| HI02 peptide is a hydrophilic peptide composed of 10 amino acids with molecular weight of 1289.4. Previous studies have confirmed that it has the abilities to stabilize the space structure of Aβ42, prevent deposition of amyloid plaques and improve the spatial memory impairment, which indicates that H102 is a promising drug for Alzheimer’s disease (AD) treatment. However, in previous studies, lateral ventricle injection was chose for adminisrtation of H102, which was complicated and non-compliant to patients. Moreover, the unstability and poor ability of H102 to across BBB limited its use for treating AD. Thus, it is important to find a noninvasive and effective way with pleasant compliance for administration to treat AD.Intranasal administration is one of the most studied routes for its non-invasion, pleasant compliance and the possibilities of drugs, especially peptides and proteins being deliveried to brain via nose-brain direct pathway. However, because of the poor capacity of penetration through nasal mucosa, the enzymatic degradation and the rapid mucociliary clearance, the total amount of drug absorbed is too low to achieve the therapeutic level. Thus, in this study, we designed and prepared two formulations: a nasal solution of H102 containing absorption enhancer, stabilizer and bioadhesive agents to improve the absorption of H102 through nasal mucosa and its stability in nasal cavity; a nasal liposome encapsulating H102 peptide to improve its stability in vivo and enhance the absorption amount via intranasal administration.In the first part, a HPLC and HPLC-MS method were set up for the detection of H102 peptide both in vitro and in vivo. The in vitro stability studies showed that H102 peptide was sensitive to light, high heat and enzyme in nasal cavity, and was extremely unstable in plasma and brain homogenate. The optimal pH value for H102 was 6-7. Besides, H102 peptide was found to degrade dramatically in blood and could barely cross the BBB via intravenous administration, while it could neither cross nasal mucosa alone after intranasal administration. The results above indicated that proper absorption enhancer or dosage form should be chose to improve the ability of HI02 to cross nasal mucosa. At the same time, H102 peptide was confirmed to have no stimulation to nasal mucosa.According to the properties and biopharmaceutics features of H102 peptide obtained in the first part, proper preparations were designed and prepared in the second part. Firstly, to prepare a nasal solution of H102 peptide,1% chitosan was chose as absorption enhancer by transporting experiment of Calu-3 cell models in vitro and in vivo absorption experiment in SD rats; 0.1% EDTA-2Na was selected as stabilizer for its strong ability to decrease the degradation of HI02 peptide in nasal lavage fluid and its low cytotoxicity. Besides,3.5% mannitol was added to adjust the isotonicity of the preparation, chlorhexidine acetate and human serum albumin were chosen as preservative and antitack agent, which contributes to the final prescription of the H102 peptide nasal solution.Secondly, a nasal liposome was studied.Film dispersion method was selected to prepare liposomes. A single-factor test was carried out to determine the optimal conditions of preparation. The lipid materials were dissolved under the conditions of 40℃ and 10 r/min after forming the phospholipid membrane and in PBS with the pH of 6.5. After 2 hours for hydration and 90 s for ultrasound under 200 W, the H102 liposomes were prepared. The best prescriptions of liposome were optimized by uniform experimental design as followed, the mass ratios of phospholipid:H102 peptide, phospholipid:cholesterol and phospholipid:DSPE-mPEG 2000 were 40:1, 4:1 and 20:1, respectively. Finally, reproducibility H102 peptide liposomes with stable particle size (140-160 nm) and approving encapsulation efficiency (>70%) were prepared. The releasing characteristic of prepared liposomes in artificial nasal fluid fitted the first-order equation. Furthermore, to improve the stability of liposomes, 10% trehalose was chose as freeze-drying protective agents. Freeze-dried powders of liposomes were prepared and were proved to improve the stability of liposomes significantly in vitro.In the third part, the results of pharmacokinetics and brain delivery characteristic study showed that the absolute bioavailability of HI02 nasal solution, H102 nasal liposome and HI02 nasal liposome (containing 1% chitosan) were 7.12%,16.16% and 25.71%, respectively. The AUC of H102 liposome in olfactory bulb, cerebrum, cerebellum and hippocampus was respectively 1.67、1.68、1.59 and 2.92 times that of HI02 solution, indicating that the nasal liposome could enhance the drug delivery from nose to brain than solution. Besides, after combining liposomes with 1% chitosan, the absorption enhanced dramatically. The AUC of H102 liposome (with 1% chitosan) in different brain tissues was 2.55-3.78 times that of H102 solution, and 1.29-1.87 times that of H102 liposomes.In the fourth part, the AD model was established by bilateral injection of Aβ1-40 into rat hippocampus. The Morris water maze experiment was subsequently conducted to evaluate the improving effect of H102 nasal solution, H102 nasal liposome, H102 liposome (with 1% chitosan) and H102 intravenous injection on rat spatial memory impairment. The results showed that except intravenous group, all the nasal formulations of H102 peptide could effectively ameliorate spatial memory impairment of AD model rats. The enzyme activities of acetylcholinesterase (AchE) and choline acetyltransferase (ChAT) in rat hippocampus were evaluated and the results showed that both H12 nasal solution and liposome could protect central cholinergic neurons to some degree. The results of western blot of insulin degrading enzyme (IDE) and immunohistochemistry in hippocampal sections demonstrated that both H102 nasal solution and liposome, especially high dose of H102 solution and liposome, showed a protective effect on the hippocampal cells and could prevent amyloid from deposition.In the last part, the obtained freeze-dried powders of H102 nasal solution and liposome were subjected to quality evaluation and preliminary stability test. The indexes such as appearance, pH, content, total spray times/bottle, average content/spray, particle sizes, encapsulation efficiency and biological activity were all met their specified requirements. Nasal mucosa toxicity examination showed that H102 nasal formulations had no toxicity on nasal mucosa, which indicated that they had good biological safety.3-month accelerated test and long-term test results revealed that both H102 nasal solution and liposome were of stable quality. |
PDF Full Text Request