Nanocrystals of Novel Valerolactam-Fenbendazole Hybrid with Improved in vitro Dissolution Performance.

Valero-fenbendazole (VAL-FBZ) is a novel hybrid compound with in vitro anthelmintic activity, designed and synthesized to address the global problem of resistance to anthelmintic compounds. This new molecule derives from fenbendazole (FBZ), a well-known commercially available benzimidazole used in veterinary medicine despite its poor water solubility. In this work, we report for the first time a strategy to solve the solubility problems of FBZ and VAL-FBZ by means of self-dispersible nanocrystals (SDNC). Nanocrystals were prepared by media milling followed by a spray-drying step, and a comprehensive and exhaustive structural and physicochemical characterization was carried out, in order to understand the systems and their behavior. The formulation poloxamer 188 (P188):FBZ 1:1 turned out with the best process yield (53%) and re-dispersability properties, particle size average of 258 nm, and polydispersity index of 0.2 after redispersion in water. The dissolution profile showed a markedly increased dissolution rate compared with the simple mixture of the components (80% FBZ dissolved in 15 min from the SDNC vs 14% from the control formulation). FTIR spectroscopy, thermal analysis, and X-Ray Powder Diffraction (XRPD) studies showed no chemical interactions between components and an extensive confocal Raman microscopy analysis of the formulations showed very homogeneous spatial distribution of components in the SDNC samples. This manufacturing process was then successfully transferred for preparing and characterizing VAL-FBZ:P188 (1:1) SDNC with similar results, suggesting the promising interest of a novel anthelmintic with improved biopharmaceutical behavior. In conclusion, new FBZ and VAL-FBZ SDNC with improved dissolution rate were successfully prepared and characterized. Graphical abstract.

Purification of native M. vogae and H. contortus tubulin by TOG affinity chromatography.

Microtubules are non-covalent cylindrical polymers formed by alpha- and beta-tubulin heterodimer units, crucial for cell division, intracellular transport, motility and differentiation. This makes them very attractive pharmacological targets exploited to develop different drugs such as anthelmintics, antifungals, and antineoplastics. In this work, in order to establish an in vitro target-based screen to integrate to the search for new anthelmintics, we explored the extraction of native assembly-competent tubulin from two helminth parasites: Mesocestoides vogae tetrathyridia (syn. corti, Cestoda: Cyclophyllidea), a useful cestode biological model, and Haemonchus contortus, a sheep gastrointestinal nematode of interest in livestock production. For this purpose, a novel tubulin affinity chromatography procedure was employed, based on the binding capacity of TOG (Tumor Overexpressed Gene) domain from MAPs (microtubule-associated proteins). The TOG domain of the protein Stu2 from Saccharomyces cerevisiae fused to GST (glutathione S- transferase) were produced in E. coli, and the immobilized recombinant proteins allowed for native tubulin extraction from parasites. The binding capacity of TOG1 affinity column (3.6%) was estimated using commercial porcine brain tubulin. A total amount of up to 126 µg of M. vogae tubulin was purified, whereas H. contortus tubulin co-eluted with glutamate dehydrogenase enzyme. The identity of tubulins was confirmed by western blotting and mass spectrometry. The abundance of tubulin estimated in M. vogae was 10% soluble extract, which probably could explain differences observed between tubulin purification results of both helminth parasites.

Development of novel valerolactam-benzimidazole hybrids anthelmintic derivatives: Diffusion and biotransformation studies in helminth parasites.

In the search for new anthelmintics able to overcome the resistance problem against all available drugs in livestock, the synthesis of novel valerolactam-benzimidazole hybrid compounds was reported. This allowed us to obtain these in vitro and in vivo bioactive compounds using Nippostrongylus brasiliensis rat model by integrating physiology-based assays and ex vivo diffusion studies. In order to further study those novel hybrid molecules, Haemonchus contortus (a sheep gastrointestinal nematode of interest) and Mesocestoides vogae tetrathyridia (a useful system to study the efficacy of anthelmintic drugs against cestoda) were used as parasite models to compare the ex vivo patterns of diffusion and biotransformation of benzimidazoles and their valerolactam-benzimidazole hybrid derivatives. On average, a nine-fold higher intraparasitic concentration of compounds was found in M. vogae compared with H.contortus, with similarities regarding the order of entry of compounds, highlighting febendazole (FEB) and its hybrid compound 10, while valerolactam compound 2 practically did not penetrate the parasites. Interestingly, sulphoxidation drug metabolism was observed and measured, revealing percentages of oxidation of 8.2% and 14.5% for albendazole (ABZ) and febendazole respectively in M. vogae, while this effect was more relevant in H. contortus parasite. More importantly, significant differences were observed between anthelmintic-susceptible adult parasites (Hc S) and those from sheep farms (Hc U). In fact, the percentages of oxidation of FEB and the hybrid compound 8 were higher in Hc U (25.5%, 54.1%, respectively) than in Hc S (8.8%, 38.2%). Interestingly, sulphoxidation of hybrid compound 10 was neither observed in M. vogae nor in H. contortus parasites, suggesting that increased drug metabolism (oxidation reactions) could not be used by these parasites as a defense mechanism against this novel drug.

Sensitivity of Haemonchus contortus to anthelmintics using different in vitro screening assays: a comparative study.

BACKGROUND: Helminthiasis and resistance to commercial anthelmintic compounds are major causes of economic losses for livestock producers, resulting in an urgent need for new drugs and reliable in vitro screening tests capable of detecting potentially active products. Considering this, a series of novel benzimidazole derivatives (5-methylbenzimidazole 1,2-disubstituted, 5-carboxybenzimidazole, 5-methylbenzimidazole 2-one) was screened on exsheathed L3 (xL3) and on the adult stage of Haemonchus contortus (Kirby anthelmintic-susceptible McMaster isolate). METHODS: This work presents the set-up of an automated motility assay on the xL3 stage of H. contortus using an infrared tracking device (WMicrotracker One) together with a larval development test (xL3 to L4) and a motility assay on the adult stage of H. contortus. A comparative study of the sensitivity of these in vitro assays using commercial anthelmintics with different mechanisms of action was carried out, also evaluating anthelmintic activity of a series of novel benzimidazole derivatives. RESULTS: The automated xL3 assay had the great advantage of being able to analyze many compounds simultaneously, but it showed the limitation of having lower sensitivity, requiring higher concentrations of the commercial anthelmintics tested compared to those needed for the adult motility or development assays. Although none of the novel 1,2,5-tri-substituted benzimidazole derivatives could significantly decrease the motility of xL3s, one of them (1e) significantly affected the development of xL3s to L4, and five new compounds (1b, 1d, 1e, 2a and 2c) reduced the motility of H. contortus adult stage. CONCLUSIONS: The analysis of the results strongly suggests that the in vitro xL3 to L4 development test, particularly for the L4 stage, could be closer to the pharmacological sensitivity of the adult stage of H. contortus (target of interest) for commercial anthelmintic selected, with different mechanisms of action, and for the series of benzimidazole derivatives assayed. Therefore, an automated motility assay on L4 using the infrared tracking device is being set up. Further studies will be conducted to evaluate the in vivo anthelmintic activity of the most active novel benzimidazole derivatives.

Improving the in vitro dissolution rate and pharmacokinetic performance of fenbendazole in sheep using drug nanocrystals.

Benzimidazole methylcarbamate anthelmintics, including fenbendazole (FBZ), have only limited water solubility and small differences in drug solubility may have a major influence on their absorption, pharmacokinetic behavior and anthelmintic efficacy. To improve FBZ water solubility and dissolution rate, novel self-dispersible nanocrystals (SDNCs) of FBZ were recently described. In this work, the pharmacokinetic behavior of the SDNCs of FBZ and Poloxamer 188 was compared against a physical mixture (PM) of its components. The experiment was conducted following a crossover design with two different experimental phases. In phase I, sheep were treated with the SDNC (n = 3) or the PM (n = 3) formulations by the intraruminal route at the same dose rate (5 mg/kg). The treatment groups were reversed after a 7-days washout period. A non-compartmental analysis of the concentration in plasma versus time results showed that the calculated Cmax and AUC0-T were significantly higher (p < 0.05) for FBZ and its metabolites after the SDNC treatment compared to the PM (for FBZ: Cmax 0.346 µg/mL and AUC0-T 10.1 µg.h/mL after the SDNC vs Cmax 0.157 µg/mL and AUC0-T 5.1 µg.h/mL after the PM treatment). Additionally, population pharmacokinetic parameters of FBZ were estimated for the first time in sheep. In conclusion, the formulation of FBZ as SDNCs is a promising approach to improve FBZ dissolution reaching a higher drug plasma exposure in ruminants.

Chemical characterization and in vitro anthelmintic activity of Citrus bergamia Risso and Citrus X paradisii Macfad essential oil against Haemonchus contortus Kirby isolate.

Haemonchus contortus, a blood-sucking parasite of small ruminants, produces very important economic losses in the productive sector. This abomasum parasite has become resistant to most commercial drugs worldwide, and alternatives to fight this problem are urgently needed. Essential oils (EO) are a complex mixture of volatile secondary metabolites, composed mainly by terpenoids and phenolic compounds, from plants that have several pharmacological properties, including anthelmintic activity. Particularly, citrus peel is a source of cold-pressed EO, where limonene is its major component, and can be used as an additional food component for ruminants. The aim of the present work was to determine the in vitro anthelmintic activity of EO from Citrus bergamia (EOB), C. x paradisii (EOG) and limonene against the benzimidazole-susceptible Kirby isolate of H. contortus, using the egg hatch test (EHT) and the exsheathed third stage larval motility test (XLMT) using a WMicroTracker equipment. Albendazole (ABZ) and monepantel (MON) were used as positive controls. The 50% inhibitory concentrations (IC50) in XLMT were 8.77 and 13.88 µg/ml for EOB and EOG respectively, after an incubation of 72 h. An interesting observation on XLMT resulted when the positive controls were tested on the same plate, but in different well of the EOB. The volatile components of the EO significantly influenced (P < 0.05) the percentage of larval motility, reducing values from 66.9 to 19.6% for ABZ, and from 72.8 to 33.7% for MON, when comparing the activity of positive controls in a control plate without EO. The in vitro anthelmintic activity of EOB and EOG shows that they could be interesting candidates for nematode control. It is still necessary additional studies against the adult stage of H. contortus in efficacy trials in infected animals to validate their anthelmintic activity.

Molecular analysis of benzimidazole-resistance associated SNPs in Haemonchus contortus populations of Uruguay.

Haemonchus contortus is one of the most important parasite nematodes of small ruminants around the world and causes great economic losses in livestock production. Control of gastrointestinal nematode infections, like haemonchosis, relies mainly on anthelmintic drugs, but its excessive and inappropriate use has caused serious drug resistance issues in many countries, including Uruguay, where sheep production occupies an important place in the country's economy. Benzimidazole (BZ) anthelmintics have been used for decades to treat sheep against H. contortus infection and resistance to this anthelmintic group has been widely described. Molecularly, BZ resistance in H. contortus has been correlated with single nucleotide polymorphisms (SNPs) of the ß-tubulin isotype 1 gene at codon 200 and 167 (both TTC to TAC, F167Y and F200Y) and at codon 198 (GAA to GCA, E198A).The aim of this work was to explore the presence of these tubulin SNPs in H. contortus adult worms recovered from sheep abomasa from a slaughterhouse in Uruguay. The mean resistant allelic frequencies at positions F167Y and F200Y were 20.25 and 47.45%, respectively, for worms recovered from naturally infected sheep slaughtered in 2013, while those that were slaughtered in 2014 presented only F200Y SNP with a frequency of 86.89%. Also H. contortus Kirby adult worms (anthelmintic- susceptible McMaster isolate), recovered from artificially infected sheep, were analyzed as reference for comparative purposes This analysis showed susceptible genotype at 167 and 198 position, and a low level of the resistance allele at the 200 position (3.66%). This is the first study for the presence of SNPs in the isotype-1 ß-tubulin gene of H. contortus populations in Uruguay, which is consistent with the previous epidemiological studies carried out through the method of fecal egg count reduction test (FECRT), thus confirming the serious resistance levels to BZ anthelmintics also in this country.

Excursión diafragmática como predictor de éxito para el retiro de la ventilación mecánica en el paciente obeso / Diaphragmatic excursion as a predictor of success in the removal of mechanical ventilation in obese patient / Mobilidade diafragmática como preditor de sucesso na remoção da ventilação mecânica no paciente obeso

Resumen: Introducción: El soporte respiratorio es una de las principales indicaciones para los pacientes que ingresan a la Unidad de Cuidados Intensivos (UCI). La liberación de la ventilación mecánica invasiva (VMI) representa hasta 40% del tiempo en la VMI; comorbilidades como obesidad son más frecuentes en la UCI; existen recomendaciones en la liberación de la VMI en este grupo de pacientes, sin embargo, no existen predictores de éxito que esté ajustado para la población mexicana. Objetivo: Evaluar la excursión diafragmática (ED) como predictor de éxito para el retiro de la VMI en pacientes obesos. Material y métodos: Se realizó un estudio transversal, prospectivo, comparativo y analítico, en pacientes obesos ingresados a las UCI con VMI por más de 48 horas. Se analizaron variables demográficas, índice de ventilación de respiración superficial (IVRS) y ED. El punto de desenlace fue el éxito en la liberación de la VMI. Resultados: La distribución por género fue 42% de mujeres y 58% de hombres; la edad fue de 41.3 ± 12.8 años; estancia hospitalaria de 9.6 ± 4.6 días; el tiempo de VMI: 8.8 ± 4.8 días, e índice de masa corporal (IMC): 33.3 ± 2.7 kg/cm2; representados en grado I: 76%, grado II: 2%, grado III: 22%, IVRS: 59 ± 9.4; ED: 1.55 ± 0.11. El área bajo la curva (ABC) para IRS fue 0.60 y ED de 0.77. Adicionalmente, el mejor punto de corte para éxito fue IVRS < 44 con sensibilidad y especificidad de 100%, y la ED > 1.9 cm con sensibilidad y especificidad de 100%. Conclusión: El mejor predictor de éxito fue la ED con respecto al IVRS. Se proponen nuevos puntos de corte para IVRS y ED.

Abstract: Introduction: Respiratory support is an indication of admission to Intensive Care Units (ICU). The release of invasive mechanical ventilation (VMI), represents up to 40% of the time in VMI; comorbidities such as obesity are more frequent in the ICU; there are recommendations on the release of the VMI (LVMI) in this group of patients, however, there are no adjusted predictors of success for the Mexican population. Objective: To evaluate the diaphragmatic excursion (ED) as a predictor of success in the removal of IMV in obese patients (PO). Material and methods: A cross-sectional, prospective, comparative and analytical study was conducted in POs admitted to ICUs with IMV for more than 48 hours, demographic variables, surface respiration index (RSBI), ED were analyzed, the outcome was the success in LVMI. Results: The distribution by gender was women 42% and men 58%; age: 41.3 ± 12.8 years; hospital stay: 9.6 ± 4.6 days; VMI time: 8.8 ± 4.8 days, l body mass index (BMI): 33.3 ± 2.7; represented in grade I: 76%, grade II: 2%, grade III: 22%, RSBI: 59 ± 9.4; ED: 1.55 ± 0.11. The area under the curve (ABC) for RSBI was 0.60 and ED 0.77. Additionally, the best cut-off point for success was: RSBI < 44 with 100% sensitivity and specificity, and ED > 1.9 cm with 100% sensitivity and specificity. Conclusion: The best predictor of success was the ED with respect to the RSBI, new cut-off points for RSBI and ED are proposed.

Resumo: Introdução: O suporte respiratório é uma indicação de admissão em Unidades de Terapia Intensiva (UTI), a liberação de ventilação mecânica invasiva (VMI) representa até 40% do tempo na VMI; comorbidades como obesidade são mais frequentes na UTI; existem recomendações sobre a liberação do VMI (LVMI) nesse grupo de pacientes, no entanto, não há preditores ajustados de sucesso para a população mexicana. Objetivo: Avaliar a mobilidade diafragmática (MD) como preditor de sucesso na remoção de VMI em pacientes obesos (PO). Material e métodos: Foi realizado um estudo transversal, prospectivo, comparativo e analítico em PO admitidos em UTI com VMI por mais de 48 horas, foram analisadas variáveis demográficas, índice de ventilação da respiração superficial (IVRS), MD. O resultado foi bem-sucedido em LVMI. Resultados: A distribuição por gênero foi de mulheres 42%, homens 58%; idade 41.3 ± 12.8 anos internação 9.6 ± 4.6 dias, tempo de VMI: 8.8 ± 4.8 dias, índice de massa corporal (IMC) 33.3 ± 2.7; representado em Grau I 76%, Grau II 2%, Grau III 22%, IRS 59 ± 9.4; MD 1.55 ± 0.11. A área sob a curva (ABC) para IRS foi de 0.60 e MD 0.77. Além disso, o melhor ponto de corte para o sucesso foi: IVRS < 44 com 100% de sensibilidade e especificidade e ED > 1.9 cm com sensibilidade e especificidade de 100%. Conclusão: O melhor preditor de sucesso foi o MD em relação ao IVRS, novos pontos de corte para IVRS e MD são propostos.