Applicability of cerebral open flow microperfusion and microdialysis to quantify a brain-penetrating nanobody in mice

The use of biologics in the therapeutic landscape has increased exponentially since the last 3 decades. Nevertheless, patients with central nervous system (CNS) related disorders could not yet benefit from this revolution because the blood-brain barrier (BBB) severely hampers biologics from entering the brain. Considerable effort has been put into generating methods to modulate or circumvent the BBB for delivery of therapeutics to the CNS. A promising strategy is receptor-mediated transcytosis (RMT). Recently, Wouters et al. (2020) discovered a mouse anti-transferrin receptor nanobody that is able to deliver a biologically active peptide to the brain via RMT. The present study aims to sample a derivative of this brain-penetrating nanobody (Nb105) in the CNS. Therefore, we compared the applicability of cerebral open flow microperfusion (cOFM) and microdialysis as sampling techniques to directly obtain high molecular weight substances from the cerebral interstitial fluid. A custom AlphaScreen™ assay was validated to quantify nanobody concentrations in the samples. In vitro microdialysis probe (AtmosLM™, 1 MDa cut-off) recovery by gain and by loss for Nb105 was 18.3 ± 3.2% and 27.0 ± 2.5% respectively, whereas for cOFM it was 87.2 ± 4.0% and 97.3 ± 1.6%. Although a large difference in in vitro recovery is observed between cOFM and microdialysis, in vivo similar results were obtained. Immunohistochemical stainings showed an astrocytic and microglial reaction in the immediate vicinity along the implantation track for both probe types. Coronal sections showed higher fluorescein isothiocyanate-dextran and immunoglobulin G extravasation around the microdialysis probe track than after cOFM sampling experiments, however this leakage was clearly limited compared to a positive control where the BBB was disrupted. This is the first study that samples a bispecific nanobody in the brain's interstitial fluid in function of time, providing a pharmacokinetic profile of nanobodies in the CNS. Furthermore, this is the first time a cOFM study is performed in awake freely moving mice, providing data on inflammation and blood-brain barrier integrity in the mouse brain. Overall, this work demonstrates that, while taking into account the (bio)analytical considerations, both microdialysis and cOFM are suitable in vivo sampling techniques for quantification of nanobodies in the CNS.