*4.5. Study Drugs*

Naloxone Hydrochloride provided in pre-filled syringes (International Medication Systems Ltd., South El Monte, CA, USA) was utilized for IM injections of the two primates at doses of 0.06 mg/kg, 0.14 mg/kg, and 0.28 mg/kg. Synthesis and quality control testing of [11C]carfentanil was conducted as previously described [13]. Briefly, [11C]CFN was prepared in a TRACER lab FXC-Pro synthesis module (GE Healthcare, Uppsala, Sweden) fitted with an Agilent Bond Elut C2 cartridge (Agilent Technologies, Santa Clara, CA, USA). [11C]MeOTf (~1 Ci) was bubbled into a solution of desmethyl carfentanil TBA salt (0.5 mg) (MilliporeSigma, Burlington, MA, USA, prepared as described in [13]) in ethanol, USP (100 μL) (Akorn, Lake Forest, IL, USA) at room temperature for 3 min. After this time, 1% NH4OH (1 mL) (Fisher Scientific, Hampton, NH, USA) was added to the reaction vessel. This crude reaction mixture was further diluted with 1% NH4OH (5 mL), and the resulting mixture was passed through the Agilent Bond Elut C2 cartridge to trap [11C]CFN. The cartridge was washed with 20% EtOH (3 mL) (Decon Laboratories, King of Prussia, PA, USA), followed by Milli-Q water (10 mL), to remove unreacted precursor and impurities from the cartridge, and dried for 1.0 min with He gas. [11C]CFN was eluted with EtOH, USP (0.5 mL) and diluted with Sterile Water for Injection, USP (9.5 mL) (Hospira, Lake Forest, IL). The formulated product was then passed through a Millipore-GV 0.22-μm filter (MilliporeSigma, Burlington, MA, USA) into a sterile dose vial (Jubillant Hollister-Stier, Spokane, WA) and analyzed for pH, radiochemical purity, mass of CFN, and molar activity as previously described [13].

#### *4.6. Plasma Naloxone Assay*

A quantitative LC-MS/MS method with an internal standard was developed for use in determination of the concentration of NLX in monkey plasma. Specificity, range, and linearity were assessed. For specificity, extracted ion chromatograms of monkey plasma, and monkey plasma spiked with an internal standard, demonstrated that monkey plasma does not interfere with NLX or internal standard quantification. For linearity and range, the analytical calibration curve was constructed with 8 non-zero standards by plotting the peak area ratio of NLX to the internal standard versus the concentration. The concentration range was evaluated from 0.5 to 100 ng/mL for quantification. A blank sample (matrix sample processed without internal standard) was used to exclude contamination or interference. The curve was assessed with weighted linear regression (1/X2). The linearity of the relationship between peak area ratio and concentration was demonstrated by the correlation coefficient (r = 0.9974).
