Studies On The Oral Absorption Mechanism Of Nimodipine Nanocrystals

Nimodipine(NMD)exhibited favorable permeability but poorly aqueous solubility.Dissolution was the rate-limiting process for its oral absorption.In addition,the obvious first pass effect was the other important hurdle for its bioavailability improvement.The aim of this paper was to improve the aqueous solubility and oral bioavailability of NMD by applying nanocrystal drug delivery system.NMD nanocrystals were prepared by a hybrid method of microprecipitation and high pressure homogenization.The preparation process and formulation composition were optimized with particle size as an index,and the mean particle size of the optimized formulation was about 833.3 ± 20.6 nm.Nanocrystals of different particle sizes(503.0 ± 88.6 nm and 159.0 ± 7.9 nm)were obtained by centrifuging.The NMD nanocrystals were further lyophilized to improve the physical stability.The lyophilized powders of NMD nanocrystals showed favorable appearance and flowability,and it was easy to be reconstituted.It remained the same particle size as it was lyophilized before.The physical stability test showed that the lyophilized powders of NMD nanocrystals were stable in 24 months.The physicochemical properties of NMD nanocrystals were characterized.The transmission electron microscope showed that the nanocrystals were rod-shaped.X-ray powder diffraction showed that NMD had undergone crystal form transition during microprecipitation process,but experienced no conversion under the high pressure homogenization.The aqueous solubility was significantly improved and displayed a particle size dependent manner.All the formulations,including Nimotop(?),dissolved rapidly under sink condition,but the nanocrystals exhibited lower dissolution performances than Nimotop(?)under non-sink condition.A sensitive and selective LC-MS-MS method was developed for the determination of NMD in biological specimens.The pharmacokinetics was performed in beagle dogs in a crossover experimental design.The results showed that the bioavailability of the nanocrystals(833.3 ± 20.6 nm and 159.0 ± 7.9 nm)was bioequivalent,but about 2.6-fold higher than that of Nimotop(?).Such results led to an unfavorable in vitro in vivo correlation.The gut permeability was evaluated using everted gut sac models,and it was suggested that special mechanism may be involved in the absorption of NMD nanocrystals.The uptake mechanism was studied using Caco-2 cell monolayers.The crude NMD and Nimotop(?)showed a concentration dependent uptake manner,indicating passive transport.On the contrary,the amount of uptake for NMD nanocrystals was not varied over the concentration range of 5～25 μg/mL.Further,the cellular entry mechanism for nanocrystals was evaluated in the presence of different endocytosis inhibitors.The results indicated that the nanocrystals were taken up via macropinocytosis and caveolin-mediated endocytosis.Mesenteric lymph duct cannulation was performed in male rats.Exudates of nanocrystals from enterocytes were detected in mesenteric lymphatic fluids using transmission electron microscope,demonstrating that the NMD nanocrystals were transported via the lymph route.We also quantitatively investigated the contribution of lymphatic route to the bioavailability of NMD nanocrystals by blocking the chylomicron flow in rats.It revealed that mesenteric lymphatic route played an important role in the absorption of NMD nanocrystals.In summary,it was impossible to establish a favorable in vitro in vivo correlation for NMD nanocrystals of different particle sizes and Nimotop(?),because portions of the nanocrystals underwent macropinocytosis and caveolin-mediated endocytosis by enterocytes as intact nanocrystal forms,then enter the mesenteric lymph duct,bypassed the liver and avoided first-pass metabolism.NMD microcrystals with particle sizes of 16296.7 ± 161.7 nm and 4060.0 ± 31.2 nm were prepared using a microprecipitation method.Compared with NMD nanocrystals,the colloidal dispersions did not show any difference in aqueous equilibrium solubility.Additionally,the three formulations also displayed similar dissolution curves in purified water and 0.05%SDS.But it showed a particle size dependent manner for the AUC0-12h of the three formulations tested.Thus,the critical particle size was found to be within the range of 833.3～4060.0 nm in improving the bioavailability of NMD,and dissolution performance was not an effective index in evaluating the bioavailability for NMD colloidal dispersions.