Sensibilidad in vitro a benznidazol, nifurtimox y posaconazol de cepas de Trypanosoma cruzi de Paraguay / In vitro susceptibility to benznidazole, nifurtimox and posaconazole of Trypanosoma cruzi isolates from Paraguay

Resumen: Introducción. Trypanosoma cruzi, agente causal de la enfermedad de Chagas, exhibe una sustancial heterogeneidad fenotípica y genotípica que puede influir en las variaciones epidemiológicas y clínicas de la enfermedad, así como en la sensibilidad a los fármacos utilizados en el tratamiento. Objetivo. Evaluar la sensibilidad in vitro al benznidazol, el nifurtimox y el posaconazol de 40 cepas clonadas de T. cruzi de Paraguay, con distintos genotipos, huéspedes y localidades de origen. Materiales y métodos. En su estado epimastigote, los parásitos se incubaron en medio de cultivo LIT (Liver Infusion Tryptose) con diferentes concentraciones de cada fármaco en ensayos por triplicado. El grado de sensibilidad se estimó a partir de las concentraciones inhibitorias del 50 y el 90% (IC50 e IC90) y se obtuvieron los valores promedio y la desviación estándar de cada cepa y fármaco. La significación estadística entre grupos se determinó mediante análisis de varianzas con el test no paramétrico de Wilcoxon/Kruskal-Wallis y valores de p<0,05. Resultados. Se observó un amplio rango de respuesta a los fármacos. Se identificaron dos grupos de parásitos (A y B) con diferencias significativas en la sensibilidad al benznidazol (p<0,0001), y tres grupos (A, B, C) en cuanto a la sensibilidad al nifurtimox y el posaconazol (p<0,0001). Conclusiones. En general, las cepas fueron más sensibles al nifurtimox que al benznidazol y el posaconazol. Estas diferencias evidencian la heterogeneidad de las poblaciones de T. cruzi que circulan en Paraguay, lo que debe considerarse en el tratamiento y el seguimiento de las personas afectadas.

Abstract: Introduction: Trypanosoma cruzi, the causative agent of Chagas disease, shows substantial phenotypic and genotypic heterogeneity, which can influence the epidemiological and clinical variations of the disease and the sensitivity to the drugs used in the treatment. Objective: To assess the in vitro susceptibility to benznidazole, nifurtimox, and posaconazole of 40 cloned strains of T. cruzi isolated in Paraguay belonging to different genotypes, hosts, and localities. Materials and methods: We incubated the parasites in their epimastigote stage in LIT culture medium with different concentrations of each drug in triplicate assays. The degree of susceptibility was estimated by the inhibitory concentrations of 50 and 90% (IC50 and IC90) to obtain the average values and the standard deviation for each strain and drug. We determined the statistical significance between groups by analysis of variances with the Wilcoxon/Kruskal-Wallis non-parametric test and values of p<0.05. Results: A wide range of drug response was observed. Two groups of parasites (A and B) were identified as having significant differences in susceptibility to benznidazole (p<0.0001), and three groups (A, B, C) to nifurtimox and posaconazole (p<0.0001). Conclusions: Overall, the isolates were more susceptible to nifurtimox than benznidazole and posaconazole. Such differences highlight the heterogeneity of T. cruzi populations circulating in Paraguay, an aspect to consider in the treatment and follow up of patients.

In vitro genotoxicity of nitroimidazoles as a tool in the search of new trypanocidal agents

BACKGROUND Only benznidazole (Bnz) (1) and nifurtimox (Nfx) (2) are licensed for the treatment of Chagas disease although their safety and efficacy profile are far from ideal. Farmanguinhos from Fiocruz has developed seven nitroimidazole compounds (4-10) analogs of megazol (3). OBJECTIVES To evaluate whether the genotoxic effect of 3 was abolished in the seven nitroimidazoles (4-10) analogs using the in vitro alkaline comet assay (CA) and the in vitro cytokinesis-block micronucleus assay (CBMN) in whole human blood cells (WHBC) and correlate this effect with their trypanocidal activity using bloodstream trypomastigote forms of Trypanosoma cruzi. METHODS The toxicity of 3-10 to WHBC in the in vitro CA was determined using the fluorescein diacetate/ethidium bromide assay. DNA damage in the in vitro CA was evaluated according to tail size in four classes (0-3) and methyl methane-sulfonate (MMS) was used as a positive control. The cytotoxicity of 3-10 to WHBC in the CBMN was measured using the cytokinesis-block proliferation index and the replication index. The number of the micronucleate cells in 2,000 binucleate cells by experimental group was determined. Mitomycin C and N-deacetyl-N-methylcolchicine were used as positive controls. FINDINGS Compound 3 showed a significant DNA strand break effect through the in vitro CA and highly significant clastogenic and/or aneugenic effect in the CBMN. Compounds 5, 6, 8, 9 and 10 showed negative results in the CBMN and positive results in the in vitro CA, while the inverse effect was observed for 4 and 7. MAIN CONCLUSIONS Compound 10 was the most promising to proceed with the development as a drug candidate in the treatment of Chagas disease showing absence of chromosomal cytogenetic damage and high activity against T. cruzi, about two times higher than 3 and the clinical drug 1.

Preliminary exploration of 18 F-FMISO PET/CT SUVmax with relevant factors for nasopharyngeal carcinoma / 中华核医学与分子影像杂志

Objective To explore the relationship between the maximum standardized uptake value ( SUVmax ) of 18 F-fluoromisonidazole ( FMISO) PET/CT and the pathological classification, differentiation, T stage and primary tumor volume of nasopharyngeal carcinoma ( NPC) . Methods A retrospective analysis was performed on 41 patients with NPC (31 males, age 18-74 years;10 females, age 35-67 years) who underwent head and neck 18 F-FMISO PET/CT from 2012 to 2015. The relationship between the clinicopath-ological parameters (pathological classification, differentiation, T stage, tumor volume) of primary lesion and SUVmax were analyzed. Mann-Whitney u test, approximate t test and Spearman correlation were used for data analysis. Results There was no significant difference in SUVmax between non-keratinizing carcinoma and squamous cell carcinoma ( u=183.5, P>0.05) , nor between the differentiated carcinoma and undiffer-entiated carcinoma( t'=-1.23, P>0.05) . SUVmax of T1-T2 primary tumor was significantly lower than that of T3-T4 tumor (1.52±0.43 vs 2.05±0.85; t'=-2.60, P<0.05), and SUVmax was correlated with primary tumor volume ( rs=0.488, P<0.05) . Conclusions The hypoxic degree is related with T stage and primary tumor volume in NPC. The combination analysis of T stage and tumor size will contribute to the assessment of oxygen level and prognosis of primary NPC.

Hypoxic condition monitoring and treatment evaluation for non-small cell lung cancer before and after radiotherapy by 18F-FMISO PET/CT / 中华核医学与分子影像杂志

Objective To evaluate the changes of hypoxic conditions in non-small cell lung cancer (NSCLC) patients before and after radiotherapy and assess the value of 18F-fluoromisonidzaole (FMISO)PET/CT for radiotherapy efficacy evaluation.Methods A total of 21 NSCLC patients (15 males,6 females,age 30-74 years) from January 2014 to October 2016 were prospectively enrolled.18F-FMISO PET/CT was performed before and after radiotherapy,and all patients underwent 18F-fluorodeoxyglucose (FDG)PET/CT before radiotherapy.Routine chest CT was performed at the 3rd and 6th month after radiotherapy.The maximum standardized uptake value (SUVmax) of tumor and muscle,tumor volume and hypoxic volume (HV) were measured.Tumor-to-muscle (T/M) value of 18F-FMISO was calculated,and T/M ≥ 1.3 was considered as the hypoxia cut-off value.Data were analyzed using Pearson correlation,paired t test,signed rank sum test and Wilcoxon rank sum test.Results Totally 81.0％(17/21) of NSCLC patients had hypoxia.There were significant positive correlations between 18F-FMISO T/M value and tumor volume or 18F-FDG SUVmax(r:0.72,0.60,both P＜0.05).The T/M value after radiotherapy was significantly lower than that before radiotherapy (1.42± 1.12 vs 2.08±0.71;t =3.62,P＜0.05),and median HV was also significantly lower than that before radiotherapy (6.53 vs 12.41 cm3;z =-3.83,P＜0.05).The median T/M values of effective group (n =14) and ineffective group (n =7) before radiotherapy were significantly different (2.14 vs 2.87;z=-2.27,P＜0.05),and the median HV of 2 groups before radiotherapy was also significantly different (6.43 vs 10.20 cm3;z=-2.14,P＜0.05).Conclusions Most NSCLC patients have hypoxia before radiotherapy.The larger tumor volume,the higher degree of hypoxia.Radiotherapy can alleviate the hypoxia of tumors.18F-FMISO PET/CT imaging before radiotherapy can be used to predict the efficacy of patients with NSCLC.

Radiosensitization of sodium glycididazole in esophageal cancer evaluated by 18F-FMISO PET/CT / 中华核医学与分子影像杂志

Objective To investigate the feasibility of sodium glycididazole(CMNa)on the radio-sensitization of esophageal cancer(EC)by 18F-fluoromisonidazole(FMISO)PET/CT.Methods A total of 60 patients with EC from December 2016 to December 2017 were enrolled prospectively and were divided into control group(n = 30;21 males,9 females;age:(56.6±10.0)years)and experimental group(n=30;20 males,10 females;age:(59.3±9.0)years)using completely randomized grouping design.Patients in the control group received radiotherapy alone,and those in the experimental group were treated with conventional radiotherapy and CMNa.All patients underwent 18F-FMISO PET/CT imaging 1 week before and after radiotherapy.The imaging results were visually and semi-quantitatively analyzed.The maximum standardized uptake value(SUVmax),tumor/muscle ratio(T/M),and hypoxia volume(HV)were calculated.Paired t test,two-sample t test and/x2 test were used to analyze the data.Results T/M and HV in the control group before and after radiotherapy were 3.92±0.57 vs 1.66±0.35,(1.84±0.31)vs(1.04±0.15)mm3,respectively;T/M and HV in the experimental group before and after radiotherapy were 4.01±0.68 vs 1.27±0.11,(2.01±0.22)vs(0.90±0.09)mm3,respectively.The primary tumor T/M,HV after radiotherapy in 2 groups were significantly lower than those before radiotherapy(t values:12.15-24.43,all P<0.05)and the amplitudes of T/M and HV in the experimental group were significantly higher than those in the control group(?T/M:2.77±0.60vs 2.25±0.49,?HV:(1.12±0.18)vs(0.81±0.26)mm3;t values:3.00 and 1.80,both P<0.05).Meanwhile,the local control rate of EC after 3 months in the experimental group was higher than that in the control group(80.0%(24/30)vs 53.3%(16/30);x2=4.80,P<0.05).Conclusion CMNa has the radiosensitizing effect on EC and the 18F-FMISO PET/CT can evaluate the radiosensitization effect.

Evaluation of radiosensitivity enhancement by irisquinone with 18F-FETNIM microPET/CT imaging in breast cancer bearing nude mice / 中华核医学与分子影像杂志

Objective To investigate the effect of irisquinone (IR) on the radiosensitization of MDA-MB-231 breast cancer in nude mice using 18F-fluoroerythronitroimidazole (18F-FETNIM) microPET/ CT.Methods Thirty-two nude mice bearing MDA-MB-231 breast cancer were divided into 4 groups by random number table method (n =8 for each group):group A was treated with radiotherapy alone,group B was treated with radiotherapy and IR,group C was treated with IR alone,and group D was fed with distilled water.18F-FETNIM microPET/CT images were obtained to monitor the maximum standardized uptake value (SUVmax) of tumor before radiotherapy and 24 h after radiotherapy.The mice were sacrificed and tumor tissues were removed for HE staining.The expression of hypoxia inducible factor-1α (HIF-1α) was detected by immunohistochemical (IHC) method.Data were analyzed by paired t test,one-way analysis of variance,the least significant different t test and Pearson correlation analysis.Results There was no significant difference in SUVmax among the 4 groups before radiotherapy (1.429±0.090,1.430±0.076,1.445±0.071,1.432± 0.074;F=0.072,P＞0.05).At 24 h after radiotherapy,the SUVmax of group A and B decreased significantly (1.075±0.098,0.890±0.076;t values:12.888,33.217,both P＜0.05),and there was significant difference between group B and group A (t =4.197,P＜0.05).However,the SUVmax of group C and D increased significantly (1.617±0.090,1.644±0.063;t values:-11.009,-16.061,both P＜0.05).Pathological results showed that tumor cells in group A and B were reduced.IHC results showed that the expression of HIF-1α was lower in group B than others ((26.75±7.19)％;F=46.745,t values:2.898-9.743,all P＜ 0.05).The expression of HIF-1α was positively correlated with SUVmax(r =0.89,P＜0.05).Conclusion 18F-FETNIM microPET/CT could evaluate the radiosensitization effect of IR on MDA-MB-231 breast cancer in nude mice.

Preparation and Application of Ornidazole Magnetic Imprinted Polymers with Dual Functional Monomers / 分析化学

The magnetic molecularly imprinted polymers ( MMIPs ) was synthesiZed by suspension polymers technique using ornidaZole ( ONZ) as template, and methacrylic acid ( MAA) and acrylamide ( AA) as the binary functional monomers.The MMIPs were characteriZed by means of scanning electron microscopy ( SEM ) , Fourier transform infrared spectroscopy ( FT-IR ) , vibrating sample magnetometer ( VSM ) , thermogravimetric analysis ( TGA) and ultraviolet-visible ( UV-Vis) spectrophotometer.The results indicated that the adsorption capacity of MMIPs using the binary functional monomers to ONZ was higher than that using single functional monomer or that of its structural analogues.The Scatchard plot revealed that the template polymer system had a two-site binding behavior and the MMIPs exhibited the maximum rebinding to ONZ at 48.96 μmol/g and 10.60 μmol/g.Combined with high performance liquid chromatographic analysis technology, the prepared MMIPs were successfully applied to extract and enrich trace nitroimidaZoles from river water samples with recoveries range of 85.4%-104.3%.The precision of the results was good, and the manipulation of the developed method was simple and fast comparing to other methods.

In vitro anti-tuberculosis activity of azole drugs against Mycobacterium tuberculosis clinical isolates / Actividad in vitro de azoles contra aislamientos clínicos de Mycobacterium tuberculosis

Background: Latent tuberculosis has been associated with the persistence of dormant Mycobacterium tuberculosis in the organism of infected individuals, who are reservoirs of the bacilli and the source for spreading the disease in the community. New active anti-TB drugs exerting their metabolic action at different stages and on latent/dormant bacilli are urgently required to avoid endogenous reactivations and to be part of treatments of multi- and extensively-drug resistant tuberculosis (M/XDR-TB). It was previously reported that azole drugs are active against M. tuberculosis. For that reason, the aims of this study were to determine the in vitro activity of azole drugs, imidazole (clotrimazole, CLO and econazole, ECO) and nitroimidazole (metronidazole, MZ and ipronidazole, IPZ), against a collection of MDR M. tuberculosis clinical isolates; and to analyze their potential use in both the LTB and the active forms of M/XDR-TB treatments. Methods: A total of 55 MDR M. tuberculosis isolates and H37Rv were included. MZ and IPZ activity against M. tuberculosis isolates were tested using anaerobic culture conditions. The activity of ECO and CLO was measured by the minimal inhibitory concentration (MIC) using a microdilution colorimetric method. Results: MZ and IPZ showed bacteriostatic activity against M. tuberculosis strains. MIC5o and MIC90 to ECO was 4.0 µg/ml, while MIC50 to CLO was 4.0 µg/ml and MIC90 was 8.0 µg/ml respectively. Conclusion: All azole compounds tested in the study showed inhibitory activity against MDR M. tuberculosis clinical isolates.

Introducción: La tuberculosis (TB) latente ha sido asociada a la persistencia de Mycobacterium tuberculosis durmientes en el organismo de las personas infectadas, las cuales constituyen un reservorio del bacilo y una fuente de diseminación de la enfermedad en la comunidad. Urge la necesidad de contar con nuevos fármacos antituberculosos con acción sobre el bacilo en estado latente/durmiente, a fin de evitar reactivaciones endógenas y para ser incluidas en el tratamiento de la TB multirresistente y extensivamente resistente (M/XDR-TB). Se ha reportado que los azoles son activos contra M. tuberculosis. Por esta razón, los objetivos del presente estudio fueron determinar la actividad in vitro sobre aislamientos clínicos de M/XDR-TB de distintos azoles, incluyendo los imidazoles econazol (ECO) y clotrimazol (CLO) y los 5-nitro-imidazoles ipronidazol (IPZ) y metronidazol (MZ), así como analizar su potencial uso contra las formas latente y activa de esta enfermedad. Métodos: Fueron incluidos 55 aislamientos clínicos de M. tuberculosis MDR y la cepa de referencia H37Rv. Se evaluó la actividad del MZ y el IPZ sobre los aislamientos en condiciones de cultivo anaeróbico, mientras que la actividad del ECO y el CLO fue estimada determinando la concentración inhibitoria mínima (CIM) mediante el método colorimétrico de microdilución en placa. Resultados: El MZ y el IPZ presentaron actividad bacteriostática frente a las cepas de M. tuberculosis. La CIM50 y CIM90 del ECO fue de 4 µg/ml, mientras que el CLO presentó una CIM50 de 4 µg/ml y una CIM90 de 8 µg/ml. Conclusión: Todos los compuestos azólicos evaluados presentaron actividad inhibitoria frente a aislamientos clínicos de M. tuberculosis.

Biodistribution of 99 Tcm O-labeled MAG3-2-nitroimidazole hypoxia imaging agents with different spacers in S180 scarcoma-bearing mice / 中华核医学与分子影像杂志

Objective To evaluate the hypoxia selectivity of 99 Tcm O?labeld MAG3?nitroimidazole complexes with different spacers. Methods Four kinds of 99 Tcm O?labeled MAG3?2?nitroimidazole hypoxia imaging agents with different spacers were synthesized and radiolabeled. The stability and lipid solubility of BzMAG3?2NIEA(1), BzMAG3?2NIPA(2), BzMAG3?2NIHA(3) and BzMAG3?2NIUA(4) were measured. The uptake was investigated by biodistribution experiment using S180?bearing mice. Two?sample t test was used for statistical analysis. Results All 4 99 Tcm O?MAG3 complexes were stable and negatively charged, showing an increasing trend in fat solubility with the increase of spacer length. In biodistribution study, tumor uptake of 99TcmO?1 and 99TcmO?2 with medium? and short?carbon chain were (0.67±0.18) and (0?65±0.18) %ID/g 2 h post injection, which were (0.19±0.03) and (0.39±0.05) %ID/g for 99TcmO?3 and 99TcmO?4 with long?carbon chain (t=2.78-5.88, all P<0.05). Conclusion Molecular structure of spacers has a significant effect on the physicochemical properties and tumor targeting of 99 Tcm O?labeled MAG3?2?nitroimidazole hypoxia imaging agents, such that the medium?and short?carbon chain spacers show the best hypoxia?selective property.

Studies of genotoxicity and mutagenicity of nitroimidazoles: demystifying this critical relationship with the nitro group

Nitroimidazoles exhibit high microbicidal activity, but mutagenic, genotoxic and cytotoxic properties have been attributed to the presence of the nitro group. However, we synthesised nitroimidazoles with activity against the trypomastigotes of Trypanosoma cruzi, but that were not genotoxic. Herein, nitroimidazoles (11-19) bearing different substituent groups were investigated for their potential induction of genotoxicity (comet assay) and mutagenicity (Salmonella/Microsome assay) and the correlations of these effects with their trypanocidal effect and with megazol were investigated. The compounds were designed to analyse the role played by the position of the nitro group in the imidazole nucleus (C-4 or C-5) and the presence of oxidisable groups at N-1 as an anion receptor group and the role of a methyl group at C-2. Nitroimidazoles bearing NO2 at C-4 and CH3 at C-2 were not genotoxic compared to those bearing NO 2 at C-5. However, when there was a CH3 at C-2, the position of the NO2 group had no influence on the genotoxic activity. Fluorinated compounds exhibited higher genotoxicity regardless of the presence of CH3 at C-2 or NO2 at C-4 or C-5. However, in compounds 11 (2-CH3; 4-NO2; N-CH2OHCH2Cl) and 12 (2-CH3; 4-NO2; N-CH2OHCH2F), the fluorine atom had no influence on genotoxicity. This study contributes to the future search for new and safer prototypes and provide.

Application of 18F-FMISO for tumor hypoxia imaging / 中华核医学与分子影像杂志

Hypoxia is the combined result of tumor over-growth and vascular structural abnormalities.Hypoxia is related to tumor invasiveness,radioresistance,and chemoresistance.18F-fluoromisoni-dzole (18F-FMISO) can selectively bind to hypoxic tissues of malignant tumors,and is thus useful for imaging of tumor hypoxia in cancer diagnosis and treatment.This review summarizes the state-of-art research of 18F-FMISO in comparison with other hypoxic agents when applied to animals and clinical studies.

Determination of Nitroimidazoles and Their Metabolites Residues in Honey by TurboFlow Online Purification-Ultra Performance Liquid Chromatography-Tandem Mass Sepctrometry and Istopes Dilution Technique / 分析化学

A TurboFlow ( TF ) online purification-ultra performance liquid chromatography-tandem mass spectrometric method was developed for the determination of nitroimidazoles and their metabolites ( ornidazole, IPZ, MNZ, MNZOH, DMZ, RNZ, IPZOH and HMMNI) in honey. The honey sample was extracted by H2 O containing 0. 1% formic acid solution. Then, the extraction solution was analyzed by TF-UPLC-MS/MS. The main factors influencing the purification efficiency including TF column, mobile phases and elution solutions were optimized. The compounds were detected by selective reaction monitoring ( SRM) in positive ion mode electrospray ionization ( ESI+) . The linear range of the method ranged from 0 . 1 to 50μg/L for nitroimidazoles and their metabolites, with the correlation coefficient ( R2 ) of over 0. 997. The limits of quantification were 0. 1 μg/kg for ornidazole, IPZ, 0. 2 μg/kg for MNZ, MNZOH, DMZ, RNZ and IPZOH, and 1. 0 μg/kg for HMMNI, respectively. The recoveries were in the range of 73. 7% to 116. 4% at four spiked levels with the relative standard deviations ranged from 1. 1% to 9. 1% in honey samples. The results indicate that the developed method is simple, efficient and precise, and can be used for the determination of nitroimidazoles and their metabolites in the actual honey samples.

Detection and spectrophotometric determination of tinidazole.

A direct spectrophotometric method was developed by the authors for the quantitative determination of tinidazole in a pure form and also in other pharmaceutical formulations. The method was based on the diazotization reaction between nitro group of the drug sample, sulphanilamide and NEDA. In the present method, the reddish-purple colour dye formed, exhibited a maximum absorbance at 540nm. Beer’s law was found to be obeyed in the range of 100-600μgmL-1for tinidazole with detection limits of 0.04μgmL-1. The present method was found to be precise, accurate for the qualitative and quantitative determinations.