Reverse phase high-performance liquid chromatography for quantification of hydroxymethylnitrofurazone in polymeric nanoparticles

Hydroxymethylnitrofurazone (NFOH) is a new compound with potential leishmanicidal and trypanocidal activity. Despite its effectiveness, the formulators have to overcome its poor aqueous solubility. Recently, polymeric nano-scale drug delivery systems have proposed for the treatment of neglected diseases. As several studies have confirmed the advantages of such formulations, and this approach provides new analytical challenges, including the need to detect trace amounts of the drug. A suitable method was developed and validated for NFOH determination bound to poly (n-butylcyanoacrylate) (PBCA) nanoparticles. The chromatographic separation was achieved using a C18 column maintained at 25 ºC and an isocratic mobile phase consisting of water and acetonitrile: 80:20 (v/v) at a flow rate of 1.2 mL min-1 and UV-detection at 265 nm. Investigated validation parameters included selectivity, linearity, accuracy, precision and robustness (changes in column temperature, mobile phase composition and flow). The method was specific, the peak of NFOH had no interference with any nanoparticle excipients and no co-elution with main degradation product (nitrofurazone). Linearity was over the range of 0.94 13.11 μg mL-1 (r2=0.999). The method was accurate and precise, recovery of 100.7%, RSD of 0.4%; intra-day and inter-day RSD range 9.98-9.99 μg mL-1 and 0.3% to 0.5%, respectively. Robustness confirmed that method could resist the applied changes. Application of the optimized method revealed an encapsulation efficiency of 64.4% (n=3). Therefore, the method was successfully developed and validated for the determination of the encapsulation efficiency of NFOH-PBCA nanoparticles.

Hidroximetilnitrofural (NFOH) é um novo composto que possui atividade leishmanicida e tripanomicida potencial. Um método apropriado foi desenvolvido e validado para a determinação de NFOH em nanopartículas de poli(n-butil cianoacrilato) (PBCA). A separação cromatográfica foi obtida usando uma coluna C18 (5 µm de tamanho de partícula, 4,6 mm de diâmetro e 150 mm de comprimento), mantida a 25 °C, fase móvel composta de água e acetonitrila 80:20 (v/v), fluxo de 1,2 mL min- 1 e detecção por UV a 265 nm. Investigaram-se os seguintes parâmetros de validação: seletividade, linearidade, exatidão, precisão e robustez (mudanças na temperatura de coluna, proporção da fase móvel e fluxo). O método mostrou-se específico, o pico de NFOH não apresentou interferência dos picos provenientes dos excipientes das nanopartículas e separado do principal produto de degradação (nitrofural). A linearidade foi obtida na faixa de 0,94-13,11 μg mL- 1 (r2=0,999). O método mostrou exatidão (recuperação de 100,7%, DPR de 0,4 %) e precisão (intra-dia e inter-dia, 9,98-9,99 μg mL- 1 e DPR 0,3% a 0,5%, respectivamente). A robustez provou que o método pode resistir às mudanças propostas. Aplicação do método otimizado revelou eficiência de encapsulação de 64,4% (n=3). Portanto, o método foi desenvolvido e validado com sucesso para a determinação da eficiência de encapsulação de nanopartículas de NFOH-PBCA.

Pharmacokinetics of hydroxymethylnitrofurazone, a promising new prodrug for Chagas' disease treatment.

Hydroxymethylnitrofurazone (NFOH) is a trypanocidal prodrug of nitrofurazone (NF), devoid of mutagenic toxicity. The purpose of this work was to study the chemical conversion of NFOH into NF in sodium acetate buffer (pH 1.2 and 7.4) and in human plasma and to determine preclinical pharmacokinetic parameters in rats. At pH 1.2, the NFOH was totally transformed into NF, the parent drug, after 48 h, while at pH 7.4, after the same period, the hydrolysis rate was 20%. In human plasma, 50% of NFOH was hydrolyzed after 24 h. In the investigation of kinetic disposition, the concentration of drug in serum versus time curve was used to calculate the pharmacokinetic parameters after a single-dose regimen. NFOH showed a time to maximum concentration of drug in serum (Tmax) as 1 h, suggesting faster absorption than NF (4 h). The most important results observed were the volume of distribution (V) of NFOH through the tissues, which showed a rate that is 20-fold higher (337.5 liters/kg of body weight) than that of NF (17.64 liters/kg), and the concentration of NF obtained by in vivo metabolism of NFOH, which was about four times lower (maximum concentration of drug in serum [Cmax] = 0.83 µg/ml; area under the concentration-time curve from 0 to 12 h [AUC0-12] = 5.683 µg/ml · h) than observed for administered NF (Cmax = 2.78 µg/ml; AUC0-12 = 54.49 µg/ml · h). These findings can explain the superior activity and lower toxicity of the prodrug NFOH in relation to its parent drug and confirm NFOH as a promising anti-Chagas' disease drug candidate.

Preparação e caracterização inicial de complexo de inclusão entre nitrofurazona e 2-hidroxipropil-Beta-ciclodextrina / Preparation and initial characterization of inclusion complex between nitrofurazone and 2-hydroxypropyl-Beta-cyclodextrin

Nitrofurazona(NF), 5-nitro-2-furaldeído semicarbazona, é um antibiótico de amplo espectro, que apresenta diversos efeitos tóxicos e baixa solubilidade aquosa. A complexação da NF com ciclodextrinas é de grande interesse para o desenvolvimento de uma formulação para este antibiótico que seja mais segura e eficiente. Neste trabalho foi realizada a preparação e caracterização inicial do complexo de inclusão entre NF e hidroxipropil-Beta-ciclodextrina (HP-Beta-CD) através de experimentos para determinação da cinética de complexação, medidas de fotoestabilidade, medidas de constante de afinidade fármaco: ciclodextrina, ensaios de liberação in vitro, estequiometria de formação do complexo e morfologia do complexo por microscopia eletrônica de varredura. Os ensaios de cinética de complexação mostram que para o complexo atingir o equilíbrio são necessárias 17,3h. As isotermas de solubilidade determinadas para a NF em função da temperatura mostraram perfis do tipo A e B indicando que a temperatura é um fator importante na complexação da NF com ciclodextrina. Os experimentos de fotoestabilidade indicam que a inserção da molécula de NF na cavidade interna da ciclodextrina protege o fármaco da fotodecomposição. A cinética de liberação mostra que o perfil de liberação do fármaco é modificado pela presença da ciclodextrina no meio. A estequiometria de complexação entre NF e HP-Beta-CD determinada foi de 1:1 NF:HP-Beta-CD. Os resultados demicroscopia eletrônica de varredura indicam alterações na estrutura cristalina da NF em presença deciclodextrina. Este estudo está baseado na caracterização físico-química da complexação entre NF e HP-Beta-CD podendo ser uma nova potencial opção para utilização terapêutica do NF.

Nitrofurazone (NF), 5-nitro-2-furaldehyde semicarbazone, a broad-spectrum antibiotic, has reported toxic effects and low solubility in water. It would be of great interest to form inclusion complexes between NF and a cyclodextrin, to develop more effective and safer antibiotic formulations. This paper focuses on the preparation of inclusion complexes of NF with 2- hydroxypropyl- -cyclodextrin (HP- -CD) and their initial characterization by evaluating rates of complex formation, photostability, solubility isotherms, release rate profiles, stoichiometry of the complexes and their morphology, as revealed by scanning electron microscopy. The kinetic tests of complex formation revealed that 17,3 h is enough for stabilization of the NFcyclodextrin complex. The solubility isotherm studies showed that the isotherm changes from type A to type B, as a function of temperature. The photostability experiments showed that the insertion of the NF in the HP- -CD cavity protects the drug from photodecomposition. The release kinetic tests showed that the profile of NF release from the complex is altered by the presence of HP- -CD in the medium. A Job's plot indicated that the stoichiometry of the complex was 1:1 NF:HP- -CD. The scanning electron micrographs showed changes in the crystal structure of NF in the complex. This study focused on the physicochemical properties of drug-delivery formulations that could potentially be developed into a novel type of therapy with NF.

Effect of dental adhesives on the exudative phase of the inflammatory process in subcutaneous tissue of rats.

The vascular changes in the subcutaneous connective tissue of rats induced by dentin bonding systems (one step) was studied and compared to those induced by saline solution (negative control) and Furacin (positive control), during the exudative phase of the inflammatory process. Twenty mg/kg of Evan's blue were injected intravenously in the vein of the rats' penises; 0.1 ml of each substance tested was inoculated in the subcutaneous tissue. After a 3 hour period the animals were sacrificed and their skins were excised and punched out with a standard steel 2.5 cm in diameter. The specimens were immediately immersed in 8 ml of formamide and taken to a double boiler for 72 hours at 37 C, to remove the dye. The liquid containing the overflowed dye was filtered, analyzed in the spectrophotometer (620 nm) and classified according to the criteria established by Nagem-Filho, Pereira (1976). After statistical analysis, the irritative potential of the substances was ranked as follows: Furacin (severe) > Single Bond and Bond 1 (moderate - no significant differences between the dentin bonding systems tested) > saline solution (not significant as regards the irritation degree).

Effect of dental adhesives on the exudative phase of the inflammatory process in subcutaneous tissue of rats

The vascular changes in the subcutaneous connective tissue of rats induced by dentin bonding systems (one step) was studied and compared to those induced by saline solution (negative control) and Furacin (positive control), during the exudative phase of the inflammatory process. Twenty mg/kg of Evan's blue were injected intravenously in the vein of the rats' penises; 0.1 ml of each substance tested was inoculated in the subcutaneous tissue. After a 3 hour period the animals were sacrificed and their skins were excised and punched out with a standard steel 2.5 cm in diameter. The specimens were immediately immersed in 8 ml of formamide and taken to a double boiler for 72 hours at 37ºC, to remove the dye. The liquid containing the overflowed dye was filtered, analyzed in the spectrophotometer (620 nm) and classified according to the criteria established by Nagem-Filho, Pereira (1976). After statistical analysis, the irritative potential of the substances was ranked as follows: Furacin (severe) > Single Bond and Bond 1 (moderate - no significant differences between the dentin bonding systems tested) > saline solution (not significant as regards the irritation degree)