Fentanyl citrate

Biolayer interferometry (BLI) was performed on an Octet Red 96e system (Sartorius). Streptavidin-coated biosensors were pre-hydrated with PBS-T (phosphate buffered saline pH 7.5, 0.05% Tween 20), and baseline responses were measured in PBS-T. Biosensors were loaded with biotinylated fentanyl-hapten (where the biotin replaces the poly-gly sequence from fen-G4; biotinylated fentanyl derivative syntheses are described in^{33 (link)}), 0.2 μg/mL for 60 s. Association of antibody with biotinylated hapten was measured in PBS-T with 5 sample concentrations ranging from 5 nM–200 nM for 180 s, followed by dissociation in PBS-T for 300 s. Isothermal titration calorimetry (ITC) experiments were performed using Microcal PEAQ-ITC (Malvern, UK). For all experiments, PBS was used to dissolve fentanyl-citrate (Sigma, 2 mg/mL), fentanyl-hapten, and for the final purification step of Fabs. Titrations were performed at 25°C by injecting consecutive (1–3 μL) aliquots of fentanyl or fentanyl-hapten (both 50 μM) into Fab fragment (4–7 μM) with 120 s intervals. The binding data was corrected for the heat of dilution and fit to a one-site binding model to calculate the K_d, and the binding parameters, N and ΔH. Binding sites were assumed to be identical.

The phospholipids, DMPC and DMPG (both > 99% purity) were purchased from Sygena, Inc. (Cambridge, MA). Palmitoylated peptide Gly-Arg-Gly-Asp-Ser (GRGDS) (> 99.9% purity) was custom synthesized by United Biochemical Research, Inc. 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl); NBD-PE (> 99% purity) was purchased from Avanti Polar Lipids (Alabaster, AL). Fentanyl citrate was purchased from Sigma-Aldrich (St. Louis, MO). Beuthanasia-D (Schering-Plough Animal Health Corp, North Chicago, IL) was used for euthanasia at the end of the distribution studies. Fentanyl was dissolved in 0.9% saline solution (Hospira Inc, Lake Forest, IL). All other chemicals used were of analytical grade.

We purchased fentanyl citrate from Sigma, dissolved it in sterile bacteriostatic saline at 12.5mg/ml, and diluted the drug further in sterile saline to a concentration of 44.65 μg/ml/kg. Injection duration controlled the dose, and the rate of infusion was constant at 28 μl/second. Injection duration was 2 seconds during acquisition and intermittent access phases of the experiment (2.5μg/kg/injection, using the weight of the salt - fentanyl citrate). We based the acquisition doses on prior studies^{20 (link)} and our pilot studies in which we observed robust drug lever responding at this dose. We obtained 2,5-dimethoxy-4-iodoamphetamine hydrochloride (DOI) from Cayman Chemicals, dissolved it in saline, and injected it i.p. at a volume of 1.0 ml/kg. We obtained M100907 from MedChem Express and dissolved it in absolute ethanol (1mg/100μl), followed by the addition of emulphur oils at equal volume. We mixed these vehicles thoroughly with sonication and vortexing before adding saline at 18X the volume of ethanol. M100907 solutions were prepared the day of use and injected at a volume of 0.6 ml/kg at a final concentration of 0.5mg/ml. The M100907 dose of 0.3mg/kg was based on a previous study^{21 (link)} using this dose for effective 5-HT_2A blockade. Vehicle for DOI was saline and vehicle for M100907 experiments was 5% EtOH, 5% Emulphur, 90% Saline.