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EFFICACY OF NEW ALBENDAZOLE FORMULATIONS IN TREATING Trichinella spiralis INFECTED MICE García A1, Barrera MG1, Vasconi MD2, Di Masso RJ2, Hinrichsen, LI2, Leonardi D1, Lamas MC 1 1Departamento de Farmacia, Facultad de Ciencias Bioquímicas y Farmacéuticas, Universidad Nacional de Rosario. Suipacha 570, Rosario, CP: S2002LRK. 2Instituto de Genética Experimental, Facultad de Ciencias Médicas, Universidad Nacional de Rosario. Santa Fe 3100, Rosario, CP: S2000KTR Introduction Albendazole (ABZ) is a benzimidazole derivative with a broad spectrum of activity against intestinal and systemic parasitosis. However, ABZ is classified as class II/IV by the Biopharmaceutical Classification System (BCS) because it has a very poor aqueous solubility (1µg/mL) that limits oral absorption.(1) As a consequence, the bioavailability is very low and erratic. Furthermore, this aqueous insolubility reduces the pharmaceutical alternatives for drug formulation and administration, especially for pediatric patients.(2) The aim of this study was to determine whether a microparticle formulation system improved ABZ anthelmintic efficacy in vivo in a murine model of trichinellosis. Materials and methods Design of the microparticle systems. Preparation using the spraying method (MP 1)- ABZ (100 mg) was dissolved at room temperature in acetic acid (25 mL) and water (25 mL). Chitosan (CH) (3.0%, w/v) was dispersed in the acidic solution. The microspheres were formed by spraying the ABZ?CH solution into a precipitation bath containing sodium lauryl sulphate solution (5.0%, w/v), under magnetic stirring. The polymeric microparticles were filtrated, washed with water, centrifuged twice and finally collected in a drying chamber at 40 ºC. Preparation using the spray drying method (MP 2)- ABZ (100 mg) was dissolved at room temperature in 30 mL of glacial acetic acid and distilled water was added to obtain a concentration of 30% v/v. CH (1.0 %, w/v) was dispersed in the acetic acid solution. Carboxymethylcellulose (0.2 %, w/v) and pectin (0.1 %, w/v) solutions were prepared by dissolving in 100 mL of water with stirring for a fixed time (2 h). The spraying procedure was carried out with a Buchi Mini dryer B-290. Preparation using the spray drying method (MP 3)- ABZ (100 mg) was dissolved at room temperature in 30 mL of glacial acetic acid and distilled water was added to obtain a concentration of 30% v/v. CH (1.0 %, w/v) was dispersed in the acetic acid solution. Sodium lauryl sulphate (% w/v), solutions were prepared by dissolving in 100 mL of water with stirring for a fixed time (2 h). The spraying procedure was carried out with a Buchi Mini dryer B-290. Physicochemical parameters such as yield, encapsulation efficacy, size and morphology of the microparticles (observed by scanning electron microscopy) were evaluated. In addition, dissolution profiles following USP conditions were carried out. Physicochemical parameters such as yield, encapsulation efficacy, size and morphology of the microparticles (observed by scanning electron microscopy) were evaluated. In addition, dissolution profiles following USP conditions were carried out. Antiparasitic activity assay for Trichinella spiralis- Susceptible CBi+ mice of the CBi colony from the Animal Facilities of the Instituto de Genética Experimental, Facultad de Ciencias Médicas, Universidad Nacional de Rosario (CBi/IGE stock) were used to evaluate the in vivo effect of ABZ formulations, on Trichinella spiralis (Ts) muscle cyst formation.(3) Adult CBi+ males (90-110 days old) were weighed (mean weight 54 ± 3 g) and orally infected with 2 Ts infective muscle larvae per g of body weight. The larvae used in the infection were recovered by artificial digestion in a solution of 1% pepsin-1% HCl at 37°C, from the muscles of a CBi mouse that had been infected 2-3 months earlier; the infection dose for each animal was prepared by counting individual larvae. After infection, the animals were divided in four groups (n = 4 per group) to test the effect of the different ABZ formulations on Ts infectivity: I) ABZ suspended in distilled water; II) MP1; III) MP2; IV) infection control without treatment. Groups I, II, and III were treated with 50 mg ABZ per kg of body weight on days 5 and 6 post-infection. The therapeutic activity of each ABZ preparation was assessed by the number of muscle encysted parasites (muscle parasitic burden) in the host, on the chronic phase of the infection (32±2 days post-infection). The parasitic burden was determined by counting the parasites encysted in the tongue. Briefly, after euthanizing the mouse, the tongue was excised, weighed and submitted to digestion in 2 mL of digestion fluid (pepsin 1:1000, 0.7 g; HCl, 0.9 g; water, up to 100 mL). The supernatant was removed immediately and 5 mL of 10% v/v formalin were admixed in order to inactivate and preserve intact the structure of the larvae. Finally, all larvae on each sample were counted under a microscope with a 100X magnification. Parasite burden was calculated as total number of larvae per g of muscle tissue (PBr). The reduction percentage of PBr for each treated animals was calculated as follows: Mouse ?A? PBr percentage reduction = PBr control group mean ? mouse ?A? PBr x 100 PBr control group mean The statistical significance of the differences in PBr among groups was assessed by an analysis of variance, and the Bonferroni post-test was used for comparisons between groups.(4) Variables expressed as percentages were studied with the non parametric Kruskall?Wallis test; differences between groups were analyzed with Dunn?s test (2). Differences were considered significant if p < 0.05. Results Antiparasitic activity assay for Trichinella spiralis- Table II shows the effect of the different ABZ formulations on the parasitic burdens of the treated animals. ABZ treatment, in any of its formulations, was associated with a significant decrease in the number of muscle encysted Ts parasites as compared to group IV, the infection control. This result is confirmed by the high percentage reduction seen in PBr in all treated groups. No significant differences in worm load or percentage reduction were observed among the treated groups. Nevertheless, it should be noted that MP 2 had the lowest parasitic burden as well as the lowest variance. The lack of statistical significance may be due to a small sample size. The low variance denotes homogeneous treatment results; this low variability on the therapeutic response of MP 2 might be due to an improvement on the availability of the drug in this formulation, as derived from the dissolution test (table I). Conclusions Oral microparticles systems obtained by two different procedures significantly improved the dissolution, particularly in the MP 2 system, which was associated with a better therapeutic efficacy of ABZ in Ts infected mice. References 1) Kawabata Y, Wada K, Nakatani M, Yamada S, Onoue S. Formulation design for poorly water-soluble drugs based on biopharmaceutics classification system: Basic approaches and practical applications. International Journal of Pharmaceutics. 2011;420(1):1-10. 2) Horton J. Human gastrointestinal helminth infections: are they now neglected diseases? Trends in Parasitology. 2003;19(11):527-31. 3) Hinrichsen L, Di Masso RJ. Empleo de un modelo murino original de Argentina en la caracterización de fenotipos complejos. Journal of Basic & Applied Genetics 2010;21(7):1-12. 4) Sheskin DJ. Handbook of parametric and non-parametric statistical procedures. 5º ed. USA: Chapman & Hall/CRC, Taylor & Francis Group; 2011. Table I. Percentage ABZ dissolved at 60 min of dissolution test (Q60) ABZ 6.1 MP 1 29.8 MP 2 72.6 Table II. Efficacy of different Albendazole formulations on muscle parasitic load in Trichinella spiralis infected CBi+ males PBr* PBr reduction percentage' Infection control 2853± 620.2a ---- ABZ 658 ± 139.5b 75 (67.5 ? 90.3)a MP1 592 ± 154.3b 78 (69.7 ? 92.3)a MP2 406 ± 28.8b 86 (83.3 ? 87.9)a *Mean ± SEM 'Median (range) For each variable, differences between groups not sharing the same superscript are significant at the 0.001 level.