An Update on the Biologic Effects of Fenbendazole

Abstract

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.

An accurate estimate of the prevalence of Aspiculuris tetraptera and Syphacia spp. infection in rats and mice is not available for recent years, but these agents likely remain problematic for many animal facilities despite effective treatment regimens using fenbendazole-medicated diets.^6,7,9,39,52 Although many facilities may still depend on microscopic examination of fecal floats and tape tests, the advent of PCR testing has enhanced detection despite reports of false-negative and false-positive results.^8,23,32 Infestations may still occur due to poor decontamination procedures, nonvigorous use of diagnostics procedures, animal movement within facilities by investigators, and sharing of animals between facilities.⁷ These events can result in lengthy and expensive treatment regimens. including the use of fenbendazole and mechanical disinfection, that may fail due to inadequate ovicidal effects. In addition, fenbendazole is often an integral part of quarantine protocols when mice are shared between facilities and institutions.

Fenbendazole is widely used in many species for the eradication of various nematode infections.⁵² Although therapeutic levels of fenbendazole generally are tolerated without grossly evident toxic or physiologic effects, the use of medicated feed in facilities with diverse research programs can worry researchers. Over the past 40 y, studies published on the specific effects of fenbendazole both support and refute these concerns. Serendipitous findings from varied research models during fenbendazole therapeutic regimens reinforce a basis for this apprehension.

In 2007, a comprehensive overview of the biologic effects of fenbendazole on mice and rats was published.⁵² At the time, conflicting reports were summarized regarding the drug’s influence on the immune system, although no significant effects had been reported on reproduction or behavior. Since that time, the literature has continued to reflect a discordance in overall effects of therapeutic treatment using fenbendazole. A brief summary of the more recent literature is presented here. We recommend that readers refer to the original review paper and to specific publications that may best address any issues of interest.

Bone Marrow

Myelosuppression is considered to be a side effect of fenbendazole treatment in many species, including birds, reptiles, porcupines, and many other mammals.⁵² Albendazole and fenbendazole are commonly used to treat Encephalitzoon cuniculi infection in rabbits. Toxicosis, including lethargy, hemorrhage, and death were reported in 13 cases involving pet rabbits, especially in association with high doses or prolonged administration.²⁴ A CBC count conducted early after the start of treatment has been recommended as being helpful in the detection of changes consistent with toxicosis, including anemia, leukopenia, and thrombocytopenia.²⁴

In rodents, only a few reports have studied the effects of fenbendazole on bone marrow. In an in vitro study,³⁶ LPS was used to induce inflammation in mouse bone marrow cells. Concurrent application of fenbendazole led to apoptosis of activated cells, with greater effects on granulocytes than on B cells.³⁶ In an extensive in vivo study, an alternating-week fenbendazole therapeutic regimen adversely affected precursor B cells in young and old BALB/c mice.³¹ Specifically, levels of E47, a factor necessary for the generation of antibody diversity, including immunoglobulin rearrangement and class switching, were reduced. Protein levels normalized in old mice within 3 wk after the cessation of fenbendazole treatment. However, E47 levels remained low in young mice through the end of the study, which ended 4 wk after treatment.

Immune System

Considerable interest remains in the effects of fenbendazole on the immune system. Results in the older literature are mixed on this subject in rodents and other mammals; this disparity continues in the few newer studies available.⁵²

Peripheral blood lymphocytes from squirrel monkeys treated with fenbendazole for Giardia infection were altered as compared with those from Giardia-negative untreated control monkeys.³⁴ The alterations included changes in numbers of circulating B and NK cells and significant decreases in circulating cytokines after fenbendazole treatment. In addition, several hematologic changes, including leukopenia and erythropoiesis, were present initially but decreased over a 6-wk period after treatment.

The effects of on–off and continuous therapeutic regimens were examined in an extensive study of several immune parameters in BALB/c mice.¹⁰ Broad immune measures including flow cytometric evaluation of splenic T and B cells, spleen cell proliferation to mitogens, primary and secondary humoral immune responses, and skin graft rejection revealed no differences between treated and untreated mice at several time points during each treatment regimen. Moreover, no differences in colony-forming units for granulocytes, erythrocytes, and pre-B cells emerged, thus contrasting with a concurrent study.³¹ However, in the latter study, some very specific measures of stimulated and purified precursor B cells were altered after 4 wk of continuous treatment.

A few studies have focused on defining the effects of fenbendazole in models of immune-medicated disease. The nonobese diabetic (NOD) mouse model previously reported to be unaffected by the use of fenbendazole diet for a period of 23 wk.¹⁹ The NOD model is based on autoreactive T_h1 cells that recognize islet-specific antigens. However, the cited study¹⁹ was based on a single experiment involving a small number of mice. In contrast, in a more extensive study, continuous and on–off fenbendazole regimens had no effect on the development of a B cell–driven model of systemic lupus erythematosus in (NZB × NZW)F1 mice.¹¹ Like the NOD model, (NZB × NZW)F1 mice are a model of naturally occurring disease based on the recognition of self-antigens.

Two well-described mouse models of experimental autoimmune encephalitis were examined during the use of a 13-wk therapeutic regimen to eradicate pinworms.⁴¹ These models involve the injection of a peptide to induce T_h1-driven disease. As quantified using a clinical scoring system, fenbendazole reduced the incidence of disease, clinical onset, duration, and severity in 75% of the mice as compared with untreated control animals. However, the pattern and incidence of disease induction returned to normal 3 wk after the cessation of fenbendazole treatment.

Investigators using a mouse model of spinal cord injury studied possible effects of colony-wide treatment with fenbendazole due to pinworm infection.⁵⁷ After 4 wk of continuous use of a fenbendazole-containing diet, a thoracic level spinal injury was induced in a few C57BL/6 mice. Fenbendazole exposure significantly improved locomotor scores until termination of the experiment at day 42 after injury. In addition, immunohistochemistry revealed lower levels of IgG at the injury site. The investigators proposed that fenbendazole affected the activation of B cells and thus the production of autoantibody, which is integral to the pathogenesis of this model. In a related and more controlled study, the same research group examined the effects of flubendazole in a rat model of spinal cord contusion injury.⁵⁶ Flubendazole was administered intraperitoneally on a daily basis for a period of 3 wk beginning 3 h after injury. As compared with a control group, flubendazole-treated rats showed significantly more locomotor function by day 7 after injury. Histology revealed a smaller lesion size and more normal tissue in the spinal cord lesion in the treatment group as compared with untreated controls; spinal cord injury biomarkers were also significantly affected. These changes were associated with less astrogliosis; reactive and proliferating astrocytes promote enhanced inflammation and tissue damage. As with the previous fenbendazole study, B cell activation and proliferation were also diminished.

In a model of allergic airway disease induced by ovalbumin exposure, an unreported number of BALB/c mice were treated in utero and before and after weaning with a therapeutic dose of fenbendazole.⁵ Although neither leukocytes (percent composition in blood) nor goblet cells in lung sections were affected, the number of eosinophils in the bronchoalveolar lavage fluid was lower with treatment. Levels of ovalbumin-specific IgG1 antibody were also lower. In follow up in vitro studies, fenbendazole decreased T_h2-derived cytokines and inhibited the activation of CD4⁺ T cells. These observations were present until the termination of the study at 4 wk after the return to normal diet.

Effects on Tumors

A serendipitous finding supported potential antitumorigenic effects of fenbendazole. During a therapeutic fenbendazole regimen in SCID mice, investigators observed that a lymphoma xenograft model failed to grow.²¹ A well-defined follow-up study used standard diet, standard diet with vitamins, fenbendazole-containing diet, and fenbendazole diet supplemented with a 1.25- to 25-fold greater vitamin content. The diet change began 2 wk before tumor cell implantation; significant inhibition of tumor growth occurred only in the mice fed fenbendazole with vitamin supplements. This group had significantly lower total WBC and neutrophil counts than did the other groups. A pathway for synergistic effects of fenbendazole and vitamins is unknown, but the investigators hypothesized that the vitamin antioxidants enhance the activity of fenbendazole and influence factors that have a role in tumorigenesis.

A second report described a prospective study to address the possible effects of fenbendazole on a tumor model, which the investigators initiated when a therapeutic regimen of fenbendazole was scheduled in their animal facility. BALB/cRw mice were placed on fenbendazole-containing diet or unmedicated feed for 1 wk before the implantation of mammary tumor cells.¹⁶ The therapeutic diet did not affect tumor growth, invasion, or metastasis. In addition, as part of the experimental protocol, local irradiation was applied to the tumor; fenbendazole still did not alter the growth of the tumor. In a second study, no changes were observed in mice that received fenbendazole intraperitoneally.¹⁷ However, additional studies conducted in vitro found that fenbendazole concentrations of 1 µM and greater inhibited the growth of tumor cells. Follow-up studies found that fenbendazole had both cytotoxic and cytostatic effects.¹⁷ However, this concentration is 10 times that found in the tissues of rodents fed fenbendazole-containing diet.⁵²

In a third report, an unreported number of nude mice with tumors of a human nonsmall cell lung carcinoma cell line received 1 mg fenbendazole orally every other day for 12 d.¹⁵ Fenbendazole significantly decreased tumor size and vascularity. Concurrent in vitro studies showed that fenbendazole targeted the microtubules of the tumor cells, resulting in mitotic arrest, and also interfered with glucose metabolism.

Although these 3 studies^15,17,52 suggest that the effects of therapeutic doses of fenbendazole mat have limited effects on tumorigenesis, benzimidazoles have garnered continued interest in cancer research.^15-17,46 Flubendazole and mebendazole induced cell death in a myeloma cell line in vitro.⁴⁶ In addition, 1 to 3 µM flubendazole had similar effects on several leukemia cell lines, other myeloma cell lines, and primary culture from patients with acute myeloid leukemia. SCID mice treated intraperitonally with flubendazole and implanted with myeloma cells showed less tumor growth than did untreated mice; this inhibition was significantly greater when flubendazole was used in tandem with vinblastine and vincristine.

Mebendazole has been the focus of several preclinical studies.^1,44,53,54 In an orthotopic model of thyroid cancer in nude mice, mebendazole-treated mice had small nonpalpable tumors, with no local invasion and no distant metastasis.⁵⁴ This finding was consistent among several aggressive cell lines. In a study of colon cancer, daily treatment with mebendazole reduced both the growth of colon cancer cell line xenografts in nude mice and tumor formation in a transgenic model of polyposis.⁵³ Similar results have been reported in models of glioblastoma multiforme and malignant meningioma.^1,44

Research on the effects of the benzimidazoles has focused on their known affinity for binding tubulin.²⁸ That action, which is responsible for their antiparasitic effects, is similar to that of antimitotic drugs used in cancer research and therapies; the disruption of tubulin aids in the induction of apoptosis.¹⁵ In a study of the effects on a human nonsmall cell lung carcinoma cell line,¹⁵ fenbendazole was observed to distort the microtubules, resulting in mitotic cell death. This distortion occurred concurrently with the induction of elevated levels of p53 and reduced glucose uptake, both of which occur during apoptosis.

These studies have resulted in the consideration of benzimidazoles as a drug of interest in cancer research. Conceptually proposed as a repurposing of drugs from veterinary medicine, several studies have been published in this area.^18,45,49 In clinical trials, albendazole showed some efficacy in the treatment of refractory and metastatic tumors, with actions that include tumor stabilization, regression, and reduction in tumor markers; neutropenia was reported as a side effect.⁴⁵ Fenbendazole has not been a focus of clinical trials to date, mostly due to a lack of safety studies, although the drug receives considerable coverage on the web because it is readily available for cancer patients seeking alternative self-driven therapies.⁵⁵

Reproduction, Teratologic, and Behavioral Studies

The effects of fenbendazole treatment on reproductive performance continue to be a concern given the diverse and transgenic strains that are now available. Previous reports both did and did not support effects on reproduction.⁵² An extensive study examined the use of the 2 common treatment regimens (continuous and every other week) on reproductive parameters of C57BL/6J mice.²⁵ No differences were observed in litter size, survival, or weaning weight.

Some significant behavioral changes were observed in C57BL/6N mice (n = 12) treated with 4 wk of fenbendazole or with 2 wk of fenbendazole followed by 2 wk of regular diet as compared with mice on nonmedicated feed.²⁰ The investigators used a panel of open-field, maze, and rotarod tests to assess activity, anxiety, and motor coordination; these tests are commonly used in neuroscience animal research. Mice in both treatment groups showed significantly reduced balance but no changes in appetite or histopathologic lesions, including in the middle ear. Subtle changes in wheel running and maze tests in fenbendazole-treated Sprague–Dawley rats were previously reported.²

Food intake was examined in adult male Sprague–Dawley rats.⁵⁰ No significant differences were observed in body weight or food intake over a 12-d period between rats fed fenbendazole diet or standard diet. In a second experiment, rats were switched to the alternative diet midway through the protocol. The results were consistent with those previously reported in rats.²⁹ When both foods were offered, the animals avoided the medicated diet.⁵⁰ Although this preference did not influence the study results, the observation should be considered when research studies monitoring food intake are undertaken concomitantly with fenbendazole therapy.

Other Studies of Note

The possible effect of fenbendazole on experimental studies involving other drugs was addressed in a study of acetaminophen administration in C57BL/6J mice.²² Acetaminophen hepatotoxicity has been well characterized in mice. In a small study, mice were maintained on a fenbendazole-containing diet for 7 d prior before the intraperitoneal injection of acetaminophen. Overall, as compared with control mice, the medicated group showed 63% mortality within 24 h (as compared with 0% in control mice). By 12 h, treated mice had more hepatic necrosis as observed by microscopy, greater increases in serum ALT and AST levels; and greater decreases in levels of the antioxidant glutathione than did control mice. In total, these observations reflected a marked increase in toxicity.

The continuous application of fenbendazole diet in C57BL/6 was reported to result in some variation in gut microbiota.³⁰ In a small study, mice were purchased from 2 different suppliers and received fenbendazole-medicated diet for 8 wk; some changes in microbiota α diversity and richness were detected, especially within 4 wk of the start of treatment. However, as compared with control groups given nonmedicated feed, these changes were viewed as smaller than the changes associated with acclimation to a new facility and thus were not attributed to the use of fenbendazole.

In an experimental model, concurrent infection with Syphacia muris promoted the immune response to a trematode (Ecinostoma caproni) infection.⁴⁸ This large study using Wistar rats revealed changes, including elevated local antibody levels and alterations of the ileum, that were proposed to hinder the development of infection from a second challenge with the same agent.

Discussion

Treatment of pinworm infections is necessary to maintain a controlled research environment. Animal age, strain, sex, and genetic mutations affecting the immune system may influence the prevalence and severity of infection.^7,12 Although infection may be subclinical, weight loss, rectal prolapse, digestion, and enteritis have been reported.^7,38 In addition, several studies report effects on the immune system, including the development of lymphoma in athymic mice, the induction of T_h2-mediated autoimmunity, increased antibody production, and alteration of an adjuvant-induced arthritis model.^3,37,43 After an outbreak of pinworms, CBA mice were observed to have increased myelopoiesis and erythropoiesis.⁴ Concurrent experimental Syphacia infection altered a novel rat model of type 2 diabetes, lowering levels of serum IL1 and local IL1α expression in the cecum and delaying the onset of hyperglycemia.⁴⁷ The authors proposed that pinworm infection may regulate macrophage activity, as suggested by genomic studies of Syphacia.³⁵ These types of observations are consistent with the proposition that parasites have evolved to modulate the immune system to benefit their survival.³³ This potential for effects on the immune system, together with the overall welfare of research animals, necessitates the treatment of pinworm infections. When faced with disagreements over initiating pinworm treatment in research colonies, laboratory animal veterinarians should educate investigators regarding the possible effects of enzootic infection and outbreaks on study results.

The effects of antiparasitic drug treatments can vary.⁴⁰ Ivermectin, another drug that is effective against pinworms, has been reported to have toxic effects in rodents.⁷ Topical selamectin did not show any toxicities but was therapeutically ineffective in rats and mice.²⁶ Fenbendazole provides excellent efficacy, and commercially-available fenbendazole-medicated diets have made this type of treatment easy to deploy and maintain.⁷ Alleles for resistance to benzimidazoles have been identified and are concerning, given the widespread use of these drugs in veterinary medicine.¹⁴ Fenbendazole resistance has been addressed in rodents in an experimental model of Aspiculuris infection in mice.²⁷ In that study, diets containing fenbendazole, piperazine or both were all effective against a pinworm inoculum obtained from enzootically infected mice.²⁷ Syphacia infection in mice has been used as an experimental model for evaluation of novel anthelmintic drugs.^13,51

Immunomodulatory effects of antihelminth treatments have been the focus of many studies in other mammals and humans.⁴² In total, the diverse studies of fenbendazole effects in rodents and rodent models published during the past 30 y support the ability of fenbendazole to alter the immune system. Significant changes have been identified through very specific assays, such as those addressing precursor B cell function, leading to the report of significant changes in models of autoimmunity.^31,41,57 Some parameters appeared to be affected even after termination of treatment, although these factors were often assessed at the endpoint of experiments rather than at a later time point that truly addressed the permanence of the dysfunction. Although many studies did not use currently recommended treatment regimens, the potential for fenbendazole to affect immunity must be acknowledged and discussed with researchers before commencement of facility-wide treatment of a confirmed pinworm infection. In some extreme cases, a potential solution may be to use only the offspring of treated mice to avoid possible research complications from long-term or permanent effects of treatment.

The serendipitous finding of the effect of a therapeutic fenbendazole regimen on a mouse lymphoma model is notable for its potentiation of further research on benzimidazoles as an anticancer therapeutic.²¹ Although follow-up studies used much higher doses of fenbendazole, they found similar effects on other tumor models, both in vitro and in vivo models with both flubendazole and mebendazole, providing strong preclinical evidence supporting the translation of these findings into ongoing clinical trials.¹⁷

The catalog of fenbendazole effects likely will continue to grow. The continued use of fenbendazole will lead to other unforeseen effects in research rodents. Given that the goal of complete pinworm eradication is likely unattainable, the research veterinary community must acknowledge the possible unintended effects of treatment to best advise their research colleagues. When possible, researchers should be encouraged to undertake controlled studies using therapeutic regimens to further define the effects of fenbendazole use in research animals.