AbstractAlzheimer disease (AD) is a neurodegenerative and age related disease characterised by gradual decrease in memory, caused by the abnormal accumulation of beta-amyloid (Aβ) in the brain. Recently, the incidence of AD has increased with 47 million people worldwide having this disease. Current available drugs are only capable of improving symptoms, but do not have profound effects on the underlying cause of the disease. Lack of a true treatment is due to limitations imposed by the blood brain barrier (BBB) that blocks the passage of drugs to the brain. Flurbiprofen is an example of a drug that has the
potential to be used in the treatment of AD as it has shown to decrease the formation of Aβ, but the drug’s efficacy is hampered due to its poor permeability across the BBB. A promising approach for improved drug permeability is the integration of the drug with specific carriers, such as dendrimers, that have the ability to pass through the BBB.
Dendrimers are hyper branched macromolecules, have a unique structure that can be easily bifunctionalised with both the drug and a ligand such as ApoE derived peptide facilitating the biochemical integration with cell membranes. This favours the recognition by the low density lipoprotein receptors on the brain endothelial cells, enhancing cellular uptake and brain targeting of the carrier system. The present project aims to improve brain permeability of Flurbiprofen by developing novel, biocompatible and biodegradable dendrimer-based carrier systems utilising an ApoE-derived peptide to facilitate brain
delivery and targeting.The novel dendronised molecules and the functionalisation with ApoE-derived peptide were produced and integrated with the Flurbiprofen using solid phase peptide synthesis.
Mass spectra, Fourier transform infra-red spectroscopy, high performance liquid
chromatography (HPLC) and thin layer chromatography were used for characterisation and verifying the purity of the products. A bEnd.3 brain endothelial cell line was used as an in vitro BBB model to determine the biocompatibility, uptake and permeability of the synthesised molecules. The cellular biochemical response upon exposure to the novel materials was studied using MTT, LDH, and Hoechst-propidium iodide assays. The cellular permeability of each material was assessed using an in vitro model based on a monolayer system utilising bEnd.3 cells grown in a Transwell support, while confocal
microscopy, flow cytometry and HPLC were used for quantitative and qualitative analysis.
In addition, the activity of the drug conjugates was investigated biochemically by quantification of γ-secretase enzyme and the biodegradability of these conjugates was also verified.
The results of this study indicated that the dendronised carrier systems were successfully produced and efficiently conjugated with the Flurbiprofen via amide linkage. These dendrons, both denuded and functionalised with the drug and ApoE peptide, were also found non-toxic. Moreover, the uptake and permeability results demonstrated the potential of the dendronised ApoE peptide to improve the drug permeability significantly across the in-vitro model of the BBB after the validity of this model was verified. Furthermore, the results showed that the Flurbiprofen-bound carriers still retain the drug action in decreasing the activity of γ-secretase enzyme on C6 glial cells alongside with evidence of the biodegradability of the drug conjugates into their building units.
This novel study provides the first demonstration of drug-laden, ApoE peptidefunctionalised dendrimers with the potential ability to cross the BBB and targeting the brain via receptor mediation for enhanced drug therapy of AD.
|Date of Award
|Matteo Santin (Supervisor)