NMR Longitudinal Rotating Frame Relaxation Time (T1ρ) with a Weak Spin Locking Field as an Approach to Characterize Solid-State Active Pharmaceutical Ingredients: Proof of Concept.

Nuclear magnetic resonance (NMR) longitudinal rotating frame relaxation time (T1ρ), rarely used in low-field NMR, can be more effective than conventional T1 and T2 relaxation times to differentiate polymorphic forms of solid pharmaceuticals. This could be attributed to T1ρ sensibility to structural and molecular dynamics that can be enhanced by changing the strength of the oscillating magnetic field (B1) of spinlock pulses. Here, we compared the capacity of T1, T2, and T1ρ to differentiate inactive (A) and active (C) crystalline forms of the World Health Organization essential drug Mebendazole. The results showed that T1 and T2 values of both forms were statistically identical at 0.47 T. Conversely, T1ρ of both forms measured with weak spinlock B1 fields, ranging from 0.08 to 0.80 mT were statistically different in the same spectrometer. The T1ρ also has the limit of detection to detect the presence of at least 10% of inactive A form in the active C form. Therefore, T1ρ, measured with weak spinlock B1 fields can be an effective, streamlined, and complementary approach for characterizing not only solid active pharmaceutical ingredients but other solid-state materials as well.

Biodegradable and biocompatible subcutaneous implants consisted of pH-sensitive mebendazole-loaded/folic acid-targeted chitosan nanoparticles for murine triple-negative breast cancer treatment.

BACKGROUND: Mebendazole (MBZ) is a well-known anti-parasite drug with significant anti-cancer properties. However, MBZ exhibits low solubility, limited absorption efficacy, extensive first-pass effect, and low bioavailability. Therefore, multiple oral administration of high dose MBZ is required daily for achieving the therapeutic serum level which can cause severe side effects and patients' non-compliance. METHOD: In the present study, MBZ-loaded/folic acid-targeted chitosan nanoparticles (CS-FA-MBZ) were synthesized, characterized, and used to form cylindrical subcutaneous implants for 4T1 triple-negative breast tumor (TNBC) treatment in BALB/c mice. The therapeutic efficacy of the CS-FA-MBZ implants was investigated after subcutaneous implantation in comparison with Control, MBZ (40 mg/kg, oral administration, twice a week for 2 weeks), and CS-FA implants, according to 4T1 tumors' growth progression, metastasis, and tumor-bearing mice survival time. Also, their biocompatibility was evaluated by blood biochemical analyzes and histopathological investigation of vital organs. RESULTS: The CS-FA-MBZ implants were completely degraded 15 days after implantation and caused about 73.3%, 49.2%, 57.4% decrease in the mean tumors' volume in comparison with the Control (1050.5 ± 120.7 mm3), MBZ (552.4 ± 76.1 mm3), and CS-FA (658.3 ± 88.1 mm3) groups, respectively. Average liver metastatic colonies' number per microscope field at the CS-FA-MBZ group (2.3 ± 0.7) was significantly (P < 0.05) lower than the Control (9.6 ± 1.7), MBZ (5.0 ± 1.5), and CS-FA (5.2 ± 1) groups. In addition, the CS-FA-MBZ treated mice exhibited about 52.1%, 27.3%, and 17% more survival days after the cancer cells injection in comparison with the Control, MBZ, and CS-FA groups, respectively. Moreover, the CS-FA-MBZ implants were completely biocompatible based on histopathology and blood biochemical analyzes. CONCLUSION: Taking together, CS-FA-MBZ implants were completely biodegradable and biocompatible with high therapeutic efficacy in a murine TNBC model.

Flubendazole Plays an Important Anti-Tumor Role in Different Types of Cancers.

Flubendazole, belonging to benzimidazole, is a broad-spectrum insect repellent and has been repurposed as a promising anticancer drug. In recent years, many studies have shown that flubendazole plays an anti-tumor role in different types of cancers, including breast cancer, melanoma, prostate cancer, colorectal cancer, and lung cancer. Although the anti-tumor mechanism of flubendazole has been studied, it has not been fully understood. In this review, we summarized the recent studies regarding the anti-tumor effects of flubendazole in different types of cancers and analyzed the related mechanisms, in order to provide the theoretical reference for further studies in the future.

In vitro efficacies of solubility-improved mebendazole derivatives against Echinococcus multilocularis.

Recently, we introduced an epoxy group to mebendazole by a reaction with epichlorohydrin and obtained two isoforms, mebendazole C1 (M-C1) and mebendazole C2 (M-C2). The in vitro effects of mebendazole derivatives at different concentrations on Echinococcus multilocularis protoscoleces and metacestodes as well as cytotoxicity in rat hepatoma (RH) cells were examined. The results demonstrated that the solubility of the two derivatives was greatly improved compared to mebendazole. The mortality of protoscoleces in vitro reached to 70-80% after 7 days of exposure to mebendazole or M-C2, and M-C2 showed higher parasiticidal effects than mebendazole (P > 0.05). The parasiticidal effect of M-C1 was low, even at a concentration of 30 µm. The percentage of damaged metacestodes that were treated with mebendazole and M-C2 in vitro at different concentrations were similar, and M-C1 exhibited insignificant effects on metacestodes. Significant morphological changes on protoscoleces and metacestodes were observed after treatment with mebendazole and M-C2. In addition, the introduction of an epoxy group to mebendazole also reduced its cytotoxicity in RH cells. Our results demonstrate that the introduction of an epoxy group not only improved the solubility of mebendazole, but also increased its parasiticidal effects on E. multilocularis and reduced its cytotoxicity in RH cells.

Targeted Drug Delivery and Treatment of Endoparasites with Biocompatible Particles of pH-Responsive Structure.

Biomaterials conceived for vectorization of bioactives are currently considered for biomedical, biological, and environmental applications. We have produced a pH-sensitive biomaterial composed of natural source alginate and chitosan polysaccharides for application as a drug delivery system via oral administration. The composite particle preparation was in situ monitored by means of isothermal titration calorimetry. The strong interaction established between the macromolecules during particle assembly led to 0.60 alginate/chitosan effective binding sites with an intense exothermic effect and negative enthalpy variation on the order of a thousand kcal/mol. In the presence of model drugs mebendazole and ivermectin, with relatively small and large structures, respectively, mebendazole reduced the amount of chitosan monomers available to interact with alginate by 27%, which was not observed for ivermectin. Nevertheless, a state of intense negative Gibbs energy and large entropic decrease was achieved, providing evidence that formation of particles is thermodynamically driven and favored. Small-angle X-ray scattering provided further evidence of similar surface aspects independent of the presence of drug. The physical responses of the particles to pH variation comprise partial hydration, swelling, and the predominance of positive surface charge in strong acid medium, whereas ionization followed by deprotonation leads to compaction and charge reversal rather than new swelling in mild and slightly acidic mediums, respectively. In vivo performance was evaluated in the treatment of endoparasites in Corydoras fish. Systematically with a daily base oral administration, particles significantly reduced the infections over 15 days of treatment. The experiments provide evidence that utilizing particles granted and boosted the action of the antiparasitic drugs, leading to substantial reduction or elimination of infection. Hence, the pH-responsive particles represent a biomaterial with prominent characteristics that is promising for the development of targeted oral drug delivery.

Oral bioavailability enhancement of flubendazole by developing nanofibrous solid dosage forms.

The bioavailability of the anthelminthic flubendazole was remarkably enhanced in comparison with the pure crystalline drug by developing completely amorphous electrospun nanofibres with a matrix consisting of hydroxypropyl-ß-cyclodextrin and polyvinylpyrrolidone. The thus produced formulations can potentially be active against macrofilariae parasites causing tropical diseases, for example, river blindness and elephantiasis, which affect altogether more than a hundred million people worldwide. The bioavailability enhancement was based on the considerably improved dissolution. The release of a dose of 40 mg could be achieved within 15 min. Accordingly, administration of the nanofibrous system ensured an increased plasma concentration profile in rats in contrast to the practically non-absorbable crystalline flubendazole. Furthermore, easy-to-grind fibers could be developed, which enabled compression of easily administrable immediate release tablets.

Phenolic Metabolites of Dalea ornata Affect Both Survival and Motility of the Human Pathogenic Hookworm Ancylostoma ceylanicum.

Hookworms are ubiquitous human parasites, infecting nearly one billion people worldwide, and are the leading cause of anemia and malnutrition in resource-limited countries. Current drug treatments rely on the benzimidazole derivatives albendazole and mebendazole, but there is emerging resistance to these drugs. As part of a larger screening effort, using a hamster-based ex vivo assay, anthelmintic activity toward Ancylostoma ceylanicum was observed in the crude extract of aerial parts of Dalea ornata. These studies have led to the isolation and characterization of phenolic metabolites 1-10. The structures were determined by 1D and 2D NMR spectroscopy, and the absolute configuration of 1 was assigned using electronic circular dichroism data. The new compound, (2S)-8-(3-methylbut-2-en-1-yl)-6,7,4'-trihydroxyflavanone (1), was weakly active at 7.3 µM, with 17% reduction in survival of the hookworms after 5 days. The rotenoids deguelin (9) and tephrosin (10), predictably perhaps, were the most active, with complete worm mortality observed by day 4 (or earlier) at 6.3 and 6.0 µM, respectively. The effects of 1-10 on hookworm motility and on toxicity to hamster splenocytes were also explored as important measures of treatment potential.

Development of a capillary zone electrophoresis method for determination of mebendazole and levamisole hydrochloride in a combined tablet and a comparison with a LC method.

The separation and determination of mebendazole (MEB) and levamisole hydrochloride (LH) from a combination tablet by capillary zone electrophoresis (CZE) was developed and validated. The separation was performed in a 60 cm (50.5 cm to detection window) x 75 microm internal diameter fused silica capillary using a background electrolyte of NaH2PO4 (20 mM/L, pH 3.0 ) at 25 kV. Good separation was obtained in less than 8 min. The LOQ values for LH and MEB were 1.43 and 2.50 microg/mL, respectively; calibration curves were linear from 10 to 500 microg/mL with r2 > 0.999. Mean recoveries of the analytes were greater than 96%. In addition, a comparison with the LC method described in the 2010 Chinese Pharmacopoeia demonstrated that the developed CZE method was equally comparable with regard to linearity, sensitivity, precision, and accuracy, but the analysis time and reagent consumption were decreased. The CZE method is an effective and lower-cost alternative to the LC method.

Development and in vitro characterization of mebendazole delayed release tablet for colonic drug delivery.

The main objective behind this study was to formulate delayed release colon targeted tablet of Mebendazole by using different polymers. The precompressional parameters of powder blend were studied. The wet granulation method was used for the preparation of tablets. The tablets of all formulation were subjected for different physicochemical evaluation. The drug-excipient interaction study was carried out by using Fourier transforms Infrared spectroscopy (FTIR). The in vitro evaluation was carried out at different pH ranges (0.1M HCl, 6.8 and 7.4 Phosphate buffer) for the prepared tablets. From the stability, Fourier transform infra-red spectroscopy studies Mebendazole tablet does not show any interaction between drug and polymer. The prepared tablets were complied all the physicochemical test as per official limit. The formulated (M3) batch shows better sustained release 99.89% over a period of 12 hours and the data was fitted into Korsemeyer-Peppas kinetic equation. The result indicates that Mebendazole colon targeted matrix tablet remain stable in the stomach and shows better release into the colon with the help of pH dependent synthetic polymers.

Nucleotides as new co-formers in co-amorphous systems: Enhanced dissolution rate, water solubility and physical stability.

Developing co-amorphous systems is an attractive strategy to improve the dissolution rate of poorly water-soluble drugs. Various co-formers have been investigated. However, previous studies revealed that it is a challenge to develop satisfied acidic co-formers, e.g., acidic amino acids showed much poorer co-former properties than neutral and basic amino acids. Only a few acidic co-formers have been reported, such as aspartic acid, glutamic acid, and some other organic acids. Thus, this study aims to explore the possibility of adenosine monophosphate and adenosine diphosphate used as acidic co-formers. Mebendazole, celecoxib and tadalafil were used as the model drugs. The drug-co-former co-amorphous systems were prepared via ball milling and confirmed using XRPD. The dissolution study suggested that the solubility and dissolution rate of the drug-co-formers systems were increased significantly compared to the corresponding crystalline and amorphous drugs. The stability study revealed that using the two nucleotides as co-formers enhanced the physical stability of pure amorphous drugs. Molecular interactions were observed in MEB-co-former and TAD-co-former systems and positively affected the pharmaceutical performance of the investigated co-amorphous systems. In conclusion, the two nucleotides could be promising potential acidic co-formers for co-amorphous systems.

The activity of drug-metabolizing enzymes and the biotransformation of selected anthelmintics in the model tapeworm Hymenolepis diminuta.

The drug-metabolizing enzymes of some helminths can deactivate anthelmintics and therefore partially protect helminths against these drugs' toxic effect. The aim of our study was to assess the activity of the main drug-metabolizing enzymes and evaluate the metabolism of selected anthelmintics (albendazole, flubendazole, mebendazole) in the rat tapeworm Hymenolepis diminuta, a species often used as a model tapeworm. In vitro and ex vivo experiments were performed. Metabolites of the anthelmintics were detected and identified by HPLC with spectrofluorometric or mass-spectrometric detection. The enzymes of H. diminuta are able to reduce the carbonyl group of flubendazole, mebendazole and several other xenobiotics. Although the activity of a number of oxidation enzymes was determined, no oxidative metabolites of albendazole were detected. Regarding conjugation enzymes, a high activity of glutathione S-transferase was observed. A methyl derivative of reduced flubendazole was the only conjugation metabolite identified in ex vivo incubations of H. diminuta with anthelmintics. The results revealed that H. diminuta metabolized flubendazole and mebendazole, but not albendazole. The biotransformation pathways found in H. diminuta differ from those described in Moniezia expanza and suggest the interspecies differences in drug metabolism not only among classes of helminths, but even among tapeworms.

Exploring flubendazole formulations for use in sheep. Pharmacokinetic evaluation of a cyclodextrin-based solution.

BACKGROUND: Flubendazole (FLBZ) is a poor water solubility broad-spectrum BZD methylcarbamate anthelmintic compound. Cyclodextrins (CDs) are usually used to increase aqueous solubility of poor hydrosoluble compounds. The comparative in vitro aqueous solubility of FLBZ and other BZD anthelmintics in the presence of hydroxypropyl-ß-cyclodextrin (HPßCD) was evaluated in the current work. Additionally, the comparative pharmacokinetic behaviour of FLBZ (and its metabolites) administered by the intraruminal (i.r.) or intraabomasal (i.a.) routes to sheep as either an aqueous CDs-based solution or a conventional carboximethylcellulose (CMC) suspension was assessed. Drug solubility studies involving albendazole, mebendazole, oxfendazole and FLBZ were performed in an aqueous solution (pH 1.2 or 7.4) with or without HPßCD (10%, w/v). The pharmacokinetic study involved two experiments. Experiment 1: In a crossover study, sheep received either a FLBZ-CDs solution (n = 3) or a FLBZ-CMC suspension (n = 3) by the i.r. route (3.8 mg/kg). The treatment Groups were reversed after a 21-days washout period. Experiment 2: sheep (n = 4) were treated by the i.a. route with the FLBZ-CDs solution (3.8 mg/kg). Plasma and abomasal fluid samples were collected between 0 and 72 h post-treatment. Samples were analysed by HPLC. RESULTS: Improvement of FLBZ aqueous solubility due to CDs resulted markedly higher than that observed for mebendazole and albendazole. However, oppositely to what was expected, the absorption-related pharmacokinetic parameters did not show any marked formulation-dependant effect. After the i.a. administration of FLBZ, the AUC and the Tmax of the parent compound were significantly (P < 0.05) reduced, which is consistent with ruminal bypass. CONCLUSION: The administration of FLBZ as a CDs-based solution, does not seem to achieve great practical relevance for parasite control in sheep.

Enhanced bioavailability and cysticidal effect of three mebendazole-oil preparations in mice infected with secondary cysts of Echinococcus granulosus.

The aim of the present study is to explore the possibility to increase the efficacy of mebendazole (MBZ) against secondary cysts of Echinococcus granulosus harbored in mice by augmenting the solubility and bioavailability of the drug. Firstly, the saturated solubility of MBZ in nine kinds of oil was determined by high performance liquid chromatography (HPLC), and MBZ was found exhibiting the highest, secondary, and lowest solubility in oleic acid (OA), glycerol trioleate (GT), and soybean oil (SB), respectively. Secondly, MBZ-OA suspension, MBZ-GT suspension, MBZ-SB suspension, and MBZ suspended in 1 % tragacanth (MBZ-1 % tragacanth) were selected for further studies on pharmacokinetics and experimental therapy in mice. Four groups of mice were treated orally with one of aforementioned four MBZ preparations at a single dose of 25 mg/kg, and concentrations of MBZ in plasma obtained from each mouse at various intervals within 24 h postadministration were determined by HPLC. The major pharmacokinetic parameters calculated by MBZ plasma concentration-time curve demonstrated that the peak concentration of the drug (C (max) ) values obtained from three MBZ-oil preparation groups was 1.6-2.8 times higher than that of MBZ-1 % tragacanth group. The same was true that the area under the drug concentration-time curve (AUC(0-∞)) values of 19.8 (2.5)-28.2 (2.5) µg/ml × h revealed in the three MBZ-oil preparation groups was significantly higher than that of 11.6 (2.0) µg/ml × h in MBZ-1 % tragacanth group, and the bioavailability of the three MBZ-oil preparation groups was 71-143 % higher than that of MBZ-1 % tragacanth group. In mice infected with secondary cysts of E. granulosus for 8 months treated orally with MBZ-1 % tragacanth at a daily dose of 25 mg/kg for 14 consecutive days, the mean cyst weight was lower than that of untreated control, but the difference was not statistically significant with cyst weight reduction of 48 %. When the infected mice received three MBZ-oil preparations at the same oral dose schedule as aforementioned, the mean cyst weights were significantly lower than those in MBZ-1 % tragacanth group or control group with cyst weight reductions of 71.2-84.7 %. The results indicate that the solubility of MBZ in oils may increase to various degrees according to the kinds of oil used. Meanwhile, three MBZ-oil (OA, GT, and SB) preparations administered orally to mice not only improve the bioavailability of MBZ relative to that of MBZ suspended in 1 % tragacanth, but their effects against hydatid cysts also significantly enhance.

Transfer of flubendazole and tylosin at cross contamination levels in the feed to egg matrices and distribution between egg yolk and egg white.

Chemical residues may be present in eggs from laying hens' exposure to drugs or contaminants. These residues may pose risks to human health. In this study, laying hens received experimental feed containing flubendazole or tylosin at cross contamination levels of 2.5, 5, and 10% of the therapeutic dose. Eggs were collected daily and analysis of the whole egg, egg white, and egg yolk was performed using liquid chromatography tandem mass spectrometry. Highest concentrations of the parent molecule flubendazole, as well as the hydrolyzed and the reduced metabolite, were detected in egg yolk. Residue concentrations of the parent molecule were higher compared with those of the metabolites in all egg matrices. No tylosin residue concentrations were detected above the limit of quantification for all concentration groups and in all egg matrices. Neither molecule exceeded the set maximum residue limits.

Transfer of flubendazole and tylosin from feed at cross-contamination levels to various poultry matrices.

Residues of veterinary drugs and feed additives used extensively in animal husbandry are sometimes found in edible matrices. In this study, broilers received experimental feed, containing either flubendazole or tylosin, at cross-contamination levels of 2.5%, 5%, and 10% of the therapeutic dose to determine the transfer ratio of these molecules from feed to poultry matrices. Breast and thigh muscle and liver samples were collected during treatment and depletion periods and then analyzed using liquid chromatography-tandem mass spectrometry. The parent molecule flubendazole and its 2 major metabolites were quantified. After 3 to 5 d, a plateau phase was reached, and a few days after withdrawal of the experimental feed, a depletion of residues was noted. Significant difference between both muscle types was noted for flubendazole. Strong metabolization of flubendazole in the liver was seen. For tylosin, no residue concentrations above the limit of quantification could be detected in muscle. None of the residue concentrations for either molecule exceeded the corresponding maximum residue limits.

Preparation, characterization and application of a new stir bar sorptive extraction based on poly(vinylphthalimide-co-N,N'-methylenebisacrylamide) monolith.

In this study, a new stir bar sorptive extraction (SBSE) coating based on poly(vinylphthalimide-co-N,N'-methylenebisacrylamide) monolith (SBSE-VPMB) was prepared. The influences of the contents of monomer in polymerization mixture and the percentage of porogen solvent on the extraction performance were investigated thoroughly. Several characteristic techniques, such as elemental analysis, scanning electron microscopy, mercury intrusion porosimetry and infrared spectroscopy, were used to characterize the monolithic material. The analysis of oxfendazole (OFZ) and mebendazole (MBZ) in milk and honey samples by the combination of SBSE with HPLC with diode array detection was selected as paradigms for the practical evaluation of the new coating. Under the optimized extraction conditions, the limits of detection (S/N=3) for OFZ and MBZ were 0.23-0.60 µg/L in milk and 0.24-1.08 µg/L in honey, respectively. The method also showed good linearity, repeatability, high feasibility and acceptable recoveries for real samples. At the same time, the extraction performance and the distribution coefficients (K(VPMB/W)) of OFZ and MBZ on SBSE-VPMB were compared with other SBSEs based on porous monoliths and commercial SBSE.

Solubility behavior and prediction for antihelmintics at several temperatures in aqueous and nonaqueous mixtures.

A model based on solubility parameters is proposed to predict the solubility curves of antihelmintic drugs at several temperatures, including aqueous and non-aqueous mixtures. The solubility of the drugs was measured in ethanol-water and ethanol-ethyl acetate mixtures at 15-35 degrees C (mebendazole) and at 25 degrees C (thiabendazole and metronidazole). The solid phases were analyzed by differential scanning calorimerty. The polymorphic form A of mebendazole was also characterized from infrared spectroscopy. Markedly different solubility profile shapes were obtained against the solubility parameter of the mixtures: two symmetrical peaks (metronidazole), two maxima of different height (mebendazole) and a single peak (thiabendazole). The solubility parameter of the drugs was related to the co-solvent action of both mixtures and to the solubility peaks. The single equation proposed was able to predict solubility profiles of different shape, including both mixtures and all temperatures, providing reasonable physical meaning for the regression coefficients. The model was successfully tested for its predictive capability using a limited number of experimental data. More than 100 solubilities were predicted at several temperatures using 20 data point for each drug.

Continuous twin screw granulation: A complex interplay between formulation properties, process settings and screw design.

Due to the numerous advantages over batch manufacturing, continuous manufacturing techniques such as twin screw wet granulation are rapidly gaining importance in pharmaceutical production. Since a large knowledge gap on the importance of formulation variables exists, this study systematically assessed the impact of different screw configurations and process settings on eight model formulations, varying in filler type, active pharmaceutical ingredient (API) characteristics and drug load. Although liquid to solid (L/S) ratio was the most influential variable for all formulations, also a large effect of the kneading element thickness was observed. Narrow kneading elements with a length to diameter ratio (L/D) of 1/6 had a significant detrimental effect on granule size, flow and strength compared to 1/4 L/D elements. The effects of kneading element distribution and barrel fill level were less pronounced. At low drug load, both filler types could be used to obtain granules with acceptable critical quality attributes (CQAs) for both APIs. Granulation at high drug load of the poorly soluble, poorly wettable API mebendazole proved challenging as it could not be processed using lactose as filler, in contrast to lactose/MCC. As formulations containing lactose/MCC as filler were less influenced by different screw configurations, process settings and API characteristics than formulations without MCC, lactose/MCC/HPMC was considered a promising platform formulation.

Continuous twin screw granulation: Impact of binder addition method and surfactants on granulation of a high-dosed, poorly soluble API.

Despite the recent commercialization of several drug products manufactured through continuous manufacturing techniques, knowledge on the formulation aspect of these techniques, such as twin screw wet granulation, is still rather limited. Previous research identified lactose/MCC/HPMC as a robust platform formulation for several model formulations, although granulation of the high-dosed, poorly soluble API mebendazole proved challenging. Therefore, current research evaluated the binder addition method (wet or dry) as well as surfactant (SLS) addition when using PVP, instead of HPMC. Compared to the previous formulation, using HPMC as binder, all four formulations with PVP yielded significantly stronger granules at similar to significantly lower liquid to solid (L/S) ratios. Through the combination of four replicate center composite circumscribed designs, each evaluating the impact of screw speed and L/S ratio on granule quality attributes, the effect of the formulation variables was assessed. Overall, L/S ratio had the most significant impact on granule characteristics whereas the effect of screw speed was negligible. Similar granule quality attributes were obtained for each formulation, although the addition of SLS and wet binder addition significantly reduced the required L/S ratio to achieve the desired characteristics. This significant reduction could prove useful for processing other formulations requiring high amounts of moisture, which could otherwise not be dried at a high throughput due to the limited drying capacity of the dryer unit of the Consigma system.

In silico molecular target prediction unveils mebendazole as a potent MAPK14 inhibitor.

The concept of polypharmacology involves the interaction of drug molecules with multiple molecular targets. It provides a unique opportunity for the repurposing of already-approved drugs to target key factors involved in human diseases. Herein, we used an in silico target prediction algorithm to investigate the mechanism of action of mebendazole, an antihelminthic drug, currently repurposed in the treatment of brain tumors. First, we confirmed that mebendazole decreased the viability of glioblastoma cells in vitro (IC50 values ranging from 288 nm to 2.1 µm). Our in silico approach unveiled 21 putative molecular targets for mebendazole, including 12 proteins significantly upregulated at the gene level in glioblastoma as compared to normal brain tissue (fold change > 1.5; P < 0.0001). Validation experiments were performed on three major kinases involved in cancer biology: ABL1, MAPK1/ERK2, and MAPK14/p38α. Mebendazole could inhibit the activity of these kinases in vitro in a dose-dependent manner, with a high potency against MAPK14 (IC50  = 104 ± 46 nm). Its direct binding to MAPK14 was further validated in vitro, and inhibition of MAPK14 kinase activity was confirmed in live glioblastoma cells. Consistent with biophysical data, molecular modeling suggested that mebendazole was able to bind to the catalytic site of MAPK14. Finally, gene silencing demonstrated that MAPK14 is involved in glioblastoma tumor spheroid growth and response to mebendazole treatment. This study thus highlighted the role of MAPK14 in the anticancer mechanism of action of mebendazole and provides further rationale for the pharmacological targeting of MAPK14 in brain tumors. It also opens new avenues for the development of novel MAPK14/p38α inhibitors to treat human diseases.