After the 24 h period, the mice were sacrificed by cervical dislocation. A total of 20 female BALB/c inbred mice were obtained from a professional stockbreeder (Harlan Laboratories, Netherlands) and quarantined for two weeks prior to the start of the experiment. The mice were divided into 7 groups, A, B, C, D, E, F (n = 3) and G (n = 2). The mice in groups A and C were injected with a mixture of saline solution and Iodine-123-Sodium Iodine (123I-NaI) or with a cocaine analogue Iodine-123-(2-beta-carbomethoxy-3-beta-(4-iodophenyl)-tropane) (123I-β-CIT) (MAP Medical Technologies Oy, Finland), respectively. The mice in groups B and D were injected with a 5:1 mixture of 1% NFC and 123I-NaI or

123I-β-CIT, respectively (final mixture of 0.83% NFC hydrogel with added study compound). Group E was injected with a mixture of 123I-NaI Birinapant and 99mTc-NFC for dual-radionuclide SPECT/CT.

Groups F and G were injected similarly with 5:1 mixture of 1% NFC and 99mTc-labeled human serum albumin (HSA) (Sigma–Aldrich, Finland) or 99mTc-labeled HSA in a saline solution, respectively (final mixture of 0.83% NFC hydrogel with added study compound). All mice received 50–60 MBq/200 μl injections. 99mTc-HSA was prepared, and radiochemical purity was tested according to the manufacturer’s instructions (Vasculocis®, CIS bio international, France). Radiochemical impurities were found below the allowed 5% of the total activity. SPECT/CT imaging was performed with a four-headed small animal scanner (NanoSPECT/CT®, Bioscan, USA), outfitted INCB018424 cost with 1.0 mm multipinhole apertures. All mice were sedated with isoflurane, and SPECT images were acquired 0 h (with 5 or 6 acquisitions at 15 min intervals), 5 h and 24 h post-injection in 16 projections using time per projection of 45, 90 and 180 s, respectively. CT imaging was accomplished with 45 kVp tube voltage in 180 projections. For 3D co-registration and analysis,

the SPECT images were reconstructed with HiSPECT NG software (Scivis GmbH, Germany) and fused with CT datasets by using the molecular imaging suite InVivoScope™ (Bioscan Inc., USA). In the analysis, volumes of interests (VOI’s) were drawn at the injection site (whole NFC implant), thyroid glands, stomach, left kidney, heart, and around CYTH4 the striatum depending on the study compound, respectively. Counts within each VOI were recorded, corrected for radioactive decay, and normalized to the activity at the time of injection. 99mTc-HSA release kinetics was described using the built-in 1-compartmental models of Phoenix® WinNonlin® (Pharsight, Mountain View, USA). The saline preparations were assumed to be 100% available for absorption immediately after injection. The pharmacokinetic (PK) data obtained from the saline injections were observed against the data obtained from the hydrogels.