The optimized formulation also exhibited sustained release of CLA over 24 hours during in vitro release testing and CLA release was bes t described using the Baker - Lonsdale model . The cumulative % CLA released over 24 hours was 56.13 ± 0.23% and mass balance analysis revealed 41.38 ± 0.02% CLA had been retained in the NLC. In vitro cytotoxicity testing revealed that the optimized CLA - NLC w ere less cytotoxic to HeLa cells when compared to CLA alone and further confirmed that the lipids and excipients used in these studies were of GRAS status . Stability studies revealed that the EE reduced over 28 days by 14.42% and 5.14% when stored at 4 °C and 22 °C , respectively. In addition, the particle size increased from the nm to μm range for samples stored at 22 °C. The findings are a good starting point but require further optimization to ensure prolongation of stability. In addition , the technology requires additional developmental studies and a powder for reconstitution for use as a single - dose considered as single dose packaging may be a solution to the compromised formulation stability observed in these studies. The CLA - NLC produced in these stu dies exhibit sound product attributes which serve as a useful foundation for the novel delivery of antibiotics to the eye. The results suggest that the optimized NLC have the potential to enhance precorneal retention and increase ocular availability of CLA , which in turn may be useful to reduce the required dose and dosing frequency when administering CLA as a reconstituted solution to treat susceptible organisms that infect ocular tissues.
- Full Text:
- Date Issued: 2021
Formulation development, manufacture and evaluation of a lamivudine-zidovudine nano co-crystal thermo-responsive suspension
- Authors: Witika, Bwalya Angel
- Date: 2020
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/140546 , vital:37897 , http://dx.doi.org/10.21504/10962/140546
- Description: Expected release date-April 2021
- Full Text:
- Date Issued: 2020
- Authors: Witika, Bwalya Angel
- Date: 2020
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/140546 , vital:37897 , http://dx.doi.org/10.21504/10962/140546
- Description: Expected release date-April 2021
- Full Text:
- Date Issued: 2020
Development and validation of a health literacy measure for limited literacy public sector patients in South Africa
- Authors: Marimwe, Chipiwa
- Date: 2018
- Subjects: Health literacy -- South Africa , Patient education -- South Africa , Communication in medicine -- South Africa , Health literacy -- Social aspects -- South Africa , Poor -- Medical care -- South Africa , Analysis of variance , Multidimensional Screener of Functional Health Literacy (MSFHL)
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/62661 , vital:28227
- Description: The growing complexity of healthcare demands greater patient involvement and skills to navigate this complex system. It has therefore become increasingly important to identify individuals with inadequate health literacy, by using efficient, short and reliable measures for doing so. Most research on the development and validation of health literacy tests has been conducted in high-income countries, with very little reported from low-and middle-income countries (LMICs). Existing health literacy measures have come under scrutiny for their lack of cultural sensitivity, bias towards certain population groups and failure to acknowledge health literacy as a multidimensional concept. These measures usually have limited application in LMICs due to the significantly different structuring of healthcare systems, they overlook the extreme discrepancies in educational levels, and rely too heavily on the ability to read health information. No health literacy data for South Africa are available, and only a few health literacy-based research papers have been published in this country. The aim of the study was to develop and validate a health literacy measure that is contextually and culturally appropriate to measure health literacy in limited literacy public sector patients in South Africa. An Item Bank of 30 questions was developed with the input of a diverse expert consultant panel, and included skills-based and self-reported questions which ensured cultural, contextual and educational level appropriateness. The Information and Support for Health Actions Questionnaire (ISHA-Q) is a health literacy measure developed to assess health literacy for LMICs which includes 14 core scales. These were useful in ensuring coverage of a range of health literacy constructs within the Item Bank. The 30 questions were then allocated to one of three health literacy domains: Procedural knowledge, Factual knowledge and Access to healthcare, health services and social support. Ethical approval for the study was obtained. The questions were translated into isiXhosa and underwent pilot testing. Following pilot testing, 120 isiXhosa first-language speakers, at least 18 years old, who attended public sector facilities and had a maximum 12 years of education were recruited from a primary healthcare clinic in Grahamstown. An interpreter was trained and he participated in all interviews. A questionnaire was used to collect data on the 30-question Item Bank. The Multidimensional Screener of Functional Health Literacy (MSFHL) was used as the primary comparator.The second phase of the study involved the refinement of the 30 questions in the Item Bank, which involved a multi-stage process. Data were analysed statistically using t-test, correlations, chi-square and ANOVA tests at a 5% level of significance, in order to identify problematic questions. Item Response Theory was used to ascertain difficulty and discriminatory ability of the questions. Each question was further subjected to in-depth interrogation by a panel of healthcare professionals to ensure that questions were supported by the conceptual framework and the definitions of health literacy adopted for this study. The number of questions was reduced from 30 to 12, and formed the new Health Literacy Test - Limited Literacy (HELT-LL). To validate the HELT-LL, 210 patients with the same inclusion criteria as previously noted, were recruited from four primary healthcare clinics in the Eastern Cape Province. Individual interviews were conducted with the assistance of the interpreter to collect sociodemographic data as well as data from the HELT-LL, the primary comparator (MSFHL), and a secondary comparator which was a South African modified version of the Newest Vital Sign (NVS-SA). The HELT-LL was re-administered to 40 patients in a follow-up interview two weeks later. The HELT-LL categorised only 17.6% of the patients as having adequate health literacy, just over a third with inadequate health literacy, and the majority with marginal health literacy. Questions in the cognitively demanding Procedural knowledge domain were the most poorly answered, with a mean score of 48.6±24.9%. Patients had great difficulty performing the basic numeric tasks in this domain. The overall mean score for the HELT-LL was 52.8±18.4%, compared with the more cognitively demanding NVS-SA with a mean of 28.6±21.1%, and clearly illustrated the impact of the strategy to include in the HELT-LL a variety of questions with differing cognitive load. The MSFHL, which is based on demographic characteristics and perceived difficulties with reading and writing, had an overall mean score of 44.4±26.2%. Demographic characteristics including age, education and English literacy, were found to be good predictors of limited health literacy, with significant correlations being found between these variables and the mean HELT-LL score. An acceptable value for Cronbach’s alpha, excellent test-retest reliability and excellent concurrent validity show that the HELT-LL is a valid and reliable measure of health literacy in our target population. As there is a paucity of health literacy research emanating from developing countries, this study presents a significant contribution to literature. It is the first study to report the development and validation of a health literacy measure to address the dearth of available health literacy measures applicable for South Africa. If implemented for use in clinical settings and for research purposes, it could provide valuable South African health literacy data which could inform the development of interventions focusing on improving health literacy and health outcomes.
- Full Text:
- Date Issued: 2018
- Authors: Marimwe, Chipiwa
- Date: 2018
- Subjects: Health literacy -- South Africa , Patient education -- South Africa , Communication in medicine -- South Africa , Health literacy -- Social aspects -- South Africa , Poor -- Medical care -- South Africa , Analysis of variance , Multidimensional Screener of Functional Health Literacy (MSFHL)
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/62661 , vital:28227
- Description: The growing complexity of healthcare demands greater patient involvement and skills to navigate this complex system. It has therefore become increasingly important to identify individuals with inadequate health literacy, by using efficient, short and reliable measures for doing so. Most research on the development and validation of health literacy tests has been conducted in high-income countries, with very little reported from low-and middle-income countries (LMICs). Existing health literacy measures have come under scrutiny for their lack of cultural sensitivity, bias towards certain population groups and failure to acknowledge health literacy as a multidimensional concept. These measures usually have limited application in LMICs due to the significantly different structuring of healthcare systems, they overlook the extreme discrepancies in educational levels, and rely too heavily on the ability to read health information. No health literacy data for South Africa are available, and only a few health literacy-based research papers have been published in this country. The aim of the study was to develop and validate a health literacy measure that is contextually and culturally appropriate to measure health literacy in limited literacy public sector patients in South Africa. An Item Bank of 30 questions was developed with the input of a diverse expert consultant panel, and included skills-based and self-reported questions which ensured cultural, contextual and educational level appropriateness. The Information and Support for Health Actions Questionnaire (ISHA-Q) is a health literacy measure developed to assess health literacy for LMICs which includes 14 core scales. These were useful in ensuring coverage of a range of health literacy constructs within the Item Bank. The 30 questions were then allocated to one of three health literacy domains: Procedural knowledge, Factual knowledge and Access to healthcare, health services and social support. Ethical approval for the study was obtained. The questions were translated into isiXhosa and underwent pilot testing. Following pilot testing, 120 isiXhosa first-language speakers, at least 18 years old, who attended public sector facilities and had a maximum 12 years of education were recruited from a primary healthcare clinic in Grahamstown. An interpreter was trained and he participated in all interviews. A questionnaire was used to collect data on the 30-question Item Bank. The Multidimensional Screener of Functional Health Literacy (MSFHL) was used as the primary comparator.The second phase of the study involved the refinement of the 30 questions in the Item Bank, which involved a multi-stage process. Data were analysed statistically using t-test, correlations, chi-square and ANOVA tests at a 5% level of significance, in order to identify problematic questions. Item Response Theory was used to ascertain difficulty and discriminatory ability of the questions. Each question was further subjected to in-depth interrogation by a panel of healthcare professionals to ensure that questions were supported by the conceptual framework and the definitions of health literacy adopted for this study. The number of questions was reduced from 30 to 12, and formed the new Health Literacy Test - Limited Literacy (HELT-LL). To validate the HELT-LL, 210 patients with the same inclusion criteria as previously noted, were recruited from four primary healthcare clinics in the Eastern Cape Province. Individual interviews were conducted with the assistance of the interpreter to collect sociodemographic data as well as data from the HELT-LL, the primary comparator (MSFHL), and a secondary comparator which was a South African modified version of the Newest Vital Sign (NVS-SA). The HELT-LL was re-administered to 40 patients in a follow-up interview two weeks later. The HELT-LL categorised only 17.6% of the patients as having adequate health literacy, just over a third with inadequate health literacy, and the majority with marginal health literacy. Questions in the cognitively demanding Procedural knowledge domain were the most poorly answered, with a mean score of 48.6±24.9%. Patients had great difficulty performing the basic numeric tasks in this domain. The overall mean score for the HELT-LL was 52.8±18.4%, compared with the more cognitively demanding NVS-SA with a mean of 28.6±21.1%, and clearly illustrated the impact of the strategy to include in the HELT-LL a variety of questions with differing cognitive load. The MSFHL, which is based on demographic characteristics and perceived difficulties with reading and writing, had an overall mean score of 44.4±26.2%. Demographic characteristics including age, education and English literacy, were found to be good predictors of limited health literacy, with significant correlations being found between these variables and the mean HELT-LL score. An acceptable value for Cronbach’s alpha, excellent test-retest reliability and excellent concurrent validity show that the HELT-LL is a valid and reliable measure of health literacy in our target population. As there is a paucity of health literacy research emanating from developing countries, this study presents a significant contribution to literature. It is the first study to report the development and validation of a health literacy measure to address the dearth of available health literacy measures applicable for South Africa. If implemented for use in clinical settings and for research purposes, it could provide valuable South African health literacy data which could inform the development of interventions focusing on improving health literacy and health outcomes.
- Full Text:
- Date Issued: 2018
Synthesis and biological evaluation of novel thiazole-based compounds
- Authors: Olawode, Emmanual Oladayo
- Date: 2016
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/62955 , vital:28325
- Description: Thesis embargoed for one-year period. Expected date of release: April 2019
- Full Text:
- Date Issued: 2016
- Authors: Olawode, Emmanual Oladayo
- Date: 2016
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/62955 , vital:28325
- Description: Thesis embargoed for one-year period. Expected date of release: April 2019
- Full Text:
- Date Issued: 2016
Synthesis and biological evaluation of truncated sarganaphthoquinoic acid derivatives as Hsp90 inhibitors
- Authors: Chiwakata, Maynard T
- Date: 2015
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/64708 , vital:28594
- Description: Hsp90 inhibition has been at the centre of attention in current research due to the possibility of “cracking down” on the entire process leading to the development of malignant cancers. Small underlying principles common in all types of cancers have been determined that govern the transformation of normal human cells into cancerous cells, with all relying on the ATPase activity of Hsp90 protein. Hsp90 protein is therefore an attractive drug target that if successfully inhibited can result in the remission of cancer tumours by one form of treatment. To date, no Hsp90 inhibitor has been sanctioned for cancer treatment as most are still in clinical development. Our research was therefore inspired by reports that indicated the potential of quinones / naphthoquinones to act as Hsp90 inhibitors. Preliminary results of a few selected marine natural product quinone systems i.e. sargaquinoic acid (SQA) (2.47) and lapachol (3.6) showed moderate cytotoxicity and weak interactions with the Hsp90 molecular chaperone, and evidence suggested C-terminal binding of these molecules. No correlation has been determined yet between cytotoxicity and Hsp90 inhibition, hence we aimed to develop natural product inspired molecules that exhibit both cytotoxic and Hsp90 inhibition properties. Due to limited amounts of the natural product that can be acquired from natural sources, synthetic analogues were opted for. Isolation of a few selected quinones was conducted to have material that could be used in biological assays. For structural modifications, a series of truncated naphthoquinone systems were prepared adopting the sarganaphthoquinoic acid (3.5) scaffold. The naphthoquinones were prepared via Diels-Alder reactions of relevant benzoquinones with myrcene, followed by aromatization reactions using MnO2. Various alkyl and aryl amines were then coupled to the C-2/3 position of the naphthoquinone using Michael’s addition reactions. Tricyclic naphthoquinones were also synthesized from reactions with hypotaurine and citral. Design of the analogues incorporated functionalities from known Hsp90 inhibitors e.g. geldanamycin (2.28) and its analogues. Preliminary results obtained showed that coupling of naphthoquinones with aryl-amines resulted in the most cytotoxic compounds (4.14-4.19) with IC50 values as low as 0.3 μM against Hs578T breast cancer carcinoma (triple negative). Most of the alkyl amines (4.20-4.25) had IC50 values greater than 50 μM except for 4.20 and 4.21 that showed IC50 values of 7.6 μM and 2.6 μM respectively. Tricyclic naphthoquinones (4.28-4.29) showed moderate cytotoxic activity of approximately 10 μM. Hsp90 inhibition was assessed by client protein degradation assays, of which SQA (2.47), showed the best Hsp90 inhibition properties, followed by compound 4.20. The most cytotoxic arylamino-naphthoquinone (4.16) and tricyclic naphthoquinones (4.28-4.29) showed only moderate inhibition. None of the compounds led to Hsp70 induction, suggesting possible binding to the C-terminus of Hsp90. Interactions at the binding site were assessed by molecular docking studies and saturation transfer difference (STD) NMR. Docking studies were conducted on the N-terminus of Hsp90 and better binding was observed for arylamino naphthoquinones (4.14-4.19) than for other series of compounds. Unfortunately, the co-crystal structure for the C-terminus of Hsp90 is unavailable, hence docking study comparisons on both domains could not be conducted. However, STD NMR offered a platform to assess binding interactions between the naphthoquinones and the N- or C-terminal domains of Hsp90. However no interactions were observed at both the N- and C- termini of Hsp90 due to either weak binding of ligands to the protein or poor water solubility of the ligands. From these preliminary results, naphthoquinones bind to Hsp90 protein but conclusive remarks to which terminal domain they bind to could not be made. The best candidate from amongst the series of naphthoquinones prepared that showed moderate cytotoxicity and promising Hsp90 inhibition was compound 4.20. We therefore succeeded in developing a new series of naphthoquinones that possess moderate cytotoxicity and show Hsp90 inhibition.
- Full Text:
- Date Issued: 2015
- Authors: Chiwakata, Maynard T
- Date: 2015
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/64708 , vital:28594
- Description: Hsp90 inhibition has been at the centre of attention in current research due to the possibility of “cracking down” on the entire process leading to the development of malignant cancers. Small underlying principles common in all types of cancers have been determined that govern the transformation of normal human cells into cancerous cells, with all relying on the ATPase activity of Hsp90 protein. Hsp90 protein is therefore an attractive drug target that if successfully inhibited can result in the remission of cancer tumours by one form of treatment. To date, no Hsp90 inhibitor has been sanctioned for cancer treatment as most are still in clinical development. Our research was therefore inspired by reports that indicated the potential of quinones / naphthoquinones to act as Hsp90 inhibitors. Preliminary results of a few selected marine natural product quinone systems i.e. sargaquinoic acid (SQA) (2.47) and lapachol (3.6) showed moderate cytotoxicity and weak interactions with the Hsp90 molecular chaperone, and evidence suggested C-terminal binding of these molecules. No correlation has been determined yet between cytotoxicity and Hsp90 inhibition, hence we aimed to develop natural product inspired molecules that exhibit both cytotoxic and Hsp90 inhibition properties. Due to limited amounts of the natural product that can be acquired from natural sources, synthetic analogues were opted for. Isolation of a few selected quinones was conducted to have material that could be used in biological assays. For structural modifications, a series of truncated naphthoquinone systems were prepared adopting the sarganaphthoquinoic acid (3.5) scaffold. The naphthoquinones were prepared via Diels-Alder reactions of relevant benzoquinones with myrcene, followed by aromatization reactions using MnO2. Various alkyl and aryl amines were then coupled to the C-2/3 position of the naphthoquinone using Michael’s addition reactions. Tricyclic naphthoquinones were also synthesized from reactions with hypotaurine and citral. Design of the analogues incorporated functionalities from known Hsp90 inhibitors e.g. geldanamycin (2.28) and its analogues. Preliminary results obtained showed that coupling of naphthoquinones with aryl-amines resulted in the most cytotoxic compounds (4.14-4.19) with IC50 values as low as 0.3 μM against Hs578T breast cancer carcinoma (triple negative). Most of the alkyl amines (4.20-4.25) had IC50 values greater than 50 μM except for 4.20 and 4.21 that showed IC50 values of 7.6 μM and 2.6 μM respectively. Tricyclic naphthoquinones (4.28-4.29) showed moderate cytotoxic activity of approximately 10 μM. Hsp90 inhibition was assessed by client protein degradation assays, of which SQA (2.47), showed the best Hsp90 inhibition properties, followed by compound 4.20. The most cytotoxic arylamino-naphthoquinone (4.16) and tricyclic naphthoquinones (4.28-4.29) showed only moderate inhibition. None of the compounds led to Hsp70 induction, suggesting possible binding to the C-terminus of Hsp90. Interactions at the binding site were assessed by molecular docking studies and saturation transfer difference (STD) NMR. Docking studies were conducted on the N-terminus of Hsp90 and better binding was observed for arylamino naphthoquinones (4.14-4.19) than for other series of compounds. Unfortunately, the co-crystal structure for the C-terminus of Hsp90 is unavailable, hence docking study comparisons on both domains could not be conducted. However, STD NMR offered a platform to assess binding interactions between the naphthoquinones and the N- or C-terminal domains of Hsp90. However no interactions were observed at both the N- and C- termini of Hsp90 due to either weak binding of ligands to the protein or poor water solubility of the ligands. From these preliminary results, naphthoquinones bind to Hsp90 protein but conclusive remarks to which terminal domain they bind to could not be made. The best candidate from amongst the series of naphthoquinones prepared that showed moderate cytotoxicity and promising Hsp90 inhibition was compound 4.20. We therefore succeeded in developing a new series of naphthoquinones that possess moderate cytotoxicity and show Hsp90 inhibition.
- Full Text:
- Date Issued: 2015
The isolation, characterisation and chemotaxonomic significance of secondary metabolites from selected South African Laurencia spp. Rhodophyta
- Authors: Fakee, Jameel
- Date: 2015
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/64696 , vital:28593
- Description: Bioprospection of marine organisms as a potential source for lead drugs is becoming increasingly popular. The secondary metabolome of these organisms consists of structurally diverse molecules possessing unprecedented carbon skeletons, the biosynthesis of which occurs via complex metabolomic pathways driven by specialist enzymes. This structural novelty is highly influential on the favourable bioactivity these compounds display. A prominent example of such a compound is trabectedin marketed as Yondelis®. Registered for the treatment of soft tissue sarcomas, this marine drug was developed from extracts of the tunicate Ecteinascidia turbinata. South Africa is renowned for possessing a highly diverse marine biota including several endemic species of marine red algae belonging to the Laurencia sensu stricto genus, which falls within the Laurencia complex. Despite having a good reputation for fascinating secondary metabolites, the taxonomy of Laurencia natural products is proving challenging for reasons including the presence of cryptic species, as well as individual species displaying morphological variability. The aim of this study was thus to isolate secondary metabolites from various South African Laurencia spp. and subsequently assess their chemotaxonomic significance by analysis of a parallel plastid rbcL phylogeny study of Laurencia spp. This study reports the first phycochemical investigation into Laurencia natalensis Kylin, Laurencia cf. corymbosa J.Agardh, Laurencia complanata (Suhr) Kützing, Laurencia sodwaniensis Francis, Bolton, Mattio and Anderson submitted, Laurencia multiclavata Francis, Bolton, Mattio and Anderson submitted, and a South African specimen of Laurenciella marilzae Gil-Rodríguez, Sentíes, Díaz-Larrea, Cassano and M.T. Fujii (basionym: Laurencia marilzae) originally described from Spain. Additionally, the chemical profiles of previously explored species Laurencia flexuosa Kützing and Laurencia glomerata Kützing were re-investigated. The organic extracts of the above species afforded 31 compounds belonging to a wide array of structural classes including halo-chamigranes, linear C15 acetogenins, indole alkaloids, cuparanes and cyclic bromo-ethers. A new tri-cyclic keto-cuparane (4.4) was isolated from L.cf. corymbosa alongside the new cuparanes 4.1 and 4.7. Algoane (5.9), a unique marker compound isolated from L. natalensis, was previously only reported from a sea-hare. Such marker compounds which are exclusive to an individual algal species increase the ease of their subsequent identification. The feasibility of chemotaxonomy as an additional tool to classify Laurencia spp. Was established as broad predictions of a specimen’s phylogeny, based on representatives of its secondary metabolome, proved viable. The study specimens were shown to possess similar chemical profiles to their sister species e.g. L. complanata, L. sodwaniensis and L. multiclavata produced similar metabolites to their sister species as inferred by an rbcL phylogeny tree. Finally, a 1H NMR profiling study on the crude organic extracts of various Laurencia spp. generated distinctive, reproducible spectra, exposing the value of NMR spectroscopy as a rudimentary species discernment tool.
- Full Text:
- Date Issued: 2015
- Authors: Fakee, Jameel
- Date: 2015
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/64696 , vital:28593
- Description: Bioprospection of marine organisms as a potential source for lead drugs is becoming increasingly popular. The secondary metabolome of these organisms consists of structurally diverse molecules possessing unprecedented carbon skeletons, the biosynthesis of which occurs via complex metabolomic pathways driven by specialist enzymes. This structural novelty is highly influential on the favourable bioactivity these compounds display. A prominent example of such a compound is trabectedin marketed as Yondelis®. Registered for the treatment of soft tissue sarcomas, this marine drug was developed from extracts of the tunicate Ecteinascidia turbinata. South Africa is renowned for possessing a highly diverse marine biota including several endemic species of marine red algae belonging to the Laurencia sensu stricto genus, which falls within the Laurencia complex. Despite having a good reputation for fascinating secondary metabolites, the taxonomy of Laurencia natural products is proving challenging for reasons including the presence of cryptic species, as well as individual species displaying morphological variability. The aim of this study was thus to isolate secondary metabolites from various South African Laurencia spp. and subsequently assess their chemotaxonomic significance by analysis of a parallel plastid rbcL phylogeny study of Laurencia spp. This study reports the first phycochemical investigation into Laurencia natalensis Kylin, Laurencia cf. corymbosa J.Agardh, Laurencia complanata (Suhr) Kützing, Laurencia sodwaniensis Francis, Bolton, Mattio and Anderson submitted, Laurencia multiclavata Francis, Bolton, Mattio and Anderson submitted, and a South African specimen of Laurenciella marilzae Gil-Rodríguez, Sentíes, Díaz-Larrea, Cassano and M.T. Fujii (basionym: Laurencia marilzae) originally described from Spain. Additionally, the chemical profiles of previously explored species Laurencia flexuosa Kützing and Laurencia glomerata Kützing were re-investigated. The organic extracts of the above species afforded 31 compounds belonging to a wide array of structural classes including halo-chamigranes, linear C15 acetogenins, indole alkaloids, cuparanes and cyclic bromo-ethers. A new tri-cyclic keto-cuparane (4.4) was isolated from L.cf. corymbosa alongside the new cuparanes 4.1 and 4.7. Algoane (5.9), a unique marker compound isolated from L. natalensis, was previously only reported from a sea-hare. Such marker compounds which are exclusive to an individual algal species increase the ease of their subsequent identification. The feasibility of chemotaxonomy as an additional tool to classify Laurencia spp. Was established as broad predictions of a specimen’s phylogeny, based on representatives of its secondary metabolome, proved viable. The study specimens were shown to possess similar chemical profiles to their sister species e.g. L. complanata, L. sodwaniensis and L. multiclavata produced similar metabolites to their sister species as inferred by an rbcL phylogeny tree. Finally, a 1H NMR profiling study on the crude organic extracts of various Laurencia spp. generated distinctive, reproducible spectra, exposing the value of NMR spectroscopy as a rudimentary species discernment tool.
- Full Text:
- Date Issued: 2015
Adherence to antiretroviral therapy in children in Zimbabwe: a randomized control trial to validate a new self-reported adherence monitoring tool
- Authors: Mugore, Linnetie
- Date: 2014
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/54734 , vital:26607
- Description: Background: Among children taking antiretroviral therapy (ART), self-reports have been widely reported to over-estimate adherence levels. Pill count adherence levels are often lower than self-reported levels, with unannounced home pill count adherence being lower than facility based pill count adherence. There is often poor agreement between pill count adherence levels and those measured using other objective adherence measuring methods such as Medication Event Monitoring Systems (MEMS®), which is widely viewed as the gold standard for adherence measurement. Objectives: The aim of this study was to design and evaluate a new self-reported paediatric adherence monitoring tool, assess the feasibility of using pill count methods in monitoring adherence and identify challenges to reporting adherence among children on ART in rural and urban Zimbabwe. Methods A dual centre, superiority, parallel design RCT was conducted to evaluate the newly-developed visually- and verbally-cued „past 10 days‟ tool for the assessment of adherence in children on ART at two sites in Zimbabwe; Harare Central Children‟s Hospital in an urban setting, and Murambinda Mission Hospital, a rural site. Child-caregiver pairs presenting to one of these facilities for the child‟s review of ART and refill of the medication were recruited, signed informed consent obtained, and were randomised for self-reported adherence monitoring into either the experimental group („new 10-day tool‟) or the control group („PACTG-style‟ self-report tool). Data (demographic, socioeconomic, and reported adherence) were collected in individual interviews with child-caregiver pairs. Additional adherence monitoring methods used for both groups included the Morisky-8-Item Medication Adherence Scale (MMAS-8) and a facility based pill count. FGDs were held with groups of caregivers and groups of children ≥13 years of age to understand reasons for non-adherence as well as issues around reporting non-adherence. Superiority testing was conducted by comparing adherent proportions and their confidence intervals (95% CI). Further concurrent validity test was done using the Mann-Whitney U test to evaluate the relationship between the new tool and the MMAS-8 scores. Agreement between the child and caregiver reports of adherence was used as a test of reliability of the new tool using the kappa statistic. Socio-demographic, clinical and care-related factors associated with adherence were identified using reported adherence in both child and caregiver groups in a logistic regression model. Two pill count methods were assessed for feasibility using the proportions of children with complete data for calculating adherence levels, and their CI and a comparison of the two methods, a routinely-used method and one that incorporated the reported residual quantity (RRQ) of medication at last refill. Results : Analysis included 245 child-caregiver pairs, 123 in the experimental group and 122 in the control group. The median age for children was 9 years. In the experimental group, adherence by caregiver and child reports ranged from 94.3% - 98.4% and 78.4% - 96.1%, and those in the control group ranged from 89.2% - 97.5% and 71.2% - 98.1%, respectively. There was no significant difference between adherence levels in the two groups. Adherence levels measured by both the experimental and control tools were found to be associated with MMAS-8 adherence levels (p <0.05). Agreement between child- and caregiver-reported adherence was moderate though significant (kappa; 0.407, p <0.05). Only about half of the children had adequate data to compute pill counts. Proportions adherent at 95% cut-off were 39% by the „routine pill count‟ and 58% by the „Pill count RRQ‟. Being an orphan was associated with child reported-adherence whereas use of non-human reminders, having a maternal relative as a primary caregiver and knowledge of dose frequency, were all associated with caregiver-reported adherence. Major causes of non-adherence mentioned during the FGDs included interference of medication administration times with scheduling of routine socio-economic activities and lack of support from some non-biological caregivers. Reporting of non-adherence appeared to be hampered by perceptions of negative reactions by healthcare workers to these reports and by caregivers being unaware that the child missed some doses. Conclusions: The „new 10-day tool‟ was not shown to be superior to the „PACTG-style tool‟ in detecting non-adherence, however this new tool was found to be a valid and reliable adherence monitoring tool that included a moderately long recall period of 10 days, can be applied without the need for the respondent to remember names of individual medicines in the
- Full Text:
- Date Issued: 2014
- Authors: Mugore, Linnetie
- Date: 2014
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/54734 , vital:26607
- Description: Background: Among children taking antiretroviral therapy (ART), self-reports have been widely reported to over-estimate adherence levels. Pill count adherence levels are often lower than self-reported levels, with unannounced home pill count adherence being lower than facility based pill count adherence. There is often poor agreement between pill count adherence levels and those measured using other objective adherence measuring methods such as Medication Event Monitoring Systems (MEMS®), which is widely viewed as the gold standard for adherence measurement. Objectives: The aim of this study was to design and evaluate a new self-reported paediatric adherence monitoring tool, assess the feasibility of using pill count methods in monitoring adherence and identify challenges to reporting adherence among children on ART in rural and urban Zimbabwe. Methods A dual centre, superiority, parallel design RCT was conducted to evaluate the newly-developed visually- and verbally-cued „past 10 days‟ tool for the assessment of adherence in children on ART at two sites in Zimbabwe; Harare Central Children‟s Hospital in an urban setting, and Murambinda Mission Hospital, a rural site. Child-caregiver pairs presenting to one of these facilities for the child‟s review of ART and refill of the medication were recruited, signed informed consent obtained, and were randomised for self-reported adherence monitoring into either the experimental group („new 10-day tool‟) or the control group („PACTG-style‟ self-report tool). Data (demographic, socioeconomic, and reported adherence) were collected in individual interviews with child-caregiver pairs. Additional adherence monitoring methods used for both groups included the Morisky-8-Item Medication Adherence Scale (MMAS-8) and a facility based pill count. FGDs were held with groups of caregivers and groups of children ≥13 years of age to understand reasons for non-adherence as well as issues around reporting non-adherence. Superiority testing was conducted by comparing adherent proportions and their confidence intervals (95% CI). Further concurrent validity test was done using the Mann-Whitney U test to evaluate the relationship between the new tool and the MMAS-8 scores. Agreement between the child and caregiver reports of adherence was used as a test of reliability of the new tool using the kappa statistic. Socio-demographic, clinical and care-related factors associated with adherence were identified using reported adherence in both child and caregiver groups in a logistic regression model. Two pill count methods were assessed for feasibility using the proportions of children with complete data for calculating adherence levels, and their CI and a comparison of the two methods, a routinely-used method and one that incorporated the reported residual quantity (RRQ) of medication at last refill. Results : Analysis included 245 child-caregiver pairs, 123 in the experimental group and 122 in the control group. The median age for children was 9 years. In the experimental group, adherence by caregiver and child reports ranged from 94.3% - 98.4% and 78.4% - 96.1%, and those in the control group ranged from 89.2% - 97.5% and 71.2% - 98.1%, respectively. There was no significant difference between adherence levels in the two groups. Adherence levels measured by both the experimental and control tools were found to be associated with MMAS-8 adherence levels (p <0.05). Agreement between child- and caregiver-reported adherence was moderate though significant (kappa; 0.407, p <0.05). Only about half of the children had adequate data to compute pill counts. Proportions adherent at 95% cut-off were 39% by the „routine pill count‟ and 58% by the „Pill count RRQ‟. Being an orphan was associated with child reported-adherence whereas use of non-human reminders, having a maternal relative as a primary caregiver and knowledge of dose frequency, were all associated with caregiver-reported adherence. Major causes of non-adherence mentioned during the FGDs included interference of medication administration times with scheduling of routine socio-economic activities and lack of support from some non-biological caregivers. Reporting of non-adherence appeared to be hampered by perceptions of negative reactions by healthcare workers to these reports and by caregivers being unaware that the child missed some doses. Conclusions: The „new 10-day tool‟ was not shown to be superior to the „PACTG-style tool‟ in detecting non-adherence, however this new tool was found to be a valid and reliable adherence monitoring tool that included a moderately long recall period of 10 days, can be applied without the need for the respondent to remember names of individual medicines in the
- Full Text:
- Date Issued: 2014
The design, synthesis and antiplasmodial activity of a series of halogenated fosmidomycin analogues and hybrid drugs
- Authors: Afolayan, Anthonia Folake
- Date: 2012
- Subjects: Uncatalogued
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/64370 , vital:28538
- Description: Malaria continues to be a devastating disease and a major cause of death in sub-Saharan Africa. With resistance against most of the available antimalarial drugs, there is a need for ongoing research and development of antimalarial agents. Fosmidomycin and its acetyl analogue FR900098 have been identified as potent inhibitors of Plasmodium falciparum, the causative agent of the most deadly form of malaria. Clinical trials of these agents have revealed poor absorption due to their high hydrophilicity. In the present studies the effect of halogenation of the acyl chain as well as the biological effect of extending the acyl sidechain was explored. This provided the basis on which fosmidomycin hybrids were designed to investigate the feasibility of hybrid extending into NADPH binding pocket. Synthesis of a series of halogenated FR900098 analogues was carried out in three stages. This included i) The introduction of the phosphonate group by reaction with 1,3dibromopropane in an Arbuzov reaction, ii) The introduction of a hydroxamate group by reaction of the propyl phosphonate by means of a nucleophilic substitution reaction with BocNHOBn and iii) The introduction of a halogenated acyl side chain on a protected fosmidomycin backbone. The synthesis of fosmidomycin-hybrids for which chloroquinefosmidomycin hybrids were used as the prototype, involved convergence of the two separately constructed moieties i.e. fosmidomycin and the quinoline moieties in a covalent linkage. The quinoline moiety was easily synthesized from the reaction of 4,7dichloroquinoline with 1,2-diamino ethane. The aminoquinoline so formed resulted in chloroquine-fosmidomycin hybrids 3.8 and 3.9 when reacted with halogenated FR900098 analogues. Antiplasmodial assays were conducted on the chloroquine-fosmidomycin hybrids and the halogenated fosmidomycin derivatives against the chloroquine resistant Gambian FCR-3 strain of P. falciparum. The most potent iodoacetyl fosmidomycin analogues 2.21 gave an IC50 value of 5.54 µM which is eight times more potent than the known antiplasmodial FR900098 which gave an IC50 value of 41.67 µM. All the halogenated FR900098 analogues showed better antiplasmodial activity than their non-halogenated derivatives. This indicated that the presence of halogens in the FR900098 analogues contributes to their biological Chapter 1 Literature review activity. The acetyl and propyl linked hybrids 3.8 and 3.9 showed potent antiplasmodial activity with IC50 values of 0.18 and 0.82 µM respectively. These were by far the most potent hybrids synthesized and provided leads for a new class of promising antimalarial agents. Preliminary E. coli DXR enzyme inhibition assays were carried out on the halogenated fosmidomycin analogues. The results showed good inhibition of the enzyme by the phosphonic acids of the chloroacetyl and chloropropyl analogues 2.1 and 2.2 respectively. Molecular modelling of the compounds on E. coli (PDB code: 2EGH) and P. falciparum (PDB code: 3AUA) DXR showed strong binding of the halogenated fosmidomycin analogues while the hybrids in the absence of docked NADPH showed minimum binding to the enzymes.
- Full Text:
- Date Issued: 2012
- Authors: Afolayan, Anthonia Folake
- Date: 2012
- Subjects: Uncatalogued
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/64370 , vital:28538
- Description: Malaria continues to be a devastating disease and a major cause of death in sub-Saharan Africa. With resistance against most of the available antimalarial drugs, there is a need for ongoing research and development of antimalarial agents. Fosmidomycin and its acetyl analogue FR900098 have been identified as potent inhibitors of Plasmodium falciparum, the causative agent of the most deadly form of malaria. Clinical trials of these agents have revealed poor absorption due to their high hydrophilicity. In the present studies the effect of halogenation of the acyl chain as well as the biological effect of extending the acyl sidechain was explored. This provided the basis on which fosmidomycin hybrids were designed to investigate the feasibility of hybrid extending into NADPH binding pocket. Synthesis of a series of halogenated FR900098 analogues was carried out in three stages. This included i) The introduction of the phosphonate group by reaction with 1,3dibromopropane in an Arbuzov reaction, ii) The introduction of a hydroxamate group by reaction of the propyl phosphonate by means of a nucleophilic substitution reaction with BocNHOBn and iii) The introduction of a halogenated acyl side chain on a protected fosmidomycin backbone. The synthesis of fosmidomycin-hybrids for which chloroquinefosmidomycin hybrids were used as the prototype, involved convergence of the two separately constructed moieties i.e. fosmidomycin and the quinoline moieties in a covalent linkage. The quinoline moiety was easily synthesized from the reaction of 4,7dichloroquinoline with 1,2-diamino ethane. The aminoquinoline so formed resulted in chloroquine-fosmidomycin hybrids 3.8 and 3.9 when reacted with halogenated FR900098 analogues. Antiplasmodial assays were conducted on the chloroquine-fosmidomycin hybrids and the halogenated fosmidomycin derivatives against the chloroquine resistant Gambian FCR-3 strain of P. falciparum. The most potent iodoacetyl fosmidomycin analogues 2.21 gave an IC50 value of 5.54 µM which is eight times more potent than the known antiplasmodial FR900098 which gave an IC50 value of 41.67 µM. All the halogenated FR900098 analogues showed better antiplasmodial activity than their non-halogenated derivatives. This indicated that the presence of halogens in the FR900098 analogues contributes to their biological Chapter 1 Literature review activity. The acetyl and propyl linked hybrids 3.8 and 3.9 showed potent antiplasmodial activity with IC50 values of 0.18 and 0.82 µM respectively. These were by far the most potent hybrids synthesized and provided leads for a new class of promising antimalarial agents. Preliminary E. coli DXR enzyme inhibition assays were carried out on the halogenated fosmidomycin analogues. The results showed good inhibition of the enzyme by the phosphonic acids of the chloroacetyl and chloropropyl analogues 2.1 and 2.2 respectively. Molecular modelling of the compounds on E. coli (PDB code: 2EGH) and P. falciparum (PDB code: 3AUA) DXR showed strong binding of the halogenated fosmidomycin analogues while the hybrids in the absence of docked NADPH showed minimum binding to the enzymes.
- Full Text:
- Date Issued: 2012
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