RESUMO
A quantitative and confirmatory multiresidue method for determining the presence of avermectins, benzimidazoles and nitroimidazoles in bovine muscle tissue by ultra-high performance liquid chromatography tandem mass spectrometry (UHPLC-MS/MS) was developed, optimized and validated, using a QuEChERS extraction. The evaluated performance parameters were linearity, selectivity, matrix effect, decision limits (CCα), detection capability (CCß), limits of detection (LOD), limits of quantification (LOQ), accuracy, precision and robustness. The validated method exhibited linearity with coefficient of determination (R2) higher than 0.90 in the working range from 0.5 to 2.0 times the maximum residue limit (MRL) or the minimum required performance level (MRPL) for the studied analytes, except for closantel, for which the linear study range was defined from 50 to 200µgkg-1. The method was selective in the presence of macrolides and lincosamides for all the studied analytes. The LOD varied from 0.007 to 66.715µgkg-1, whereas LOQ values ranging from 0.011 to 113.674µgkg-1 were found. The results of the evaluation of the accuracy and precision were satisfactory for all the studied analytes, and according to the assessment of the robustness, the method was not robust only for the analytes abamectin, moxidectin, doramectin fenbendazole sulfone, closantel, thiabendazole, hydroxyl-metronidazole and ronidazole. The performance parameters demonstrated total method adequacy for the detection and quantification of avermectins, benzimidazoles and nitroimidazoles residues in bovine muscle tissues.
Assuntos
Benzimidazóis/análise , Benzimidazóis/isolamento & purificação , Ivermectina/análogos & derivados , Músculos/química , Nitroimidazóis/análise , Nitroimidazóis/isolamento & purificação , Métodos Analíticos de Preparação de Amostras , Animais , Bovinos , Fracionamento Químico , Cromatografia Líquida de Alta Pressão , Resíduos de Drogas/análise , Resíduos de Drogas/isolamento & purificação , Ivermectina/análise , Ivermectina/isolamento & purificação , Limite de Detecção , Espectrometria de Massas em TandemRESUMO
The aim of this work was the development of an analytical procedure using spectrophotometry for simultaneous determination of benznidazole (BNZ) and itraconazole (ITZ) in a medicine used for the treatment of Chagas disease. In order to achieve this goal, the analysis of mixtures was performed applying the Lambert-Beer law through the absorbances of BNZ and ITZ in the wavelengths 259 and 321 nm, respectively. Diverse tests were carried out for development and validation of the method, which proved to be selective, robust, linear, and precise. The lower limits of detection and quantification demonstrate its sensitivity to quantify small amounts of analytes, enabling its application for various analytical purposes, such as dissolution test and routine assays. In short, the quantification of BNZ and ITZ by analysis of mixtures had shown to be efficient and cost-effective alternative for determination of these drugs in a pharmaceutical dosage form.
Assuntos
Antifúngicos/análise , Itraconazol/análise , Nitroimidazóis/análise , Espectrofotometria/métodos , Tripanossomicidas/análise , Doença de Chagas/tratamento farmacológico , Química Farmacêutica/métodos , Combinação de Medicamentos , Humanos , Limite de Detecção , Controle de QualidadeRESUMO
Chagas disease constitutes a major public health problem in Latin America. Human breast milk is a biological sample of great importance for the analysis of therapeutic drugs, as unwanted exposure through breast milk could result in pharmacological effects in the nursing infant. Thus, the goal of breast milk drug analysis is to inquire to which extent a neonate may be exposed to a drug during lactation. In this work, we developed an analytical technique to quantify benznidazole and nifurtimox (the two antichagasic drugs currently available for medical treatment) in human breast milk, with a simple sample pretreatment followed by an ionic-liquid-based dispersive liquid-liquid microextraction combined with high-performance liquid chromatography and UV detection. For this technique, the ionic liquid 1-octyl-3-methylimidazolium hexafluorophosphate has been used as the "extraction solvent." A central composite design was used to find the optimum values for the significant variables affecting the extraction process: volume of ionic liquid, volume of dispersant solvent, ionic strength, and pH. At the optimum working conditions, the average recoveries were 77.5 and 89.7%, the limits of detection were 0.06 and 0.09 µg/mL and the interday reproducibilities were 6.25 and 5.77% for benznidazole and nifurtimox, respectively. The proposed methodology can be considered sensitive, simple, robust, accurate, and green.
Assuntos
Doença de Chagas , Líquidos Iônicos/química , Microextração em Fase Líquida , Leite Humano/química , Nifurtimox/análise , Nitroimidazóis/análise , Tripanossomicidas/análise , Cromatografia Líquida de Alta Pressão , Humanos , Imidazóis/química , Estrutura Molecular , Raios UltravioletaRESUMO
BACKGROUND: Due to migration, Chagas disease is a significant public health problem in Latin America, and in other nonendemic regions. The 2 drugs currently available for the treatment, nifurtimox and benznidazole (BNZ), are associated with a high risk of toxicity in therapeutic doses. Excretion of drug into human breast milk is a potential source of unwanted exposure and pharmacologic effects in the nursing infant. However, this phenomenon was not evaluated until now, and measurement techniques for both drugs in milk were not developed. METHODS: In this work, we described the development of a simple and fast method to quantify BNZ in human milk using a pretreatment that involves acid protein precipitation followed by tandem microfiltration, and reverse phase high-performance liquid chromatography/ultraviolet analysis. It is simple because it takes only 3 steps to obtain a clean extracted solution that is ready to inject into the high-performance liquid chromatography equipment. It is fast because a complete analysis of a sample takes only 36 minutes. RESULTS: Although the human breast milk composition is very variable, and lipids are one of the most difficult compounds to clean up on a milk sample, the procedure has proven to be robust and sensitive with a limit of detection of 0.3 µg/mL and quantization of 0.9 µg/mL. Despite a 70% recovery value, which could be considered a relatively low result, this recovery is reproducible (coefficient of variation <10%) and the analytical response under the linear range is very good (r = 0.9969 adjusted). Real samples of human breast milk from patients in treatment with BNZ were dosed to support the validation process of the method. CONCLUSIONS: The method described is fast, specific, accurate, precise, and sufficiently sensitive in the clinical context for the quantification of BNZ in human milk. For all these reasons, it is suitable for clinical risk evaluation studies.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Leite Humano/química , Nitroimidazóis/análise , Monitoramento de Medicamentos/métodos , Feminino , Voluntários Saudáveis , Humanos , Lactação/metabolismo , Nitroimidazóis/química , Nitroimidazóis/farmacocinética , Tripanossomicidas/análise , Tripanossomicidas/química , Tripanossomicidas/farmacocinéticaRESUMO
Benznidazole (BNZ) is traditionally used to treat Chagas disease. Despite its common use, BNZ has a poor water solubility and a variable bioavailability. The purpose of this study was to prepare BNZ microcrystals by solvent change precipitation and to study the effects of BNZ micronisation on therapeutic efficiency using a murine model of Chagas disease. The solvent change precipitation procedure was optimised in order to obtain stable and homogeneous particles with a small particle size, high yield and fast dissolution rate. The thermal and crystallographic analysis showed no polymorphic change in the microcrystals, and microscopy confirmed a significant reduction in particle size. A marked improvement in the drug dissolution rate was observed for micronised BNZ particles and BNZ tablets in comparison with untreated BNZ and commercial Rochagan. In vivo studies showed a significant increase in the therapeutic efficacy of the BNZ microparticles, corroborating the dissolution results.
Assuntos
Doença de Chagas/tratamento farmacológico , Composição de Medicamentos/métodos , Nitroimidazóis/química , Tripanossomicidas/química , Animais , Precipitação Química , Modelos Animais de Doenças , Sistemas de Liberação de Medicamentos , Excipientes , Feminino , Testes de Dureza , Derivados da Hipromelose , Metilcelulose/análogos & derivados , Metilcelulose/química , Camundongos , Nitroimidazóis/análise , Nitroimidazóis/uso terapêutico , Polímeros/química , Solubilidade , Solventes/química , Comprimidos , Tripanossomicidas/análise , Tripanossomicidas/uso terapêutico , Trypanosoma cruzi/efeitos dos fármacosRESUMO
This study evaluated the association of N-hexyl-2-methyl-4-nitroimidazol, a model drug, to aggregates formed by anionic polyelectrolytes on aqueous solution. The alternating copolymers of maleic anhydride and N-vinyl-2-pyrrolidone were synthesized and then modified by reaction of the anhydride groups with aliphatic amines and alcohols of varying length of the alkyl chain. The partition of the model drug between water and the hydrophobic microdomains provided by the copolymers was studied using the pseudo-phase model to determinate the distribution coefficient K S, and the standard free energy of transfer ∆µ°t. The results indicate that all copolymers assessed are potential pharmaceutical reservoirs of the model drug. Nevertheless, the solubility of N-hexyl-2-methyl-4-nitroimidazol on the polymeric solutions is independent from the length of the alkyl chain of the copolymer.
Realizou-se estudo sobre a associação da N-hexil-2-metil-4-nitroimidazol, fármaco modelo, aos agregados formados por polieletrólitos aniônicos em solução aquosa. Os copolímeros alternados de anidrido maléico e N-vinil-2-pirrolidona foram sintetizados e, em seguida, modificados pela reação dos grupos de anidrido com aminas e álcoois alifáticos de duração variável da cadeia alquílica. A partição do fármaco modelo entre a água e os microdomínios hidrofóbicos fornecido pelos copolímeros foi estudada usando o modelo de pseudo-fase, a fim de determinar a distribuição do coeficiente K S e a energia livre padrão de transferência ∆µ°t. Os resultados indicam que todos os copolímeros avaliados são potenciais reservatórios farmacêuticos do fármaco. No entanto, a solubilidade do N-hexil-2-metil-4-nitroimidazol sobre as soluções poliméricas é independente do comprimento da cadeia alquílica do copolímero.
Assuntos
Química Farmacêutica , Nitroimidazóis/análise , Copolímero de Pirano , Anidridos MaleicosRESUMO
Two nitroheterocyclic drugs, nifurtimox (NFX) and benznidazole (BZ), used in the treatment of Chagas' disease have serious side effects attributed to their nitroreduction to reactive metabolites. Here, we report that these drugs reach the mammary tissue and there they could undergo in situ bioactivation. Both were detected in mammary tissue from female Sprague-Dawley rats after their intragastric administration. Only NFX was biotransformed by pure xanthine-oxidoreductase and from tissue cytosol. These activities were purine dependent and were inhibited by allopurinol. Also, only NFX was biotransformed by microsomes in the presence of ß-nicotinamide adenine dinucleotide phosphate, reduced form (NADPH), and was inhibited by carbon monoxide and partially by diphenyleneiodonium. NFX treatment produced significant decrease in protein sulfhydryl content after 1, 3 and 6 hours; no increases in protein carbonyl content at any time tested and significantly higher levels of lipid hydroperoxides at 3 and 6 hours; besides, ultrastructural observations after 24 hours showed significant differences in epithelial cells compared to control. These findings indicate that NFX might be more deleterious to mammary tissue than BZ and could correlate with early reports on its ability to promote rat mammary tissue toxicity.
Assuntos
Glândulas Mamárias Animais/metabolismo , Nifurtimox/farmacocinética , Nitroimidazóis/farmacocinética , Tripanossomicidas/farmacocinética , Alopurinol/farmacologia , Animais , Fracionamento Celular , Cromatografia Líquida de Alta Pressão , Feminino , Peroxidação de Lipídeos/efeitos dos fármacos , Glândulas Mamárias Animais/química , Glândulas Mamárias Animais/ultraestrutura , Microscopia Eletrônica de Transmissão , Microssomos/efeitos dos fármacos , Microssomos/metabolismo , Nifurtimox/análise , Nifurtimox/metabolismo , Nitroimidazóis/análise , Nitroimidazóis/metabolismo , Nitrorredutases/metabolismo , Carbonilação Proteica/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Compostos de Sulfidrila/análise , Compostos de Sulfidrila/metabolismo , Tripanossomicidas/análise , Tripanossomicidas/metabolismoRESUMO
Increasing evidence indicates that hypoxia-inducible factor 1alpha (HIF-1alpha) can be upregulated in different cell types by nonhypoxic stimuli such as growth factors, cytokines, nitric oxide, lipopolysaccharides and a range of infectious microorganisms. In this study, the ability of the following mononuclear phagocytes to express HIF-1alpha is reported: mouse macrophages (mMPhi), human macrophages (hMPhi) and human dendritic cells (DC), parasitized in vitro with Leishmania amazonensis; as assessed by immunofluorescence microscopy. A logical explanation for HIF-1alpha expression might be that the mononuclear phagocytes became hypoxic after L. amazonensis infection. Using the hypoxia marker pimonidazole, observation revealed that L. amazonensis-infected cells were not hypoxic. In addition, experiments using a HIF-1alpha inhibitor, CdCl(2), to treat L. amazonensis-infected macrophage cultures showed reduced parasite survival. These studies indicated that HIF-1alpha could play a role in adaptative and immune responses of mononuclear phagocytes presenting infection by the parasite L. amazonensis.
Assuntos
Células Dendríticas/metabolismo , Células Dendríticas/parasitologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Leishmania mexicana/fisiologia , Macrófagos Peritoneais/imunologia , Macrófagos Peritoneais/parasitologia , Animais , Cloreto de Cádmio/farmacologia , Hipóxia Celular , Células Cultivadas , Células Dendríticas/imunologia , Leishmania mexicana/imunologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Nitroimidazóis/análiseRESUMO
Ischemia-induced acute renal failure (ARF) is a disorder with high morbidity and mortality. ARF is characterized by a regeneration phase, yet its molecular basis is still under study. Changes in gene expression have been reported in ARF, and some of these genes are specific for nephrogenic processes. We tested the hypothesis that the regeneration process developed after ischemia-induced ARF can be characterized by the reexpression of important regulatory proteins of kidney development. The distribution pattern and levels of nephrogenic proteins in rat kidneys after ischemia were studied by immunohistochemistry and immunoblot analysis. Ischemic damage was assessed by conventional morphology, serum creatinine, and the apoptotic markers terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) and caspase 3. The hypoxia levels induced by ischemia were assessed by specific markers: hypoxia induced factor (HIF)-1alpha and 2-pimonidazole. In kidneys with ARF, an important initial damage was observed through periodic acid Schiff staining, by the induction of damage markers alpha-smooth muscle actin (alpha-SMA) and macrophages (ED-1) and by apoptosis induction. In agreement with diminishing renal damage at the initial reparation phase, the expression of the mesenchymal proteins vimentin, neural cell adhesion molecules (Ncam), and the epithelial markers, Pax-2, Noggin, and basic fibroblast growth factor was observed; after, in a second phase, the tubular markers bone morphogen protein 7, Engrailed, and Lim-1, as well as the transcription factors Smad and p-Smad, were observed. Additionally, the endothelial markers VEGF and Tie-2 were induced at the initial and middle stages of regeneration phase, respectively. The expression of these proteins was restricted in time and space, as well as spatially and temporally. Because all of these proteins are important in maintaining a functional kidney, these results suggest that during the regeneration process after induced hypoxia, these nephrogenic proteins can be reexpressed in a similar fashion to that observed during development, thus restoring mature kidney function.
Assuntos
Injúria Renal Aguda/metabolismo , Proteínas Morfogenéticas Ósseas/metabolismo , Proteínas de Transporte/metabolismo , Túbulos Renais/metabolismo , Rim/irrigação sanguínea , Injúria Renal Aguda/etiologia , Injúria Renal Aguda/patologia , Animais , Biomarcadores/análise , Proteína Morfogenética Óssea 7 , Caspase 3 , Caspases/metabolismo , Morte Celular , Hipóxia/etiologia , Imuno-Histoquímica , Rim/metabolismo , Túbulos Renais/patologia , Masculino , Nitroimidazóis/análise , Fosforilação , Ratos , Ratos Sprague-Dawley , Regeneração , Traumatismo por Reperfusão/complicações , Proteínas Smad/metabolismoRESUMO
Giardia intestinalis is a common parasite in our country and the rest of the world and is responsible for several clinical disturbances that include dysentery type diarrheas, recurrent abdominal pain, duodenitis, jejunitis, cholecystitis and in some cases toxemias and convulsions. In this paper we review recent concepts of intestinal giardiasis, considering the basic aspects of the biology and physiology of Giardia intestinalis, its morphology and its relationship the parasite pathogenicity. We detail the physiopathological mechanisms responsible for the different clinic manifestations of giardiasis, the specific laboratory and endoscopic methods of diagnosis and the most recent advances in the treatment and prophylaxis of this disease.
Assuntos
Giardíase , Adulto , Animais , Antiprotozoários/uso terapêutico , Criança , Contraindicações , Feminino , Furazolidona/uso terapêutico , Giardia lamblia/crescimento & desenvolvimento , Giardia lamblia/imunologia , Giardia lamblia/fisiologia , Giardia lamblia/ultraestrutura , Giardíase/diagnóstico , Giardíase/tratamento farmacológico , Giardíase/epidemiologia , Giardíase/imunologia , Giardíase/prevenção & controle , Humanos , Leite Humano/química , Nitroimidazóis/análise , Nitroimidazóis/uso terapêutico , Paromomicina/uso terapêutico , Gravidez , Poluição da ÁguaRESUMO
Benznidazole is a drug used commonly as a therapeutic agent against Chagas' disease in Brazil. To clarify the cytotoxic action of benznidazole the electrochemical reduction of benznidazole has been investigated using a DNA-electrochemical biosensor, prepared by modification of a glassy carbon electrode with DNA, and the results compared with reduction at a bare glassy carbon electrode. The dependence of peak potential with pH follows slopes of 59 and 52 mV per pH unit in acid media, respectively, which corresponds to a mechanism involving the same number of electrons and protons. In neutral and alkaline solution no significant dependence of peak potential with pH was found. During the electrochemical reduction of benznidazole the formation of the hydroxylamine derivative occurs, involving a total of four electrons. The potentials for reduction were less negative when using the DNA-modified glassy carbon electrode than at the bare glassy carbon electrode although the mechanism was the same, and at pH 7.51 the peak current was four times higher than that obtained with the bare electrode. The DNA-biosensor enabled pre-concentration of the drug onto the electrode surface and the in situ damage caused to the DNA on the electrode surface by the product of benznidazole reduction could be detected electrochemically. The results are in agreement with the hypothesis that the hydroxylamine derivative is the reactive species responsible for the cytotoxic action of benznidazole.
Assuntos
Técnicas Biossensoriais/métodos , Nitroimidazóis/análise , Carbono , Dano ao DNA , DNA de Cadeia Simples , Eletroquímica , EletrodosRESUMO
Giardia intestinalis is a common parasite in our country and the rest of the world and is responsible for several clinical disturbances that include dysentery type diarrheas, recurrent abdominal pain, duodenitis, jejunitis, cholecystitis and in some cases toxemias and convulsions. In this paper we review recent concepts of intestinal giardiasis, considering the basic aspects of the biology and physiology of Giardia intestinalis, its morphology and its relationship the parasite pathogenicity. We detail the physiopathological mechanisms responsible for the different clinic manifestations of giardiasis, the specific laboratory and endoscopic methods of diagnosis and the most recent advances in the treatment and prophylaxis of this disease.