4.3.2. In Vitro Stability and Plasma Protein Binding for [18F]6

A total of 40 μL of [18F]**6** was incubated in 0.01 M PBS at room temperature, in a microtube for 90 min, with mixing (400 rpm). At selected time-points (*t* = 5, 30, 60 and 90 min, *n* = 1), the samples were injected into an HPLC with PDA- and radiodetector, for analysis. Radiotracer [18F]**6** (40 μL) was incubated in 50% human plasma (anonymous donor FFP-24 plasma provided by the Finnish Red Cross Blood Service, Helsinki, Finland) in PBS at 37 ◦C, and in mouse plasma (separated from CD-1 mouse blood). At selected time-points (5, 60, 120, and 240 min, *n* = 2 for each), 100 μL of the samples were taken, diluted with 200 μL of cold acetonitrile, and centrifuged at 10,000 g for 5 min. The radioactivity in the precipitated pellet (protein-bound fraction) and supernatant (free fraction) were measured with a gamma counter and 100 μL of the samples were injected into HPLC, for radio-HPLC analysis.

#### *4.4. Biological Studies*

All animal experiments were conducted under a project license approved by the National Board of Animal Experimentation in Finland (license number ESAVI/12132/04.10.07/2017). The animals were group-housed in standard polycarbonate cages, on aspen bedding, in a HEPA-filtered housing unit (UniProtect, Ehret, Emmendingen, Germany), with food (Envigo Teklad Global Diet 2016) and tap water, available ad libitum. Conditions were maintained at 21 ± 1 ◦C and 55 ± 15% relative humidity, with a 12:12 lighting cycle. The biodistribution studies were conducted in healthy, female CD-1 mice (weight 25–33 g, 11 to 12 weeks, Charles River). The radiotracers [18F]**6** and [18F]**10** were injected via the tail vein to CD-1 mice, in the following formulations—10% EtOH and 0.5% Solutol HS 15 in 0.01 M PBS pH 7.4 for [18F]6 and 0.01 M PBS pH 7.4 for [18F]10. At selected time-points (5, 60, 120, and 240 min), the mice were euthanized with CO2 asphyxiation, followed by cervical dislocation, and selected tissues were collected, weighed, and the radioactivity was measured on an automated gamma counter.
