Animals
The animals for this study were four Maccaca mulatta (1 female, 11 years old, 12.0 kg; 3 males, 6–13 years old, 10.3–18.0 kg). A total of 10 PET scans were conducted, including 8 brain-only acquisitions and 2 whole body acquisitions for dosimetry estimates. On the day of scans, subjects were initially sedated with combination of alphaxalone (1–2 mg/kg), midazolam (0.3 mg/kg) and dexmedetomidine (0.01 mg/kg) at least 2 h prior to radiotracer administration. Subjects were then maintained on oxygen and 1.5-3% isoflurane for the duration of scans. Vital signs, SpO2, and end tidal CO2 were continuously monitored and recorded, including respiration rate, blood pressure, heart rate, and temperature. All experiments followed institutional guidelines and were approved by the Yale University Institutional Animal Care and Use Committee.
Radiochemistry
Radiosynthesis of [18F]TZ4877 was accomplished through nucleophilic radiofluorinaton starting with between the tosylate precursor to reach with [18F]KF/Kryptofix 222 in acetonitrile, followed by removal of the methoxymethyl (MOM) protecting group. [18F]Fluoride was produced via the 18O(p.n)18F nuclear reaction utilizing H218O (Huayi Isotopes, Toronto, Canada) within a 16.5 MeV GE PETtrace cyclotron (Uppsala, Sweden). Following bombardment, the resultant activity was promptly transferred to a shielded hot cell and trapped on an anionic exchange resin cartridge (Chromafix PS-HCO3, Macherey-Nagel, Dueringen, Germany). The trapped [18F]F− was subsequently eluted in a 2 mL borosilicate glass reaction vial using 1 mL solution comprising Kryptofix 222 (0.7 mL, 10 mg/mL in acetonitrile) and K2CO3 (0.3 mL, 2 mg/mL in deionized (D.I.) water. The solution was azeotropically dried for 5 min at 110°C under argon gas, followed by two rounds of drying with 1.0 mL MeCN under argon gas. To the dried [18F]F− was then added a solution of the tosylate precursor (2 mg precursor in 0.4 anhydrous acetonitrile). The reaction mixture was stirred at 110°C for 15 min, followed by addition of 6 M HCl (150 µL) and heating at 110 oC for another 10 min. The reaction was cooled, quenched with 6 M NaOH (150 µL), and diluted with 1.2 mL of the HPLC mobile phase (47% acetonitrile in 0.1 M ammonium formate buffer, pH = 4.2). The mixture was then filtered through a Alumina N light Sep-Pak® Cartridges (Pat No. WAT023561) and injected onto the semi-preparative HPLC (Waters Xbridge®BEH C18 column, 10 mm x 250 mm, 5 µm, UV = 254 nm, 4.0 mL/min). The HPLC fraction containing [18F]TZ4877 (retention time around 22–24 min) was collected, diluted with 50 mL of DI water, and then passed through a light C18 Sep-Pak™ cartridge (Pat No. WAT023501). The C18 cartridge was washed with 0.001 N HCl (10 mL) and dried with argon flow. The product was eluted off the C18 cartridge sequentially with 1 mL of USP ethanol and 3 mL of USP saline, and passed through a 0.22 µm membrane filter (GV, Millipore, Sigma) into a 10 mL dose vial pre-charged with 7 mL of USP saline and 8.4% NaHCO3 solution (20 µL). An aliquot of the final production solution was injected onto an analytic HPLC to determine radiochemical purity and molar activity. Tracer authentication was performed by co-injection with non-radiolabeled standard TZ4877 sample solution. The analytic HPLC conditions were: Phenomenex Luna C18(2) column, UV absorbance at 260 nm, with mobile phase of 55% CH3CN in 0.1 M ammonium formate with 5% AcOH (pH = 4.2) at a flow rate of 2.0 mL/min. The retention time of [18F]TZ4877 was 7.4 min.
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Brain PET Scanning Procedures
Brain-dedicated scans consisting of a baseline scan, preblocking scans with ponesimod and TZ82112, and an endotoxin challenge (see below) were acquired in each of two animals for a total of 10 brain studies. These data were acquired with a Focus 220 PET scanner (Simens/CTI, Knoxville, TN). A transmission scan was first acquired using a continuously rotating 57Co source for 9 min. [18F]TZ4877 was administered as a 118–163 MBq slow bolus injection over 3 min with a Harvard syringe pump (PHD 22/2000, Harvard Apparatus, Holliston, MA). PET data were then continuously acquired in list-mode for 120–180 min. An arterial line for blood sampling was inserted in a radial or femoral artery on the limb opposite the tracer administration line. Discrete arterial blood samples were acquired throughout PET scanning, with rapid (45 s) sampling immediately post-injection and gradually slowing to 30 min sampling at the end of scans.
Arterial Input Function Measurement
Radioactivity assay of arterial blood samples was performed with a cross calibrated well-type gamma counter (Wizard 1480, Perkin Elmer, Waltham, MA). Whole blood samples were assayed and then centrifuged (2,930 g for 5 min). Plasma samples were then separated and assayed for radioactivity. Select plasma samples (drawn at 3, 8, 15, 30, 60, 90, 120, 180 min post-injection) were analyzed with HPLC to measure radioligand metabolism.
For metabolite analysis, plasma samples were mixed with urea to a final concentration of 8 M and filtered through 1.0 µm Whatman 13 mm CD/X filters (GE, Florham Park, NJ). Samples were then analyzed on an adapted column-switching HPLC system20. Upon injection, samples were first trapped on a C18 sorbent capture column (Strata-X, Phenomenex, Torrance CA) with a mobile phase of 1:99 (v:v) MeCN:H2O at 2 mL/min for 4 min. The capture column was then backflushed with a mobile phase of 45% acetonitrile and 55% 20 mM ammonium bicarbonate (v/v), and the eluent passed through a Phenomenex Gemini-NX analytical column (5 µm, 4.6 × 250 mm) at a flow rate of 1.65 mL/min. The eluent was collected with a fraction collector (CF-1 Fraction Collector, Spectrum Chromatography, Houston, TX) in discrete 2 min bins and counted in a gamma counter (Wizard 1480, Perkin Elmer, Waltham, MA). The fraction of unmetabolized parent was measured as the ratio of the eluted parent (retention time of ~ 12 min) to the total radioactivity collected. The time course of this parent fraction was fitted to an inverted gamma function and corrected for filtration efficiency. Finally, the input function was calculated as the product of the assayed radioactivity concentration in plasma and the unmetabolized parent fraction.
In addition, the free fraction (fP) was determined from plasma samples with ultrafiltration techniques. An arterial blood sample drawn prior to radiotracer injection (3.0 mL) was vigorously mixed with ~ 3 kBq of [18F]TZ4877. After partitioning the plasma from red blood cells via centrifugation, the plasma sample was extracted, loaded onto an ultrafiltration cartridge (Millipore Centrifree UF devices), and centrifuged at 1,228 g for 20 min. The free fraction was calculated as the ratio of radioactivity in the ultrafiltrate to the total radioactivity in the plasma sample. Measurements of fP were performed in triplicate for each scan.
Challenge Studies with [18F]TZ4877
To evaluate [18F]TZ4877 specific binding, blocking studies were acquired with each of ponesimod, and TZ8112, a compound with high affinity (IC50 value of 9 nM) and selectivity for S1PR121. Ponesimod (0.047 or 0.063 mg/kg) was infused 15–18 min before [18F]TZ4877 injection, with PET image data acquired for 120 min. TZ8112 (0.4 or 0.8 mg/kg) was infused 33 min before [18F]TZ4877 injection, with PET image data acquired for 120 min.
To evaluate the effects of an acute immune stimulus on [18F]TZ4877 uptake, the classic acute immune stimulus lipopolysaccharide (LPS; NIH Clinical Center Reference E. coli serotype O:113) was used. A dose of 1 ng/kg, previously shown to trigger acute immune stimulus22,23, was administered 167–195 min before [18F]TZ4877 injection, with PET image data acquired for 180 min. Cytokine levels for TNF-α, IL–1β, IL-6, IL-8, IL-18, IL-22, IL-28A, and MCP-1 were measured in plasma samples taken 10 minutes prior to LPS administration and 90, 180, and 300 min after LPS injection. Cytokine levels were measured in triplicate using MILLIPLEX panel assay (MillporeSigma, Burlington, MA, USA).
Anatomical MRI Acquisition
High-resolution T1-weighted images were acquired for image co-registration and region of interest (ROI) identification. MR data were acquired prior to PET image acquisition with a Siemens 3T Trio scanner, with an extremity head coil in the coronal direction and the following spin echo sequence (TE = 3.34 ms, TR = 2530 ms, flip angle = 7°, thickness = 0.50 mm, field-of-view = 140 mm, image matrix = 256 × 256 × 176, voxel size = 0.547 × 0.547 × 0.500 mm). Non-brain structures were removed with FMRIB’s Brain Extraction Tool (http://www.fmrib.ox.ac.uk/fsl/BET).
Brain PET Data Processing
Raw list-mode PET data was histogrammed (frames of 6 × 0.5 min; 3 × 1 min; 2 × 2 min; and N×5 min to scan termination) and reconstructed with Fourier rebinning followed by 2D filtered back projection, using a Shepp filter and including corrections for scanner normalization, detector deadtime, randoms, scatter, and attenuation. This resulted in a reconstructed image resolution of ~ 3.2 mm. The PET images were then registered to MR image space with a 6-parameter rigid body registration24. The MR native space was then normalized using nonlinear affine registration to a high-resolution rhesus monkey atlas25 using BioImage Suite 3.01 (http://www.bioimagesuite.org/index.html). Time-activity curves were extracted by mapping atlas-defined regions to PET native space using the optimal transformation matrices calculated in the registration and normalization steps. ROIs extracted included caudate, cerebellum, frontal cortex, hippocampus, pons, putamen, temporal cortex, and thalamus.
[18F]TZ4877 Kinetic Analysis
For all PET scans, the primary outcome measure was the total volume of distribution26, both uncorrected (VT) and corrected (VT/fP) by the plasma free fraction. Regional VT values were estimated using both one-tissue (1TCM) and two-tissue (2TCM) compartment models (see 27 for review). Model suitability was compared with the corrected Akaike Information Criterion (cAIC; 28). Additionally, the multilinear analysis method (MA1; 29) was assessed as a more stable, data-driven analysis method. To visualize [18F]TZ4877 VT images, MA1 was also used to calculate VT on the voxel level. Receptor occupancy (Occ) and nondisplaceable volume of distribution (VND) was estimated with occupancy plots using the following Eq. 30:
V T/fP(baseline) - VT/fP(block) = Occ(VT/fP(baseline) – VND/fP)
Radiation Dosimetry Study
The 2 whole body biodistribution scans were acquired in two animals (18.0 kg Male, 12.0 kg F). These data were acquired with a Biograph Vision PET/CT system (Siemens Medical Systems, Knoxville, TN) after i.v. injection of 94 MBq and 86 MBq [18F]TZ4877. Animals were imaged for approximately 3 hours in a sequence of 22 passes from top of the head to the mid-thigh. Images were reconstructed and visually inspected for organ activity concentrations exceeding background level. The organs included were heart, bladder, testes, kidney, liver, and gallbladder. Regions of interest were hand-delineated on these organs to compute mean time activity curves.
Within-pass decay correction was removed to reflect the actual activity in each organ, and the tail portions of each curve beyond the end of the scan were extrapolated assuming only physical decay of the radiotracer. The cumulative activity (Bq⋅h/cm3) was computed by integrating these data. These values were multiplied by the organ volumes of standard 60 kg adult female and 73 kg adult male reference mathematical phantoms31 and normalized to injected activity to obtain organ residence times. Residence times were then entered into OLINDA/EXM 2.0 software to compute absorbed doses in all organs32, which were computed without voiding.