Quantitative chromatographic determination of several benzimidazole anthelmintic molecules in parasite material

A specific, precise and accurate high-performance liquid chromatographic (HPLC) analytical method has been developed for the quantitative determination of different benzimidazole (BZD) anthelmintics in parasite material (Moniezia benedeni). Mebendazole (MBZ), oxibendazole (OBZ), flubendazole (FLBZ), albendazole (ABZ) ricobendazole (RBZ), albendazole sulphone (ABZSO(2)), fenbendazole (FBZ), oxfendazole (OFZ) and fenbendazole sulphone (FBZSO(2)) were measured simultaneously in M. benedeni, a sheep and cattle cestode parasite used as a model of the biological matrix. The recovery, linearity, precision, accuracy, limits of detection and quantification of the method were determined. Drug extraction from the parasite's tissue homogenate was performed using methanol (liquid phase extraction), and after solvent evaporation, the residual material was cleaned up by solid phase extraction prior to analysis by reversed-phase HPLC. The resolution of all the BZD molecules assayed was obtained on a C(18) reversed-phase (5 microm, 250 mm x 4.6 mm) column using acetonitrile and ammonium acetate as the mobile phase and ultraviolet (UV) detection at 292 nm. Regression analyses for all the BZD compounds assayed were linear at concentrations ranging from 1.61 to 64.21 nmol/100mg protein (triplicate determinations) showing correlation coefficients greater than 0.9922. The developed method is efficient for the simultaneous determination of several benzimidazole anthelmintic molecules in parasite material and useful for the ex vivo and in vivo characterisation of the kinetics of drug uptake/diffusion in target parasites, which seems to be relevant to optimise parasite control both in human and veterinary medicine.     

Dose-dependent activity of albendazole against benzimidazole-resistant nematodes in sheep: relationship between pharmacokinetics and efficacy

The relationship between the pharmacokinetic behaviour and the anthelmintic efficacy of albendazole (ABZ) against benzimidazole (BZD)-resistant nematodes was studied in sheep. A micronized ABZ suspension was orally administered at two different dose levels to sheep naturally infected with BZD-resistant gastrointestinal (GI) nematodes. The experimental animals were allocated into the following groups (n = 8): (a) untreated control; (b) orally treated with ABZ at 3.8 mg/kg b.w.; and (c) orally treated with ABZ at 7.5 mg/kg b.w. Plasma samples were obtained serially over 72 h post-treatment from both treated groups and analysed by HPLC to measure the concentrations of ABZ and its sulphoxide (ABZSO) and sulphone (ABZSO(2)) metabolites. Faecal egg counts were performed prior to treatment and at the necropsy day. All experimental animals were sacrificed 10 days after treatment to perform GI worm counts. While ABZ parent drug was not recovered in the bloodstream, ABZSO and ABZSO(2) were the molecules found in plasma. ABZSO was the metabolite measured at the highest concentrations in the bloodstream for up to 36 (treatment at 3.8 mg/kg) or 60 h (treatment at 7.5 mg/kg) post-administration. There was a proportional relationship between the administered ABZ dose and the measured plasma concentrations of both ABZ metabolites. Over a 100% increment on the plasma AUC values for the anthelmintically active ABZSO metabolite was observed at the 7.5 mg/kg compared to the 3.8 mg/kg treatment. The low efficacy patterns (< 24%) observed against the GI nematodes investigated indicate a high level of resistance to ABZ given at 3.8 mg/kg an efficacious therapeutic dose rate recommended in some countries. However, the higher and prolonged plasma drug concentration measured after the 7.5 mg/kg treatment resulted in an improved efficacy pattern (estimated by both faecal egg and adult worm counts) against most of the GI nematodes studied compared to that obtained at the lower dose rate. A direct relationship between drug pharmacokinetic behaviour and anthelmintic efficacy against BZD-resistant nematodes in sheep was shown in the current work, although individual variation precluded the observation of statistically significant differences in worm counts.     

Influence of hydrophilic polymers on celecoxib complexation with hydroxypropyl beta-cyclodextrin

Complexation of celecoxib with hydroxypropyl beta-cyclodextrin (HPbetaCD) in the presence and absence of 3 hydrophilic polymers-polyvinyl pyrrolidone (PVP), hydroxypropyl methylcellulose (HPMC), and polyethylene glycol (PEG)-was investigated with an objective of evaluating the effect of hydrophilic polymers on the complexation and solubilizing efficiencies of HPbetaCD and on the dissolution rate of celecoxib from the HPbetaCD complexes. The phase solubility studies indicated the formation of celecoxib-HPbetaCD inclusion complexes at a 1:1M ratio in solution in both the presence and the absence of hydrophilic polymers. The complexes formed were quite stable. Addition of hydrophilic polymers markedly enhanced the complexation and solubilizing efficiencies of HPbetaCD. Solid inclusion complexes of celecoxib-HPbetaCD were prepared in 1:1 and 1:2 ratios by the kneading method, with and without the addition of hydrophilic polymers. The solubility and dissolution rate of celecoxib were significantly improved by complexation with HPbetaCD. The celecoxib-HPbetaCD (1:2) inclusion complex yielded a 36.57-fold increase in the dissolution rate of celecoxib. The addition of hydrophilic polymers also markedly enhanced the dissolution rate of celecoxib from HPbetaCD complexes: a 72.60-, 61.25-, and 39.15-fold increase was observed with PVP, HPMC, and PEG, respectively. Differential scanning calorimetry and X-ray diffractometry indicated stronger drug amorphization and entrapment in HPbetaCD because of the combined action of HPbetaCD and the hydrophilic polymers.     

Transtegumental diffusion of benzimidazole anthelmintics into Moniezia benedeni: correlation with their octanol-water partition coefficients

The experiments described here report on the correlation between the ex vivo diffusion of different benzimidazole (BZD) anthelmintics into the cestode parasite Moniezia benedeni, and their octanol-water partition coefficients (P.C.). The characterisation of the drug diffusion process into target parasites is relevant to understand the mechanism of drug penetration and the pharmacological activity of anthelmintic drugs. Specimens of the tapeworm M. benedeni, used as a helminth parasite model, were obtained from untreated cattle killed at the local abattoir. The collected parasites were incubated (5-210 min) with either fenbendazole (FBZ), albendazole (ABZ), ricobendazole (RBZ), oxfendazole (OFZ), mebendazole (MBZ), oxibendazole (OBZ), or thiabendazole (TBZ), in a Kreb's Ringer Tris buffer medium at a final concentration of 5 nmol/ml. After the incubation time elapsed, samples of parasite material were chemically extracted and prepared for high performance liquid chromatography (HPLC) analysis to measure drug/metabolite concentrations. Additionally, the octanol-water P.C. for each molecule was estimated as an indicator of drug lipophilicity, using reversed phase HPLC analysis. All the incubated drugs were recovered from the tapeworms as early as 5 min post incubation. There was a high correlation (r=0.87) between drug lipophilicity, expressed as octanol-water P.C. (Log P), and drug availability within the parasite. The most lipophilic BZD compounds (FBZ, ABZ, and MBZ), with P.C. values higher than 3.7, were measured at significative higher concentrations within the tapeworm compared to those drugs with the lowest P.C. values. Considering the results from the current and previous studies, it is clear that passive diffusion is a major mechanism of BZD penetration into cestode parasites, where lipid solubility is a determinant factor influencing the diffusion of these anthelmintic molecules through the parasite tegument.     

Inclusional complex study between 6-p-toluidinylnaphthalene-2-sulfonate and 2-hydroxypropyl-beta-cyclodextrin

6-p-Toluidinylnaphthalene-2-sulfonate (TNS) is used as a fluorescent probe for exploring hydrophobic regions of several biological substances, such as proteins, and studying solution state folding behaviour. The current study examines the complexation of TNS with 2-hydroxypropyl-beta-cyclodextrin (HPbetaCD) in aqueous solution, mainly by ultraviolet spectrophotometry using various concentrations of HPbetaCD. The structure of HPbetaCD was confirmed by using positive-ion electrospray ionization (ESI+) mass spectrometry. The complex was examined for its stoichiometry applying the continuous variation (Job plot) method. Also, the kinetics of the complex formation was monitored and the determination of the stability constant was calculated. For this purpose, the spectrophotometric properties of TNS were observed in the presence of increasing concentrations of HPbetaCD applying the transformed Benesi-Hildebrand linear model as well as a nonlinear one. The results suggest that TNS forms a stable complex of 1:1 molar ratio, at least at the examined concentrations. Furthermore, from the measurements of 1H nuclear magnetic resonance (1H NMR) spectra, interactions between protons of TNS with HPbetaCD were determined.     

Exploring the Potential of Flubendazole in Filariasis Control: Evaluation of the Systemic Exposure for Different Pharmaceutical Preparations

The goal of elimination of the human filariases would benefit greatly from the use of a macrofilaricidal agent. In vivo trials in humans and many experimental animal models suggest that flubendazole (FLBZ) is a highly efficacious macrofilaricide. However, since serious injection site reactions were reported in humans after parenteral FLBZ administration, the search for alternative pharmaceutical strategies to improve the systemic availability of FLBZ and its metabolites has acquired urgency in both human and veterinary medicine. The goal of the current work was to compare the systemic exposure of FLBZ formulated as either an aqueous hydroxypropyl-β-cyclodextrin (CD) or aqueous carboxymethyl cellulose (CMC) suspension or a Tween 80-based formulation (TWEEN) in rats and jirds (Meriones unguiculatus). Healthy animals of both species were allocated into four experimental groups of 44 animals each: FLBZ-CD_oral and FLBZ-CD_sc, treated with the FLBZ-CD formulation by the oral or subcutaneous routes, respectively; FLBZ-TWEEN_sc, dosed subcutaneously with the FLBZ-TWEEN formulation; and FLBZ-CMC_oral, treated orally with the FLBZ suspension. The FLBZ dose was 5 mg/kg. FLBZ and its hydrolyzed (H-FLBZ) and reduced (R-FLBZ) metabolites were recovered in plasma samples collected from rats and jirds treated with the different FLBZ formulations. In both species, FLBZ parent drug was the main analyte recovered in the bloodstream. In rats, FLBZ systemic exposure (AUC_0-LOQ) was significantly (P<0.05) higher after the FLBZ-CD treatments, both oral (4.8±0.9 µg.h/mL) and subcutaneous (7.3±0.6 µg.h/mL), compared to that observed after oral administration of FLBZ-CMC suspension (0.93±0.2 µg.h/mL). The same differences were observed in jirds. In both species, parenteral administration of FLBZ-TWEEN did not improve the systemic availability of FLBZ compared to FLBZ-CD_oral treatment. In conclusion, formulation approaches that enhance the availability of flubendazole in the rat and jird may have therapeutic implications for a drug with poor or erratic bioavailability.     

A comparative study of different albendazole and mebendazole regimens for the treatment of intestinal infections in school children of Usigu Division, western Kenya

A clinical trial to compare the effectiveness of 4- and 6-mo repeated treatment with albendazole 600 mg (Zentel, SmithKline Beecham) or mebendazole 600 mg (Vermox, Janssen) on geohelminth infections was carried out on children in 6 primary schools; the study included 1,186 children, ages 4 to 19 yr. Kato-Katz examination was performed on stool samples before and after treatment. Overall, albendazole produced better cure rates and egg reduction rates for geohelminths. The cure rates for albendazole were 92.4% for hookworm infection, 83.5% for Ascaris lumbricoides, and 67.8% for Trichuris trichiura. Mebendazole given either 2 or 3 times in a year had cure rates of 50 and 55.0% (respectively) for hookworm, 79.6 and 97.5% for A. lumbricoides, and 60.6 and 68.3% for T. trichiura infection. The geometric mean intensity of hookworm eggs per gram (epg) of stool decreased by 96.7% after albendazole treatment compared with 66.3 and 85.1%, respectively, for 2 or 3 doses of mebendazole (P < 0.05) over the same period. Reductions in epg for A. lumbricoides and T. trichiura were comparable for both drugs. Our results indicate that treatment with albendazole at a 6-mo interval was more effective than mebendazole regimens and may be the best choice for use in the control of the 3 geohelminths.     

Relating the structure,activity, and physical properties of ultrashort-acting benzodiazepine receptor agonists

The ultrashort-acting benzodiazepine (USA BZD) agonists reported previously have been structurally modified to improve aqueous solubility. Lactam-to-amidine modifications, replacement of the C5-haloaryl ring, and annulation of heterocycles are presented. These analogues retain BZD receptor potency and full agonism profiles.     

Effect of two formulations of benzimidazole carbamates on the viability of cysts of Echinococcus granulosus in vivo

Two different preparations, solution and suspension, of three benzimidazole carbamate drugs, mebendazole, albendazole and ricobendazole, were compared by analyzing their in vivo activity against Echinococcus granulosus cysts in a mouse model. Polyvinylpyrrolidone was used for the elaboration of drug solutions and these formulations manifested better results in terms of reduction of number of viable hydatid cysts in mice than the reference drug suspensions. The effect was more prominent on mebendazole-treated mice, at doses of 25-50 mg/kg. There was a correlation between ED50 and pharmacokinetical parameters of AUC0-infinity and Cmax, showing that a significant improvement on solubility affects the in vivo activity of these drugs.     

Low efficacy of single-dose albendazole and mebendazole against hookworm and effect on concomitant helminth infection in Lao PDR

Background

Albendazole and mebendazole are increasingly deployed for preventive chemotherapy targeting soil-transmitted helminth (STH) infections. We assessed the efficacy of single oral doses of albendazole (400 mg) and mebendazole (500 mg) for the treatment of hookworm infection in school-aged children in Lao PDR. Since Opisthorchis viverrini is co-endemic in our study setting, the effect of the two drugs could also be determined against this liver fluke.

Methodology

We conducted a randomized, open-label, two-arm trial. In total, 200 children infected with hookworm (determined by quadruplicate Kato-Katz thick smears derived from two stool samples) were randomly assigned to albendazole (n = 100) and mebendazole (n = 100). Cure rate (CR; percentage of children who became egg-negative after treatment), and egg reduction rate (ERR; reduction in the geometric mean fecal egg count at treatment follow-up compared to baseline) at 21–23 days posttreatment were used as primary outcome measures. Adverse events were monitored 3 hours post treatment.

Principal Findings

Single-dose albendazole and mebendazole resulted in CRs of 36.0% and 17.6% (odds ratio: 0.4; 95% confidence interval: 0.2–0.8; P = 0.01), and ERRs of 86.7% and 76.3%, respectively. In children co-infected with O. viverrini, albendazole and mebendazole showed low CRs (33.3% and 24.2%, respectively) and moderate ERRs (82.1% and 78.2%, respectively).

Conclusions/Significance

Both albendazole and mebendazole showed disappointing CRs against hookworm, but albendazole cured infection and reduced intensity of infection with a higher efficacy than mebendazole. Single-dose administrations showed an effect against O. viverrini, and hence it will be interesting to monitor potential ancillary benefits of a preventive chemotherapy strategy that targets STHs in areas where opisthorchiasis is co-endemic.

Clinical Trial Registration

Current Controlled Trials ISRCTN29126001     

Effect of a single dose (600 mg) of albendazole on Loa loa microfilaraemia

The problem of Loa-encephalopathy, which may occur after ivermectin treatment of patients harbouring high Loa microfilarial loads, might be solved if one could find a treatment regimen bringing about a significant but progressive decrease in the Loa microfilaraemia. A trial was performed in Central Cameroon, whose aim was to follow up for 10 months, and to compare the changes in the Loa microfilarial loads in two groups of patients, one treated with a single dose (600 mg) of albendazole (Alben, SmithKline Beecham) given with fatty food, and the other treated with mebendazole (100 mg, twice a day, generic tablets) at a fasting state. The microfilarial loads remained stable in the mebendazole group, whereas a significant decrease in microfilaraemia was recorded in the albendazole group (initial median load: 230 microfilariae per 50 microliters; median load ten months after: 84 microfilariae per 50 microliters). This should encourage further trials to evaluate the effects and the safety of two- or three-day albendazole regimens in patients infected with Loa loa.     

Mebendazole induces apoptosis via Bcl-2 inactivation in chemoresistant melanoma cells

Most metastatic melanoma patients fail to respond to available therapy, underscoring the need for novel approaches to identify new effective treatments. In this study, we screened 2,000 compounds from the Spectrum Library at a concentration of 1 micromol/L using two chemoresistant melanoma cell lines (M-14 and SK-Mel-19) and a spontaneously immortalized, nontumorigenic melanocyte cell line (melan-a). We identified 10 compounds that inhibited the growth of the melanoma cells yet were largely nontoxic to melanocytes. Strikingly, 4 of the 10 compounds (mebendazole, albendazole, fenbendazole, and oxybendazole) are benzimidazoles, a class of structurally related, tubulin-disrupting drugs. Mebendazole was prioritized to further characterize its mechanism of melanoma growth inhibition based on its favorable pharmacokinetic profile. Our data reveal that mebendazole inhibits melanoma growth with an average IC(50) of 0.32 micromol/L and preferentially induces apoptosis in melanoma cells compared with melanocytes. The intrinsic apoptotic response is mediated through phosphorylation of Bcl-2, which occurs rapidly after treatment with mebendazole in melanoma cells but not in melanocytes. Phosphorylation of Bcl-2 in melanoma cells prevents its interaction with proapoptotic Bax, thereby promoting apoptosis. We further show that mebendazole-resistant melanocytes can be sensitized through reduction of Bcl-2 protein levels, showing the essential role of Bcl-2 in the cellular response to mebendazole-mediated tubulin disruption. Our results suggest that this screening approach is useful for identifying agents that show promise in the treatment of even chemoresistant melanoma and identifies mebendazole as a potent, melanoma-specific cytotoxic agent.     

Effects of three benzimidazoles on growth, general morphology and ultrastructure of Tritrichomonas foetus

Tritrichomonas foetus is a venereal pathogen of cattle, which causes infertility, early embryonic death or abortion. In order to evaluate the potential trichomonicidal activity of benzimidazoles, the effects of thiabendazole, mebendazole and albendazole were analyzed on the multiplication, general morphology and ultrastructure of T. foetus. It was found that mebendazole presented the highest IC(50%) (2.3 microM), when compared with albendazole (IC(50%)=9.4 microM) and thiabendazole (IC(50%)=142.6 microM), and that such effects were irreversible. Concerning microscopic analysis, thiabendazole- and mebendazole-treated cells presented increased volume, internalization of the flagella, disruption or multiplication of the nucleus, multiple organelles and cytoplasmic vacuolization. Albendazole-treated cells exhibited slight alterations, because the parasite became slightly rounded, its flagella were not internalized but the cytoplasm was vacuolated. Mebendazole was indeed highly effective as an in vitro trichomonicidal agent, and this might open up new possibilities for the use of mebendazole in the therapy of bovine trichomoniasis.     

Synergistic effect of PEG-400 and cyclodextrin to enhance solubility of progesterone

Conclusion  PEG-400, polysorbate 80, and 2 CDs (Trappsol HPB and Captisol) were used in an attempt to improve the aqueous solubility of a model hydrophobic drug, progesterone. The aqueous solubility of progesterone improved significantly from 0.007 mg/mL by the addition of PEG-400, CDs, and polysorbate 80. In systems containing various amounts of PEG-400 and 3% Trappsol HPB in water (% wt/wt), the theoretical solubility was calculated by adding the solubilities in the individual systems. The observed solubility values were up to 96% higher than the theoretical values. The effect of synergism was significant in 5% to 50% PEG-400/water systems containing Trappsol HPB. Systems containing Captisol did not show such synergistic effects. In general, the addition of polysorbate 80 to the PEG-400/water systems containing CDs affected synergism negatively.     

Synthesis and hydrolytic stability studies of albendazole carrier prodrugs

Three N-acyl (2, 3, and 4), two N-alkoxycarbonyl (5 and 6), and one N-acyloxymethyl (7) derivatives of albendazole (1) have been prepared and assessed as potential prodrugs. The determination of the aqueous solubility and partition coefficient, as well as the conversion of these derivatives to 1 in buffer solution, human plasma, and pig liver esterase were determined.     

Diversity in the supramolecular interactions of 5,6-dichloro-2-(trifluoromethyl)-1H-benzimidazole with modified cyclodextrins: Implications for physicochemical properties and antiparasitic activity

The molecular interactions of 5,6-dichloro-2-(trifluoromethyl)-1H-benzimidazole (G2), an antiprotozoa with poor aqueous solubility, with 2-hydroxypropyl-α-cyclodextrin (HPαCD), methyl-β-cyclodextrin (MβCD) and 2-hydroxypropyl-β-cyclodextrin (HPβCD) were examined. The aqueous solubility enhancement by cyclodextrins (CDs) was evidenced in phase-solubility diagrams, and the stoichiometry of G2/CD systems was determined by Job's plots. Two-dimensional NMR spectroscopic data revealed that a different mode of interaction took place between G2 and CDs in solution. With HPαCD, a non-inclusion complex was generated. In the case of MβCD, a typical host-guest system was obtained and with HPβCD a partial inclusion complex through the narrow side of the macrocycle was formed. ESI-mass spectrometric data confirmed the stoichiometry and mode of interaction of these systems in solution. Solid-state characterization (scanning calorimetry and powder X-ray diffraction) supported the inclusion complex formation. The leishmanicidal activity, trypanocidal activity and non-toxic profile of G2/MβCD showed the advantages of using this inclusion complex to promote the biological assays extension of G2.     

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.     

Aggregation of cyclodextrins as an important factor to determine their complexation behavior

Here, we report a study on the complexation behavior of carotenoids with cyclodextrins (CDs) using solubility experiments and molecular-modelling methods. Carotenoids are an important group of naturally occurring dyes found in vegetables and fruits. Their antioxidant property has initiated investigations on their possible use as drugs. However, carotenoids are lipophilic molecules with very little inherent aqueous solubility. Cyclodextrin complexation has been widely used in order to increase the potential applications of hydrophobic compounds. Thus, the aim of our investigation was to design carotenoids with enhanced water solubility by cyclodextrin complexation. Molecular modelling of carotenoid-cyclodextrin complexes with a 1 : 1 stoichiometry successfully explained the experimentally observed capability of beta-cyclodextrins (beta-CDs) to form complexes with carotenoids as opposed to alpha-cyclodextrins (alpha-CDs) and gamma-cyclodextrins (gamma-CDs). Furthermore, molecular-dynamics calculations revealed that the aggregation properties of CD derivatives significantly influence their complexation behavior. Our docking calculations showed that RAMEB (random methylated beta-CD) is the beta-CD derivative that possesses the lowest tendency to aggregate. Solubility experiments yielded the same results, namely, RAMEB complexes possess the best water solubility. Our results showed that complexation of a ligand not buried inside of the CD cavity is dependent on two factors: i) the geometry of the inclusion part of the complex; ii) the self-aggregation property of the CD itself. The lower affinity the CDs possess for self-aggregation, the more likely are they involved in interactions with carotenoids. These results suggest that self-aggregation of CDs should be considered as an important parameter determining complexation in general.     

Comparative study on gastric absorption of albendazole and mebendazole in rats.

1. A study was carried out to determine the kinetics of the gastric absorption of two wide spectrum benzimidazole anthelmintics, albendazole and mebendazole. 2. The method used was gastric recirculation of solutions containing the drugs in concentrations ranging from 0.5 to 100 mM. 3. The results obtained showed that absorption corresponds to first order kinetics, with diffusion constants of 0.0087 min-1 for albendazole and 0.0077 min-1 for mebendazole. 4. Blood levels of the drugs for the whole range of concentrations were always higher in the case of albendazole.