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
Implementation of the dual therapy prevention of mother-to-child transmission protocol
- Authors: Singh, Vikesh
- Date: 2010
- Subjects: HIV infections -- Transmission -- South Africa , AIDS (Disease) in pregnancy -- Prevention -- South Africa , Antiretroviral agents
- Language: English
- Type: Thesis , Masters , MPharm
- Identifier: vital:10138 , http://hdl.handle.net/10948/1374 , HIV infections -- Transmission -- South Africa , AIDS (Disease) in pregnancy -- Prevention -- South Africa , Antiretroviral agents
- Description: Antiretroviral drugs taken during pregnancy, reduce the rates of mother-to-child transmission from 35 percent to as low as 1 to 2 percent (UNAIDS, 2009). In 2002, the Prevention of Mother-to-Child Transmission (PMTCT) programme was implemented in South Africa. Studies on the implementation of the PMTCT programme have shown that understaffed and under-developed health care facilities were key barriers to the provision of PMTCT services (Health Systems Trust, 2002: 6; Skinner et al., 2003). The aim of this study was to assess the challenges experienced by health care workers working in public sector facilities in the Nelson Mandela Metropole after implementation of the dual therapy PMTCT programme. Four areas were investigated: Infrastructure; Drug Supply Management; Clinic Procedures and Staffing. A quantitative descriptive study was conducted in August 2009 at nine public health care facilities in the Nelson Mandela Metropole, South Africa. Questionnaires were issued to 81 nurses and 41 pharmacy personnel (pharmacists and pharmacist assistants). Checklist audit forms were issued to the Facility Manager of each facility and completed with the researcher. The key findings for Infrastructure were lack of space at patient waiting rooms (9; 100 percent n=9), counselling area (5; 55.5 percent; n=9), nurse consultation rooms (6; 66.6 percent; n=9), storage areas (5; 55.5 percent; n=9) and filing areas (7; 77.7 percent; n=9). The key findings for Drug Supply Management were none of the dispensaries (0 percent; n=10) were fully compliant with Good Pharmacy Practice, pharmacy personnel indicated that there were no stock cards for medication (13; 31.7 percent; n=41); there was less than two weeks supply of buffer stock kept for zidovudine and nevirapine (13; 35.1percent; n=37) and medication orders were placed without any reference to minimum and maximum levels of medication (15; 36.5 percent; n=41) . The key findings for Clinic Procedures were only two facilities followed up on patients that had missed appointments (22.2 percent; n=9) and four facilities (44.4 percent; n=9) had a tracking system for patients that had defaulted. Of the nine facilities only three (33.3 percent; n=9) updated patient demographic details regularly. The key findings for Staffing were a shortage of doctors, nurses, counsellors and pharmacists at the facilities. One of the major challenges identified was the lack of training offered on new PMTCT protocols with 56.2 percent (45; n=80) of the nurses stating that no training was provided on the dual PMTCT protocol. Only 54.3 percent (44; n=81) of nurses stated that they knew the criteria to start the mother on dual PMTCT therapy. In conclusion there is an urgent need for barriers such as lack of staff, lack of space, lack of training on PMTCT and standard procedures for follow up of patients to be addressed in order to ensure the successful scaling up of PMTCT.
- Full Text:
- Date Issued: 2010
- Authors: Singh, Vikesh
- Date: 2010
- Subjects: HIV infections -- Transmission -- South Africa , AIDS (Disease) in pregnancy -- Prevention -- South Africa , Antiretroviral agents
- Language: English
- Type: Thesis , Masters , MPharm
- Identifier: vital:10138 , http://hdl.handle.net/10948/1374 , HIV infections -- Transmission -- South Africa , AIDS (Disease) in pregnancy -- Prevention -- South Africa , Antiretroviral agents
- Description: Antiretroviral drugs taken during pregnancy, reduce the rates of mother-to-child transmission from 35 percent to as low as 1 to 2 percent (UNAIDS, 2009). In 2002, the Prevention of Mother-to-Child Transmission (PMTCT) programme was implemented in South Africa. Studies on the implementation of the PMTCT programme have shown that understaffed and under-developed health care facilities were key barriers to the provision of PMTCT services (Health Systems Trust, 2002: 6; Skinner et al., 2003). The aim of this study was to assess the challenges experienced by health care workers working in public sector facilities in the Nelson Mandela Metropole after implementation of the dual therapy PMTCT programme. Four areas were investigated: Infrastructure; Drug Supply Management; Clinic Procedures and Staffing. A quantitative descriptive study was conducted in August 2009 at nine public health care facilities in the Nelson Mandela Metropole, South Africa. Questionnaires were issued to 81 nurses and 41 pharmacy personnel (pharmacists and pharmacist assistants). Checklist audit forms were issued to the Facility Manager of each facility and completed with the researcher. The key findings for Infrastructure were lack of space at patient waiting rooms (9; 100 percent n=9), counselling area (5; 55.5 percent; n=9), nurse consultation rooms (6; 66.6 percent; n=9), storage areas (5; 55.5 percent; n=9) and filing areas (7; 77.7 percent; n=9). The key findings for Drug Supply Management were none of the dispensaries (0 percent; n=10) were fully compliant with Good Pharmacy Practice, pharmacy personnel indicated that there were no stock cards for medication (13; 31.7 percent; n=41); there was less than two weeks supply of buffer stock kept for zidovudine and nevirapine (13; 35.1percent; n=37) and medication orders were placed without any reference to minimum and maximum levels of medication (15; 36.5 percent; n=41) . The key findings for Clinic Procedures were only two facilities followed up on patients that had missed appointments (22.2 percent; n=9) and four facilities (44.4 percent; n=9) had a tracking system for patients that had defaulted. Of the nine facilities only three (33.3 percent; n=9) updated patient demographic details regularly. The key findings for Staffing were a shortage of doctors, nurses, counsellors and pharmacists at the facilities. One of the major challenges identified was the lack of training offered on new PMTCT protocols with 56.2 percent (45; n=80) of the nurses stating that no training was provided on the dual PMTCT protocol. Only 54.3 percent (44; n=81) of nurses stated that they knew the criteria to start the mother on dual PMTCT therapy. In conclusion there is an urgent need for barriers such as lack of staff, lack of space, lack of training on PMTCT and standard procedures for follow up of patients to be addressed in order to ensure the successful scaling up of PMTCT.
- Full Text:
- Date Issued: 2010
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