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.

Biotransformation of flubendazole and fenbendazole and their effects in the ribwort plantain (Plantago lanceolata).

Although veterinary anthelmintics represent an important source of environmental pollution, the fate of anthelmintics and their effects in plants has not yet been studied sufficiently. The aim of our work was to identify metabolic pathways of the two benzimidazole anthelmintics fenbendazole (FBZ) and flubendazole (FLU) in the ribwort plantain (Plantago lanceolata L.). Plants cultivated as in vitro regenerants were used for this purpose. The effects of anthelmintics and their biotransformation products on plant oxidative stress parameters were also studied. The obtained results showed that the enzymatic system of the ribwort plantain was able to uptake FLU and FBZ, translocate them in leaves and transform them into several metabolites, particularly glycosides. Overall, 12 FLU and 22 FBZ metabolites were identified in the root, leaf base and leaf top of the plant. Concerning the effects of FLU and FBZ, both anthelmintics in the ribwort plantain cells caused significant increase of proline concentration (up to twice), a well-known stress marker, and significant decrease of superoxide dismutase activity (by 50%). In addition, the activities of four other antioxidant enzymes were significantly changed after either FLU or FBZ exposition. This could indicate a certain risk of oxidative damage in plants influenced by anthelmintics, particularly when they are under other stress conditions.

Genotoxicity of flubendazole and its metabolites in vitro and the impact of a new formulation on in vivo aneugenicity.

The anti-parasitic benzimidazole flubendazole has been used for many years to treat intestinal infections in humans and animals. Previous genotoxicity studies have shown that the compound is not a bacterial mutagen and a bone marrow micronucleus test, using a formulation that limited systemic absorption, was negative. The purpose of this study is to explore the genotoxicity of flubendazole and its main metabolites in in vitro micronucleus studies and to test a new oral formulation that improves systemic absorption in an in vivo micronucleus test. The isolated metabolites were also screened using the Ames test for bacterial mutagenicity. It was found that flubendazole, like other chemically related benzimidazoles used in anti-parasitic therapies, is a potent aneugen in vitro The hydrolysed metabolite of flubendazole is negative in these tests, but the reduced metabolite (R- and S-forms) shows both aneugenic and clastogenic activity. However, in vitro micronucleus tests of flubendazole in the presence of rat liver S9 gave almost identical signals for aneugenicity as they did in the absence of S9, suggesting that any clastogenicity from the reduced metabolite is not sufficient to change the overall profile. Like flubendazole itself, both metabolites are negative in the Ames test. Analysis of dose-response curves from the in vitro tests, using recently developed point of departure approaches, demonstrate that the aneugenic potency of flubendazole is very similar to related anti-parasitic benzimidazoles, including albendazole, which is used in mass drug administration programmes to combat endemic filarial diseases. The in vivo micronucleus test of the new formulation of flubendazole also showed evidence of induced aneugenicity. Analysis of the in vivo data allowed a reference dose for aneugenicity to be established which can be compared with therapeutic exposures of flubendazole when this has been established. Analysis of the plasma from the animals used in the in vivo micronucleus test showed that there is increased exposure to flubendazole compared with previously tested formulations, as well as significant formation of the non-genotoxic hydrolysed metabolite of flubendazole and small levels of the reduced metabolite. In conclusion, this study shows that flubendazole is a potent aneugen in vitro with similar potency to chemically related benzimidazoles currently used as anti-parasitic therapies. The reduced metabolite also has aneugenic properties as well as clastogenic properties. Treatment with a new formulation of flubendazole that allows increased systemic exposure, compared with previously used formulations, also results in detectable aneugenicity in vivo. Based on the lack of carcinogenicity of this class of benzimidazoles and the intended short-term dosing, it is unlikely that flubendazole treatment will pose a carcinogenic risk to patients.

Acute Toxicity and Environmental Risks of Five Veterinary Pharmaceuticals for Aquatic Macroinvertebrates.

Due to the high use of antibiotics and antiparasitics for the treatment of livestock, there is concern about the potential impacts of the release of these compounds into freshwater ecosystems. In this context, the present study quantified the acute toxicity of two antibiotics (sulfadiazine and sulfadimidine), and three antiparasitic agents (flubendazole, fenbendazole, ivermectin) for nine freshwater invertebrate species. These experiments revealed a low degree of toxicity for the sulfonamide antibiotics, with limited implications in the survival of all test species at the highest test concentrations (50 and 100 mg/L). In contrast, all three antiparasitic agents indicated on the basis of their acute toxicity risks for the aquatic environment. Moreover, chronic toxicity data from the literature for antiparasitics, including effects on reproduction in daphnids, support the concern about the integrity of aquatic ecosystems posed by releases of these compounds. Thus, these pharmaceuticals warrant further careful consideration by environmental risk managers.

Flubendazole induces mitotic catastrophe and apoptosis in melanoma cells.

Flubendazole (FLU) is a widely used anthelmintic drug belonging to benzimidazole group. Recently, several studies have been published demonstrating its potential to inhibit growth of various tumor cells including those derived from colorectal cancer, breast cancer or leukemia via several mechanisms. In the present study we have investigated cytotoxic effects of FLU on malignant melanoma using A-375, BOWES and RPMI-7951 cell lines representing diverse melanoma molecular types. In all three cell lines, FLU inhibited cell growth and proliferation and disrupted microtubule structure and function which was accompanied by dramatic changes in cellular morphology. In addition, FLU-treated cells accumulated at the G2/M phase of cell cycle and displayed the features of mitotic catastrophe characterized by formation of giant cells with multiple nuclei, abnormal spindles and subsequent apoptotic demise. Although this endpoint was observed in all treated melanoma lines, our analyses showed different activated biochemical signaling in particular cells, thus suggesting a promising treatment potential of FLU in malignant melanoma warranting its further testing.

Mixture toxicity of flubendazole and fenbendazole to Daphnia magna.

Nowadays, residual amounts of many pharmaceuticals can be found in various environmental compartments including surface and ground waters, soils and sediments as well as biota. Even though they undergo degradability, their environmental discharge is relatively continuous, thus they may be regarded as quasi-persistent contaminants, and are also frequently regarded as emerging organic pollutants. Benzimidazoles, especially flubendazole (FLU) and fenbendazole (FEN), represent two anthelmintic drugs belonging to this group. Although their presence in environmental matrices has been reported, there is relatively little data concerning their (eco)toxicological impact. Furthermore, no data is available on their mixture toxicity. FLU and FEN have been found to have a strong impact on an environmentally important non-target organism - Daphnia magna. Moreover, these compounds are usually present in the environment as a part of pharmaceutical mixtures. Therefore, there is a need to evaluate their mixture toxicity, which was the main aim of this study. Single substance toxicity tests were carried out in parallel with mixture studies of FLU and FEN, with the application of two well established concepts of Concentration Addition (CA) and Independent Action (IA). As a result, both models (CA and IA) were found to underestimate the toxicity of mixtures, however CA yielded more accurate predictions.

Safety of Mebendazole Use During Lactation: A Case Series Report.

INTRODUCTION: Mebendazole is an effective drug widely used in the treatment of parasitic infections. Although theoretically considered as safe during lactation, no studies have evaluated its potential adverse effects in infants of breastfeeding mothers. OBJECTIVES: We aimed to evaluate the safety of mebendazole in infants of lactating women treated with the drug. METHODS: Women referred for consultation regarding mebendazole use were invited to participate in the study. Overall 45 lactating women treated with various protocols of mebendazole were recruited in this case series study. RESULTS: Regardless of the treatment protocol used (single or repeated doses) mebendazole was well tolerated and was not associated with any adverse effects in infants of lactating mothers. There was mild GI irritability in two treated women. CONCLUSION: This study provides first evidence in humans as to the safety of mebendazole in breastfeeding.

Toxicity of anthelmintic drugs (fenbendazole and flubendazole) to aquatic organisms.

Flubendazole (FLU) and fenbendazole (FEN) belong to benzimidazoles-pharmaceuticals widely used in veterinary and human medicine for the treatment of intestinal parasites as well as for the treatment of systemic worm infections. In recent years, usage of these drugs increased, which resulted in a larger contamination of the environment and possible negative effects on biota. Hence, in our research, we investigated an aquatic ecotoxicity of these pharmaceuticals towards: marine bacteria (Vibrio fischeri), green algae (Scenedesmus vacuolatus), duckweed (Lemna minor) and crustacean (Daphnia magna). Ecotoxicity tests were combined with chemical analysis in order to investigate the actual exposure concentration of the compounds used in the experiment as well as to stability and adsorption studies. As a result, study evaluating sensitivity of different aquatic organisms to these compounds and new ecotoxicological data is presented. The strongest negative impact of FLU and FEN was observed to D. magna.

Indications for a threshold of chemically-induced aneuploidy in vitro in human lymphocytes.

The possible existence of a threshold for compounds inducing chromosomal loss was investigated for four known aneugens (colchicine, COL; carbendazim, MBC; mebendazole, MEB; nocodazole, NOC) and two clastogens (methyl methanesulfonate, MMS; mitomycin C, MMC) using the micronucleus (MN) test in human lymphocytes. The presence of a whole chromosome in the MN was studied by fluorescent in situ hybridization (FISH) using a synthetic pancentromeric oligonucleotide probe. FISH was applied on two different MN preparations: cytokinesis-blocked MN (MNCB) assay, and MN sorted by flow cytometry. At subtoxic concentrations analyzed by MNCB and FISH, COL, MEB, MBC, and NOC induced a concentration-dependent increase in centromere-positive MN (MNCen+). MMC seemed to induce an increase in both types of MN (MNCen- and MNCen+), while MMS induced only MNCen-. On the sorted micronuclei (in a wide range of low to subtoxic concentrations), the concentration-effect profile for MNCen+, with the four aneugens tested, showed a statistically nonsignificant increase over a range of concentrations, followed by a second range of high concentrations with a statistically significant increase. To analyze the existence of a threshold, a piecewise linear regression was applied to the data. The first concentration that showed a statistically significant increase in MNCen+ was chosen as a breakpoint (0.037 microM for COL, 2.62 microM for MBC, 0.27 microM for MEB, and 0.066 microM for NOC). The statistical correlation between observed and predicted values showed a high correlation (r = 0.99), indicating a clear threshold for aneuploidy induction. However, for MMS the concentration-effect profile for MNCen+ showed a continuous concentration-dependent decrease with no threshold. With the two cytotoxicity assays used (Bio-Rad and MTT), no significant reduction was detected either in the protein content or in mitochondrial succinate dehydrogenase activity with all chemicals tested for MN induction. Therefore, our data suggest that the observed thresholds were not due to indirect toxic effects but to real aneugenic effects.

Albendazole, mebendazole and praziquantel. Review of non-clinical toxicity and pharmacokinetics.

The pharmacokinetics and toxicity of albendazole, mebendazole and praziquantel are extensively reviewed, drawing on original published work and reviews in the open scientific literature and on assessments by international agencies and official regulatory bodies in Europe and the USA. Information about human and veterinary medical uses and adverse reactions is evaluated. The totality of the non-clinical information available about these long-established drugs may not comply with current official guidelines for new medicines but reasons are given why the "deficiencies" are only apparent and the data gaps can be replaced by other results, largely obtained from the target species and the many years of clinical experience of safe use of these drugs in humans and animals.

Benzimidazole treatment of cystic echinococcosis.

Hydatidosis (cystic echinococcosis, CE) constitutes a serious public health problem worldwide. Total surgical removal of a hydatid cyst is still considered the gold standard treatment for CE. Percutaneous treatment (PAIR), using either hypertonic saline or alcohol as a larvacidal agent, appears to be an additional effective form of treatment. Benzimidazoles (albendazole, ABZ; mebendazole, MBZ), given either alone or combined with praziquantel (PZ) are currently used for the treatment of non-surgical cases and as a supplementary treatment prior and post-surgery. Combined chemotherapy was found to be more effective than either of the agents given alone. ABZ is easily absorbed and more effective than MBZ. ABZ (12-15 mg/kg/day) and MBZ (30-70 mg/kg/day) given for 14-20 days prior to surgery and continued for an additional 3-24 months in a cyclic monthly form were found effective against the disease. Either increased or decreased circulating antigen levels, which consequently cause changes in the humoral (IgG, IgG1, IgG4, IgE) immune responses, have a prognostic value in successfully treated CE cases. However, although the cellular immune response to echinococcal antigens decreased in improved or cured CE patients, it was not considered of practical use in determining treatment efficacy. In certain cases successful treatment was also followed by elevated eosinophilia and erythrocyte sedimentation rates. In the present article, the mechanism of drug activities as well as the development of resistance against the drugs available are further discussed.

Teratogenicity of flubendazole in rats.

Flubendazole, the p-fluoroderivative of mebendazole, was suspended in deionized water, and administered by gavage once daily to pregnant rats on days 8 through 15 of pregnancy at 0 (control), 2.5, 10, 40 or 160 mg/kg. Fetuses were removed on day 21 of pregnancy by caesarian section, and examined by routine teratological methods. The highest dose (160 mg/kg) was embryocidal and resulted in a significant increase in the fetal resorption rate. There was a dose-dependent decrease in fetal body weights which was significant at 40 mg/kg or more. The 40 and 160 mg/kg doses induced significant fetal (gross, skeletal and internal) malformations. A variety of gross malformations occurred, i.e. encephalocele, cranial meningocele, omphalocele, ectrodactyly, club foot, defects in tail, anal atresia, shortened backbone and Spina bifida occulta. The skeletal malformations mainly affected the vertebrae and ribs. The most frequently observed internal malformation was hydrocephaly, followed by anophthalmia and/or microphthalmia.

Effect of oral dosing vehicles on the developmental toxicity of flubendazole in rats.

Flubendazole was suspended in deionized water or olive oil and administered by gavage once daily to pregnant rats on Days 8-15 of pregnancy to examine if the embryolethal and teratogenic doses were affected by the vehicles used. Flubendazole in olive oil caused a statistically significant increase in embryolethality at doses of 7.83 mg/kg per day and higher, with complete resorption in all dams at 31.33 mg/kg per day. When flubendazole was suspended in deionized water, a significant increase in embryolethality occurred only at a maternal dose of 125.32 mg/kg per day. The proportion of litters with anomalous fetuses was significantly increased at doses of 31.33 mg/kg per day and above when flubendazole was administered in deionized water, but increased at doses at four times lower when flubendazole was administered as in olive oil. Administered as a single dose in olive oil on any one of Days 6-12 of pregnancy, a flubendazole dose of 31.33 mg/kg caused significant increases in embryolethality and decreased fetal body weights on Days 7-9, with an 82.7% incidence of embryolethality on Day 8, with complete resorption in 5 of the 8 dams. The critical periods for teratogenic effects were between Days 8 and 11 of pregnancy, with Day 9 being the most critical. Fetuses with gross, skeletal, or internal anomalies were seen in dams given a single dose of as low as 7.83 mg/kg.

Genotoxic activity of mebendazole in Aspergillus nidulans.

Mebendazole is an anthelmintic drug widely used in Cuba and in Mexico. Its interaction with tubulin interferes with the assemblage of the mitotic apparatus in the parasite cells, thus suggesting a possible genotoxic activity leading to chromosomal malsegregation. The heterozygous diploid strain D30 of Aspergillus nidulans was used to establish the ability of mebendazole to induce mitotic recombination and/or chromosomal non-disjunction, and the haploid strain FGSC #219 of A. nidulans was used to study the ability of mebendazole to induce point mutations in the methG suppressor system. Our results show that mebendazole can induce chromosomal non-disjunction but it fails to promote point mutations.

An in vivo evaluation of induction of abnormal sperm morphology by some anthelmintic drugs in mice.

Using the murine sperm-head abnormality test, the mutagenicity of pyrantel pamoate, levamisole, albendazole, mebendazole and niridazole was evaluated. Pyrantel pamoate and niridazole induced increases in sperm-head abnormalities statistically significant over the negative controls at all the dose levels that were considered; the induction was dose-dependent indicating that both drugs might be mutagenic. Levamisole, albendazole, mebendazole and thiabendazole, all were unable to induce statistically significant increases in sperm-head abnormalities over the negative controls at all the dose levels tested; there was no correlation between dose level of administered drugs and incidence of abnormal sperms, indicating that the drugs might not be mutagenic.

A comparison of the genetic activity of pyrvinium pamoate with that of several other anthelmintic drugs in Saccharomyces cerevisiae.

Several anthelmintic drugs that are used routinely in oxyuriasis therapy were analyzed for genotoxicity in a diploid mitotic recombination and gene conversion assay (strain D5 of Saccharomyces cerevisiae), and in a haploid yeast reversion assay (strain XV185-14C). Piperazine citrate, piperazine adipate, mebendazole and thiabendazole did not appear to be genotoxic in either yeast strain. Pyrvinium pamoate induced the reversion of the missense, nonsense and frameshift alleles in strain XV185-14C, whereas pyrantel pamoate induced only the reversion of the frameshift allele. Pyrvinium pamoate was recombinogenic in strain D5, and there is an indication that pyrantel pamoate, at the lowest dose that was tested, might induce gene conversion or aneuploidy.

Multiple anthelmintic resistance in Haemonchus contortus on a sheep farm in India.

Multiple resistance to benzimidazoles (fenbendazole, albendazole and mebendazole) in a strain of Haemonchus contortus in sheep was detected on a farm where fenbendazole resistance had already been identified. Following a faecal egg count reduction test, this was confirmed by both critical and controlled anthelmintic tests. Different groups of sheep infected naturally or given an experimental infection with the fenbendazole-resistant strain were treated with the recommended doses of various anthelmintics. Compared to the control group, percentage reductions in faecal egg counts of sheep treated with fenbendazole, albendazole, mebendazole, levamisole and morantel varied between 56% and 81% and worm counts between 71% and 86%. The results indicate the presence of multiple anthelmintic resistance in this strain of H. contortus on this farm. Sheep treated with ivermectin and closantel showed 100% reductions in faecal egg and worm counts, suggesting high efficacy of these drugs against the population of H. contortus on this farm.

Assessing behavioral toxicity with Caenorhabditis elegans.

Behavior, even in simple metazoans, depends upon integrated processes at the subcellular, cellular, and organismal level, and thus is susceptible to disruption by a broad spectrum of chemicals. Locomotor behavior (movement) of the small free-living nematode Caenorhabditis elegans has proven to be useful in assessing toxicity. Recently reported observations suggest that behavioral change (reduced movement) occurs after 4 h of exposure to heavy metals, and that with abbreviated exposure, the concentration-response relationship for Pb (a known neurotoxic metal) differs from that for Cu. In this study, movement was evaluated after 4-h exposures for nine compounds from three chemical classes: organic pesticides, organic solvents, and heavy metals. Concentration-dependent reduction of movement was observed for all test compounds with the exception of mebendazole, for which test concentrations were limited by solubility. Within each chemical class, movement was more sensitive to the neurotoxic compounds than to substances not believed to be neurotoxic, as evidenced by behavioral effective concentration to reduce average worm movement to 50% of the control movement values (e.g., levamisole and chlorpyrifos < mebendazole, ethanol and acetone < dimethylsulfoxide, and Pb and Al < Cu). These observations are discussed as they relate to the use of acute behavioral tests in assessing general chemical toxicity, and the enhanced value of 4-h testing for the detection of neural toxicants.

Evaluation of the safety of flubendazole premix in swine.

Forty-eight pigs were used to evaluate the safety of flubendazole, given as a 6% rice-hull premix. In 1 experiment, flubendazole was administered orally to 16 pigs daily for 5 days at dosages of 10 or 20 times the recommended rate. In a 2nd experiment, flubendazole was administered in the feed to 18 pigs daily for 15, 16 or 17 consecutive days at dosages of 1, 3 and 5 times the recommended rate. The remaining 14 pigs received a placebo premix and served as controls. Clinical observations were made each day, and body weights and clinical laboratory values were monitored before, during and after the treatment period. In the 2nd experiment, all pigs were necropsied 1 day after the last day of treatment. Clinical signs during the experiments consisted of soft/fluid feces, observed in pigs from all groups, and occasional vomiting from 3 pigs in the group given the 3X-exaggerated dose. Statistical differences in the frequency of loose feces and vomiting were not detected between the placebo group and flubendazole-treated groups. There were no other abnormal signs noted throughout either experiment. Results of serum chemistry analysis, hematologic examination, and histopathologic evaluation revealed no evidence of drug-related toxicity.

Clinical toxicosis and erythrocyte cholinesterase inhibition of trichlorfon combined with mebendazole in horses.

Thirty adult horses were used to compare the toxicity and cholinesterase (ChE) inhibition of various dosages of a combination anthelmintic, trichlorfon + mebendazole. Single oral doses of up to 5 times the effective dosage (39.7 mg of trichlorfon and 8.8 mg of mebendazole/kg of body weight) did not result in deaths. Horses given a placebo and horses treated at the recommended dosage rate showed few or no side effects, whereas horses given higher dosages showed dosage-related increases in the severity of clinical signs of organophosphate toxicosis. Dosage-related inhibition of erythrocyte ChE activity was also detected. A 2nd treatment of some of the horses at the recommended dosage 35 days after the initial treatment resulted in minimal or no side effects, despite the fact that erythrocyte ChE activity was still depressed before the repeat treatment. Changes indicating organ toxicosis were not detected in any of the clinical pathologic determinations in any of the dosage groups.