Drug Delivery of Oligonucleotides at the Blood-Brain Barrier: a Therapeutic Strategy for Inflammatory Diseases of the Central Nervous System
In this project a vector for drug delivery into brain endothelial cells, consisting of an anti-transferrin receptor antibody and a complex of polyethylenimine (PEI) and an oligodeoxynucleotide (ODN) drug was evaluated. NF-kB decoys are short double-stranded ODNs, which contain a recognition sequence...
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|In this project a vector for drug delivery into brain endothelial cells, consisting of an anti-transferrin receptor antibody and a complex of polyethylenimine (PEI) and an oligodeoxynucleotide (ODN) drug was evaluated. NF-kB decoys are short double-stranded ODNs, which contain a recognition sequence for binding to the transcription factor. By binding to the transcription factor NF-kB they prevent the transcription of several genes involved in inflammatory processes, such as adhesion molecules and pro-inflammatory enzymes. Adhesion molecules promote the transmigration of activated T lymphocytes across the blood-brain barrier (BBB), leading to demyelination of nerve fibers within the brain, as seen in inflammatory diseases like Multiple Sclerosis (MS). Inhibition of lymphocyte transmigration by downregulation of the expression of these genes may be therapeutically beneficial.
The rat anti mouse anti-transferrin receptor antibody 8D3 was purified from hybridoma supernatant and was conjugated to recombinant streptavidin (SA) in order to bind to biotinylated ligands. In vivo pharmacokinetics and in vitro binding studies were performed to evaluate the 8D3-SA vector. Pharmacokinetic studies showed a comparable half-life and area under the curve (AUC) of antibody conjugate and free 8D3 antibody in plasma. Immunohistochemistry with a brain endothelioma cell line (bEnd5) revealed a similar binding behavior for both, antibody and its streptavidin conjugate. The specificity of uptake of 3H-biotin-8D3SA by bEnd5 cells could be observed in experiments carried out either with an acid wash step to remove all surface bound antibody, or by using the mouse anti rat transferrin receptor antibody OX26.
The biotinylated drug applied in this project was a polymer polyethylenimine (PEI), which is able to complex a double-stranded 20bp ODN sequence (NF-kB decoy). The low molecular weight PEI (LMW PEI) of 2,700 Da was chosen due to such favorable properties as low cytotoxicity, high transfection efficiency, and narrow size distribution. A co-polymer of PEI and biotinylated PEG was synthesized to allow coupling to the vector. Stability studies were carried out to examine the characteristics of bioPEGPEI/ODN complexes in salt solution, under addition of 10% or 20% plasma, or by incubation of complex in medium over a long time frame. Particle size measurements in PBS pH 7.4 revealed an optimal size distribution of bioPEGPEI/NF-kB complex at a ratio PEI-amine/ODN-phosphate of 6:1 with an average particle size of 120nm, which was stable for at least 1 week. Gel retardation assays also showed a complete complexation of bioPEGPEI and ODN at this ratio. The addition of 8D3-SA increased the complex size by 40nm.
In vitro pharmacological effects of bioPEGPEI/NF-kB could be observed after treatment of bEnd5 cells with different concentrations of NF-kB decoy over a time frame of 48 hours. An inhibition of VCAM-1 (isoform 1 and 2) expression was already visible after 12 hours with a steady increase of this inhibitory effect to 48 hours. ODN-drug concentrations of 5uM in complex with bioPEGPEI showed a nearly complete suppression of VCAM-1 expression. Coupling of bioPEGPEI/NF-kB decoy to the vector decreased the concentration required for a reduction of VCAM-1 mRNA to 0.5uM - 1uM, suggesting an increase in potency by targeting to the transferring receptor.
Pharmacokinetic studies in mice showed the behavior of bioPEGPEI/NF-kB complexes with different N/P ratios. At a N/P ratio of 6:1 the AUC (area under the plasma curve) could be increased 2-fold compared to free NF-kB. A coupling to the targeting vector did not further change the pharmacokinetic characteristics.
The drug targeting approach investigated in this project promises to be an excellent model for in vitro applications. The in vivo administration must be further evaluated with the goal to improve the pharmacokinetic behavior (enhance AUC). This may require additional modifications of the bioPEGPEI copolymer.