Naloxone’s dose-dependent displacement of [11C]carfentanil and duration of receptor occupancy in the rat brain

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. The study was designed in accordance with ARRIVE guidelines. [11C]CFN PET studies were performed in male Long Evans rats (n = 63) using a small animal PET scanner (MicroPET Focus 220, Siemens). For the displacement study, nine rats underwent PET scans (290.3 ± 26.4 g, Charles River Laboratories), while for the RO study, 48 rats were used (310.7 ± 72.3 g, Charles River Laboratories). Six rats were performed for the plasma pharmacokinetic analysis (225.5 ± 28.6 g, Charles River Laboratories).

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) injections were performed using a programmable pump (Pump 11 Elite, Harvard Apparatus). Blood plasma was obtained by centrifugation (MiniSpin, Eppendorf). [11C]CFN was synthesized according to the reported procedure with minor modifications40,41 (Supplementary materials).

Rodent PET studies

Anesthesia in rats was 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: 40, 60, 90, 110, 210 min). List-mode data was acquired over 80 min after a 10 min transmission scan with a Co-57 point source for attenuation correction. PET data was reconstructed into 22 frames (6 × 20 s, 5 × 60 s, 4 × 120 s, 3 × 300 s, 3 × 600 s, and 1 × 1200 s) 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 MORs42 and high specific binding, and the cerebellum, which was used as a reference region mostly devoid of specific binding43.

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, the value of (SUV_{r} – 1) was used as an apparent specific binding measurement. B/CI method achieved constant radioactivity levels in the ROIs and in the reference region44,45 and consistent with this we obsereved that activity curves were plateaued after 15 min. Therefore, the value of SUVr was calculated directly from the concentration ratio of thalamus to cerebellum (15–40 min). Receptor occupancy [RO(%)] was calculated using Eq. (1)46:

$$RO;(% ) = 100 times frac{{baseline; (SUV_{r} – 1) – post, drug ;(SUV_{r} – 1) }}{{baseline ;(SUV_{r} – 1)}}.$$


Plasma pharmacokinetics assessment of IV NLX

To determine plasma concentrations of NLX over time, a NLX bolus was administered to 6 male rats (225.5 ± 28.6 g; n = 3, 0.035 mg/kg; n = 3, 0.17 mg/kg) via penile vein, and arterial whole blood samples (250 μL) 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 of 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, Cu, and the Kd values for the MOR, RO was calculated according to the reaction kinetics between a MOR and NLX, as follows47:

$$Occupancy;(% ) = frac{{C_{u} }}{{C_{u} + K_{d} }}.$$

Statistical analysis

The descriptive statistics and computations for data analysis were performed using MATLAB and Statistics Toolbox Release 2012b, The MathWorks, Inc., Natick, Massachusetts, United States. In all analyses, the statistical significance (alpha level) was set at p < 0.05.