2.2.1. Additional Approval of Target Specificity of (*S*)-(−)-[18F]Fluspidine PET

Due to access to a limited number of sig1R-knockout mice [42], we performed a single head-to-head dynamic PET study with (*S*)-(−)-[18F]fluspidine in sig1R-knockout (*<sup>n</sup>* <sup>=</sup> 1) and control (*<sup>n</sup>* <sup>=</sup> 3) mice with the main focus on the tracer kinetics in the striatum. Mean injected activity was 3.5 MBq and 1.9 MBq with a molar activity of 24 GBq/μmol and 21 GBq/μmol at the time of injection, resulting in a mean chemical concentration of 4.7 nmol/kg and 3.2 nmol/kg in the control group and the sig1R-knockout mouse respectively. The corresponding time-activity curves (TACs) are presented in Figure 3. Both the sig1R-knockout and the control animals showed a rapid uptake of activity within the first minutes after i.v. injection of (*S*)-(−)-[18F]fluspidine (initial peak between 1 and 2 min p.i. of 1.45 and 1.12 SUV, respectively) along with a much stronger and faster washout observed in the sig1R-knockout mouse in comparison to the control animals (SUV at 10, 20, and 40 min p.i. of 0.84, 0.64, and 0.25 vs. 1.07, 1.03, and 0.67, respectively) confirming the selectivity in vivo of (*S*)-(−)-[18F]fluspidine for sig1R (Figure 3A). In order to correct for potential model-related differences in the brain perfusion, we calculated an SUV ratio (SUVR) from the TAC data obtained in striatum and blood at each time points. The corresponding SUVR curves, presented in Figure 3B, indicate for both animal models nearly stable SUVR values at 15 to 60 min p.i., albeit with clearly different values. While for the control animals SUVR values in the range of 0.8 to 1 (corresponding to an area under the curve (AUC) value of 48.6 ± 8.3) has been estimated, the notably lower value of about 0.4 (corresponding to an AUC value of 23.4) determined in the single sig1R-knockout mouse confirms the target specificity of (*S*)-(−)-[18F]fluspidine.

**Figure 3.** Positron Emission Tomography/ Magnetic Resonance (PET/MR) imaging of control mice (*n* = 3) and of sig1R-knockout mouse (*n* = 1) after i.v. administration of (*S*)-(−)-[18F]fluspidine. (**A**) Average striatal time-activity curves for control mice (black squares) and sig1R-knockout mouse (orange dots). (**B**) Average time-varying SUVRs of the striatum over the blood (defined from the image-derived input function (idif)) of control mice (black squares) and sig1R-knockout mouse (orange dots) (**C**) Average time-varying SUV of the blood (defined from the idif) of control mice (black squares) and sig1R-knockout mouse (orange dots).
