STSM - Blood brain barrier – derived peptide shuttles for drug delivery to the CNS -

Summary

The objective of this STSM project was to perform an in vivo infusion study to determine the extent of transport to the brain of a previously designed, synthesized and in vitro screened cyclic peptide. During this STSM project rats were used as model organism and the peptide was administered for four hours to reach steady state conditions. Terminal plasma and brain samples were taken for LC-MS analysis, which will be performed at the home institution. After quantification of the peptide, the partition coefficient to the brain (Kp,brain) will be calculated, which is necessary for the calculation of Kp,uu,brain (unbound brain-to-plasma concentration ratio coefficient). If transport is observed in vivo, the peptide will be further developed and tested as a shuttle for transporting compounds into the brain or to be used as a tool to study therapeutic applications like leishmania infiltrating the CNS.

Outcome

This STSM was performed as a part of my PhD project, which aims to study blood-brain barrier (BBB) transport and stability of peptide BBB shuttles. These probes were generated by incorporating a BBB transport-mediating peptide sequence into a natural stabilized peptide scaffold. During my stay at the group of Assoc. Prof. Irena Loryan from 13/01/2024 to 24/01/2024, I focused on the CNS pharmacokinetic characterization of a sunflower trypsin inhibitor 1 (SFTI-1) based cyclic peptide shuttle. The CNS pharmacokinetics of a drug can essentially be described with the Kp,uu,brain, a parameter describing the fraction of unbound drug distributing into the CNS. Therefore, three main experiments have to be performed, that are building blocks to derive the Kp,uu,brain. During previous work,  1) Brain slice assay to calculate the Vu,brain 2) Equilibrium dialysis to determine the fraction unbound (fu) in plasma and brain homogenate and a 10 minute in vivo infusion to study systemic pharmacokinetic (PK) parameters, were conducted in preparation of the planned equilibrium infusion experiment. All studies were performed with rats or tissues derived from Sprague-Dawley rats. During this STSM, the final experiment was performed 3) a 4-hour steady-state infusion study to derive the Kp,brain. This parameter describes the brain-to-plasma concentration ratio at steady-state conditions. The main goal from this work is to evaluate the transport of the test compound into the brain. Assuming that after 4 hours equilibrium between the compartments is reached, which is the case for most substances within this period, all influx and efflux mechanisms are at steady state and the fraction of peptide graft in brain vs. plasma can be measured. For this study, the peptide graft was administered to 3 rats via infusion via a femoral vein catheter. First, a 10-minute fast infusion (0.333 mg/kg) was given to quickly achieve the target plasma concentration. Afterwards, the maintenance dose (5.6 mg/kg) was administered via constant infusion, to hold target concentration (1000ng/kg) in plasma for 4 hours. The target concentration was chosen based on the previously determined Kp,brain of SFTI-1 ~0.06 and the limit of quantification of the LC-MS method. Blood samples were collected from a femoral artery catheter every 60 minutes for monitoring plasma concentrations. After taking the samples, they were centrifuged immediately to receive the plasma. After the 4 hours of administration of the maintenance dose brain samples and final plasma samples were collected. In parallel to that blank matrix, standard curves and quality control samples for LC-MS analysis were prepared using the relevant matrix. 

Results

I will follow up the STSM related work by analyzing the peptide content of the collected samples with an in-house developed bioanalytical method by utilizing the high-end LC-MS equipment provided by my home institution (Medical University of Vienna, Institute of Pharmacology). There, the samples will be processed for analysis and amount of peptide in each sample will be quantified using LC-MS. From these data the Kp,brain will be calculated. The outcomes will be used to determine the Kp,uu,brain, by using all the measured parameters (fu,plasma, Vu,brain, Kp,brain) for calculation. The anticipated outcomes of my experiments will show if the chimeric peptide probes, based on the SFTI-1 scaffold and BBB transport active sequences, is able to improve the PK characteristics of SFTI-1 so that more unbound substance will be located in the CNS. Ultimately, the knowledge generated from the results of this STSM will contribute to the better understanding of how grafting transport-mediating sequences into cyclic scaffolds influences BBB transport and pharmacokinetic properties. The most active probe in an in vitro endothelial cell transwell assay, was tested in vivo in this STSM and also showed to be an inhibitor of the enzymatic activity of prolyl oligopeptidase (POP), an enzyme, which was proposed as molecular target with anti-parasitic potential. If successful, this will lead to the generation of a CNS permeable POP inhibitor, which would be a useful tool to study therapeutic applications for leishmania infiltrating the CNS in vivo. In addition to that, my work will support the application of peptides in drug development. Based on the interdisciplinary nature of the project, I had chance to expand my knowledge of brain pharmacokinetic concepts and methodology. This research stay has allowed me to establish international contacts, which lead to a transfer of knowledge, which will be valuable for the further work on this project and for my further professional career in science. Participating in STSM and joining the COST Action allowed me to integrate myself into a team of internationally recognized scientists and my work will further contribute to a high-impact research publication.