Thesis (M.Sc. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2010.

Orientador: Iguatemy Lourenço Brunetti / Banca: Rosângela Gonçalves Peccinini Machado / Banca: Regina Lúcia de Moraes Moreau / Resumo: Este estudo teve como objetivo avaliar os efeitos da administração simultânea de PYR sobre os parâmetros farmacocinéticos da INH e a produção de seus metabólitos, em um grupo de animais sob tratamento com INH+PYR e outro com INH+RMP+PYR, bem como de avaliar o comportamento dos biomarcadores de hepatotoxicidade, as transaminases AST e ALT, nesses grupos. Na primeira fase do protocolo experimental foi coletado sangue da cauda dos animais (ratos Wistar, machos, peso~250g) para análise dos biomarcadores como valores basais, em seguida realizou-se a administração, por gavagem, de doses múltiplas de INH (100mg/Kg) (grupo I), INH+PYR (350mg/Kg) (grupo II), INH+PYR+RMP (100mg/Kg) (grupo III), PYR (grupo IV) e água estéril (grupo V – controle) por 21 dias. Para os animais que receberam INH (grupo I, grupo II e grupo III) construiu-se a curva de sua concentração plasmática x tempo. Amostras seriadas de sangue foram coletadas em 10 tempos diferentes entre 0-24h; para cada tempo de coleta foram empregados 5 ratos. As amostras foram desproteinizadas com ácido tricloroacético a 10%; para análise dos metabólitos acetilados promovendo a hidrólise com HCl 6M; em seguida, a INH e a Hz foram derivatizadas com cinamaldeído. As amostras foram analisadas por HPLC usando coluna Resolve TM C18 e detector UV-VS, operando a 340nm. Os parâmetros farmacocinéticos da INH apresentam diferenças estatisticamente significativas (p<0,05, teste de Tukey) entre os grupos, e sua média e EPM foram: Grupo INH vs Grupo INH+PYR: t1/2 = 1,4 (0,070) vs 1,0 (0,100); K = 0,51 (0,024) vs 0,77 (0,100); t1/2 = 3,4 (0,026) vs 7,2 (0,910); Kel = 0,21 (0,015) vs 0,10 (0,018); ClT/F = 0,68 (0,016) vs 0,90 (0,047); Vd/F = 3,32 (0,19) vs 9,52 (1,52); AUC0-24 ss INH = 146,36 (3,25) vs 111,96 (5,60); AUC0-24 ss Hz = 2,87 (0,07) vs 4,60 (0,09)... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of the present study was to evaluate the hepatotoxicity effect, pharmacokinetic response and biotransformation of isoniazid of rats treated with i) isoniazid (INH), ii) INH + pyrazinamide (PYR) and iii) INH + PYR + rifampicin (RMP). Daily doses of the tuberculostatic drugs were administrated intragastrically to the animals (Wistar rats) for one period of 21 days as follow: INH (100mg/Kg) (group I), INH (100mg/Kg) + PYR (350mg/Kg) (group II), INH (100mg/Kg) + PYR (350mg/Kg) + RMP (100mg/Kg) (group III), PYR (350mg/Kg) (group IV) and sterile water (group V, control). The serum levels of the biomarkers aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were determined before the administration of the drugs (basal) and after the 21 days treatments. On day 21, blood samples were obtained before and 0,25; 0,5; 0,75; 1; 1,5; 3; 6; 12 and 24 hours after the dose (five animals for each point). The blood samples were deproteinized with 10% trichloroacetic acid, derivazed by 1% cinnamaldehyde. For the determination of the acetylated metabolites acetylisoniazid (AcINH) and acetylhydrazine (AcHz) a previous hydrolysis with 6 M hydrochloride acid was performed. Then, the samples were centrifuged and the supernatant analyzed by liquid chromatograph. The results are presented as mean and SEM. The pharmacokinetic parameter of the INH and its metabolites AcINH and hydrazine (Hz) were compared between the groups (p < 0.05, Turkey’s test). The results significantly different were: Group INH vs Group INH+PYR: t1/2 = 1,4 (0,070) vs 1,0 (0,100); K = 0,51 (0,024) vs 0,77 (0,100); t1/2 = 3,4 (0,026) vs 7,2 (0,910); Kel = 0,21 (0,015) vs 0,10 (0,018); ClT/F = 0,68 (0,016) vs 0,90 (0,047); Vd/F = 3,32 (0,19) vs 9,52 (1,52); AUC0-24 ss (INH) = 146,36 (3,25) vs 111,96 (5,60); AUC0-24 ss (Hz) = 2,87 (0,07) vs 4,60 (0,09)... (Complete abstract, click electronic address below) / Mestre

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / As leishmanioses são um grupo de doenças causadas por protozoários do gênero Leishmania spp que afetam 98 países. No Brasil, no ano de 2013, foram relatados 3.253 casos de leishmaniose visceral e 18.226 casos de Leishmaniose Tegumentar Americana. O tratamento de primeira escolha continua sendo realizado com antimoniais pentavalentes, e em casos de insucessos os fármacos de segunda escolha são a pentamidina e a anfotericina B. Tais medicamentos causam intensos efeitos adversos e ultimamente têm surgido cepas resistentes aos mesmos. Em áreas endêmicas têm sido cada vez mais comum o surgimento da co-infecção Leishmania com Mycobacterium tuberculosis. O tratamento para a tuberculose com pirazinamida (PZA) e isoniazida (INZ), controla a leishmaniose. Esses dados sugerem atividade anti-leishmania da PZA e da INZ. O objetivo deste trabalho foi avaliar a atividade in vitro da INZ e da PZA e seus compostos derivados (série G e série R, respectivamente) sobre Leishmania (Viannia) braziliensis. As moléculas foram testadas em monocamadas de macrófagos peritoneais de camunongos infectados com L. (V) braziliensis durante 48h. Todas as moléculas testadas inibiram o índice de infecção de forma dose dependente em comparação aos controles. As moléculas da série R foram mais ativas do que a PZA, porém o resultado foi significativo somente para a R02 (p < 0,005). Apenas a molécula R05 (76,64M) foi relativamente tóxica para macrófagos. Os compostos mais ativos foram R02, G01 e G02, cujos índices de seletividade foram 14,31, 19 e 30, respectivamente. A dosagem de nitrito foi feita em sobrenadantes de monocamadas de macrófagos peritoniais infectados e tratados com as substâncias nas concentrações 10 e 100M. A G01 e a G02 estimularam a produção de NO2 nas duas concentrações, entretanto o resultado foi estatisticamente significativo para a G02 em 100M (p < 0,0001), a G05 só estimulou óxido nítrico na maior concentração. Todos os compostos da série R estimularam NO2, contudo, o resultado foi estatisticamente significativo para a R03 e R05 a 100M (p < 0,001). Adicionalmente, foi realizado uma análise preditiva in sílico de parâmetros farmacocinéticos das moléculas mais ativas in vitro, utilizando o software admetSAR. Os dados obtidos mostraram que de forma semelhante às suas moléculas originais a G01, G02 e R02 apresentaram alta capacidade de serem absorvidas pelo trato gastrointestinal, baixo potencial hepatotoxico e carcinogênico. Juntos, esses dados demonstram que essas moléculas são seletivamente tóxicas para o parasito com potencial para serem testadas pela via oral em estudos em modelo experimental de infecção. / Leishmaniasis are a group of diseases caused by protozoan of genus Leishmania spp affecting of 98 countries. In Brazil, in the year 2013 were 3.253 reported cases of visceral leishmaniasis and 18.226 cases of cutaneous leishmaniasis. The first choice of treatment is still performed with pentavalent antimonials and in cases of failures drugs of second line of treatment are pentamidine and anfotericin B. These drugs cause many adverse effects and has lately emerged resistant strains. In endemic areas it has been increasingly common the appearance of co-infection Leishmania and Mycobacterium tuberculosis. The treatment of tuberculosis with pyrazinamid and isoniazid control the leishmaniasis.The aim of this study was evaluate the anti-leishmania activity in vitro of INZ and PZA and yours derivatives compounds (serie G and serie R,respectively) on the Leishmania (Viannia) braziliensis. The molecules were tested on infected monolayers of peritoneal murine macrophages for 48h. All the molecules te inhibited the infection index in a dose-dependent manner in relation to controls. The molecules of the R series were more active than PZA, but the result was only significant for R02 (p < 0,005). Only R05 (CC50 = 76,64M) was relatively toxic to macrophages. The most active compounds were R02, G01 and G02 whose select index were 14,31, 19 and 30, respectively. Nitrite assay was performed in supernatants of infected monolayers of peritonial macrophages treated with the substancies at 10 and 100M. G01 and G02 stimulated NO2 prodution, however, the result was only statistically significant for G02 at 100M (p < 0,001). All the compounds of R serie stimulated NO2 production however the results were statistically significant for the R03 and R05 at 100M (p < 0,001). Additionally, a in silico preditive pharmacokinetic analysis was performed to active molecules using the admetSAR software the data showed that G01, G02 and R02 were similar to their original molecules as to high capacity to be absorbed by the gastrointestinal tract, low hepatotoxic and carcinogenic potencial. Together, these data demonstrate that these molecules are selectively toxic to the parasite with the potencial to be tested orally on studies in experimental infection.

Magister Pharmaceuticae - MPharm / Tuberculosis and HIV are arguably South Africa’s largest and most important health issues. With drug-resistant strains of tuberculosis on the increase and little research on new drugs, there is an urgent need for research around the drugs presently available to ensure their optimal use and to minimise their sometimes serious and significant side effects. Treatment of drug-resistant tuberculosis is expensive and lengthy, and is complicated by a limited choice of drugs with lower efficacies and higher toxicities. Treatment is further complicated in patients with HIV due to several factors including drug interactions. While some authors suggest that HIV and malabsorption might be associated with poor clinical outcomes, other researchers have found no link. Patients may benefit from Therapeutic Drug Monitoring in order to ensure that their doses of antituberculosis drugs are reaching the required minimum effective concentrations, without attaining toxic levels in the plasma which may cause unpleasant side effects. There is little research concerning drug levels in HIV patients with TB in South Africa, let alone in patients with drug-resistant forms of tuberculosis, and there are no studies in this country that use Liquid Chromatography-Mass Spectrometry to investigate the plasma levels of pyrazinamide in patients with MDR-TB. This study aimed to investigate whether or not there is a difference in the pharmacokinetics of PZA in MDR-TB patients with HIV, and those without HIV infection. It also aimed to establish whether LC-MS could be used to study the levels of pyrazinamide in the plasma of patients with multidrug-resistant tuberculosis with and without concurrent HIV infection. The plasma levels of pyrazinamide in 32 MDR-TB patients (23 HIV negative and 9 HIV positive), were successfully 2 analysed using LC-MS, and the pharmacokinetics of PZA in these 2 populations was described. It was established that the Tmax of pyrazinamide was significantly higher in HIV-negative patients than in HIV-positive patients. Although there was a difference between the Ka in the two populations, this difference did not quite reach statistical significance. There were no statistically significant differences between HIV-negative and HIV-positive patients with regards to the other pharmacokinetic parameters investigated. Our findings established that there was little evidence to suggest that there is a difference between the pharmacokinetics of the antimycobacterial drug pyrazinamide in HIV-positive patients and that in HIV-negative patients. We were also able to successfully develop and validate an assay for the analysis of PZA in plasma using LC-MS, and this finding could be very valuable for further studies. Although our study failed to prove this, the possibility still exists that HIV-positive patients could exhibit altered kinetics of antiTB drugs and this has not been fully investigated in South Africa. The clinical impact of low plasma levels of antimycobacterial drugs is still largely unexplored and further research with larger sample sizes should be done in order to establish which factors may contribute to low plasma levels of anti-tuberculosis drugs in MDR-TB patients, and whether or not these low levels are increasing the risk of treatment failure or other poor clinical outcomes.

Philosophiae Doctor - PhD / Tuberculosis (TB) remains a global epidemic despite the fact that treatment has been available since the 1950’s. This disease is highly contagious and spreads via transmission of the Mycobacterium Tuberculosis (MTB) tubercle via coughing, sneezing and spitting. The disease has various side effects including weight loss, fatigue and even death. To date no cure has been found for TB and thus optimisation of treatment is a constant focus in health related research. TB is highly prevalent in South Africa due to the increased level of patients who are co-infected with HIV. Treatment for TB consists of first line drugs including isoniazid (INH), ethambutol (ETH), pyrazinamide (PYR) and rifampicin (RIF). These drugs are highly effective but also produce many adverse drug reactions (ADR’s) over the 6-month course of treatment. These reactions lead to patients not completing the course, losing quality of life and ultimately adding to the development of drug resistant strains of TB. A method of minimising these ADR’s is the development of a phenotype sensor, which is able to determine the metabolic profile of patients. Metabolic profiles play a huge role in the efficacy of treatment by tailoring treatment in order for patients to stay within the therapeutic range of treatment. This would in turn minimise both toxicity and ineffective treatment. Various methods for the quantification of drugs have been developed such as high performance liquid chromatography (HPLC), mass spectrometry (MS) and ultra-violet visible spectroscopy (UV-vis). / 2023-12-01

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical chemistry and Pharmaceutical analysis Author: Petr Šlechta Supervisor: doc. PharmDr. Jan Zitko, Ph.D. Consultant: MSc. Ghada Basem Bouz, Ph.D. Title of diploma thesis: Derivatives combining the fragment of pyrazinamide and 4-aminosalicylic acid as antimycobacterial compounds According to WHO, tuberculosis (TB) is the leading cause of death from a single infectious organism worldwide and the number of cases with drug resistant TB is still increasing, creating the need for new antituberculotics. Therefore, we report design, synthesis and antimicrobial evaluation of a series of hybrid compounds combining different pyrazinamide derivates and p- aminosalicylic acid as potential antituberculotic agents. The compounds were prepared by mixing different pyrazinecarboxylic acids, after activation by 1,1'-carbonyldiimidazole, with p- aminosalicylic acid in dimethylsulfoxide as a solvent. Obtained compounds were in vitro tested for their antimycobacterial activity against M. tuberculosis H37Rv, M. tuberculosis H37Ra and four other mycobacterial strains. Prepared compounds were also in vitro screened for antibacterial, antifungal, and cytotoxic (HepG2) activity. Most compounds showed antimycobacterial activity in range of...

Charles University Faculty of Pharmacy in Hradec Králové Department of Pharmaceutical chemistry and Pharmaceutical analysis Author: Petr Šlechta Supervisor: doc. PharmDr. Jan Zitko, Ph.D. Consultant: MSc. Ghada Basem Bouz, Ph.D. Title of diploma thesis: Derivatives combining the fragment of pyrazinamide and 4-aminosalicylic acid as antimycobacterial compounds According to WHO, tuberculosis (TB) is the leading cause of death from a single infectious organism worldwide and the number of cases with drug resistant TB is still increasing, creating the need for new antituberculotics. Therefore, we report design, synthesis and antimicrobial evaluation of a series of hybrid compounds combining different pyrazinamide derivates and p- aminosalicylic acid as potential antituberculotic agents. The compounds were prepared by mixing different pyrazinecarboxylic acids, after activation by 1,1'-carbonyldiimidazole, with p- aminosalicylic acid in dimethylsulfoxide as a solvent. Obtained compounds were in vitro tested for their antimycobacterial activity against M. tuberculosis H37Rv, M. tuberculosis H37Ra and four other mycobacterial strains. Prepared compounds were also in vitro screened for antibacterial, antifungal, and cytotoxic (HepG2) activity. Most compounds showed antimycobacterial activity in range of...

Made available in DSpace on 2014-12-17T15:42:17Z (GMT). No. of bitstreams: 1 SeverinaDSO_DISSERT.pdf: 2512071 bytes, checksum: 12a15e7529d5b018959d5e9c644f10cf (MD5) Previous issue date: 2013-07-31 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / In this work a study was done using electrochemical cyclic voltammetry and differential pulse voltammetry for isoniazida (INH), ethambutol (EMB), rifampicina (RIF) and pyrazinamide (PZA) using the electrode boron-doped diamond (BDD) as working electrode. It also verified the applicability of the technique of differential pulse voltammetry in the quantification of the active compounds used in the treatment of tuberculosis, subsequently applying in samples of pharmaceutical formulation. Among the four active compounds studied, isoniazid showed the best results for the detection and quantification using differential pulse voltammetry. At pH 4 and pH 8, for the calibration curves to INH showed good linearity, with quantification limits of 6.15 mmol L-1 (0,844 ppm) and 4.08 mmol L-1 (0.560 ppm) for the respective pH. The proposed method can be used to determine drug isoniazid, for recovery values were obtained in approximately 100% / Neste trabalho foi feito um estudo eletroqu?mico utilizando a voltametria c?clica e voltametria de pulso diferencial para os f?rmacos isoniazida (INH), etambutol (EMB), rifampicina (RIF) e pirazinamida (PZA) usando o eletrodo de diamante dopado com boro (BDD) como eletrodo de trabalho. Foi tamb?m verificado a aplicabilidade da t?cnica de voltametria de pulso diferencial na quantifica??o dos princ?pios ativos usados no tratamento da tuberculose, posteriormente aplicando em amostras de formula??o farmac?utica. Dentre os quatro princ?pios ativos estudados, a isoniazida apresentou os melhores resultados de detec??o e quantifica??o com o uso da voltametria de pulso diferencial. Em pH 4 e pH 8, as curvas de calibra??o para a INH apresentaram boa linearidade, apresentando os limites de quantifica??o de 6,15 &#956;mol L-1 (0,844 ppm) e 4,08 &#956;mol L-1 (0,560 ppm), para os respectivos pH. O m?todo proposto pode ser usado para a determina??o de isoniazida em f?rmacos, pois foram obtidos valores de recupera??o em torno de 100%

>Magister Scientiae - MSc / Tuberculosis still remains one of the world’s killer diseases. Pyrazinamide (PZA) is one of the most commonly prescribed anti-tuberculosis (anti-TB) drugs due to its ability to significantly shorten the TB treatment period. However, excess PZA in the body caused hepatotoxicity and liver damage. This, together with the resistance of the bacteria to treatment drugs, poor medication and inappropriate dosing, contribute significantly to the high incidents of TB deaths and diseases (such as liver damage). This, therefore, calls for new methods for ensuring reliable dosing of the drug, which will differ from person to person due to inter-individual differences in drug metabolism. A novel biosensor system for monitoring the metabolism of PZA was prepared with a nanocomposite of multi-walled carbon nanotubes (MWCNTs), polyaniline (PANI) and cytochrome P450 2E1 (CYP2E1) electrochemically deposited on a glassy carbon electrode (GCE). The nanocomposite biosensor system exhibited enhanced electro-activity that is attributed to the catalytic effect of the incorporated MWCNTs. The biosensor had a sensitivity of 7.80 μA/ μg mL-1 PZA and a dynamic linear range (DLR) of 4.92 – 160 ng/mL PZA. Bisphenol A (BPA) is a hormone-disrupting chemical used in production of epoxy resins and polycarbonates, which produce various products used on a daily basis. However, BPA can leach out of plastic during normal use and cause health effects such as cancer or disrupt the endocrine system. Moreover, BPA has also been proven to degrade from the containers in landfills and accumulate in groundwater and streams, thereby, polluting the environment while destroying aquatic organisms. Therefore, this also calls for new selective and sensitive methods for the monitoring of BPA. A novel biosensor system for monitoring the oxidation of BPA was prepared from a nanocomposite of polyaniline, polymethyl methacrylate and titanium dioxide nanoparticles, also electrochemically deposited on the GCE. Biosensor fabrication was conducted by immobilization of the enzyme manganese peroxidase (MnP) iii onto the nanocomposite film. The nanobiosensor also revealed enhanced electro activity, attributed to the incorporation of TiO2 nanoparticles. The biosensor system had a sensitivity of 0.3 μA/nM and a detection limit of 0.12 nM. This detection limit falls within the range of the allowed daily intake of BPA as recommended by the Food and Drug Administration (FDA, USA) and other regulatory bodies.

Submitted by Alexandre Sousa (alexandre.sousa@incqs.fiocruz.br) on 2014-10-21T17:51:39Z No. of bitstreams: 1 Dissertação PatríciaCondéde Lima.pdf: 2501003 bytes, checksum: ae9bd16a44582c654071a25e36e5497e (MD5) / Made available in DSpace on 2014-10-21T17:51:39Z (GMT). No. of bitstreams: 1 Dissertação PatríciaCondéde Lima.pdf: 2501003 bytes, checksum: ae9bd16a44582c654071a25e36e5497e (MD5) Previous issue date: 2014 / Fundação Oswaldo Cruz. Instituto Nacional de Controle de Qualidade em Saúde. Departamento de Química. Rio de Janeiro, RJ, Brasil. / Oscomprimidosutilizadosno tratamento da tuberculose possuem quatro fármacos associados, Isoniazida(INH), Pirazinamida(PYA), Etambutol(EMB) e Rifampicina(RMP) e são distribuídos gratuitamente pelo Sistema Único de Saúde. Até o momento, os métodos analíticos oficiais para a análise dos teores dos fármacos presentes neste medicamento de dose fixa combinada são aqueles especificados na Farmacopeia Americana 36ª edição e na Farmacopeia Internacional 4ª edição. Porém, nenhum destes compêndios oficiais possui monografias para análise simultânea dos quatro fármacos. O desenvolvimento de métodos analíticos adequados para determinar simultaneamente as concentrações dos quatro fármacos em comprimidos dose fixa combinada é de grande importância para o controle da qualidade do medicamento em questão. O objetivo deste estudo foi desenvolver um método analítico para determinar simultaneamente todos os princípios ativos do comprimido para o tratamento da tuberculose por cromatografia a líquido de alta eficiência com detector de ultravioleta-visível. O método desenvolvido utilizou uma coluna cromatográfica com fase estacionária octadecilsila no, 250mm de comprimento, 4,6mm de diâmetro e5 μm de tamanho de partícula e fase móvel constituída de uma fase aquosa(85% tampão formiato de amônio 0,3 mol/L pH 5, 15%metanol e 0,005 mol/L de Cu2+ou 250 mg/L de CuSO4.5H2O) e fase orgânica(metanol, 0,1% de trietilamina e 0,2% de ácido fórmico). O fluxo empregado foi de 1,0 mL/min e comprimento de onda265 nm, quando o software do equipamento permitiu, para a INH, PYA e o EMB um comprimento de onda de 265 e de 335 nm para a RMP. Este estudo demonstrou que o método desenvolvido apresentou linearidade entre 0,05 a 0,10 mg/mL para INH, 0,27 a 0,52 mg/mL para PYA, 0,19 a 0,36 mg/mL para EMB e 0,05 a 0,1 mg/mL para RMPe mostrou-se satisfatório quanto ao parâmetro de precisão, com desvio padrão relativo inferior a 2,0% para os quatro fármacos estudados. / Tablets used to treat tuberculosis contain four drugs, isoniazid (INH), pyrazinamide (PYA), ethambutol (EMB) and rifampicin (RMP) and are freely distributed by the National Health System. So far, the official analytical methods for testing the amount of the ingredients present in the fixed-dose combination tablet are those described in the 36th editionof U.S. Pharmacopeia and in the 4th editionof International Pharmacopoeia. However, none of these official compendium present monographs for the simultaneous analysis of the four drugs. The development of suitable analytical methods for simultaneously determining the concentrations of the four drugs in fixed-dose combination tablets is of great importance to the quality control of the medicinal product concerned. The aim of this study was to develop an analytical method to simultaneously determine all the active ingredients of the tablet for tuberculosis treatment by high-performance liquid chromatography with ultraviolet-visible detector. The developed method used a chromatographic column with octadecylsilane stationary phase, with 250mm length, 4,6mm diameter and 5 μm particle size. The mobile phase was aqueous (85% ammonium formate buffer 0,3 mol/LpH 5, 15% methanol and 0,005 mol/Lof Cu2+or250 mg/LCuSO4.5H2O), and the organic phase(methanol, 0,1% triethylamine and 0,2 % formic acid). The used flow was 1,0mL/min, at a wavelength of 265 nm or when the equipment alloweda wavelength of 265 nm for INH, EMB, and PYA and 335nmfor RMP. This study showned that the developed method was linear from 0,05 to 0,10 mg/mL for INH, 0,27 to 0,52 mg/mL for PYA, 0,19 to 0,36 mg/mL for EMB and 0,05 to0,1 mg/mL for RMP and was satisfactory regarding the precision parameter, with relative standard deviation lower than 2,0 % for the four studied drugs.