Application of machine learning, molecular modelling and structural data mining against antiretroviral drug resistance in HIV-1
- Sheik Amamuddy, Olivier Serge André
- Authors: Sheik Amamuddy, Olivier Serge André
- Date: 2020
- Subjects: Machine learning , Molecules -- Models , Data mining , Neural networks (Computer science) , Antiretroviral agents , Protease inhibitors , Drug resistance , Multidrug resistance , Molecular dynamics , Renin-angiotensin system , HIV (Viruses) -- South Africa , HIV (Viruses) -- Social aspects -- South Africa , South African Natural Compounds Database
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/115964 , vital:34282
- Description: Millions are affected with the Human Immunodeficiency Virus (HIV) world wide, even though the death toll is on the decline. Antiretrovirals (ARVs), more specifically protease inhibitors have shown tremendous success since their introduction into therapy since the mid 1990’s by slowing down progression to the Acquired Immune Deficiency Syndrome (AIDS). However, Drug Resistance Mutations (DRMs) are constantly selected for due to viral adaptation, making drugs less effective over time. The current challenge is to manage the infection optimally with a limited set of drugs, with differing associated levels of toxicities in the face of a virus that (1) exists as a quasispecies, (2) may transmit acquired DRMs to drug-naive individuals and (3) that can manifest class-wide resistance due to similarities in design. The presence of latent reservoirs, unawareness of infection status, education and various socio-economic factors make the problem even more complex. Adequate timing and choice of drug prescription together with treatment adherence are very important as drug toxicities, drug failure and sub-optimal treatment regimens leave room for further development of drug resistance. While CD4 cell count and the determination of viral load from patients in resource-limited settings are very helpful to track how well a patient’s immune system is able to keep the virus in check, they can be lengthy in determining whether an ARV is effective. Phenosense assay kits answer this problem using viruses engineered to contain the patient sequences and evaluating their growth in the presence of different ARVs, but this can be expensive and too involved for routine checks. As a cheaper and faster alternative, genotypic assays provide similar information from HIV pol sequences obtained from blood samples, inferring ARV efficacy on the basis of drug resistance mutation patterns. However, these are inherently complex and the various methods of in silico prediction, such as Geno2pheno, REGA and Stanford HIVdb do not always agree in every case, even though this gap decreases as the list of resistance mutations is updated. A major gap in HIV treatment is that the information used for predicting drug resistance is mainly computed from data containing an overwhelming majority of B subtype HIV, when these only comprise about 12% of the worldwide HIV infections. In addition to growing evidence that drug resistance is subtype-related, it is intuitive to hypothesize that as subtyping is a phylogenetic classification, the more divergent a subtype is from the strains used in training prediction models, the less their resistance profiles would correlate. For the aforementioned reasons, we used a multi-faceted approach to attack the virus in multiple ways. This research aimed to (1) improve resistance prediction methods by focusing solely on the available subtype, (2) mine structural information pertaining to resistance in order to find any exploitable weak points and increase knowledge of the mechanistic processes of drug resistance in HIV protease. Finally, (3) we screen for protease inhibitors amongst a database of natural compounds [the South African natural compound database (SANCDB)] to find molecules or molecular properties usable to come up with improved inhibition against the drug target. In this work, structural information was mined using the Anisotropic Network Model, Dynamics Cross-Correlation, Perturbation Response Scanning, residue contact network analysis and the radius of gyration. These methods failed to give any resistance-associated patterns in terms of natural movement, internal correlated motions, residue perturbation response, relational behaviour and global compaction respectively. Applications of drug docking, homology-modelling and energy minimization for generating features suitable for machine-learning were not very promising, and rather suggest that the value of binding energies by themselves from Vina may not be very reliable quantitatively. All these failures lead to a refinement that resulted in a highly sensitive statistically-guided network construction and analysis, which leads to key findings in the early dynamics associated with resistance across all PI drugs. The latter experiment unravelled a conserved lateral expansion motion occurring at the flap elbows, and an associated contraction that drives the base of the dimerization domain towards the catalytic site’s floor in the case of drug resistance. Interestingly, we found that despite the conserved movement, bond angles were degenerate. Alongside, 16 Artificial Neural Network models were optimised for HIV proteases and reverse transcriptase inhibitors, with performances on par with Stanford HIVdb. Finally, we prioritised 9 compounds with potential protease inhibitory activity using virtual screening and molecular dynamics (MD) to additionally suggest a promising modification to one of the compounds. This yielded another molecule inhibiting equally well both opened and closed receptor target conformations, whereby each of the compounds had been selected against an array of multi-drug-resistant receptor variants. While a main hurdle was a lack of non-B subtype data, our findings, especially from the statistically-guided network analysis, may extrapolate to a certain extent to them as the level of conservation was very high within subtype B, despite all the present variations. This network construction method lays down a sensitive approach for analysing a pair of alternate phenotypes for which complex patterns prevail, given a sufficient number of experimental units. During the course of research a weighted contact mapping tool was developed to compare renin-angiotensinogen variants and packaged as part of the MD-TASK tool suite. Finally the functionality, compatibility and performance of the MODE-TASK tool were evaluated and confirmed for both Python2.7.x and Python3.x, for the analysis of normals modes from single protein structures and essential modes from MD trajectories. These techniques and tools collectively add onto the conventional means of MD analysis.
- Full Text:
- Date Issued: 2020
- Authors: Sheik Amamuddy, Olivier Serge André
- Date: 2020
- Subjects: Machine learning , Molecules -- Models , Data mining , Neural networks (Computer science) , Antiretroviral agents , Protease inhibitors , Drug resistance , Multidrug resistance , Molecular dynamics , Renin-angiotensin system , HIV (Viruses) -- South Africa , HIV (Viruses) -- Social aspects -- South Africa , South African Natural Compounds Database
- Language: English
- Type: text , Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/115964 , vital:34282
- Description: Millions are affected with the Human Immunodeficiency Virus (HIV) world wide, even though the death toll is on the decline. Antiretrovirals (ARVs), more specifically protease inhibitors have shown tremendous success since their introduction into therapy since the mid 1990’s by slowing down progression to the Acquired Immune Deficiency Syndrome (AIDS). However, Drug Resistance Mutations (DRMs) are constantly selected for due to viral adaptation, making drugs less effective over time. The current challenge is to manage the infection optimally with a limited set of drugs, with differing associated levels of toxicities in the face of a virus that (1) exists as a quasispecies, (2) may transmit acquired DRMs to drug-naive individuals and (3) that can manifest class-wide resistance due to similarities in design. The presence of latent reservoirs, unawareness of infection status, education and various socio-economic factors make the problem even more complex. Adequate timing and choice of drug prescription together with treatment adherence are very important as drug toxicities, drug failure and sub-optimal treatment regimens leave room for further development of drug resistance. While CD4 cell count and the determination of viral load from patients in resource-limited settings are very helpful to track how well a patient’s immune system is able to keep the virus in check, they can be lengthy in determining whether an ARV is effective. Phenosense assay kits answer this problem using viruses engineered to contain the patient sequences and evaluating their growth in the presence of different ARVs, but this can be expensive and too involved for routine checks. As a cheaper and faster alternative, genotypic assays provide similar information from HIV pol sequences obtained from blood samples, inferring ARV efficacy on the basis of drug resistance mutation patterns. However, these are inherently complex and the various methods of in silico prediction, such as Geno2pheno, REGA and Stanford HIVdb do not always agree in every case, even though this gap decreases as the list of resistance mutations is updated. A major gap in HIV treatment is that the information used for predicting drug resistance is mainly computed from data containing an overwhelming majority of B subtype HIV, when these only comprise about 12% of the worldwide HIV infections. In addition to growing evidence that drug resistance is subtype-related, it is intuitive to hypothesize that as subtyping is a phylogenetic classification, the more divergent a subtype is from the strains used in training prediction models, the less their resistance profiles would correlate. For the aforementioned reasons, we used a multi-faceted approach to attack the virus in multiple ways. This research aimed to (1) improve resistance prediction methods by focusing solely on the available subtype, (2) mine structural information pertaining to resistance in order to find any exploitable weak points and increase knowledge of the mechanistic processes of drug resistance in HIV protease. Finally, (3) we screen for protease inhibitors amongst a database of natural compounds [the South African natural compound database (SANCDB)] to find molecules or molecular properties usable to come up with improved inhibition against the drug target. In this work, structural information was mined using the Anisotropic Network Model, Dynamics Cross-Correlation, Perturbation Response Scanning, residue contact network analysis and the radius of gyration. These methods failed to give any resistance-associated patterns in terms of natural movement, internal correlated motions, residue perturbation response, relational behaviour and global compaction respectively. Applications of drug docking, homology-modelling and energy minimization for generating features suitable for machine-learning were not very promising, and rather suggest that the value of binding energies by themselves from Vina may not be very reliable quantitatively. All these failures lead to a refinement that resulted in a highly sensitive statistically-guided network construction and analysis, which leads to key findings in the early dynamics associated with resistance across all PI drugs. The latter experiment unravelled a conserved lateral expansion motion occurring at the flap elbows, and an associated contraction that drives the base of the dimerization domain towards the catalytic site’s floor in the case of drug resistance. Interestingly, we found that despite the conserved movement, bond angles were degenerate. Alongside, 16 Artificial Neural Network models were optimised for HIV proteases and reverse transcriptase inhibitors, with performances on par with Stanford HIVdb. Finally, we prioritised 9 compounds with potential protease inhibitory activity using virtual screening and molecular dynamics (MD) to additionally suggest a promising modification to one of the compounds. This yielded another molecule inhibiting equally well both opened and closed receptor target conformations, whereby each of the compounds had been selected against an array of multi-drug-resistant receptor variants. While a main hurdle was a lack of non-B subtype data, our findings, especially from the statistically-guided network analysis, may extrapolate to a certain extent to them as the level of conservation was very high within subtype B, despite all the present variations. This network construction method lays down a sensitive approach for analysing a pair of alternate phenotypes for which complex patterns prevail, given a sufficient number of experimental units. During the course of research a weighted contact mapping tool was developed to compare renin-angiotensinogen variants and packaged as part of the MD-TASK tool suite. Finally the functionality, compatibility and performance of the MODE-TASK tool were evaluated and confirmed for both Python2.7.x and Python3.x, for the analysis of normals modes from single protein structures and essential modes from MD trajectories. These techniques and tools collectively add onto the conventional means of MD analysis.
- Full Text:
- Date Issued: 2020
Synthesis of novel heterocyclic systems as potential inhibitors of HIV-1 enzymes
- Authors: Sekgota, Khethobole Cassius
- Date: 2020
- Subjects: Protease inhibitors , Heterocyclic compounds , HIV (Viruses) , Quinoline , Amides , Nuclear magnetic resonance , Antiretroviral agents , AIDS vaccines , Nitrobenzaldehyde , Propylphosphonic acid anhydride
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/146502 , vital:38531
- Description: This study has focussed on the application of Baylis-Hillman methodology in the development of efficient synthetic pathways to libraries of novel 3-[(N-cycloalkylbenzamido)methyl]-2-quinolones and indolizine-2-carboxamides and on an exploration of their medicinal potential. The approach to 3-[(N-cycloalkylbenzamido)methyl]-2(1H)-quinolones involved a six-step pathway comprising: Baylis-Hillman reaction of 2-nitrobenzaldehyde derivatives and methyl acrylate to afford nitro-Baylis-Hillman adducts; thermal cyclisation of the adducts to give a range of 3-(acetoxymethyl)-2(1H)-quinolones in good to excellent yields; hydrolysis of the acetates; conversion of the resulting alcohols to the 3-chloromethyl analogues; amination; and, finally, acylation to afford the target amides. Variable temperature NMR methods were used to facilitate analysis of the ¹H and ¹³C NMR spectra which were complicated by internal rotation and cycloalkyl ring-flipping effects. On the other hand, the indolizine-2-carboxamides were obtained in several steps commencing with the Baylis-Hillman reaction of pyridine-2-carboxaldehyde and methyl acrylate. Thermal cyclisation of the Baylis-Hillman adduct afforded indolizine esters, hydrolysis of which gave the corresponding acids which served as precursors to the target indolizine-2-carboxamides. The final amidation step, however, proved to be particularly challenging. Various coupling strategies were explored to access indolizine-2-carboxamides. These included the use of 2,2,2-trifluoroethyl borate which showed limited promise, but propylphosphonic acid anhydride (T3P) proved to be the most effective coupling agent, permitting the formation of 24 novel indolizine-2-carboxamides from hydrazines, aliphatic amines and a range of heterocyclic amines. A high-field NMR-based kinetic study of the mechanism of the Baylis-Hillman reaction of pyridine-4-carboxaldehyde and methyl acrylate in the presence of 3-hydroxyquinuclidine in deuterated chloroform was initiated, reaction progress being followed by the automated collection of ¹H and DEPT 135 NMR spectra over ca. 24 hours using a high-field (600 MHz) NMR instrument. The results have provided critical new insights into the mechanism. NMR analysis has also been used to elucidate the multiplicity of signals associated with rotameric equilibria observed at ambient probe temperature. Variable temperature 1D- and 2D-NMR spectra were used to facilitate the unambiguous characterisation of the 2-quinolone benzamides and some of the indolizine-2-carboxamides. The 3-[(N-cycloalkylbenzamido)methyl]-2(1H)-quinolones, together with selected precursors, and a number of the indolizine-2-carboxamides have been screened in vitro as potential HIV-1 enzyme inhibitors. A survey of the activity of the 2-quinolones against HIV-1 integrase, protease and reverse transcriptase revealed selective inhibition of HIV-1 integrase with the most active IN inhibitor, 3-[(cyclopentylamino)methyl-6-methoxy-2(1H)-quinolone 115e, producing residual enzyme activity of 40% at a concentration of 20 μM. Many of the 2-quinolones exhibited no significant cytotoxicity against HEK 293 cells at 20 μM concentrations. 3-[(N-Cyclohexylamino)methyl]-6-methoxy-2(1H)-quinolone 114e was the only compound to exhibit ant-plasmodial activity (55% pfLDH activity). The survey of indolizine-2-carboxamides also revealed encouraging inhibition against HIV-1 integrase. None of these compounds exhibited cytotoxicity at 20 μM against HEK 293 cells, while a number of them exhibited some activity against Plasmodium falciparum (3D7 strain) and Trypanosoma brucei. Selected indolizine-2-carboxamides exhibited significant anti-tubercular activity in the 7H9 CAS GLU Tx and 7H9 ADC GLU Tw media. In view of the inherent fluorescent character and biological potential of the synthesised indolizine-2-carboxamides, their photophysical properties were explored to establish their possible dual use as bio-imaging and therapeutic agents. The major absorption and corresponding emission bands, and the associated molar absorption coefficients (Ɛ) expressed in the form of log Ɛ were determined. Their high extinction coefficients, large Stokes shift and red-shifted emissions in the visible region indicate their potential for use as fluorophores.
- Full Text:
- Date Issued: 2020
- Authors: Sekgota, Khethobole Cassius
- Date: 2020
- Subjects: Protease inhibitors , Heterocyclic compounds , HIV (Viruses) , Quinoline , Amides , Nuclear magnetic resonance , Antiretroviral agents , AIDS vaccines , Nitrobenzaldehyde , Propylphosphonic acid anhydride
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10962/146502 , vital:38531
- Description: This study has focussed on the application of Baylis-Hillman methodology in the development of efficient synthetic pathways to libraries of novel 3-[(N-cycloalkylbenzamido)methyl]-2-quinolones and indolizine-2-carboxamides and on an exploration of their medicinal potential. The approach to 3-[(N-cycloalkylbenzamido)methyl]-2(1H)-quinolones involved a six-step pathway comprising: Baylis-Hillman reaction of 2-nitrobenzaldehyde derivatives and methyl acrylate to afford nitro-Baylis-Hillman adducts; thermal cyclisation of the adducts to give a range of 3-(acetoxymethyl)-2(1H)-quinolones in good to excellent yields; hydrolysis of the acetates; conversion of the resulting alcohols to the 3-chloromethyl analogues; amination; and, finally, acylation to afford the target amides. Variable temperature NMR methods were used to facilitate analysis of the ¹H and ¹³C NMR spectra which were complicated by internal rotation and cycloalkyl ring-flipping effects. On the other hand, the indolizine-2-carboxamides were obtained in several steps commencing with the Baylis-Hillman reaction of pyridine-2-carboxaldehyde and methyl acrylate. Thermal cyclisation of the Baylis-Hillman adduct afforded indolizine esters, hydrolysis of which gave the corresponding acids which served as precursors to the target indolizine-2-carboxamides. The final amidation step, however, proved to be particularly challenging. Various coupling strategies were explored to access indolizine-2-carboxamides. These included the use of 2,2,2-trifluoroethyl borate which showed limited promise, but propylphosphonic acid anhydride (T3P) proved to be the most effective coupling agent, permitting the formation of 24 novel indolizine-2-carboxamides from hydrazines, aliphatic amines and a range of heterocyclic amines. A high-field NMR-based kinetic study of the mechanism of the Baylis-Hillman reaction of pyridine-4-carboxaldehyde and methyl acrylate in the presence of 3-hydroxyquinuclidine in deuterated chloroform was initiated, reaction progress being followed by the automated collection of ¹H and DEPT 135 NMR spectra over ca. 24 hours using a high-field (600 MHz) NMR instrument. The results have provided critical new insights into the mechanism. NMR analysis has also been used to elucidate the multiplicity of signals associated with rotameric equilibria observed at ambient probe temperature. Variable temperature 1D- and 2D-NMR spectra were used to facilitate the unambiguous characterisation of the 2-quinolone benzamides and some of the indolizine-2-carboxamides. The 3-[(N-cycloalkylbenzamido)methyl]-2(1H)-quinolones, together with selected precursors, and a number of the indolizine-2-carboxamides have been screened in vitro as potential HIV-1 enzyme inhibitors. A survey of the activity of the 2-quinolones against HIV-1 integrase, protease and reverse transcriptase revealed selective inhibition of HIV-1 integrase with the most active IN inhibitor, 3-[(cyclopentylamino)methyl-6-methoxy-2(1H)-quinolone 115e, producing residual enzyme activity of 40% at a concentration of 20 μM. Many of the 2-quinolones exhibited no significant cytotoxicity against HEK 293 cells at 20 μM concentrations. 3-[(N-Cyclohexylamino)methyl]-6-methoxy-2(1H)-quinolone 114e was the only compound to exhibit ant-plasmodial activity (55% pfLDH activity). The survey of indolizine-2-carboxamides also revealed encouraging inhibition against HIV-1 integrase. None of these compounds exhibited cytotoxicity at 20 μM against HEK 293 cells, while a number of them exhibited some activity against Plasmodium falciparum (3D7 strain) and Trypanosoma brucei. Selected indolizine-2-carboxamides exhibited significant anti-tubercular activity in the 7H9 CAS GLU Tx and 7H9 ADC GLU Tw media. In view of the inherent fluorescent character and biological potential of the synthesised indolizine-2-carboxamides, their photophysical properties were explored to establish their possible dual use as bio-imaging and therapeutic agents. The major absorption and corresponding emission bands, and the associated molar absorption coefficients (Ɛ) expressed in the form of log Ɛ were determined. Their high extinction coefficients, large Stokes shift and red-shifted emissions in the visible region indicate their potential for use as fluorophores.
- Full Text:
- Date Issued: 2020
Genetic diversity, resistance profile of hiv and risk assessment of mother-to-child transmission in pregnant women on antiretroviral therapy in the Eastern Cape, South Africa
- Authors: Adeniyi, Oladele Vincent
- Date: 2019
- Subjects: Antiretroviral agents , HIV infections -- Treatment -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10353/15120 , vital:40183
- Description: Despite the initiation of life-long ART in HIV-infected pregnant women, the rate and determinants of infant HIV transmission are not known, especially in the poor resource settings of the Eastern Cape, South Africa. Maternal anti-retroviral therapy (ART) is crucial for elimination of mother-to-child transmission (MTCT) of HIV. However, the inevitable risks of emergence of HIV drug resistance poses significant threat to achieving this goal of HIV-free generation and keeping mothers alive. Also, it is unclear if women with high viral load at delivery have acquired clinically relevant mutations, which could confer resistance to the ART, thus, further increasing the risks of motherto-child transmission of HIV-drug resistance strains. In addition to the gaps identified in the prevention of mother-to-child transmission (PMTCT) context, the understanding of regional epidemics is crucial to the broader epidemiological profiling of HIV infections in the country. Despite the rapid influx of foreign nationals to South African and Eastern Cape Province, there has not been any molecular epidemiological studies profiling the HIV diversity in the Eastern Cape.
- Full Text:
- Date Issued: 2019
- Authors: Adeniyi, Oladele Vincent
- Date: 2019
- Subjects: Antiretroviral agents , HIV infections -- Treatment -- South Africa
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10353/15120 , vital:40183
- Description: Despite the initiation of life-long ART in HIV-infected pregnant women, the rate and determinants of infant HIV transmission are not known, especially in the poor resource settings of the Eastern Cape, South Africa. Maternal anti-retroviral therapy (ART) is crucial for elimination of mother-to-child transmission (MTCT) of HIV. However, the inevitable risks of emergence of HIV drug resistance poses significant threat to achieving this goal of HIV-free generation and keeping mothers alive. Also, it is unclear if women with high viral load at delivery have acquired clinically relevant mutations, which could confer resistance to the ART, thus, further increasing the risks of motherto-child transmission of HIV-drug resistance strains. In addition to the gaps identified in the prevention of mother-to-child transmission (PMTCT) context, the understanding of regional epidemics is crucial to the broader epidemiological profiling of HIV infections in the country. Despite the rapid influx of foreign nationals to South African and Eastern Cape Province, there has not been any molecular epidemiological studies profiling the HIV diversity in the Eastern Cape.
- Full Text:
- Date Issued: 2019
A new synthetic approach for preparation of Efavirenz
- Authors: Chada, Sravanthi
- Date: 2017
- Subjects: Antiretroviral agents , Asymmetric synthesis , Enzyme inhibitors , HIV (Viruses) -- Enzymes
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10948/15512 , vital:28265
- Description: Efavirenz, a drug that is still inaccessible to millions of people worldwide, is potent non nucleoside reverse transcriptase inhibitor (NNRTI), is one of the preferred agents used in combination therapy for first-line treatment of the human immunodeficiency virus (HIV). NNRTIs attach to and block an HIV enzyme called reverse transcriptase, by blocking reverse transcriptase; NNRTIs prevent HIV from multiplying and can reduce the amount of HIV in the body. Efavirenz can't cure HIV/AIDS, but taken in combination with other HIV medicines (called an HIV regimen) every day helps people with HIV live longer healthier lives. Efavirenz also reduces the risk of HIV transmission and can be used by children who are suffering from HIV/AIDS. All the above therapeutic uses of efavirenz prompted us to identify the novel and hopefully cost efficient synthetic methodology for the preparation of efavirenz. In this thesis a new synthetic method for asymmetric synthesis of efavirenz is described. This route started from commercially available starting materials and it is first established in traditional batch chemistry and further the parameters transferred to a semi continuous flow protocol for optimization.
- Full Text:
- Date Issued: 2017
- Authors: Chada, Sravanthi
- Date: 2017
- Subjects: Antiretroviral agents , Asymmetric synthesis , Enzyme inhibitors , HIV (Viruses) -- Enzymes
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: http://hdl.handle.net/10948/15512 , vital:28265
- Description: Efavirenz, a drug that is still inaccessible to millions of people worldwide, is potent non nucleoside reverse transcriptase inhibitor (NNRTI), is one of the preferred agents used in combination therapy for first-line treatment of the human immunodeficiency virus (HIV). NNRTIs attach to and block an HIV enzyme called reverse transcriptase, by blocking reverse transcriptase; NNRTIs prevent HIV from multiplying and can reduce the amount of HIV in the body. Efavirenz can't cure HIV/AIDS, but taken in combination with other HIV medicines (called an HIV regimen) every day helps people with HIV live longer healthier lives. Efavirenz also reduces the risk of HIV transmission and can be used by children who are suffering from HIV/AIDS. All the above therapeutic uses of efavirenz prompted us to identify the novel and hopefully cost efficient synthetic methodology for the preparation of efavirenz. In this thesis a new synthetic method for asymmetric synthesis of efavirenz is described. This route started from commercially available starting materials and it is first established in traditional batch chemistry and further the parameters transferred to a semi continuous flow protocol for optimization.
- Full Text:
- Date Issued: 2017
African traditional medicine-antiretroviral interactions : effects of Sutherlandia frutescens on the pharmacokinetics of Atazanavir
- Authors: Müller, Adrienne Carmel
- Date: 2011 , 2011-03-28
- Subjects: Antiretroviral agents , Medicinal plants , Traditional medicine , AIDS (Disease) -- Treatment , HIV infections -- Drug therapy , Drug interactions , Pharmacokinetics
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3859 , http://hdl.handle.net/10962/d1013373
- Description: In response to the urgent call for investigations into antiretroviral (ARV)-African traditional medicine (ATM) interactions, this research was undertaken to ascertain whether chronic administration of the ATM, Sutherlandia frutescens (SF) may alter the bioavailability of the protease inhibitor (PI), atazanavir (ATV), which may impact on the safety or efficacy of the ARV. Prior to investigating a potential interaction between ATV and SF in vitro and in vivo, a high performance liquid chromatography method with ultraviolet detection (HPLC-UV) was developed and validated for the bioanalysis of ATV in human plasma and liver microsomes. An improved and efficient analytical method with minimal use of solvents and short run time was achieved in comparison to methods published in the literature. In addition, the method was selective, linear, accurate and precise for quantitative analysis of ATV in these studies. Molecular docking studies were conducted to compare the binding modes and affinities of ATV and two major SF constituents, Sutherlandioside B and Sutherlandin C, with the efflux transporter, P-glycoprotein (P-gp) and the CYP450 isoenzyme, CYP3A4 to determine the potential for these phytochemicals to competitively inhibit the binding of ATV to these two proteins, which are mediators of absorption and metabolism. These studies revealed that modulation of P-gp transport of ATV by Sutherlandioside B and Sutherlandin C was not likely to occur via competitive inhibition. The results further indicated that weak competitive inhibition of CYP3A4 may possibly occur in the presence of either of these two SF constituents. The Caco-2 cell line was used as an in vitro model of human intestinal absorption. Accumulation studies in these cells were conducted to ascertain whether extracts and constituents of SF have the ability to alter the absorption of ATV. The results showed that the aqueous extract of SF significantly reduced ATV accumulation, suggesting decreased ATV absorption, whilst a triterpenoid glycoside fraction isolated from SF exhibited an opposing effect. Analogous responses were elicited by the aqueous extract and a triterpenoid glycoside fraction in similar accumulation studies in P-gp overexpressing Madin–Darby Canine Kidney Strain II cells (MDCKII-MDR1), which signified that the effects of this extract and component on ATV transport in the Caco-2 cells were P-gp-mediated. The quantitative analysis of ATV in human liver microsomes after co-incubation with extracts and components of SF was conducted to determine the effects of SF on the metabolism of ATV. The aqueous and methanolic extracts of SF inhibited ATV metabolism, whilst the triterpenoid glycoside fraction had a converse effect. Analogous effects by the extracts were demonstrated in experiments conducted in CYP3A4-transfected microsomes, suggesting that the inhibition of ATV metabolism in the liver microsomes by these SF extracts was CYP3A4-mediated. A combination of Sutherlandiosides C and D also inhibited CYP3A4-mediated ATV metabolism, which was in contrast to the response elicited by the triterpenoid fraction in the liver microsomes, where other unidentified compounds, shown to be present therein, may have contributed to the activation of ATV metabolism. The in vitro studies revealed the potential for SF to alter the bioavailability of ATV, therefore a clinical study in which the effect of a multiple dose regimen of SF on the pharmacokinetics (PK) of a single dose of ATV was conducted in healthy male volunteers. The statistical analysis showed that the 90 % confidence intervals around the geometric mean ratios (ATV + SF/ATV alone) for both Cmax and AUC0-24 hours, fell well below the lower limit of the "no-effect" boundary of 0.8 – 1.25, implying that the bioavailability of ATV was significantly reduced in this cohort of subjects. It may thus be concluded that if the reduction in bioavailability observed in this clinical study is found to be clinically relevant, co-administration of SF commercial dosage forms and ATV in HIV/AIDS patients may potentially result in subtherapeutic ATV levels, which may in turn contribute to ATV resistance and/or treatment failure. This research has therefore highlighted the potential risk for toxicity or lack of efficacy of ARV regimens which may result when ATMs and PIs are used concurrently and that patients and health care practitioners alike should be aware of these perils.
- Full Text:
- Date Issued: 2011
- Authors: Müller, Adrienne Carmel
- Date: 2011 , 2011-03-28
- Subjects: Antiretroviral agents , Medicinal plants , Traditional medicine , AIDS (Disease) -- Treatment , HIV infections -- Drug therapy , Drug interactions , Pharmacokinetics
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:3859 , http://hdl.handle.net/10962/d1013373
- Description: In response to the urgent call for investigations into antiretroviral (ARV)-African traditional medicine (ATM) interactions, this research was undertaken to ascertain whether chronic administration of the ATM, Sutherlandia frutescens (SF) may alter the bioavailability of the protease inhibitor (PI), atazanavir (ATV), which may impact on the safety or efficacy of the ARV. Prior to investigating a potential interaction between ATV and SF in vitro and in vivo, a high performance liquid chromatography method with ultraviolet detection (HPLC-UV) was developed and validated for the bioanalysis of ATV in human plasma and liver microsomes. An improved and efficient analytical method with minimal use of solvents and short run time was achieved in comparison to methods published in the literature. In addition, the method was selective, linear, accurate and precise for quantitative analysis of ATV in these studies. Molecular docking studies were conducted to compare the binding modes and affinities of ATV and two major SF constituents, Sutherlandioside B and Sutherlandin C, with the efflux transporter, P-glycoprotein (P-gp) and the CYP450 isoenzyme, CYP3A4 to determine the potential for these phytochemicals to competitively inhibit the binding of ATV to these two proteins, which are mediators of absorption and metabolism. These studies revealed that modulation of P-gp transport of ATV by Sutherlandioside B and Sutherlandin C was not likely to occur via competitive inhibition. The results further indicated that weak competitive inhibition of CYP3A4 may possibly occur in the presence of either of these two SF constituents. The Caco-2 cell line was used as an in vitro model of human intestinal absorption. Accumulation studies in these cells were conducted to ascertain whether extracts and constituents of SF have the ability to alter the absorption of ATV. The results showed that the aqueous extract of SF significantly reduced ATV accumulation, suggesting decreased ATV absorption, whilst a triterpenoid glycoside fraction isolated from SF exhibited an opposing effect. Analogous responses were elicited by the aqueous extract and a triterpenoid glycoside fraction in similar accumulation studies in P-gp overexpressing Madin–Darby Canine Kidney Strain II cells (MDCKII-MDR1), which signified that the effects of this extract and component on ATV transport in the Caco-2 cells were P-gp-mediated. The quantitative analysis of ATV in human liver microsomes after co-incubation with extracts and components of SF was conducted to determine the effects of SF on the metabolism of ATV. The aqueous and methanolic extracts of SF inhibited ATV metabolism, whilst the triterpenoid glycoside fraction had a converse effect. Analogous effects by the extracts were demonstrated in experiments conducted in CYP3A4-transfected microsomes, suggesting that the inhibition of ATV metabolism in the liver microsomes by these SF extracts was CYP3A4-mediated. A combination of Sutherlandiosides C and D also inhibited CYP3A4-mediated ATV metabolism, which was in contrast to the response elicited by the triterpenoid fraction in the liver microsomes, where other unidentified compounds, shown to be present therein, may have contributed to the activation of ATV metabolism. The in vitro studies revealed the potential for SF to alter the bioavailability of ATV, therefore a clinical study in which the effect of a multiple dose regimen of SF on the pharmacokinetics (PK) of a single dose of ATV was conducted in healthy male volunteers. The statistical analysis showed that the 90 % confidence intervals around the geometric mean ratios (ATV + SF/ATV alone) for both Cmax and AUC0-24 hours, fell well below the lower limit of the "no-effect" boundary of 0.8 – 1.25, implying that the bioavailability of ATV was significantly reduced in this cohort of subjects. It may thus be concluded that if the reduction in bioavailability observed in this clinical study is found to be clinically relevant, co-administration of SF commercial dosage forms and ATV in HIV/AIDS patients may potentially result in subtherapeutic ATV levels, which may in turn contribute to ATV resistance and/or treatment failure. This research has therefore highlighted the potential risk for toxicity or lack of efficacy of ARV regimens which may result when ATMs and PIs are used concurrently and that patients and health care practitioners alike should be aware of these perils.
- Full Text:
- Date Issued: 2011
A chemo-enzymatic process for the production of beta-thymidine, a key intermediate in antiretrovirol manufacture
- Gordon, Gregory Ernest Robert
- Authors: Gordon, Gregory Ernest Robert
- Date: 2010
- Subjects: HIV infections -- Treatment -- South Africa , HIV infections -- South Africa -- Prevention , Antiretroviral agents
- Language: English
- Type: Thesis , Doctoral , DTech
- Identifier: vital:10423 , http://hdl.handle.net/10948/d1016217
- Description: The socio-economic impact of HIV/AIDS on South Africa has resulted in lower gross domestic product, loss of skills in key sectors such as education, and increased health-care costs in providing access to treatment. Currently active pharmaceutical ingredients (API’s) such as stavudine (d4T) and azidothymidine (AZT) are imported from India and China, while formulation is conducted locally. A strategy was initiated between CSIR Biosciences and LIFElab under the auspices of Arvir Technologies to investigate the feasibility of local antiretroviral manufacture (d4T and AZT) or the manufacture of a key intermediate such as β- thymidine (dT). Several advantages associated with successful implementation of this strategy include ensuring a local supply of API’s, thus reducing reliance on procurement from foreign sources and reducing the effect of foreign exchange rate fluctuations on providing cost effective access to treatment. A local supply source would also reduce the imports and thus aid the balance of payments deficit, and in addition to this, provide stimulus in the local pharmaceutical manufacturing industry (which has been in decline for several decades), resulting in increased skills and employment opportunities. This thesis describes the development of a superior chemo-enzymatic process for the production of β-thymidine (72 percent yield, prior to isolation), a key intermediate in the preparation of anti-retrovirals. Alternative processes based purely on chemical or bioprocess transformations to prepare either 5-methyluridine (5-MU) or dT suffer from several disadvantages: lengthy transformations due to protection/deprotection strategies, low selectivties and product yields (30 percent in the chemical process) and isolation of the product from dilute process streams requiring the use of large uneconomical reactors (bioprocesss). This contributes significantly to the cost of d4T and AZT manufacture. Our novel chemoenzymatic process comprises of a biocatalytic reaction for the production of 5-MU, with subsequent chemical transformation into dT (3 steps) negating and circumventing the limitations of the chemical or bioprocess routes. During the course of this project development, the β-thymidine selling price declined from 175 $/kg (2005) to 100 $/kg (2008). However, the process described in this work is still competitive based on the current β- thymidine selling price of 100 $/kg. The process economics show that with further optimization and increasing the isolated dT yield from 70 percent to 90 percent, the variable cost decreases from 136 $/kg to 110 $/kg. The increase in isolated yield is highly probable, based on solubility data of β-thymidine. The decrease in β-thymidine selling price and technological improvement in dT manufacture should translate into lower API manufacture costs and more cost effective access to treatment. Our novel biocatalytic process producing 5-MU uses a coupled enzyme system employing PNP, Purine Nucleoside Phosphorylase and PyNP, Pyrimidine Nucleoside Phosphorylase. The overall transglycosylation reaction may be decoupled into the phosphorolysis reaction (PNP) and synthesis reaction (PyNP). During the phosphorolysis reaction, guanosine is converted into guanine and ribose-1-phosphate (R-1-P) in the presence of PNP enzyme. The reaction intermediate R-1-P is then coupled to thymine in the presence of PyNP enzyme during the synthesis reaction, producing 5-MU. The process was scaled up from lab-scale to bench-scale (10 - 20 L) and demonstrated to be robust and reproducible. This is evident from the average guanosine conversion (94.7 percent ± 2.03) and 5-MU yield (88.2 percent ± 6.21) and mole balance (104 percent ± 7.61) which were obtained at bench-scale (3 replicates, 10 L). The reaction was carried out at reactor productivities of between 7 – 11 g.L-1.h-1. The integration of the biocatalytic process and chemical processes was successfully carried out, showing that 5-MU produced using our novel biocatalytic process behaved similarly to commercially available 5- MU (ex. Dayang Chemicals, China). A PCT patent application (Ref. No. P44422PC01) on this chemo-enzymatic process has been filed and currently public private partnerships are being explored through Arvir Technologies to evaluate and validate this technology at one ton scale.
- Full Text:
- Date Issued: 2010
- Authors: Gordon, Gregory Ernest Robert
- Date: 2010
- Subjects: HIV infections -- Treatment -- South Africa , HIV infections -- South Africa -- Prevention , Antiretroviral agents
- Language: English
- Type: Thesis , Doctoral , DTech
- Identifier: vital:10423 , http://hdl.handle.net/10948/d1016217
- Description: The socio-economic impact of HIV/AIDS on South Africa has resulted in lower gross domestic product, loss of skills in key sectors such as education, and increased health-care costs in providing access to treatment. Currently active pharmaceutical ingredients (API’s) such as stavudine (d4T) and azidothymidine (AZT) are imported from India and China, while formulation is conducted locally. A strategy was initiated between CSIR Biosciences and LIFElab under the auspices of Arvir Technologies to investigate the feasibility of local antiretroviral manufacture (d4T and AZT) or the manufacture of a key intermediate such as β- thymidine (dT). Several advantages associated with successful implementation of this strategy include ensuring a local supply of API’s, thus reducing reliance on procurement from foreign sources and reducing the effect of foreign exchange rate fluctuations on providing cost effective access to treatment. A local supply source would also reduce the imports and thus aid the balance of payments deficit, and in addition to this, provide stimulus in the local pharmaceutical manufacturing industry (which has been in decline for several decades), resulting in increased skills and employment opportunities. This thesis describes the development of a superior chemo-enzymatic process for the production of β-thymidine (72 percent yield, prior to isolation), a key intermediate in the preparation of anti-retrovirals. Alternative processes based purely on chemical or bioprocess transformations to prepare either 5-methyluridine (5-MU) or dT suffer from several disadvantages: lengthy transformations due to protection/deprotection strategies, low selectivties and product yields (30 percent in the chemical process) and isolation of the product from dilute process streams requiring the use of large uneconomical reactors (bioprocesss). This contributes significantly to the cost of d4T and AZT manufacture. Our novel chemoenzymatic process comprises of a biocatalytic reaction for the production of 5-MU, with subsequent chemical transformation into dT (3 steps) negating and circumventing the limitations of the chemical or bioprocess routes. During the course of this project development, the β-thymidine selling price declined from 175 $/kg (2005) to 100 $/kg (2008). However, the process described in this work is still competitive based on the current β- thymidine selling price of 100 $/kg. The process economics show that with further optimization and increasing the isolated dT yield from 70 percent to 90 percent, the variable cost decreases from 136 $/kg to 110 $/kg. The increase in isolated yield is highly probable, based on solubility data of β-thymidine. The decrease in β-thymidine selling price and technological improvement in dT manufacture should translate into lower API manufacture costs and more cost effective access to treatment. Our novel biocatalytic process producing 5-MU uses a coupled enzyme system employing PNP, Purine Nucleoside Phosphorylase and PyNP, Pyrimidine Nucleoside Phosphorylase. The overall transglycosylation reaction may be decoupled into the phosphorolysis reaction (PNP) and synthesis reaction (PyNP). During the phosphorolysis reaction, guanosine is converted into guanine and ribose-1-phosphate (R-1-P) in the presence of PNP enzyme. The reaction intermediate R-1-P is then coupled to thymine in the presence of PyNP enzyme during the synthesis reaction, producing 5-MU. The process was scaled up from lab-scale to bench-scale (10 - 20 L) and demonstrated to be robust and reproducible. This is evident from the average guanosine conversion (94.7 percent ± 2.03) and 5-MU yield (88.2 percent ± 6.21) and mole balance (104 percent ± 7.61) which were obtained at bench-scale (3 replicates, 10 L). The reaction was carried out at reactor productivities of between 7 – 11 g.L-1.h-1. The integration of the biocatalytic process and chemical processes was successfully carried out, showing that 5-MU produced using our novel biocatalytic process behaved similarly to commercially available 5- MU (ex. Dayang Chemicals, China). A PCT patent application (Ref. No. P44422PC01) on this chemo-enzymatic process has been filed and currently public private partnerships are being explored through Arvir Technologies to evaluate and validate this technology at one ton scale.
- Full Text:
- Date Issued: 2010
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