ADL5859 HCl

2 years -20°C Powder, 2 weeks 4°C in DMSO, 2 months -80°C in DMSO

Administered via p.o.

Chinese hamster ovary (CHO) cells stably expressing human kappa, u, or delta opioid receptors

10 mg/kg - 300 mg/kg

0.8 nM (Ki), 0.8 nM (Ki), 0.8 nM (Ki), 0.8 nM (Ki), 0.8 nM (Ki), 0.8 nM (Ki)

At the screening dose of 3 mg/kg p.o., ADL5859 produces 100% reversal of hyperalgesia in the inflamed paw. The oral ED50 of ADL5859 in the FCA mechanical hyperalgesia assay is 1.4 mg/kg. The antihyperalgesia produced by ADL5859 (3 mg/kg, p.o.) is reversed by pretreatment with the delta opioid antagonist naltrindole (0.3 mg/kg s.c.), thus demonstrating a delta receptor mediated effect.In the rat forced swim assay, ADL5859 (3 mg/kg p.o.) produces robust antidepressant-like activity, as evidenced by a significant decrease in the time spent immobile and a significant increase in the time spent swimming. The bioavailability of ADL5859 (3 mg/kg p.o.) in rats and dogs is 33% and 66%, respectively.[1]ADL5859 efficiently reduces inflammatory and neuropathic pain mainly by recruiting delta-opioid receptors expressed by peripheral Nav1.8-expressing neurons.[2]

Membrane preparations from Chinese hamster ovary (CHO) cells stably expressing human kappa, u, or delta opioid receptors are prepared. The assay buffer used is composed of 50 mMtris(hydroxymethyl) aminomethaneHCl, pH 7.8, 1.0 mM ethylene glycol bis(beta-aminoethyl ether)-N, N, N', N'-tetraacetic acid (EGTA free acid), 5.0 mM MgCl2 10 mg/L leupeptin, 10 mg/L pepstatin A, 200 mg/L bacitracin, and 0.5 mg/L aprotinin. After dilution in assay buffer and homogenization in a Polytron homogenizer for 30 seconds, membrane proteins (10-80 ug) in 250 uL of assay buffer are added to mixtures containing ADL5859 and [3H]diprenorphine (0.5-1.0 nM, 25000-50000 dpm) in 250 uL of assay buffer in 96-well deep-well polystyrene titer plates and incubated at room temperature for 60 minutes. Reactions are terminated by vacuum filtration with a Brandel MPXR-96T harvester through GF/B filters that have been pretreated with a solution of 0.5% polyethylenimine and 0.1% bovine serum albumin for at least 1 hour. The filters arewashed four times with 1.0 mL each of ice-cold 50 mM Tris-HCl, pH 7.8, and 30 uL of Microscint-20 is added to each filter. Radioactivity on the filters is determined by scintillation spectrometry in a Packard TopCount. [3H]Diprenorphine with a specific activity of 50 Ci/mmolisused. The Kd values for [3H]diprenorphine binding are 0.33 nM for the kappa and u receptors and 0.26 nM for the delta receptor. Receptor expression levels, determined as Bmax values from Scatchard analyses, are 4400, 4700, and 2100 fmol/mg of protein for the kappa, u, and delta receptors, respectively. Preliminary experiments are performed to show that no specific binding is lost during the wash of the filters, that binding achieved equilibrium within the incubation time and remained at equilibrium for at least an additional 60 minutes, and that binding is linear with regard to protein concentration. Nonspecific binding, determined in the presence of 10 uM unlabeled naloxone, is less than 10% of total binding. Protein is quantified by the method of Bradford. The data from competition experiments are fit by nonlinear regression analysis with the program Prism using the four-parameter equation for one-site competition, and Ki values are subsequently calculated from EC50 values by the Cheng-Prusoff equation.

ADL5859 HCl is a delta-opioid receptor agonist with Ki of 0.8 nM.