Rokitamycin loaded microparticles based on chitosan for the therapy of amoebic infections.

OBJECTIVES The aims of this study were: i) to assess the feasibility of using biodegradable chitosan microspheres as carriers for controlled release of rokitamycin (RK), able to improve the antiamoebic activity of the drug;i ii) to investigate the preparative parameters for obtaining microspheres based on chitosan (CH) suitable for ocular or nasal administration of rokitamycin;ii iii) to verify if it was possible to maintain microsphere characteristics suitable for ocular or nasal administration using water soluble polymers such as new chitosan derivatives, diethylaminomethyl-(diethyldimethylene ammonium)n methylchitosans (C7 and C8) or CH salt, chitosan glutamate (CG);ii iv) to evaluate the influence of chitosan properties (CH vs CG) on the in vivo rokitamycin absorption, after nasal administration of spray dried microspheres.iii METHODS Preparation of spray dried microspheres. Microsphere formulations based on CH or C7 or C8 or CG were prepared by spray drying (Mini Büchi B-191, Büchi Laboratoriums-Technik AG, Flawil, Switzerland) using different drug–polymer ratios (1–3 and 1–4) and starting from feed solutions with diverse concentrations (1.5%, 1%, 0.5% and 0.25% (w/v)). Characterisation of Microspheres. Microparticles prepared were characterised in terms of yield of production, drug loading and encapsulation efficiency, particle size, morphology, in vitro drug release, water uptake, in vitro mucoadhesion, ex vivo drug permeation, Energy Dispersive X-ray Diffraction (EDXD) measurements. Determination of effects of drug and microspheres on Acanthamoeba growth. Loaded and unloaded CH microspheres were dispersed in PYG medium and applied on trophozoite grown in sterile 24-well plates (Corning). At selected time intervals (3, 4, 7, 9 and 15 days of incubation), amoebas growing in each well were counted in a Nageotte chamber, using the inverted microscope. In vivo RK administration. Nasal administration of CG and CH microparticles containing RK to each nostril of the anaesthetized Male Wistar rats was performed by use of single dose Monopowder P® insufflators (Valois Dispray). At predetermined times, blood and liquor samples were collected and analyzed. Results were compared with those obtained after intravenous infusion of RK and nasal administration of RK aqueous suspension. CONCLUSIONS The loading of RK into CH microspheres improves and prolongs the in vitro antiamoebic activity of the drug.i CH based microspheres with good morphology and narrow size distribution, able to increase the dissolution rate of RK, can be obtained by spray drying using a low concentration of feed solution and 1-4 drug–polymer ratio.ii RK encapsulation into CH microspheres increases its poor ex vivo permeability through nasal sheep mucosa. The encapsulation of RK in C7 and C8 by using the chosen parameters results in microparticles showing similar or often better properties than formulations made by CH with respect to size, in vitro release behaviour and mucoadhesiveness.ii Compared with CH particles, CG microspheres load the drug in amorphous form, leading to the best improvement of its water solubility. This property, as well as the rapid water uptake of CG particles, increases more the release rate of the drug from microspheres and only after their in vivo nasal administration, RK goes to the cerebrospinal fluid and the bloodstream.iii ACKNOWLEDGEMENTS This work was supported by MiUR through grant PRIN05 and Fondazione Banco di Sardegna. The authors thank “Farmaceutici Formenti S.p.A” and Valois Pharma for the donation of RK and the powder insufflators, respectively. REFERENCES i Rassu, G., Gavini, E., Mattana, A., Giunchedi, P. Open Drug Del J, 2, 38-43, 2008. ii Rassu, G., Gavini, E., Jonassen, H., Zambito, Y., Fogli, S., Breschi, MC, Giunchedi, P. J. Pharm. Sci., 98, 4852-4865, 2009. iii Gavini, E., Rassu, G., Ferraro, L., Generosi, A., Rau, JV., Brunetti, A., Giunchedi, P., Dalpiaz, A. J. Pharm. Sci., In Press.