(447e) Targeted Drug Delivery to the Brain Using Transferrin-Binding Peptides

Drug-delivery to the brain is a significant challenge due to the presence of the formidable blood-brain barrier (BBB). Current therapeutic interventions to treat brain disorders include surgical implants or catheters, both of which are highly invasive and carry the risk of long term neurological damage. A treatment strategy that is non-invasive provides uniform distribution of the drug in the brain at therapeutically relevant concentrations, and carries a low risk of neurological damage is highly desirable but not clinically available. We are focusing on a specific pathway called receptor-mediated transcytosis (RMT) that is facilitated by proteins expressed on the surface of endothelial cells that form the BBB and are dedicated for ferrying transport proteins across the BBB. This pathway has previously shown great promise for transport of antibodies and drug conjugates of transport proteins like transferrin (Tf), insulin etc. We are developing a novel drug delivery strategy based on short peptides as drug carriers to the brain, whereby these peptides will bind to human Tf (hTf) and deliver the attached drug via the RMT pathway of hTf. The transferrin receptor (hTfR) is of particular importance here as it is over-expressed on brain capillaries and is involved in the transfer of iron-carrying hTf to the brain. Given the high circulation half-life (~8 days) of hTf, designing peptides that enable drugs to stay conjugated to hTf increases the in vivo residence time and efficacy of the drugs as well as reduces their required dosage and side effects. This addresses another major challenge of obtaining desirable pharmacokinetics when designing therapeutics for brain diseases. High abundance (1-3 mg/ml) of hTf in blood also facilitates high drug dosing. Additionally, chaperone peptides eliminate the need for covalent conjugation of drugs to hTf outside the body and can be tailored to improve solubility and stability of drugs in circulation.

      In order to design high-affinity hTf–binding peptides that can act as drug chaperones we adopted a rational design approach that relied on naturally occurring receptors for Tf. It is known that several bacterial species have evolved surface proteins that bind to hTf in serum. These proteins, called transferrin binding proteins (Tfbp) enable the pathogens to scavenge iron from circulating hTf and use it for their own metabolic requirements. The binding interfaces of Tfbps on different pathogens do not bear any significant structural or sequence similarity to hTfR and therefore are expected not to interfere with binding of hTf to hTfR. Thus, they offer a potential search space for designing short peptides that bind to hTf with a high-affinity and enable the delivery of a drug cargo across the BBB.

      Using high-throughput peptide synthesis and microarray screening, several potential peptide candidates that mimic Tfbps have been identified from an initial library of ~1000 peptides. These peptides show very high affinity (1-10nM) for hTf in the presence of physiologically relevant concentrations of human serum albumin (HSA) – the most abundant protein in human serum. This ensures that the peptides not only have high affinity but also high selectivity for hTf. As a first step, while peptides have been identified that bind to hTf, the future validation of this drug delivery strategy in vivo in a mouse model would require peptides that bind to mouse Tf (msTf). Keeping this in mind, the entire library has also been screened with msTf in the presence of HSA in order to select peptides that bind to msTf with high affinity and selectivity. A set of candidates that bind to both hTf and msTf has been selected for in vitro and in vivostudies.

      We are currently testing a subset of these peptides in an in vitro BBB cell culture model. This, followed by studies in an animal model would further validate the lead peptide candidates as drug carriers. Finally, the fact that these peptides come from a non-human protein and therefore less likely to compete with hTfR-hTf interaction will have a significant impact on designing better peptide chaperones for drug delivery to the brain.