During neoplastic development cellular requirements for the micronutrients and macronutrients increase dramatically for the primary metabolites, sugar and amino-acids. This concept of increased cellular needs lead to the development of antimetabolites as anticancer agents. Antimetabolites, are structurally similar to that of physiological metabolites, but have the ability to interfere with normal metabolic functioning of the cell that eventually leads to cell death. One such agent used in chemotherapy is 6-thioguanine. Another group of agents that also only recently showed potential for the used in cancer therapy are the Au containing anti-arthritic gold compounds, such as Auranofin. Both these groups, antimetabolites and Au containing compounds have different intracellular targets e.g. DNA(6-Thioguanine) and thioredoxin reductase [Au(I) and Au(III)], respectively. No study thus far has attempted to combine gold to 6-thioguanine nor has it been tested on non-cancerous and cancer cell lines. In this study two novel Au(I) compounds were synthesized i.e. a Au (I) metal 6-thioguanine (EKJC56) compound and Au (I)-phoshine 6-thioguanine (EKJC80A) compound (Figure 1). The cytotoxic effect and selectivity of these novel compounds were determined on several cancerous and normal cell cultures using the MTT assay. The cells were exposed to varying concentrations of these compounds. The novel compounds (EKJC56ST, EKJC56, and EKJC80A) showed higher toxicity towards all the cancerous cell lines tested, while EKJC56ST and EKJC56 showed only slight inhibition of the proliferation of normal cell cultures. These novel compounds proved to be more selective towards the hormone dependent cell line compared to the non-hormonal cell lines. The Au-phoshine compound, EKJC80A were non selective for both cancerous cells and normal cell cultures. The mechanism of action of these compounds were evaluated and showed a higher percentage of induction early apoptosis compared to an untreated control group. These results were supported when the effect of these derivatives were evaluated on the activation of caspase-3 activity, a known marker of early apoptosis. On evaluation of the effect of these compounds on the mitochondrial membrane it was observed that the mitochondrial membrane of the HeLa cell line became hyperpolarized, but did not prove to be statistical significant. No significant changes in cell cycle phases were observed when cell cycle progression was investigated. Due to limited amounts of thioredoxin reductase enzyme an observational study was conducted to determine the possible involvement of the Au(I) side chain of the novel compounds on the functionality of this enzyme. It was observed that these novel Au(I) containing compounds were as effective as Auranofin to inhibit this enzyme at a concentration as low as 0.1ìM. Further enzymatic studies are still required to support the hypothesis. This study can assist in developing new multi-acting chemotherapeutic drugs that may have the potential to effectively and selectively treat cancer and minimize the development of resistance during treatment. / Dissertation (MSc)--University of Pretoria, 2009. / Pharmacology / unrestricted

Escherichia coli MutS is a DNA binding repair protein (97 kDa, monomer) and its biological significance arises from its recognition of mismatches which occur as errors during DNA replication. Mismatches and mutagenic bases represent a fascinating and diverse range of shapes and sizes and it is not obvious how a single protein (MutS) can recognise such molecular diversity against a huge background of canonical Watson-Crick base pairs. In this project, the structure of a 17mer mismatch GT DNA was carried out using NMR spectroscopy to identify the differences caused by the introduction of a non-canonical base pair on helical structure. The resulting structure was B-form in conformation and local helical distortions were observed about the GT mispair due primarily to its sheared orientation. The effect of mismatch orientation, sequence context and oligonucleotide length on mismatch stability was also investigated using UV absorbance melting and NMR spectroscopy. The results showed that substitution of a TG mispair for a GT mispair was accompanied by a small drop in melting temperature. It was also discovered that sequences in which purine-purine or pyrimidine-pyrimidine stacking occurred induced additional stability of the mismatch resulting in a higher melting temperature of the duplex.Affinity of mismatch GT DNA and its mismatch orientation, sequence context and length analogues for MutS was investigated by monitoring changes to the chemical shifts and linewidths of imino protons resonances during NMR titration. The results showed that MutS displayed higher affinity towards sequences which involved better stacking between the flanking base pairs and the GT/TG mispair.Analogous NMR structural investigations of 6-thioguanine modified 13mer GC DNA and its oxidised derivatives have been successfully carried out. The NMR structure was successfully determined of the former and the results obtained showed the effect on helical structure induced by the substitution of a different DNA lesion.Although the crystal structures of MutS bound to DNA mismatches have been known for a number of years, the analogous crystal structures of uncomplexed apo MutS have not been determined to date. Consequently, vital structural knowledge on the large change in conformation of MutS upon binding to the DNA mismatch is seriously lacking. We have successfully isolated the structurally and functionally important NTD of E. coli MutS and its labelled (13C, 15N) analogues and have shown that it is endowed with a stable, tertiary structural fold and well suited to NMR structure determination. This is exemplified by the assignments of several backbone amide and side chain resonances using isotope aided 3D NMR techniques.

The three thiopurine drugs azathioprine (AZA), 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are used to treat several diseases, including inflammatory bowel disease (IBD). They are pro-drugs and are believed to act through the formation of thioguanine nucleotides (TGNs). Other important metabolites are the methylthioinosine nucleotides (meTINs). These metabolites are active in the white blood cells (WBCs).Most patients respond well to the thiopurine drugs but up to a third have to modify or discontinue their treatment due to adverse events or a lack of therapeutic effects. This could be caused by inter-patient variability in the metabolism of the drugs. Therapeutic drug monitoring (TDM) of thiopurine nucleotides in red blood cells (RBCs) is used to guide treatment. Current routine assays measure the nucleotides after hydrolysation to nucleic bases and are therefore unable to distinguish between mono-, di-, and triphosphates. Recently it was shown that these assays failed to predict the clinical outcome in about 40% of the patients. It has been suggested that measuring thioguanosine triphosphate (TGTP) (believed to be the most active of the TGNs) separately might increase the clinical value.An assay suitable for measuring thioguanosine mono- (TGMP) and diphosphate (TGDP) and TGTP, as well as methylthioinosine mono- (meTIMP), di- (meTIDP) and triphosphate (meTITP) separately in RBCs in clinical samples has been developed. In clinical studies of 82 IBD patients, we found no correlation between the thiopurine dose and metabolite levels in RBCs, thus illustrating the importance of metabolite measurements in the TDM of thiopurines.The TGN peak measured by the routine assay during TDM of patients treated with thiopurines consisted of TGTP and TGDP with a small contribution from TGMP. The meTIN also consisted of mono-, di- and triphosphates, but in different proportions, indicating differences in the formation. The inter-individual differences in nucleotide distribution were very small and a strong correlation between the different nucleotides and their respective sums was observed. As a consequence, measuring the mono-, di- and triphosphates separately was not beneficial in predicting remission, which was confirmed by the results from the clinical study.Further research into the metabolism and mode of action of thiopurine drugs is needed to understand the inter-patient variability in response and metabolite formation. An assay suitable for such studies, measuring TGNs and meTINs in cultured cells, has also been developed.

A azatioprina (AZA) é um imunossupressor utilizado no tratamento de doenças autoimunes como lúpus eritematoso sistêmico, doença de Crohn, doença inflamatória intestinal e contra a rejeição em transplantes de órgãos sólidos. Após mais de 40 anos de uso a AZA continua exercendo um papel central nos regimes imunomoduladores, devido ao fato de combinar eficácia, segurança e baixo custo. Sabe-se que a atividade da tiopurina metiltransferase pode determinar, pelo menos em parte, a eficácia clínica da AZA. Esta enzima apresenta polimorfismo genético co-dominante e a distribuição dos alelos variantes é significativamente diferente entre as populações. A grande variabilidade farmacocinética no metabolismo AZA justifica a sua monitorização terapêutica. Neste trabalho otimizou-se uma metodologia para a quantificação dos metabólitos da AZA, 6-TGN e 6-MMP, por cromatografia líquida de alta eficiência (HPLC/UV-Vis), utilizando-se um detector de ultravioleta-visível em um único comprimento de onda, após a amostra passar por uma desproteinização ácida simples e ser aquecida para a conversão dos metabólitos em suas respectivas bases livres. Os valores destes metabólitos foram determinados em uma população de 124 pacientes transplantados renais. Para adequarmos o processo às legislações locais e internacionais, foram seguidas orientações da Anvisa, FDA e CLSI. A separação foi realizada em coluna de fase reversa, sendo a fase móvel A fosfato de potássio e a fase móvel B metanol. A detecção da 6-TGN e da 6-MMP foi realizada em 342 m (UV-Vis). O estudo da linearidade da 6-TGN variou entre 0,30 e 89,71 mol/L e da 6-MMP entre 0,30 e 93,86 mol/L. As recuperações, de 95,08 a 100,80% para 6-TGN e 95,38 a 105,06% para 6-MMP. Os CV da repetibilidade, de 0,04 a 5,06%, enquanto os CV da reprodutibilidade de 4,88 a 12,73% para 6-TGN e 6-MMP. Para ambos os metabólitos o LD e o LQ foram de 0,30 mol/L e 0,13 mol/L. Os eritrócitos lavados e as amostras tratadas, prontas para a injeção no HPLC, foram armazenadas abaixo de -5°C até a análise. Nesta temperatura estiveram estáveis durante 8 semanas e 1 dia, respectivamente. Os valores das concentrações de 6-TGN e 6-MMP encontrados nas amostras dos pacientes variaram entre não detectável a 1569 mol/8 x 108 RBC (mediana de 200,50) e não detectável a 113057 mol/8 x 108 RBC (mediana de 5166), respectivamente. As correlações entre os níveis de 6-TGN ou 6-MMP e as variáveis sexo, tempo pós-transplante, número de transplantes e dosagem de AZA (mg/kg) foram examinadas em diferentes grupos. O método proposto apresenta boa relação custo-benefício, é simples, preciso e rápido na determinação das concentrações intraeritrocitárias de 6-TGN e 6-MMP em pacientes sob terapia com AZA. O método validado permite que o laboratório forneça dados farmacocinéticos úteis e precisos para o ajuste do tratamento do paciente e pode ser facilmente adaptado para a análise rotineira destes metabólitos. Os resultados das amostras dos pacientes estão de acordo com os encontrados em outros estudos, atestando a utilidade dessa ferramenta analítica no acompanhamento dos pacientes / Azathioprine (AZA) is an immunosuppressant used in autoimmune pathologies like lupus erythematosus, Chrons disease, inflammatory bowel disease and against rejection in solid organs transplant. After more than 40 years of use, AZA continues exerting a central role in immunomodulatory regimens, due to the fact that it combines effectiveness, safety and low cost. It is well known that thiopurine methyltransferase activity may determine, at least in part, the clinical efficacy of AZA therapy. This enzyme exhibits codominant genetic polymorphism and the distribution of these variant alleles differs significantly among populations. The considerable pharmacokinetic variability in AZA metabolism justify the therapeutic drug monitoring of this drug. In this work a methodology was improved to quantify the metabolites of AZA, 6-TGN and 6-MMP, by high performance liquid chromatography (HPLC/UV-Vis) with an ultraviolet-visible detector, using a single wavelength reading, following a simple acid deproteinization and heating to convert the metabolites into their respective free bases. The values of these metabolites were determined in a population of 124 renal transplant recipients. To adequate the process to international and local legislation, Anvisa, FDA and CLSI guidelines were followed. Separation was achieved on a reversed-phase column; mobile phase A potassium phosphate and mobile phase B methanol. Detection of 6-TGN and 6-MMP was performed at 342 m (UV-Vis). Assay linearity for 6-TGN ranged from 0.30 to 89.71 mol/L and from 0.30 to 93.86 mol/L for 6-MMP. The recoveries were 95.08, 97.76 and 100.80% for 6-TGN and 104.79, 95.38 and 105.06% for 6-MMP. Repeatability CV were 3.50, 5.06, 1.09 and 0.04, 0.35, 1.58%, while reproducibility CV were 8.65, 7.18, 8.44 and 12.73, 6.40, 4.88% for 6-TGN and 6-MMP, respectively. LOQ and LOD of 6-TNG and 6-MMP were respectively 0.30 mol/L and 0.13 mol/L for both metabolites. The washed erythrocytes and the samples treated and ready for injection into the HPLC system were stored below -5 °C until analysis, at this temperature the samples were stable for 8 weeks and for 1 day, respectively. 6-TGN and 6-MMP patient analysis values ranged from non detectable to 1569 mol/8 x 108 RBC (median of 200.50) and non detectable to 113057 mol/8 x 108 RBC (median of 5166), respectively. The correlations between 6-TGN or 6-MMP levels and variables sex, time post-transplant, number of transplants and AZA dosage (mg/kg) were examined in different groups. The proposed HPLC method has a good cost-benefit ratio, is straightforward, precise, accurate and fast at the determining 6-TGN and 6-MMP concentrations in red blood cells of patients under AZA therapy. The validated method is good enough to enable the laboratory to routinely provide useful and accurate pharmacokinetic data in time to adjust patient regimens. It can be easily adopted for routine analysis of these drug metabolites. The results of patient samples are in agreement with others studies, thus certifying the usefulness of this analytical tool in monitoring of patients

A azatioprina (AZA) é um imunossupressor utilizado no tratamento de doenças autoimunes como lúpus eritematoso sistêmico, doença de Crohn, doença inflamatória intestinal e contra a rejeição em transplantes de órgãos sólidos. Após mais de 40 anos de uso a AZA continua exercendo um papel central nos regimes imunomoduladores, devido ao fato de combinar eficácia, segurança e baixo custo. Sabe-se que a atividade da tiopurina metiltransferase pode determinar, pelo menos em parte, a eficácia clínica da AZA. Esta enzima apresenta polimorfismo genético co-dominante e a distribuição dos alelos variantes é significativamente diferente entre as populações. A grande variabilidade farmacocinética no metabolismo AZA justifica a sua monitorização terapêutica. Neste trabalho otimizou-se uma metodologia para a quantificação dos metabólitos da AZA, 6-TGN e 6-MMP, por cromatografia líquida de alta eficiência (HPLC/UV-Vis), utilizando-se um detector de ultravioleta-visível em um único comprimento de onda, após a amostra passar por uma desproteinização ácida simples e ser aquecida para a conversão dos metabólitos em suas respectivas bases livres. Os valores destes metabólitos foram determinados em uma população de 124 pacientes transplantados renais. Para adequarmos o processo às legislações locais e internacionais, foram seguidas orientações da Anvisa, FDA e CLSI. A separação foi realizada em coluna de fase reversa, sendo a fase móvel A fosfato de potássio e a fase móvel B metanol. A detecção da 6-TGN e da 6-MMP foi realizada em 342 m (UV-Vis). O estudo da linearidade da 6-TGN variou entre 0,30 e 89,71 mol/L e da 6-MMP entre 0,30 e 93,86 mol/L. As recuperações, de 95,08 a 100,80% para 6-TGN e 95,38 a 105,06% para 6-MMP. Os CV da repetibilidade, de 0,04 a 5,06%, enquanto os CV da reprodutibilidade de 4,88 a 12,73% para 6-TGN e 6-MMP. Para ambos os metabólitos o LD e o LQ foram de 0,30 mol/L e 0,13 mol/L. Os eritrócitos lavados e as amostras tratadas, prontas para a injeção no HPLC, foram armazenadas abaixo de -5°C até a análise. Nesta temperatura estiveram estáveis durante 8 semanas e 1 dia, respectivamente. Os valores das concentrações de 6-TGN e 6-MMP encontrados nas amostras dos pacientes variaram entre não detectável a 1569 mol/8 x 108 RBC (mediana de 200,50) e não detectável a 113057 mol/8 x 108 RBC (mediana de 5166), respectivamente. As correlações entre os níveis de 6-TGN ou 6-MMP e as variáveis sexo, tempo pós-transplante, número de transplantes e dosagem de AZA (mg/kg) foram examinadas em diferentes grupos. O método proposto apresenta boa relação custo-benefício, é simples, preciso e rápido na determinação das concentrações intraeritrocitárias de 6-TGN e 6-MMP em pacientes sob terapia com AZA. O método validado permite que o laboratório forneça dados farmacocinéticos úteis e precisos para o ajuste do tratamento do paciente e pode ser facilmente adaptado para a análise rotineira destes metabólitos. Os resultados das amostras dos pacientes estão de acordo com os encontrados em outros estudos, atestando a utilidade dessa ferramenta analítica no acompanhamento dos pacientes / Azathioprine (AZA) is an immunosuppressant used in autoimmune pathologies like lupus erythematosus, Chrons disease, inflammatory bowel disease and against rejection in solid organs transplant. After more than 40 years of use, AZA continues exerting a central role in immunomodulatory regimens, due to the fact that it combines effectiveness, safety and low cost. It is well known that thiopurine methyltransferase activity may determine, at least in part, the clinical efficacy of AZA therapy. This enzyme exhibits codominant genetic polymorphism and the distribution of these variant alleles differs significantly among populations. The considerable pharmacokinetic variability in AZA metabolism justify the therapeutic drug monitoring of this drug. In this work a methodology was improved to quantify the metabolites of AZA, 6-TGN and 6-MMP, by high performance liquid chromatography (HPLC/UV-Vis) with an ultraviolet-visible detector, using a single wavelength reading, following a simple acid deproteinization and heating to convert the metabolites into their respective free bases. The values of these metabolites were determined in a population of 124 renal transplant recipients. To adequate the process to international and local legislation, Anvisa, FDA and CLSI guidelines were followed. Separation was achieved on a reversed-phase column; mobile phase A potassium phosphate and mobile phase B methanol. Detection of 6-TGN and 6-MMP was performed at 342 m (UV-Vis). Assay linearity for 6-TGN ranged from 0.30 to 89.71 mol/L and from 0.30 to 93.86 mol/L for 6-MMP. The recoveries were 95.08, 97.76 and 100.80% for 6-TGN and 104.79, 95.38 and 105.06% for 6-MMP. Repeatability CV were 3.50, 5.06, 1.09 and 0.04, 0.35, 1.58%, while reproducibility CV were 8.65, 7.18, 8.44 and 12.73, 6.40, 4.88% for 6-TGN and 6-MMP, respectively. LOQ and LOD of 6-TNG and 6-MMP were respectively 0.30 mol/L and 0.13 mol/L for both metabolites. The washed erythrocytes and the samples treated and ready for injection into the HPLC system were stored below -5 °C until analysis, at this temperature the samples were stable for 8 weeks and for 1 day, respectively. 6-TGN and 6-MMP patient analysis values ranged from non detectable to 1569 mol/8 x 108 RBC (median of 200.50) and non detectable to 113057 mol/8 x 108 RBC (median of 5166), respectively. The correlations between 6-TGN or 6-MMP levels and variables sex, time post-transplant, number of transplants and AZA dosage (mg/kg) were examined in different groups. The proposed HPLC method has a good cost-benefit ratio, is straightforward, precise, accurate and fast at the determining 6-TGN and 6-MMP concentrations in red blood cells of patients under AZA therapy. The validated method is good enough to enable the laboratory to routinely provide useful and accurate pharmacokinetic data in time to adjust patient regimens. It can be easily adopted for routine analysis of these drug metabolites. The results of patient samples are in agreement with others studies, thus certifying the usefulness of this analytical tool in monitoring of patients

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2007. / Härtill 4 uppsatser.

Les médicaments thiopuriniques que sont l'azathioprine, la 6-mercaptopurine et la 6-thioguanine sont utilisés depuis des décennies pour leurs propriétés cytotoxiques et immunosuppressives dans le traitement de certaines leucémies, de maladies inflammatoires chroniques ou auto-immunes ainsi que dans la prévention du rejet de greffe. Certains patients, traités par des doses conventionnelles de ces molécules, développent cependant des effets indésirables parfois très sévères. Le déficit d'activité, d'origine génétique, de la thiopurine S-méthyltransférase (TPMT), enzyme impliquée dans le métabolisme des thiopurines, constitue l'un des facteurs majeurs de la myélotoxicité de ces médicaments. La détermination du phénotype TPMT par génotypage, qui est une mesure préventive avant l'introduction d'un traitement thiopurinique, repose sur l'identification des mutations inactivatrices les plus fréquentes du gène TPMT. Une partie de ce travail a consisté en l'analyse fonctionnelle de quatre variants alléliques rares du gène TPMT dans un système d'expression hétérologue, la levure S. cerevisiae. Le caractère non-fonctionnel de deux d'entre eux a ainsi été démontré. Cependant, le déficit d'activité de la TPMT ne permet d'expliquer qu'environ 30 % des cas de myélotoxicité sous thiopurines, ce qui laisse supposer l'existence d'autres anomalies génétiques affectant d'autres gènes impliqués dans la réponse de l'organisme à ces molécules. Ainsi, nous avons étudié le polymorphisme génétique de deux autres protéines candidates, celui de l'inosine monophosphate déshydrogénase de type 2 (IMPDH2), enzyme-clé de la formation des métabolites actifs des thiopurines, et celui de la RhoGTPase RAC1, qui est l'une des cibles pharmacologiques de ces molécules. Certains des polymorphismes que nous avons identifiés dans ces deux gènes semblent affecter in vitro l'expression et/ou l'activité de ces protéines et pourraient, par conséquent, contribuer aux variations inter-individuelles de réponse aux thiopurines

This master's thesis is focused on the determination of 6-thioguanine (6-T) by DC voltammetry (DCV), differential pulse voltammetry (DPV) and flow injection analysis with electrochemical detection (FIA-ED) on a boron doped diamond electrode (BDDFE). The optimum conditions for determination of 6-T were found and under these conditions, concentration dependences were measured and the limits of quantification (LOQ) were calculated for each method. Medium of 60% methanol and phosphate buffer (PB) pH 2,0 was chosen as optimum for DCV and DPV determination of 6-T at BDDFE. For both DCV and DPV, the linear concentration dependences were obtained in concentration ranges of 6-T from 2 µmol·l-1 to 10 µmol·l-1 with LOQ 0,9 µmol·l-1 for DCV and 1,5 µmol·l-1 for DPV. Lower LOQ was achieved in PB pH 2,0, where LOQ was 0,6 µmol·l-1 for both DCV and DPV, but the problem was lower repeatability. In PB pH 2 6-T was determined by DCV in drinking and river water with LOQ 1,3 µmol·l-1 in both drinking and river water. The possibility of solid phase extraction was investigated as a method for preliminary separation of 6-T from urine. For FIA under optimized conditions (polarization potential 1300 mV, flow rate 5 ml·min1 and sample volume 50 µl) the linear concentrations dependences were obtained in concentration ranges...