Detec??o do composto iqg-607 : uma etapa essencial para futuros estudos de farmacocin?tica

Submitted by Setor de Tratamento da Informa??o - BC/PUCRS ([email protected]) on 2015-09-25T11:33:13Z No. of bitstreams: 1 475172 - Texto Completo.pdf: 1033533 bytes, checksum: 3695eeb1d157f2c6ed87073d37aa8b1e (MD5)Made available in DSpace on 2015-09-25T11:33:13Z (GMT). No. of bitstreams: 1 475172 - Texto Completo.pdf: 1033533 bytes, checksum: 3695eeb1d157f2c6ed87073d37aa8b1e (MD5) Previous issue date: 2015-07-10Tuberculosis (TB) caused predominantly by Mycobacterium tuberculosis is an infectious disease responsible for a significantly number of deaths worldwide. The first line TB drugs that are currently used, and others available, present several problems, such as duration and complexity of treatment, and adverse effects, factors that decrease the patient adherence to treatment regimen. These problems increase the number of cases of drug resistant TB. The pentacyano(isoniazid)ferrate(II) (IQG-607) compound is an inorganic complex designed to treat INH-resistant strains of M. tuberculosis containing mutations in catalase-peroxidase gene, katG. The compound appears to be a promising anti-TB drug candidate according to previous studies that demonstrated its effectiveness both in vitro and in vivo. However, the difficulties encountered in the development of an analytical method for detection and quantification of the compound, due to its chemical properties, were impeding further studies, such as the pharmacokinetic studies. In this context, the present study describes two analytical methods for the detection of IQG-607 for the first time. The voltammetric method was shown to be a powerful analytical tool for quantification of IQG-607 and may be used to monitoring the stability and quality control. The proposed method, together with enzymatic inhibition assay, shown that the IQG-607 is stable for at least 1week at 4 ?C. The detection of IQG-607 by high performance liquid chromatography (HPLC) was also described, and we were able to observe the retention of the compound on the stationary phase of a chromatography column for the first time in this work. In addition, the analysis of IQG-607 in mouse plasma by HPLC was also demonstrated. Therefore, this conventional method will be optimized and validated to conduct pharmacokinetics studies.A tuberculose (TB) causada predominantemente por Mycobacterium tuberculosis ? uma doen?a infecciosa respons?vel por um n?mero consider?vel de mortes em todo o mundo. Os medicamentos utilizados no tratamento de primeira linha atual, e os demais dispon?veis, apresentam diversos problemas, como a dura??o, complexidade, e efeitos adversos, dificultando a ades?o dos pacientes ao regime terap?utico. Isto favorece o aumento do n?mero de casos de TB resistente a medicamentos. O pentaciano(isoniazida)ferrato(II) (IQG-607) ? um composto proposto para agir contra casos de TB resistentes ? INH devido a infec??o com cepas que possuem muta??es no gene katG. O composto demonstrou ser um promissor agente anti-TB em estudos pr?vios, e a sua efic?cia in vitro e in vivo foi comprovada. No entanto, as dificuldades encontradas no desenvolvimento de um m?todo anal?tico para a detec??o e quantifica??o do composto devido as suas caracter?sticas qu?micas estavam impedindo a realiza??o de outros estudos, e dentre eles, a determina??o de par?metros farmacocin?ticos. Tendo isto em vista, o presente trabalho descreve, pela primeira vez, dois m?todos anal?ticos para detec??o de IQG-607. O m?todo voltam?trico mostrou potencialidade na quantifica??o do composto, podendo ser utilizado para monitorar a estabilidade e em controle de qualidade. Observamos, por meio deste m?todo, juntamente com ensaios de inibi??o enzim?tica, que o IQG-607 ? est?vel em solu??o por at? 1 semana se for mantido a 4?C. A detec??o do IQG-607 por cromatografia l?quida de alta efici?ncia (CLAE) tamb?m foi demonstrada, e, pela primeira vez, observamos a intera??o do composto em coluna cromatogr?fica. Ainda, por CLAE, demonstramos a detec??o do IQG-607 no plasma de camundongos, e por ser um m?todo convencional, ser? otimizado e validado para a realiza??o de futuros estudos de farmacocin?tica

Avalia??o farmacocin?tica pr?-cl?nica de candidatos a f?rmacos antituberculose

Submitted by PPG Biologia Celular e Molecular ([email protected]) on 2018-03-15T12:31:20Z No. of bitstreams: 1 Tese Doutorado Adilio.pdf: 2662669 bytes, checksum: 73c86d6d555c94d57c1f0714b5c2ccbc (MD5)Approved for entry into archive by Tatiana Lopes ([email protected]) on 2018-03-23T11:44:45Z (GMT) No. of bitstreams: 1 Tese Doutorado Adilio.pdf: 2662669 bytes, checksum: 73c86d6d555c94d57c1f0714b5c2ccbc (MD5)Made available in DSpace on 2018-03-23T11:52:37Z (GMT). No. of bitstreams: 1 Tese Doutorado Adilio.pdf: 2662669 bytes, checksum: 73c86d6d555c94d57c1f0714b5c2ccbc (MD5) Previous issue date: 2018-03-02Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPESTuberculosis (TB), caused mainly by Mycobacterium tuberculosis, is an infectious disease responsible for a significant number of deaths worldwide. IQG-607 is an analog of isoniazid (INH). According to several experiments carried out by our research group, IQG-607 showed both in vitro and in vivo anti-TB activity. Initial studies showed that the compound INCT-TB551 (a quinoline derivative) also presents in vitro anti-TB activity. The aim of this study was to develop analytical methods by high performance liquid chromatography (HPLC-UV) for quantification of IQG-607 and INCT-TB551 in mice plasma, and therefore to perform pharmacokinetic studies. The analytical method for the determination of IQG-607 in mice plasma showed linearity (r = 0.9992) in 0.5? 50 ?g/mL concentration range. Intra- and inter-day precision was < 15%, and the recovery ranged from 92.07 to 107.68%, showing that the method provides a precise and accurate analysis of the compound. In addition, IQG-607 was stable in plasma for at least 30 days at ?80 ?C, and after plasma processing, for 4 h in the auto-sampler (6 ?C) and maintained on ice (recovery > 85%). The applicability of the method for pharmacokinetic studies was determined after intravenous (i.v.) and oral (fasted and fed conditions) administrations to mice. IQG-607 levels in plasma were quantified at time points for up to 2.5 h. A short half-life (t1/2) (1.14 h), a high clearance (CL) (3.89 L/h/kg), a moderate volume of distribution at steady state (Vdss, 1.22 L/kg), were observed after i.v. (50 mg/kg) administration. Similar results were obtained for oral administration (250 mg/kg) under fasted and fed conditions. The oral bioavailability (F), approximately 4%, was not altered by feeding. Plasma protein binding was 88.87 ? 0.9%. Recently, experiments in mice infected with M. tuberculosis have shown that the compound INCT-TB551 has no in vivo activity, unlike previous in vitro activity studies. An analytical method was developed for the determination of INCT-TB551 in mice plasma to assess compound absorption, if any, after oral administration. The analytical method presented linearity from 0.1-10 ?g/mL (r = 0.9999). After development of the analytical method, the compound was orally administered in mice and the plasma levels were quantified at time points for up to 1 h. The compound INCT-TB551 was detected in the plasma of animals, which indicated that INCT-TB551 was absorbed. Although absorbed when orally administered, the compound is not exhibiting activity against M. tuberculosis in this animal model. This finding may be associated with the formation of inactive metabolites and/or the plasma concentration of INCT-TB551 achieved may be inadequate to exert its therapeutic effect. However, these points require further investigations. The protocols described here may serve as support to initiate pharmacokinetic studies of promising compounds, collaborating to advance in earlier stages of drug development.A tuberculose (TB) ? uma doen?a infecto-contagiosa, causada principalmente pelo Mycobacterium tuberculosis, respons?vel por um n?mero consider?vel de mortes em todo o mundo. O composto IQG-607 ? um an?logo da isoniazida (INH) que, de acordo com diversos experimentos realizados pelo grupo de pesquisa envolvido no seu estudo, apresenta atividade anti-TB in vitro e in vivo. Estudos iniciais do mesmo grupo demonstraram que o composto INCT-TB551 (um derivado quinol?nico) tamb?m apresenta promissora atividade anti-TB. O objetivo deste estudo foi desenvolver metodologias anal?ticas por cromatografia l?quida de alta efici?ncia (CLAE-UV) para a quantifica??o do composto IQG-607 e do INCT-TB551, em plasma de camundongos, para a realiza??o de estudos de farmacocin?tica. O m?todo anal?tico para a determina??o do composto IQG-607 em plasma de camundongos apresentou linearidade (r = 0.9992) na faixa de 0.5?50 ?g/mL. Os valores de precis?o intra e interensaio (CV < 15%) e de recupera??o (92.07-107.68%) demonstram que o m?todo ? preciso e exato para a an?lise do composto. Al?m disso, o IQG-607 se mostrou est?vel no plasma por at? 30 dias, quando armazenado no freezer -80 ?C, e est?vel ap?s o tratamento da amostra do plasma, por at? 4 horas (h) na bancada (gelo) e no autosampler do equipamento (6 ?C). A aplicabilidade do m?todo anal?tico para o estudo de farmacocin?tica foi determinada ap?s a administra??o i.v. e oral em camundongos (animais em jejum e animais alimentados). As concentra??es plasm?ticas de IQG-607 foram quantificadas por at? 2,5 h ap?s a administra??o nos animais. Quando administrado pela via i.v. foi observado um tempo de meia vida (t1/2) curto (1,14 h), elevado clearance (CL) (3,89 L/h/kg), e um moderado volume de distribui??o (Vdss) (1,22 L/kg). Resultados similares foram obtidos ap?s a administra??o oral (250 mg/kg) em camundongos que estavam em jejum ou alimentados. A biodisponibilidade oral (F) foi de aproximadamente 4 %, valor que n?o foi alterado pela alimenta??o. A taxa de liga??o a prote?nas plasm?ticas do IQG-607 ? de aproximadamente 88 ? 0.9%. Experimentos recentes em camundongos infectados com M. tuberculosis demonstraram que o composto INCT-TB551 n?o apresentou atividade neste modelo in vivo, ao contr?rio dos estudos de atividade in vitro realizados anteriormente. Um m?todo anal?tico para a determina??o do composto INCT-TB551 em plasma de camundongos foi desenvolvido para avaliar se o composto estava sendo absorvido ap?s a administra??o oral. O m?todo anal?tico apresentou linearidade na faixa de 0.1-10 ?g/mL (r = 0.9999). Ap?s o m?todo ter sido padronizado, o composto foi administrado por via oral em camundongos, e as concentra??es plasm?ticas foram quantificadas por at? 1 h ap?s a administra??o. O composto INCT-TB551 foi detectado no plasma dos animais, indicando que estava sendo absorvido. Apesar de absorvido quando administrado por via oral, o composto n?o est? apresentando atividade contra M. tuberculosis neste modelo animal, o que pode estar relacionado, por exemplo, com a forma??o de metab?litos inativos e/ou ainda, o n?vel plasm?tico atingido pode ser inadequado para que o composto consiga exercer o seu efeito terap?utico, e isto precisa ser melhor investigado. Os protocolos apresentados neste trabalho poder?o servir como suporte para estudos de farmacocin?ticoa de compostos que se apresentam promissores, colaborando para o avan?o nas etapas necess?rias para o desenvolvimento de poss?veis f?rmacos

Is IQG-607 a Potential Metallodrug or Metallopro-Drug With a Defined Molecular Target in Mycobacterium tuberculosis?

The emergence of strains of Mycobacterium tuberculosis resistant to isoniazid (INH) has underscored the need for the development of new anti-tuberculosis agents. INH is activated by the mycobacterial katG-encoded catalase-peroxidase, forming an acylpyridine fragment that is covalently attached to the C4 of NADH. This isonicotinyl-NAD adduct inhibits the activity of 2-trans-enoyl-ACP(CoA) reductase (InhA), which plays a role in mycolic acid biosynthesis. A metal-based INH analog, Na3[FeII(CN)5(INH)]·4H2O, IQG-607, was designed to have an electronic redistribution on INH moiety that would lead to an intramolecular electron transfer to bypass KatG activation. HPLC and EPR studies showed that the INH moiety can be oxidized by superoxide or peroxide yielding similar metabolites and isonicotinoyl radical only when associated to IQG-607, thereby supporting redox-mediated drug activation as a possible mechanism of action. However, IQG-607 was shown to inhibit the in vitro activity of both wild-type and INH-resistant mutant InhA enzymes in the absence of KatG activation. IQG-607 given by the oral route to M. tuberculosis-infected mice reduced lung lesions. Experiments using early and late controls of infection revealed a bactericidal activity for IQG-607. HPLC and voltammetric methods were developed to quantify IQG-607. Pharmacokinetic studies showed short half-life, high clearance, moderate volume of distribution, and low oral bioavailability, which was not altered by feeding. Safety and toxic effects of IQG-607 after acute and 90-day repeated oral administrations in both rats and minipigs showed occurrence of mild to moderate toxic events. Eight multidrug-resistant strains (MDR-TB) were resistant to IQG-607, suggesting an association between katG mutation and increasing MIC values. Whole genome sequencing of three spontaneous IQG-607-resistant strains harbored katG gene mutations. MIC measurements and macrophage infection experiments with a laboratorial strain showed that katG mutation is sufficient to confer resistance to IQG-607 and that the macrophage intracellular environment cannot trigger the self-activation mechanism. Reduced activity of IQG-607 against an M. tuberculosis strain overexpressing S94A InhA mutant protein suggested both the need for KatG activation and InhA as its target. Further efforts are suggested to be pursued toward attempting to translate IQG-607 into a chemotherapeutic agent to treat tuberculosis