Blood-Brain Barrier Shuttles: From Design to Application

The work of this thesis is based on research on peptides able to cross the blood-brain barrier and their use as tools to enable the delivery of drugs into the brain. The blood-brain barrier (BBB) is a permeable but selective barrier that tightly regulates the transport into the central nervous system (CNS). In this regard, therapeutic treatments at the CNS are hampered by the presence of this barrier (BBB). Thus, diverse strategies have been developed to overcome it. Blood-brain barrier shuttles are peptides able to cross this barrier and deliver drugs into the brain. Peptides are privileged structures from the therapeutic point of view, they share properties from small organic molecules and large biologics: the synthesis through solid-phase peptide synthesis (SPPS) enables a straightforward method to obtain them with high purity and at the same time they can be purified and characterized like small organic compound. In addition, their structure is present in nature and thus the risk of toxicity is lower or more predictable compared with organic compounds, and their larger structure enables to obtain more selective and stronger interactions with targets. In addition, peptides have been shown to cross the BBB by diverse transport mechanisms and thus enabling to select the best one for each therapy and drug. In this thesis a family of BBB shuttles crossing by passive diffusion (based on phenylproline) have been improved from a parent peptide shuttle (based on N-methyl-phenylalanine). The solubility was three orders of magnitude superior and the transport capacity was maintained upon cargo attachment. In addition, the role of stereochemistry in passive diffusion in biological membranes was demonstrated. A method which combined the use of MALDI-TOF MS and in vitro cell-based models of the BBB enabled the increase in sensitivity for transport quantification of three orders of magnitude compared to RP-HPLC-PDA. Additionally, a BBB shuttle library was evaluated and quantified by this novel methodology. Two new analogs showed better performance when evaluated in these in vitro cell-based models. Immunogenicity of BBB shuttle peptides made by L- or D-amino acids was evaluated and compared. Both peptide shuttles showed low immunogenic response in mice, however, the response to those made with D-amino acids was lower. Finally, the applicability of these peptide shuttles for a therapeutic use was considered for Friedreich’s Ataxia, a monogenic recessive disease. Both a protein replacement therapy and a gene therapy for the central nervous system were attempted by coupling covalently BBB shuttles to the affected protein or viruses, respectively. The protein replacement therapy was impeded by the high rate of proteolysis of the protein used. On the other hand, novel methods of conjugation of BBB shuttles into enveloped viruses (Herpes simplex Virus type 1; HSV-1) were developed. These modified viral particles were subsequently characterized through a range of methods comprising molecular biology tools (SDS-PAGE, western blots), proteomics (mass spectrometry) and biophysical tools (dynamic light scattering and z-potential).