Several investigators recently identified biased κ opioid receptor (KOP receptor) agonists. However, no comprehensive study of the functional selectivity of available KOP receptor agonists at the human and mouse KOP receptors (hKOP receptor and mKOP receptor, respectively) has been published. Here we examined the ability of over 20 KOP receptor agonists to activate G proteins and to internalize the receptor. Clonal neuro-2a mouse neuroblastoma (N2a) cells stably transfected with the hKOP receptor or mKOP receptor were used. We employed agonist-induced [(35)S]GTPγS binding and KOP receptor internalization as measures of activation of G protein and β-arrestin pathways, respectively. The method of Ehlert and colleagues was used to quantify intrinsic relative activities at G protein activation (RAi-G) and receptor internalization (RAi-I) and the degree of functional selectivity between the two [Log RAi-G - logRAi-I, RAi-G/RAi-I and bias factor]. The parameter, RAi, represents a relative estimate of agonist affinity for the active receptor state that elicits a given response. The endogenous ligand dynorphin A (1-17) was designated as the balanced ligand with a bias factor of 1. Interestingly, we found that there were species differences in functional selectivity. The most striking differences were for 12-epi-salvinorin A, U69,593, and ICI-199,441. 12-Epi-salvinorin A was highly internalization-biased at the mKOP receptor, but apparently G protein-biased at hKOP receptor. U69,593 was much more internalization-biased at mKOP receptor than hKOP receptor. ICI199,441 showed internalization-biased at the mKOP receptor and G protein-biased at the hKOP receptor. Possible mechanisms for the observed species differences are discussed.

Nalfurafine, a moderately selective kappa opioid receptor (KOR) agonist, is used in Japan for treatment of itch without causing dysphoria or psychotomimesis. Here we characterized the pharmacology of compound 42B, a 3-dehydroxy analogue of nalfurafine and compared with that of nalfurafine. Nalfurafine and 42B acted as full KOR agonists and partial μ opioid receptor (MOR) agonists, but 42B showed much lower potency for both receptors and lower KOR/MOR selectivity, different from previous reports. Molecular modeling revealed that water-mediated hydrogen-bond formation between 3-OH of nalfurafine and KOR accounted for its higher KOR potency than 42B. The higher potency of both at KOR over MOR may be due to hydrogen-bond formation between nonconserved Y^7.35 of KOR and their carbonyl groups. Both showed modest G protein signaling biases. In mice, like nalfurafine, 42B produced antinociceptive and antiscratch effects and did not cause conditioned place aversion (CPA) in the effective dose ranges. Unlike nalfurafine, 42B caused motor incoordination and hypolocomotion. As both agonists showed G protein biases, yet produced different effects on locomotor activity and motor incoordination, the findings and those in the literature suggest caution in correlating in vitro biochemical data with in vivo behavior effects. The factors contributing to the disconnect, including pharmacodynamic and pharmacokinetic issues, are discussed. In addition, our results suggest that among the KOR-induced adverse behaviors, CPA can be separated from motor incoordination and hypolocomotion.