Browsing by Author "Peltoniemi, Martta"

The glymphatic system is a waste clearance system in the brain to prevent protein accumulation that negatively affects neural functions, leading to neurogenerative diseases such as Alzheimer's. Perivascular spaces surround the brain vasculature allowing cerebrospinal fluid (CSF) inflow into the brain parenchyma during NREM sleep. The CSF moves from perivascular spaces into the white matter through AQP4 water channels, and flows towards the efflux routes, from where the interstitial solutes are drained into the lymphatic system. The existence and functions of the glymphatic system raise controversies due to the lack of quantitative data. Imaging tools that do not negatively affect the flow are required to visualize the glymphatic system in health and disease. If the glymphatic flow could be intensified in a preventive or therapeutic manner or harness for CNS drug delivery, it would be revolutionary. Positron emission tomography (PET) is a rising imaging modality in glymphatic research. It provides an efficient and non-invasive method, even with nanomolar tracer concentrations, to follow the CSF within the entire animal, being also fully translatable for human studies. Albumin is the dominant protein the CSF and can act as a carrier for tracers extending the circulation time. More recently, truncated Evans Blue (EB) and 4-(p-iodophenyl)butyric acid (IP) have shown promising results in modulating the pharmacokinetics of radiopharmaceuticals through albumin-binding. This study aimed to develop and evaluate six new albumin- binding tracers ( three NODAGA conjugated and three DFO conjugated) for the PET imaging of the glymphatic fluid flow via binding of endogenous albumin in the CSF. The precursors were synthesized using coupling reactions and radiolabeled with [68Ga]GaCl3 (0.2M sodium acetate buffer, pH=4, 95°C or 25°C, 15 min). Radiochemical purities were determined by radio-TLC and radio-HPLC and LogD (octanol:PBS) with shake-flask method. The in vitro stability assays were done in rat serum, rat CSF, 2 mM EDTA, and 0.2 mM FeCl3 solutions at 37°C over 6 h of incubation. The in vitro albumin binding affinities were investigated at physiological rat CSF albumin concentration at 37°C over 1 h of incubation using radio-SEC- HPLC. The in vivo and ex vivo experiments of the three most stable tracers were done using healthy female Swiss mice, and the experiments included blood kinetic studies, ex vivo biodistribution studies, and analysis of urine, bile, and blood fractions (plasma, blood cells, proteins) for in vivo stability. The precursors were synthesized with high yields (69–96%) and radiolabeled with high radiochemical yield (64– 82%), radiochemical purity (97–99.5%), and molar activities sufficient for low-volume infusion into the CSF (9.2– 17.2MBq/nmol). The NODAGA conjugated tracers had higher radiochemical yield and molar activity than the DFO conjugated tracers. The radiolabeled DFO conjugated products were stable in rat serum and rat CSF, but stability assays with EDTA and FeCl3 showed major transchelation. The NODAGA conjugated tracers were stable in each medium, maintaining the percent of intact labeled compound above 97%. The albumin binding affinity studies showed full binding from the first time point of 5 min incubation until the last time point of 1 h. The blood kinetic studies of the three stable NODAGA conjugates showed decreasing %ID/g with varying biological half- lives depending on the tracers’ lipophilic properties. The ex vivo analysis of urine and bile showed eliminated free tracer or some metabolite of the free tracer with similar retention time with radio-HPLC, no free gallium was observed. The analysis of blood samples after fractionation showed that each tracer had the most significant %ID/g in the protein fraction, and no activity was observed in the plasma. The in vivo investigated tracers showed promising properties for possible future use as CSF tracer due to high radiolabel stability and rapid stable binding to albumin in vivo.