Selective kappa opioid receptor (KOR) agonists are promising antipruritic agents and analgesics. However, clinical development of KOR agonists has been limited by side effects, including psychotomimetic effects, dysphoria, and sedation, except for nalfurafine, and recently. CR845 (difelikefalin). Activation of KOR elicits G protein- and β-arrestin-mediated signaling. KOR-induced analgesic and antipruritic effects are mediated by G protein signaling. However, different results have been reported as to whether conditioned place aversion (CPA) induced by KOR agonists is mediated by β-arrestin signaling. In this study, we examined in male mice if there was a connection between agonist-promoted CPA and KOR phosphorylation and internalization, proxies for β-arrestin recruitment in vivo using four KOR agonists. Herein, we demonstrated that at doses producing maximal effective analgesic and antiscratch effects, U50,488H, MOM-SalB, and 42B, but not nalfurafine, promoted KOR phosphorylation at T363 and S369 in mouse brains, as detected by immunoblotting with phospho-KOR-specific antibodies. In addition, at doses producing maximal effective analgesic and antiscratch effects, U50,488H, MOM-SalB, and 42B, but not nalfurafine, caused KOR internalization in the ventral tegmental area of a mutant mouse line expressing a fusion protein of KOR conjugated at the C-terminus with tdTomato (KtdT). We have reported previously that the KOR agonists U50,488H and methoxymethyl salvinorin B (MOM-SalB) cause CPA, whereas nalfurafine and 42B do not, at doses effective for analgesic and antiscratch effects. Taken together, these data reveal a lack of connection between agonist-promoted KOR-mediated CPA with agonist-induced KOR phosphorylation and internalization in male mice.
The kappa opioid receptor (KOR) is present in neuronal pathways involved in transmission of pain and itch sensations. KOR agonists produce analgesic and anti-itch effects; however, they also cause unwanted effects, such as dysphoria, psychotomimetic effects, motor incoordination, and sedation in humans. In rodents, KOR agonists produce analgesia, anti-itch, motor incoordination, sedation, and conditioned place aversion (CPA), an animal model of dysphoria. Clinical development of KOR agonists as analgesics and antipruritic agents has been limited by the side effects, except for nalfurafine, and recently, difelikefalin (formerly CR845). Nalfurafine has been approved in Japan for treatment of pruritus associated with chronic kidney or liver, or kidney dialysis. At the therapeutic doses of nalfurafine, dysphoria, hallucination, motor incoordination, and sedation were not reported as major adverse events in humans. In animals, whether nalfurafine causes side effects depends on the dose used. It has been demonstrated that nalfurafine produces antipruritic and antinociceptive effects in dose ranges lower than those causing side effects, including conditioned place aversion (CPA), hypolocomotion, motor incoordination, consistent with human data. However, nalfurafine did cause CPA, motor incoordination, and hypolocomotion at higher doses. Difelikefalin, a peripherally acting KOR peptide agonist, was recently approved by FDA for the treatment of systemic itch in hemodialysis patients.
The KOR belongs to the rhodopsin subfamily of the G protein-coupled receptor (GPCR) superfamily. At the cellular level, activation of the KOR stimulates Gi/o proteins and promotes receptor phosphorylation. Phosphorylated KOR recruits β-arrestins, which leads to desensitization and internalization of the receptor as well as β-arrestin-mediated signaling. Activation of Gi/o proteins inhibits adenylyl cyclases and Ca++ channel conductance and enhances activities of K+ channels and ERK1/2 (early phase), and β-arrestin-mediated signaling includes activation of ERK1/2 (late phase) and p38 MAPK.
Biased agonism or functional selectivity is a popular concept in GPCR pharmacology. Per this concept, G protein signaling and β-arrestin signaling produce different in vivo behaviors. Therefore, agonists preferentially activating G protein or β-arrestin signaling may have therapeutic advantages with fewer side effects than unbiased agonists. It is generally agreed that KOR agonists produce analgesic and anti-itch effects via G protein signaling pathways. However, literature on the role of β-arrestin signaling in CPA resulting from KOR activation is inconsistent. Investigations by Roth’s group and Bohn’s group showed that in male mice, β-arrestin2 deletion decreased impaired rotarod performance induced by KOR agonists, but did not affect KOR agonist-produced analgesia, anti-scratch effect, CPA, or hypolocomotion. Chavkin and coworkers demonstrated that GRK3 deletion in mice abolished U50,488H-induced CPA, and KOR-promoted CPA was GRK3 and p38 MAPK dependent. Both GRK3 and β-arrestin2 interact with many GPCRs; thus, the possibility that the impact of either deletion may be through indirect effects on other GPCRs cannot be excluded. In addition, β-arrestin1 may compensate for the absence of β-arrestin2, thus, obscuring the role of β-arrestin2. In this study, we examined KOR agonists for their activities in promoting KOR phosphorylation in wild-type mice to circumvent these issues and determined whether there was a relationship between KOR-mediated behaviors and KOR phosphorylation.
Agonist-induced KOR phosphorylation and internalization in brains may be used as proxies for β-arrestin recruitment. We have previously demonstrated the selective KOR agonist U50,488H-induced phosphorylation of the mouse KOR expressed in cultured cells at S356, T357, T363, and S369. We generated and purified antibodies against the mouse KOR peptides containing pS356/pT357, pT363, or pS369 and found that antibodies were specific for phosphorylated forms of KOR in cells. We further characterized pT363 and pS369 antibodies using brains of wild-type and KOR knockout mice treated with vehicle or U50,488H. We found that the antibodies were specific for KOR phosphorylated at pT363 and pS369 in brains and that U50,488H promoted KOR phosphorylation in a dose-dependent manner.
We previously generated a mutant mouse line expressing a fusion protein of KOR conjugated with the fluorescent protein tdTomato 5′ to the stop codon (KtdT). U50,488H caused internalization of KOR-tdT in the VTA of KtdT mice.
In this study, we examined whether there was a connection between KOR agonist-induced KOR phosphorylation in the mouse brain and internalization in the mouse ventral tegmental area (VTA) and KOR agonist-produced side effects, such as CPA and hypolocomotion. Four KOR agonists [U50,488H, methoxymethyl salvinorin B (MOM-SalB), nalfurafine, and 42b] were included in the study. U50,488H is the first nonpeptide selective KOR agonist and has the arylacetamide basic structure. MOM-SalB is a longer-acting analog of salvinorin A, which is a highly selective and the first non-nitrogenous KOR agonist. Nalfurafine is a moderately selective KOR agonist and has 4,5-epoxymorphinan structure. 42b is 3-deoxy nalfurafine and has moderate selectivity for the KOR.
We previously performed in vitro and in vivo pharmacological characterization of these compounds. EC 50 values and maximal effects of the four KOR agonists in stimulating KOR-mediated [35 S]GTPγS binding are shown in Supplementary Table S1. In addition, EC 50 values and maximal effects of nalfurafine and 42b in stimulating [35 S]GTPγS binding mediated by mu, delta, and nociceptin/orphanin FQ receptors (MOR, DOR, and NOR, respectively) are included in Supplementary Table S1.
