Studies On Chitosan Microspheres Of Huperzine A

Objective: Alzheimer's Disease(AD) is a degenerative disease of remembrance and cognition function, has become major one of senile dementia disease, and threatening the health and lives of human. Huperzine A (Hup-A) is a kind of alkaloid abstracted from Huperziaceae which is a strong reversible chol inesterase inhibitor found in China firstly, now mainly used to treat Alzheimer's disease through inhibiting the activity of peripheral acetylcholine esterase and raise the level of acetylcholine in brain, and it is one of the most effective drugs which can treat AD at present. Lack of selectivity on brain, the present preparation could inhibit the activity of peripheral acetylcholine esterase, which brought great side effect to patients. To study on a new type of Huperzine A preparation through changing the administration route will have significant clinical meaning, which could increase the drug concentration of brain, reinforce the efficiency and decrease the side effect.Nasal administration has the characteristic of convenience, fast absorption, targeted-brain, without first pass effect, and is becoming one of the most hot administration route in pharmaceutical domain recently. Using Huperzine A as model drug, using chitosan microsphere as carrier which has the characteristic of adhesive attraction to biomembrane, improving drug absorption, good biocompatibility, using the modern pharmaceutical preparation technique, the subject prepared the nasal chitosan microspheres brain targeting drug delivery system of Huperzine A following intranasal administration, which will have fairly important value of science technology and very prosperous perspective.Methods: Establish the assay method of Hup-A in vitro. Prepare Huperzine A chitosan microspheres using chitosan as carrier, methanal as curing agent by the method of emulsification, cross-linking and solidification. The prescription and technology were investigated by single test. Based on it, orthogonal design of four factors and three levels was adopted to optimize the prescription and technology with the size distribution of microspheres, drug loading, entrapped efficiency as combined index. Directly observe the appearance and the size distribution of microspheres by light microscope and scanning electron microscope. Investigate the release of drug in vitro, fit the release results of microsperes with classical release model, get the major parameter, and deduce the release mechanism of huperzine A chitosan microspheres. study on the primary stability of microspheres of huperzine A.Using toad maxilla mucosa as test model, study on mucosal adhesion and ciliotoxicity of huperzine A chitosan microspheres.Using rats as experimental animal model, We research the pharmacokinetics and brain targeting distribution of chitosan microspheres of huperzine A following intranasal administration, compared with the injection of huperzine A following intravenous administration. HPLC with fluorescence detector was used to determine the huperzine A in blood plasma, and the DAS software was used to handle the pharmacokinetics data of huperzine A. We got the major pharmacokinetics parameter, and calculated absolute bioavailability of chitosan microspheres of huperzine A following intranasal administration. We determine the drug content of brain tissue of rat with the injection of huperzine A as contrast, using the method of brain tissue bomogenate. Appraise the brain targeting ability of chitosan microspheres of huperzine A following intranasal administration, with drug targeting index (DTI) and brain drug direct-transport percentage (DTP) as evaluating index.Result: Based on the inspecting single factors of formula and preparation technology, we found the concertration of chitosan and dispersing stirring speed had a significant effect on appearance and grain size of chitosan microsphere, Dosage of formaldehyde and setting time influenced the entrapment mainly. We selected chitosan concertration, dispersing stirring speed, setting time and dosage of formaldehyde to orthogonal experiment with 4 factors ang 3 levels. The optimized results: chitosan concertration was 3%. dispersing stirring speed was 1200r/min, setting time was 2 hour and the dosage of formaldehyde was 1 ml. We investigated 3 batches of microsphere using the optimized conditions, and the results showed that the technology was stabile, the reproduction quality was very well.The chitosan microspheres of huperzine A prepared according to the optimized formula and preparation technology were toroid with uniformity disposition of particle size, and mean particle diameter was 42.4±0.9μm, meeting requirement of nasal cavity administration. The release process fitted to Higuchi model very well, Mucosa adhesiveness test indicated that cilium transport velocity was 2.54±0.47mm/min. When administrating chitosan microsphere in upper jaw of toad, cilium keeping swinging time was lowered compared with normal saline, which did not affect the nasal cavity administration. The test of influential factor manifested that the chitosan microspheres of huperzine A were insensitivity to light, heat, and wet. After 6 month, the chitosan microspheres of huperzine A were not obviously changed, which showed that chitosan microspheres of huperzine A had very good stability.The established method of determine blood plasma and brain tissue concentration of huperzine A was sensitive, using HPLC fluorescence detection, the lowest quantitative limit was 2ng/ml, the linear relationship was very good between 2.0～120ng/ml. Using rats as model animal, compared with the solution of huperzine A following intravenous administration, the chitosan microspheres of huperzine A were taken following intranasal administration. Pharmacokinetics test indicated that the plasma half life became 2.05h from 1.35h; and brain tissue half life became 3.33h from 2.18h; the absolute bioavailability was 76.48% following intranasal administration in rats. The AUC(0→8h) value in plasma obtained following intranasal administration was 0.76 times of that following intravenous administration, but The AUC(0→8h) value in brain tissue obtained following intranasal administration was 1.36 times of that following intravenous administration. The results of brain targeting following intranasal administration compared with intravenous injection using rat as experimental animal: DTI was 1.78, DTP was 43.96%. The results manifested nasal cavity administration possessing the ability of brain targeting, and inferred that a part of drugs accessed into brain directly avoiding BBB through direct path of nasal cavity and brain.Conclusion: The prescription and the technology screened and optimized with orthogonal design was stable and reliable. The microspheres met the quality requirements of nasal mucosa administration. The animal test in vivo proved chitosan microspheres of huperzine A after intranasal administration had good bioavailability, longer half life, targeting-brain action, which reached the test objective. The preparation with convenience, high bioavailability, better efficiency, and lower side effect has a prosperous perspective.