Development of new albumin-binding radiotracers for PET imaging of cerebrospinal fluid flow in the glymphatic system.

Abstract

Background: The glymphatic system is a recently discovered brain-wide clearance system that allows the cerebrospinal fluid (CSF) flow to clear metabolic waste, but the tools for the quantitative and non-invasive investigation of its function and activity especially in humans is lacking, hindering studies on glymphatic system physiology and therapeutic potential of glymphatic drug delivery and modulation. We postulated that albumin-binding radiotracers could be used to this end by binding to the endogenous protein in CSF, constituting a macromolecular, biological radiotracer, allowing for the visualization of CSF flow in the central nervous system non-invasively with positron emission tomography (PET).

Results: We prepared three albumin-binding tracers based on 4-(p-iodophenyl)butyric acid and truncated Evans Blue radiolabeled with gallium-68 using the NODAGA chelator for in vivo radiolabeling of CSF albumin, and an in vitro radiolabeled reference tracer Al[¹⁸F]F-RESCA-rat serum albumin (RSA) with high radiochemical yield and purity, and acceptable molar activity (A_m). The biological evaluation of the tracers showed high radiolabel stability and rapid binding with albumin in vitro and in vivo with the biological half-life in Swiss mice after intravenous administration matching serum albumin (> 18 h). Dynamic PET imaging in female Sprague Dawley rats under ketamine/dexmedetomidine anesthesia after lumbar and intracisternal infusion showed distribution of the tracers towards intracranial space and along the spinal canal from the infusion site. However, the cervical lymph nodes were only visualized after the infusion of Al[¹⁸F]F-RESCA-RSA, characteristic for macromolecular tracers, indicating that the gallium-68-labeled tracers did not bind fully to endogenous CSF albumin in vivo, but were distributing to different brain areas according to their physicochemical properties.

Conclusions: While the relatively low molar activity (A_m) of the [⁶⁸Ga]Ga-NODAGA complex achieved in our setup combined with the limited amount of endogenous albumin at the infusion site (0.012-0.024 nmol) resulted in residual unbound tracer in the rat CSF in vivo, the tracers, especially the Al[¹⁸F]F-RESCA-RSA show promise for tracking CSF flow with PET, constituting the first tailored radiotracers to this end.