General
The precursor for [11C]CFN, Desmethylcarfentanil acid, was purchased from American Biochemicals (College Station, TX, USA). All other chemicals were purchased from Millipore Sigma and used without any further purification. No-carrier-added [11C]CO2 was generated by nuclear reaction 14N(p,α)11C, bombarding a nitrogen gas target containing 1% oxygen with a proton beam using a cyclotron (PETrace, GE). Conversion to [11C]CH3I and [11C]methylation were performed using FX-MeI and FX-M automated synthesizers (GE Healthcare, Chicago, IL USA), respectively. Semi-preparative high performance liquid chromatography (HPLC) was used to purify a crude [11C]CFN mixture with a monolithic column (10x100 mm, Onyx Monolithic C18, Phenomenex; flow rate, 5 mL/min; eluent, 0.01M phosphate buffer/ethanol=51.5/48.5; pH = 7.2-7.4; UV wavelength, 218 nm; retention time, 8 min). For quality control, analytical HPLC analysis was performed using an Agilent 1100 system equipped with a ZORBAX Eclipse XDB C18 column (4.6x150mm, Agilent, Santa Clara, CA), monitoring for absorbance at 218 nm and radioactivity using a flow count radioactivity detector (Carroll Ramsey and Associates, Fort Collins, CO, USA). [11C]CFN was eluted at a flow rate of 1.0 mL/min (retention time, 5 min) with an isocratic solvent mixture (water/acetonitrile, 60/40) containing trifluoroacetic acid (0.1%).
All rat studies were approved by the Clinical Center Animal Care and Use Committee of National Institutes of Health (protocol number, NIAAA 19-01) and complied with the Guide for the Care and Use of Laboratory Animals. [11C]CFN PET studies were performed in male Long Evans rats (294.7 ± 69.6 g, Charles River Laboratories) using a small animal PET scanner (MicroPET Focus 220, Siemens). Animals were anesthetized with isoflurane (Forane, Baxter Healthcare) using an anesthesia machine (SurgiVet VaporStick, Smiths Medical) and vaporizer (SurgiVet 100 Series, Smiths Medical). Vitals (heart rate, respiratory rate, spO2, and temperature) were monitored using a pulse oximeter and heart rate monitor (MouseSTAT, Kent Scientific). A heat lamp was used to maintain body temperature (Model# 51152, Brandt Industries). Tubing for catheters (BTPE-10 for infusion, BTPU-27 for blood withdrawal) and other surgical materials were obtained from Instech Laboratories. Bolus [11C]CFN injections were performed using a syringe pump (PHD 2000, Harvard Apparatus), while bolus plus constant infusion (B/CI) were performed using a programmable pump (Pump 11 Elite, Harvard Apparatus). Blood plasma was obtained by centrifugation (MiniSpin, Eppendorf). The study is in accordance with ARRIVE guidelines.
Synthesis of [11C]Carfentanil ([11C]2)
[11C]CFN was synthesized according to the reported procedure with minor modifications [40, 41]. Briefly, anhydrous DMSO (200 µL) solution containing desmethylcarfentanil acid (1 mg, 2.64 µmol), Cs2CO3 (2 mg, 6.14 µmol) was vortexed for 1 min. After [11C]methyl iodide was transferred in a stream of helium at room temperature, the reaction mixture was heated at 120°C for 3 min. Crude mixture was purified with semi-preparative HPLC. The collected portion of [11C]CFN was adjusted to contain less than 10% ethanol content for injecitons.
Rodent PET Studies
Anensthesia in rats were initially induced with isoflurane (5.0%) in oxygen for 5 min and then was maintained at a lower level of isoflurane (1.5-2.5%), monitoring vitals throughout the experiments. Catheters were placed in the left femoral vein for [11C]CFN injection. For displacement studies, [11C]CFN was administered as a bolus (1 min), followed by IV NLX 15 min later. For RO studies, [11C]CFN was administered via a B/CI method (Kbol= 80 min) that lasted the entire duration of each scan. Before radiotracer injection, rats were pretreated with IV NLX at selected time points (0.035 mg/kg: 20, 40, 60, 87, 180 min; 0.17 mg/kg: 10, 15, 40, 60, 90, 110, 210 min). List-mode data was acquired over 90 min after a 10 min transmission scan with a Co-57 point source for attenuation correction. PET data was reconstructed into 22 frames (6 x 20 sec, 5 x 60 sec, 4 x 120 sec, 3 x 300 sec, 3 x 600 sec, and 1 x 1200 sec) using filtered back-projection. The average activity injected was 14.0 ± 8.6 MBq and the average CFN mass injected was 60.4 ± 55 ng/kg.
PET imaging processing and tracer kinetic analysis
Time-activity curves were obtained as standard uptake value (SUV, g/mL) using PMOD (3.807). Two regions of interest (ROIs) were analyzed for [11C]CFN uptake: the thalamus due to its high concentration of MORs [42] and high specific binding, and the cerebellum, which was used as a reference region mostly devoid of specific binding [43].
The ROI template was drawn using anatomical information extracted from a [18F]FDG PET scan obtained for this purpose following a [11C]CFN scan in one rat. ROIs were drawn in the cerebellum and thalamus, avoiding border regions, and were applied to generate time activity curves.
SUVr was calculated for each frame as the ratio between thalamic and cerebellar SUVs. For RO studies, BPND was obtained using the B/CI method to achieve constant radioactivity levels in the ROIs and in the reference region [44, 45]. Once equilibrium is achieved [46], the binding potential (BPND ) was calculated directly from the concentration ratio of thalamus to cerebellum (15-40 min). The value of BPND can be computed using BPND = Cthalamus/Ccerebellum – 1. Receptor occupancy [RO(%)] was calculated using Equation 1 [47]:
$$RO\left(\%\right)=100 \times \frac{{Baseline(BP}_{ND})-{Post\_drug(BP}_{ND}) }{{Baseline(BP}_{ND})-1}$$
1
Plasma pharmacokinetics assessment of IV NLX
To determine plasma concentrations of NLX over time, a NLX bolus was administered to 6 rats (n=3, 0.035 mg/kg; n=3, 0.17 mg/kg) via penile vein, and arterial whole blood samples (250 uL) were collected at 0, 1, 3, 5, 10, 15, 30, 45, 60, and 90 min after NLX injection. Each blood sample was centrifuged at 14,500 RPM for 3 min to give each plasma sample, followed by immediate freezing on dry ice until stored at -80°C. Plasma NLX concentration was determined using LC-MS/MS (Bioanalytical Shared Resource Laboratory, Virginia Commonwealth University School of Pharmacy), with a detection limit of NLX was 1 ng/mL. Pharmacokinetics parameters were estimated by non-compartmental analysis and plasma curves were fitted using two exponential clearance model.
Given the plasma concentration and the Kd values for the MOR, RO was calculated according to the reaction kinetics between a MOR and NLX, as follows [48]:
$$Occupancy\left(\%\right)= \frac{{C}_{u}}{{C}_{u}+{K}_{d}}$$