Proteomics reveals that the antifungal activity of fenbendazole against Cryptococcus neoformans requires protein kinases.

Cryptococcus neoformans is responsible for over 100 000 deaths annually, and the treatment of this fungal disease is expensive and not consistently effective. Unveiling new therapeutic avenues is crucial. Previous studies have suggested that the anthelmintic drug fenbendazole is an affordable and nontoxic candidate to combat cryptococcosis. However, its mechanism of anticryptococcal activity has been only superficially investigated. In this study, we examined the global cellular response of C. neoformans to fenbendazole using a proteomic approach (data are available via ProteomeXchange with identifier PXD047041). Fenbendazole treatment mostly impacted the abundance of proteins related to metabolic pathways, RNA processing, and intracellular traffic. Protein kinases, in particular, were significantly affected by fenbendazole treatment. Experimental validation of the proteomics data using a collection of C. neoformans mutants led to the identification of critical roles of five protein kinases in fenbendazole's antifungal activity. In fact, mutants lacking the expression of genes encoding Chk1, Tco2, Tco3, Bub1, and Sch9 kinases demonstrated greater resistance to fenbendazole compared to wild-type cells. In combination with the standard antifungal drug amphotericin B, fenbendazole reduced the cryptococcal burden in mice. These findings not only contribute to the elucidation of fenbendazole's mode of action but also support its use in combination therapy with amphotericin B. In conclusion, our data suggest that fenbendazole holds promise for further development as an anticryptococcal agent.

Investigating the Promising Anticancer Activity of Cetuximab and Fenbendazole Combination as Dual CBS and VEGFR-2 Inhibitors and Endowed with Apoptotic Potential.

In this work, the cytotoxicity of monoclonal antibody (Cetuximab, Ce) and Fenbendazole (Fen), as well as their combination therapy were tested with the MTT assay. On the other side, Ce, Fen, and a combination between them were subjected to a colchicine-tubulin binding test, which was conducted and compared to Colchicine as a reference standard. Besides, Ce, Fen, and the combination of them were tested against the VEGFR-2 target receptor, compared to Sorafenib as the standard medication. Moreover, the qRT-PCR technique was used to investigate the levels of apoptotic genes (p53 and Bax) and anti-apoptotic gene (Bcl-2) as well. Also, the effect of Ce, Fen, and the combination of them on the level of ROS was studied. Furthermore, the cell cycle analysis and Annexin V apoptosis assay were carried out for Ce, Fen, and a combination of them. In addition, the molecular docking studies were used to describe the molecular levels of interactions for both (Fen and colchicine) or (Fen and sorafenib) within the binding pockets of the colchicine binding site (CBS) and vascular endothelial growth factor-2 receptor (VEGFR-2), respectively.

The first report of triple anthelmintic resistance on a French Thoroughbred stud farm.

This study assessed the anthelmintic resistance in strongylid nematodes against commonly used anthelmintic (AH) drugs in a French galloping racehorse stud farm from March to December 2023. Faecal egg count reduction tests (FECRTs) were conducted in three different groups of Thoroughbred yearlings (a group of 6 males, a group of 13 females and a group of 8 females and 3 males) following the new World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines. The efficacy of fenbendazole was tested in two groups once during the monitoring period (in March), the efficacy of ivermectin in 3 groups twice (in March-April and in November-December) and the efficacy of pyrantel in one group once (in May-June). For each FECRT, the 90% confidence interval of the percentage faecal egg count reduction was calculated using the hybrid Frequentist/Bayesian analysis method. The resistance in strongylids was observed to fenbendazole, pyrantel and ivermectin in all the groups in which these drugs were tested. The number of animals in each group was sufficient to reach ≥80% power for the resistance test. The results highlight the first case of triple AH resistance in strongylids in France. Further studies involving more farms and equids are required to assess the prevalence of AH resistance in France and refine recommendations for owners.

Unravelling drug-drug interactions in pigs: Induction of hepatic cytochrome P450 1A (CYP1A) metabolism after the in-feed medication with the anthelmintic fenbendazole.

The anthelmintic fenbendazole (FBZ) undergoes hepatic S­oxygenation by monooxygenases belonging to the cytochrome P450 (CYP) and flavin-monooxygenase (FMO) families. The in-feed medication with FBZ induced CYP1A-dependent metabolism in pig liver. This fact may alter the metabolism of the anthelmintic itself, and of CYP1A substrates like aflatoxin B1 (AFB1). This work evaluated the effect of the in-feed administration of FBZ on CYP1A-dependent metabolism, on its own pattern of hepatic S­oxygenation, and on the metabolism of AFB1. Landrace piglets remained untreated (n = 5) or received a pre-mix of FBZ (n = 6) in feed for 9 days. Pigs were slaughtered for preparation of liver microsomes used for: CYP content determination; monitoring the CYP1A-dependent enzyme activities, 7-ethoxyresorufin O-deethylase (EROD) and 7-methoxyresorufin O-demethylase (MROD); measurement of FBZ (50 µM) S­oxygenation, and AFB1 (16 nM) disappearance from the incubation medium. In microsomes of FBZ-treated animals, EROD and MROD increased 19-fold (p = 0.002) and 14-fold (p = 0.003), respectively. An enhanced (3-fold, p = 0.004) participation of the CYP pathway in FBZ S­oxygenation was observed in the liver of piglets treated with the anthelmintic (210 ± 69 pmol/min.nmol CYP) compared to untreated animals (68 ± 34 pmol/min.nmol CYP). AFB1 metabolism was 93% higher (p = 0.009) in the liver of FBZ-treated compared to untreated pigs. Positive and significant (p < 0.05) correlations were observed between CYP1A-dependent enzyme activities and FBZ or AFB1 metabolism. The sustained administration of FBZ caused an auto-induction of the CYP1A-dependent S­oxygenation of this anthelmintic. The CYP1A induction triggered by the anthelmintic could amplify the production of AFB1 metabolites in pig liver, including the hepatotoxic AFB1-derived epoxide.+.

Effects of fenbendazole on fecal microbiome in BPH/5 mice, a model of hypertension and obesity, a brief report.

Fenbendazole (FBZ) is a common antiparasitic treatment used in research rodent colonies for biosecurity purposes. The effect of this compound has been studied in C57 mice, but never before in a strain of mice that has co-morbidities, such as the blood pressure high (BPH)/5. The BPH/5 mouse is an inbred genetic model of hypertension. While both male and female BPH/5 have high blood pressure, there is a metabolic sexual dimorphism with females displaying key features of obesity. The obese gut microbiome has been linked to hypertension. Therefore, we hypothesized that fenbendazole treatment will alter the gut microbiome in hypertensive mice in a sex dependent manner. To test the influence of FBZ on the BPH/5 gut microbiota, fecal samples were collected pre- and post-treatment from adult BPH/5 mice (males and non-pregnant females). The mice were treated with fenbendazole impregnated feed for five weeks. Post-treatment feces were collected at the end of the treatment period and DNA was extracted, and the V4 region of 16S rRNA was amplified and sequenced using the Illumina MiSeq system. The purpose was to analyze the fecal microbiome before and after FBZ treatment, the results demonstrate changes with treatment in a sex dependent manner. More specifically, differences in community composition were detected in BPH/5 non-pregnant female and males using Bray-Curtis dissimilarity as a measure of beta-diversity (treatment p = 0.002). The ratio of Firmicutes to Bacteroidetes, which has been identified in cases of obesity, was not altered. Yet, Verrucomicrobia was increased in BPH/5 males and females post-treatment and was significantly different by sex (treatment p = 5.85e-05, sex p = 0.0151, and interaction p = 0.045), while Actinobacteria was decreased in the post-treatment mice (treatment p = 0.00017, sex p = 0.5, interaction p = 0.2). These results are indicative of gut dysbiosis compared to pre-treatment controls. Lactobacillus was decreased with FBZ treatment in BPH/5 females only. In conclusion, fenbendazole does alter the gut microbial communities, most notable in the male rather than female BPH/5 mouse. This provides evidence that caution should be taken when providing any gut altering treatments before or during mouse experiments.

Redox-mediated Anticancer Activity of Anti-parasitic Drug Fenbendazole in Triple-negative Breast Cancer Cells.

BACKGROUND/AIM: An increasing number of studies are reporting anticancer activity of widely used antiparasitic drugs and particularly benzimidazoles. Fenbendazole is considered safe and tolerable in most animal species at the effective doses as an anthelmintic. Little is known about the redox-modulating properties of fenbendazole and the molecular mechanisms of its antiproliferative effects. Our study aimed to investigate the possibility of selective redox-mediated treatment of triple-negative breast cancer cells by fenbendazole without affecting the viability and redox status of normal breast epithelial cells. MATERIALS AND METHODS: The experiments were performed on three cell lines: normal breast epithelial cells (MCF-10A) and cancer breast epithelial cells (MCF7 - luminal adenocarcinoma, low metastatic; MDA-MB-231 - triple-negative adenocarcinoma, highly metastatic). Cells were treated with fenbendazole for 48-h and three parameters were analyzed using conventional assays: cell viability and proliferation, level of intracellular superoxide, and level of hydroperoxides. RESULTS: The data demonstrated that MDA-MB-231 cells were more vulnerable to fenbendazole-induced oxidative stress than MCF-7 cells. In normal breast epithelial cells MCF-10A, fenbendazole significantly suppressed oxidative stress compared to untreated controls. These data correlate with the effect of fenbendazole on cell viability and the IC50 values, which is indirect evidence of the potential targeting anticancer effect of the drug, especially in MDA-MB-231 cells. CONCLUSION: The difference in the levels of oxidative stress induced by fenbendazole in MDA-MB-231 and MCF-7 indicates that the two types of breast cancer respond to the drug through different redox-related mechanisms.

Medication with fenbendazole in feed: plasma concentrations and effects on hepatic xenobiotic metabolizing enzymes in swine.

Fenbendazole (FBZ), a benzymidazole (BZD) anthelmintic drug, is used for in-feed medication in pigs. BZD-containing drugs may induce cytochrome P450 isozymes (CYPs), particularly those members of the CYP1A subfamily. The current research evaluated the plasma and liver availability and metabolism of FBZ and its metabolites, oxfendazole (OFZ) and fenbendazole sulphone (FBZSO2), after the administration of the parent drug in feed, and characterized the effect of the sustained administration of the anthelmintic on the catalytic activities of xenobiotic metabolizing enzymes in pig liver. Five female Landrace piglets remained untreated (controls), and other six were treated with a pre-mix of FBZ, combined with feed, for 9 consecutive days as usually is recommended. Blood samples were collected from each treated animal up to day 9 and analyzed by HPLC; all animals were slaughtered for preparation of liver microsomes. Plasma concentration ratios OFZ/FBZ and FBZSO2/OFZ increased significantly (p < 0.05) from the beginning to the end of drug exposure, which may indicate an enhanced conversion of FBZ into its metabolites. FBZ represented 45.8 ± 3.4% of the total anthelmintic molecules in liver tissue. Increased CYP1A-dependent 7-ethoxy (24.5-fold, p = 0.0032) and 7-methoxyresorufin (17.2-fold, p = 0.0006) O-dealkylase activities was observed in liver microsomes from FBZ-treated animals. In addition, a 64% increase (p = 0.042) in the rate of FBZ S-oxidation was observed in pigs treated with the anthelmintic drug compared to that measured in untreated animals. Thus, the continuous FBZ administration may accelerate its own in vivo hepatic metabolism through the CYP1A pathway.

Teaching an old dog new tricks: The case of Fenbendazole.

The objective of this study is the assessment of the cytotoxic effect of fenbendazole and its commercially available formulation, which is used for its antihelmintic properties. The formulation was tested for its efficacy as well as the determination of the ingredients with proliferation assays and analytical techniques. HPLC, LC-MS and NMR confirmed the stated amount of active ingredient on the label. Dissolution studies were performed to simulate the ability of fenbendazole to dissolve adequately in the fluids of the Gastrointestinal tract, be absorbed in the circulation and reach certain areas of the human body. However, dissolution studies showed that both brands possess issues in their distribution. The in vitro drug screening exhibited potential cytotoxic effect in different types of human cancer cell lines and MDA-MB-231 human breast adenocarcinoma cells appeared to be the most sensitive with IC50 value lower than 10 µM.

Gastrointestinal helminths in dogs: occurrence, risk factors, and multiple antiparasitic drug resistance.

Helminth infections are detrimental to the overall health of dogs; therefore, this study aimed to identify antiparasitic-resistant helminths and evaluate the infection rate and risk factors for parasitism in canines. For this purpose, a parasitological evaluation of 38 randomly selected animals was performed, followed by the evaluation of the anthelminthic efficacy of three drugs: pyrantel pamoate with praziquantel (Canex Composto®), fenbendazole (Fenzol Pet®), and milbemycin oxime with praziquantel (Milbemax C®). Among the evaluated animals, 22/38 (57.89%) tested negative and 16/38 (42.71%) tested positive for Ancylostoma caninum infection. Evaluation of the efficacy of antiparasitic drugs showed that 12/16 (75%) dogs were infected with helminths that were susceptible to pyrantel pamoate with praziquantel. Among those for which pyrantel pamoate with praziquantel was not effective, 3/4 (75%) were susceptible to fenbendazole, while the remaining case resistant to both pyrantel pamoate with praziquantel and fenbendazole was sensitive to milbemycin oxime with praziquantel (100%). The odds ratio of infection in dogs inhabiting environments containing soil or grass was 6.67 times higher than that in dogs inhabiting impermeable environments. Mixed-breed dogs (SRD) were 6.54 times more likely to be infected compared to purebred dogs. A. caninum resistant to pyrantel pamoate with praziquantel (4/16, 25%) and fenbendazole (1/4, 25%) were detected. The results of this study demonstrated the importance of coproparasitological monitoring by professionals before and after treatments to assess antiparasitic drug effectiveness, ensure animal health and welfare, and minimize animal exposure to risk factors.

An Update on the Biologic Effects of Fenbendazole.

Fenbendazole remains the drug of choice to treat pinworm infection in laboratory rodents. When fenbendazole was last reviewed (15 y ago), the literature supported the drug's lack of toxic effects at therapeutic levels, yet various demonstrated physiologic effects have the potential to alter research outcomes. Although more recent reports continue to reflect an overall discordancy of results, several studies support the premise that fenbendazole affects the bone marrow and the immune system. No effects on reproduction were reported in an extensive study that assessed common treatment protocols in mice, and food intake was unchanged in rats. Behavioral studies are sparse, with only a single report of a subtle change in a rotarod performance in mice. Notably, unexpected results in tumor models during facility treatment with fenbendazole have prompted preclinical and clinical studies of the potential roles of benzimidazoles in cancer.

Fenbendazole and its synthetic analog interfere with HeLa cells' proliferation and energy metabolism via inducing oxidative stress and modulating MEK3/6-p38-MAPK pathway.

Fenbendazole, a broad-spectrum anti-parasitic drug, can be a potential anti-tumor agent. In this study, we synthesized and purified its derivative, analog 6, intending to achieve improved efficacy in cancer cells and decreased toxicity in normal cells. To evaluate in vitro anti-tumor activities of fenbendazole and analog 6 in different cancer cell lines, a CCK-8 assay was performed, and we found that human cervical cancer HeLa cells were more sensitive to analog 6 than to fenbendazole. Furthermore, we explored the associated mechanism, and our results showed that analog 6 and fenbendazole could induce oxidative stress by accumulating ROS. It not only activated the p38-MAPK signaling pathway, thereby inhibiting the proliferation of HeLa cells and enhancing the apoptosis of HeLa cells, but also significantly induced impaired energy metabolism and restrained their migration and invasion. In addition, the modified analog 6 showed reduced toxicity to normal cells without decreased anti-cancer effect. In conclusion, fenbendazole and analog 6 have multiple targets and strong anti-tumor effects on HeLa cells in vitro and in vivo. The optimized analog 6 could inhibit the viability of HeLa cells with lower toxicity than normal human cells, promising to be developed as an antitumor active compound.

Screening the pandemic response box identified benzimidazole carbamates, Olorofim and ravuconazole as promising drug candidates for the treatment of eumycetoma.

Eumycetoma is a chronic subcutaneous neglected tropical disease that can be caused by more than 40 different fungal causative agents. The most common causative agents produce black grains and belong to the fungal orders Sordariales and Pleosporales. The current antifungal agents used to treat eumycetoma are itraconazole or terbinafine, however, their cure rates are low. To find novel drugs for eumycetoma, we screened 400 diverse drug-like molecules from the Pandemic Response Box against common eumycetoma causative agents as part of the Open Source Mycetoma initiative (MycetOS). 26 compounds were able to inhibit the growth of Madurella mycetomatis, Madurella pseudomycetomatis and Madurella tropicana, 26 compounds inhibited Falciformispora senegalensis and seven inhibited growth of Medicopsis romeroi in vitro. Four compounds were able to inhibit the growth of all five species of fungi tested. They are the benzimidazole carbamates fenbendazole and carbendazim, the 8-aminoquinolone derivative tafenoquine and MMV1578570. Minimal inhibitory concentrations were then determined for the compounds active against M. mycetomatis. Compounds showing potent activity in vitro were further tested in vivo. Fenbendazole, MMV1782387, ravuconazole and olorofim were able to significantly prolong Galleria mellonella larvae survival and are promising candidates to explore in mycetoma treatment and to also serve as scaffolds for medicinal chemistry optimisation in the search for novel antifungals to treat eumycetoma.

Fenbendazole Suppresses Growth and Induces Apoptosis of Actively Growing H4IIE Hepatocellular Carcinoma Cells via p21-Mediated Cell-Cycle Arrest.

Bendimidazole anthelmintics (BAs) have gained interest for their anticancer activity. The anticancer activity is mediated via multiple intracellular changes, which are not consistent under different conditions even in the same cells. We investigated the anticancer activity of fenbendazole (FZ, one of BAs) under two different growth conditions. The growth rate of H4IIE cells was dose-dependently decreased by FZ only in actively growing cells but not in fully confluent quiescent cells. Apoptosis-associated changes were also induced by FZ in actively growing cells. Markers of autophagy were not changed by FZ. The number of cells was markedly increased in sub-G1 phase but decreased in S- and G2/M phases by FZ. FZ up-regulated p21 (an inhibitor of cyclin-CDK) but suppressed the expression of cell cycle-promoting proteins (cyclin D1 and cyclin B1). FZ did not affect integrin αV or n-cadherin expression as well as cell migration. Glycolytic changes (glucose consumption and lactate production) and the generation of reactive oxygen species (ROS) were not affected by FZ. Although the activity of mitogen-activated protein kinases (MAPKs) was altered by FZ, the inhibition of MAPKs did not affect the pro-apoptotic activity of FZ. Taken together, FZ selectively suppressed the growth of cells via p21-mediated cell cycle arrest at G1/S and G2/M, and resulted in apoptosis only in actively growing cells but not in quiescent cells. Glucose metabolism, ROS generation, and MAPKs are unlikely targets of FZ at least in H4IIE rat hepatocellular carcinoma cells used in this study.

G2/M arrest and mitotic slippage induced by fenbendazole in canine melanoma cells.

BACKGROUND: The use of fenbendazole (FBZ) in terminal cancer patients has recently increased, as anthelminthic drugs, such as FBZ and benzimidazole, exhibit anti-tubulin effects in tumour cells. OBJECTIVES: The present study evaluated the in vitro anti-cancer effects of FBZ in five canine melanoma cell lines originating from the oral cavity (UCDK9M3, UCDK9M4, UCDK9M5, KMeC and LMeC). METHODS: Five canine melanoma cell lines were treated with FBZ and analysed with cell viability assay, cell cycle analysis, western blot assay and immunofluorescence staining to identify apoptotic effect, cell cycle arrest, microtubule disruption and mitotic slippage. RESULTS: Cell viability was reduced in all melanoma cell lines in a dose-dependent manner after FBZ treatment. Through cell cycle analysis, G2/M arrest and mitotic slippage were identified, which showed a time-dependent change. All treatment concentrations induced increased cleaved PARP signals in western blot analysis compared to the control groups. Immunofluorescence of cells treated for 24 h revealed defects in microtubule structure, multinucleation or macronucleation. With the exception of UCDK9M3, the melanoma cells showed mitotic slippage and post-slippage death, indicative of mitotic catastrophe. CONCLUSIONS: These results indicate that FBZ exhibits anti-cancer effects in vitro against canine melanoma cells; however, further in vivo studies regarding the clinical applications of FBZ are required.

Comparing the performance of first-order conditional estimation (FOCE) and different expectation-maximization (EM) methods in NONMEM: real data experience with complex nonlinear parent-metabolite pharmacokinetic model.

First-order conditional estimation (FOCE) has been the most frequently used estimation method in NONMEM, a leading program for population pharmacokinetic/pharmacodynamic modeling. However, with growing data complexity, the performance of FOCE is challenged by long run time, convergence problem and model instability. In NONMEM 7, expectation-maximization (EM) estimation methods and FOCE with FAST option (FOCE FAST) were introduced. In this study, we compared the performance of FOCE, FOCE FAST, and two EM methods, namely importance sampling (IMP) and stochastic approximation expectation-maximization (SAEM), utilizing the rich pharmacokinetic data of oxfendazole and its two metabolites obtained from the first-in-human single ascending dose study in healthy adults. All methods yielded similar parameter estimates, but great differences were observed in parameter precision and modeling time. For simpler models (i.e., models of oxfendazole and/or oxfendazole sulfone), FOCE and FOCE FAST were more efficient than EM methods with shorter run time and comparable parameter precision. FOCE FAST was about two times faster than FOCE but it was prone to premature termination. For the most complex model (i.e., model of all three analytes, one of which having high level of data below quantification limit), FOCE failed to reliably assess parameter precision, while parameter precision obtained by IMP and SAEM was similar with SAEM being the faster method. IMP was more sensitive to model misspecification; without pre-systemic metabolism, IMP analysis failed to converge. With parallel computing introduced in NONMEM 7.2, modeling speed increased less than proportionally with the increase in the number of CPUs from 1 to 16.

Widespread resistance to macrocyclic lactones in cattle nematodes in Ecuador.

The aim of the present study was to assess the resistance status of bovine gastrointestinal nematodes (GINs) against ivermectin (IVM) and fenbendazole (FBZ) in Ecuador. The study involved five cattle farms located in different topographic zones of the country. Anthelmintic efficacy was assessed by calculating the percentage of fecal egg counts reduction (FECR) after treatment. Additionally, DNA from pooled larval cultures was screened to ascertain benzimidazole resistance alleles. For animals treated with IVM, FECR percentages ranged from 0 to 68%, indicating the presence of highly resistant worms. The opposite was found for animals treated with FBZ, where FECR percentages were above 90% on all the farms tested. Pooled coprocultures revealed that Cooperia spp. were the predominant species pre and post-treatment although minor proportions of Haemonchus spp. and Ostertagia spp. were also identified. No mutations conferring resistance to benzimidazoles were identified in the beta-tubulin isotype 1 gene of the isolated Cooperia spp. worms, which is in line with the results of the FECR performed with FBZ. Overall, the present study highlights widespread resistance of bovine GINs to IVM but no to FBZ in Ecuador.

Natural resistance to worms exacerbates bovine tuberculosis severity independently of worm coinfection.

Pathogen interactions arising during coinfection can exacerbate disease severity, for example when the immune response mounted against one pathogen negatively affects defense of another. It is also possible that host immune responses to a pathogen, shaped by historical evolutionary interactions between host and pathogen, may modify host immune defenses in ways that have repercussions for other pathogens. In this case, negative interactions between two pathogens could emerge even in the absence of concurrent infection. Parasitic worms and tuberculosis (TB) are involved in one of the most geographically extensive of pathogen interactions, and during coinfection worms can exacerbate TB disease outcomes. Here, we show that in a wild mammal natural resistance to worms affects bovine tuberculosis (BTB) severity independently of active worm infection. We found that worm-resistant individuals were more likely to die of BTB than were nonresistant individuals, and their disease progressed more quickly. Anthelmintic treatment moderated, but did not eliminate, the resistance effect, and the effects of resistance and treatment were opposite and additive, with untreated, resistant individuals experiencing the highest mortality. Furthermore, resistance and anthelmintic treatment had nonoverlapping effects on BTB pathology. The effects of resistance manifested in the lungs (the primary site of BTB infection), while the effects of treatment manifested almost entirely in the lymph nodes (the site of disseminated disease), suggesting that resistance and active worm infection affect BTB progression via distinct mechanisms. Our findings reveal that interactions between pathogens can occur as a consequence of processes arising on very different timescales.

A case of a dog refractory to different treatments for pulmonary capillariasis.

Pulmonary capillariasis is a parasitic disease caused by the nematode Eucoleus aerophilus which affects wild and domestic carnivores. Currently, there are no anthelmintics approved for use in the treatment of dogs infected with E. aerophilus. The use of several anthelmintics has been reported in a few case reports and field efficacy studies in cats; much less is known on the treatment of dogs infected with E. aerophilus. The paper describes a case of a 4-month-old, mixed breed intact male referred to the Veterinary Teaching Hospital (VTH) of the Department of Veterinary Medical Science of the University of Bologna for a routine vaccination and tested positive for E. aerophilus. The dog has not been responding to three different administered treatments, such as moxidectin, fenbendazole, and milbemycin oxime. Eighteen months after the first fecal examination, owner has brought in the dog for a routine visit; a coprological examination was requested and performed resulting negative for parasites. Veterinary practitioners, parasitologists, diagnostic laboratories, and dog owners need to be aware of the increased danger of possible treatment failure when attempting to control parasitic infections for which there are no approved anthelmintics with established efficacies available for use.

Molecular and phenotypic characterisation of fenbendazole resistance in a field-derived isolate of Ostertagia ostertagi.

The prevalence of anthelmintic resistance in the bovine nematode Cooperia oncophora has been well documented globally but lack of efficacy against the more pathogenic nematode species Ostertagia ostertagi is less common. The sensitivity of an O. ostertagi isolate to the benzimidazole class of anthelmintic was investigated using classical parasitological techniques following apparent clinical failure of controlled release fenbendazole capsule administration in first season grazers at pasture. A controlled efficacy test (CET) was conducted in conjunction with sequencing of the ß-tubulin isotype 1 gene of larvae pre- and post-fenbendazole administration. Twelve helminth-naïve calves were infected experimentally with 20,000 third stage larvae; six received oral fenbendazole (7.5 mg/kg bodyweight) 28 days post infection. Total abomasal nematode burdens were compared between treatment and control groups to determine efficacy. Fenbendazole resistance in O. ostertagi was confirmed with a total treatment failure in reducing worm burden: efficacy of 0%. Sequence analysis of the ß-tubulin isotype-1 gene from forty-five infective larvae from both control and treated groups was performed. The three commonest single nucleotide polymorphisms (SNPs) associated with benzimidazole resistance, namely F167Y, E198A and F200Y, were examined. The predominant resistance-associated SNPs were F200Y (78 % control and 79 % treated groups) and F167Y (remaining genotypes) and emphasises the importance of these SNPs in clinical disease in this isolate. The development of diagnostic molecular tools based on a characterised field-derived isolate of benzimidazole-resistant Ostertagia will enable future prevalence surveys to be undertaken to assess the possible risk posed by resistance in this economically important species.

Potential and mechanism of mebendazole for treatment and maintenance of ovarian cancer.

OBJECTIVE: Mebendazole and other anti-parasitic drugs are being used off-prescription based on social media and unofficial accounts of their anti-cancer activity. The purpose of this study was to conduct a controlled evaluation of mebendazole's therapeutic efficacy in cell culture and in vivo models of ovarian cancer. The majority of ovarian cancers harbor p53 null or missense mutations, therefore the effects of p53 mutations and a mutant p53 reactivator, PRIMA-1MET (APR246) on mebendazole activity were evaluated. METHODS: Mebendazole was evaluated in cisplatin-resistant high grade serous stage 3C ovarian cancer patient derived xenograft (PDX) models: PDX-0003 (p53 null) and PDX-0030 (p53 positive), and on ovarian cancer cell lines: MES-OV (p53 R282W), ES2 (p53 S241F), A2780 (p53 wild type), SKOV3 parental (p53 null) and isogenic sublines, SKOV3 R273H p53 and SKOV3 R248W p53. Drug synergy and mechanisms were evaluated in cell cultures using isobolograms, clonogenic assays and western blots. Prevention of tumor establishment was studied in a MES-OV orthotopic model. RESULTS: Mebendazole inhibited growth of ovarian cancer cell cultures at nanomolar concentrations and PDXs at doses up to 50 mg/kg, and reduced orthotopic tumor establishment at 50 mg/kg. The mechanism of mebendazole was associated with p53-independent induction of p21 and tubule depolymerization. PRIMA-1MET also inhibited tumor establishment and worked synergistically with mebendazole in cell culture to inhibit growth and induce intrinsic apoptosis through a p53- and tubule destabilization-independent mechanism. CONCLUSION: This work demonstrates the therapeutic potential of repurposing mebendazole and supports clinical development of mebendazole for ovarian cancer therapy and maintenance.