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Open in a separate window Figure 5. Plasma and brain exposure of MTf and MTfpep targeted IgG-IL-1RA fusion molecules in a mouse PK assay. unmodified NIP228 control antibody, whereas the fusion to MTf protein had significantly reduced plasma exposure most likely due to a higher tissue distribution in the periphery. Brain exposure for the MTf peptide fusions was significantly increased for the duration of the study, exceeding that of the fusions to full length MTf protein. Using a neuropathic pain model, we have demonstrated that fusions to interleukin-1 receptor antagonist (IL-1RA) are able to induce significant and durable analgesia following peripheral administration. These data demonstrate that recombinant and chemically conjugated MTf-based brain delivery vectors can deliver therapeutic levels of drug to the central nervous system. Keywords: BloodCbrain barrier, central nervous system, interleukin-1 receptor antagonist, melanotransferrin peptide, pharmacokinetic Introduction Although protective in design, the bloodCbrain barrier (BBB) presents a constant challenge to effectively deliver therapeutic drugs directed at the treatment of brain diseases. Efficient drug delivery across the BBB is most important in the treatment of neurophysiological disorders (including neuropathic pain, Alzheimers disease and lysosomal storage diseases), brain cancers, trauma and genetic diseases. Studies have shown that drugs, conjugated to antibodies that bind specific receptors on brain endothelial cells, can cross the BBB. This suggests that using ligands for these receptors as carriers of therapeutic drugs may be of value in facilitating delivery across the brain capillary endothelial cells of the BBB and into the brain.1C3 However, despite these advances, crossing the BBB remains a key obstacle in the development of drugs for the treatment of brain diseases despite decades of research.4C6 One candidate which acts as a carrier for transport across brain capillary endothelial cells is the protein melanotransferrin (MTf), a protein belonging to the transferrin (Tf) family of proteins.7 Human MTf has been found to share 37C39% protein sequence homology with human serum Tf and human lactotransferrin.8 Despite this homology, Tf receptor (TfR) has been demonstrated not to be involved in the transcytosis of MTf, but that LDL receptor-related protein-1 (LRP1) may be involved in its transcytosis.9 In addition, MTf is the only member within the family to exist in two different forms: a membrane protein attached to the cell surface via a glycosylphosphatidylinositol (GPI) anchor and a free soluble Rabbit Polyclonal to Cyclin F form NVS-PAK1-1 in the serum.10C13 The soluble form of MTf has been found to localize on the surface of normal brain endothelial cells, the main constituent of the BBB, and is able to cross NVS-PAK1-1 through the brain capillary endothelium.14,15 The soluble form of MTf functions in the transport iron across the BBB.16 Recombinant human soluble MTf is transported across brain endothelial cells at a rate of 10C15 times higher than Tf in an in?vitro model of BBB transcytosis 9,17 and 5.7-fold higher in?vivo200?L) into a Li-Hep microvette (BD Diagnostic Systems), while the second sample (600?L) was collected by cardiac puncture under isoflurane anaesthesia into a Li-hep microtainer (BD Diagnostic Systems). Following collection, blood samples were allowed to clot for 30?min and centrifuged at 10,000??for 2?min at 4 and the resultant plasma drawn off. Plasma samples were flash frozen on dry ice for subsequent analysis. After final blood collection, the mice were perfused with D-PBS for a price of 2?ml/min for 10?min before extremities appeared light. Brains had been excised, and one hemisphere processed, the various other snap iced in water nitrogen. Human brain hemisphere was homogenized in five amounts of ice-cold PBS filled with 0.5% Tween 20 and Complete? protease inhibitor cocktail tablets (Roche Diagnostics). Homogenisation was performed within a 10?ml Potter-Elvehjem mortar type cup homogeniser with polytetrafluoroethylene (PTFE) pestle, using 2??10 clockwise strokes with 5?s relax time. Homogenates had been used in LoBind pipes (Eppendorf) and rotated at 4 for 1?h just before centrifuging within a chilled bench-top centrifuge in NVS-PAK1-1 13,000?for 20?min. The supernatant was isolated for human brain antibody dimension. Five amounts of ice-cold PBS filled with 0.2% sodium dodecyl sulphate (SDS) and Complete? protease inhibitor cocktail tablets had been added to the rest of the cell pellet as well as the pellet prepared as defined above. The supernatant was once again removed and combined with initial supernatant for dimension via MesoScale Breakthrough (MSD) assay (Meso Range Diagnostics). MSD multi-array technology allows recognition of biomarkers in multiplex and one formats using electrochemiluminescent recognition. Pet husbandry as well as the techniques used were relative to the guidelines from the AstraZeneca Pet NVS-PAK1-1 Treatment Committee and complied using the Animals (Scientific Techniques) Action 1986. All tests.