Behavioral and cellular pharmacology characterization of 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(isoquinoline-3'-carboxamido)morphinan (NAQ) as a mu opioid receptor selective ligand

Abstract

Mu opioid receptor (MOR) selective antagonists and partial agonists have been used for the treatment of opioid abuse and addiction. Our recent efforts on the identification of MOR antagonists have provided several novel leads displaying interesting pharmacological profiles. Among them, 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-[(3'-isoquinolyl)acetamido]morphinan (NAQ) showed sub-nanomolar binding affinity to the MOR with significant selectivity over the delta opioid receptor (DOR) and the kappa opioid receptor (KOR). Its central nervous system penetration capacity together with marginal agonism in the MOR-GTPγS binding assay made it a very interesting molecule for developing novel opioid abuse and addiction therapeutic agents. Therefore, further pharmacological characterization was conducted to fully understand its biological profile. At the molecular and cellular level, NAQ not only induced no translocation of β-arrestin2 to the MOR, but also efficaciously antagonized the effect of DAMGO in MOR-βarr2eGFP-U2OS cells in the β-arrestin2 recruitment assay. At the in vivo level, NAQ displayed a potent inhibition of the analgesic effect of morphine in the tail-flick assay (ID50=1.19 mg/kg). NAQ (10 mg/kg) also significantly decreased the hyper-locomotion induced by acute morphine without inducing any vertical jumps. Meanwhile NAQ precipitated lesser withdrawal symptoms in morphine dependent mice than naloxone. In conclusion, NAQ may represent a new chemical entity for opioid abuse and addiction treatment.

Keywords: Antagonist; Mu opioid receptor; NAQ; Pharmacology; in vitro; in vivo.

Fig. 1

β-arrestin2 recruitment assay in opioid receptor expressing βarr2eGFP-U2OS cells. The cells are live imaged, and the βarr2 recruitment evoked by drug induced receptor activation is visualized as bright green punctae (white arrows) by confocal microscopy. (A) MOR expressing cells are treated with DAMGO or NAQ (both 10 μM) as indicated. DAMGO induces robust βarr2 recruitment, while NAQ does not. In addition, NAQ pretreatment completely blocks DAMGO induced βarr2 recruitment. These findings are consistent with NAQ acting as an antagonist at the MOR. (B) DOR and KOR expressing cells are treated with a control agonist (SNC80 or U69,593, 10 μM, as indicated) or 100 μM NAQ. The control agonist induces robust βarr2 recruitment, while NAQ induces fewer, dimmer punctae than the control agonist. These findings are consistent with NAQ acting as a partial agonist at the DOR and KOR.

Fig. 2

NAQ antinociception antagonism study in the tail-flick assay. (A) NAQ was pretreated as indicated for 10, 30 and 60 minutes, and followed by 18 mg/kg morphine. Maximal NAQ antagonism was seen at 10 and 30 minutes, and absent by 60 minutes.; (B) Using a 10 minute NAQ pretreatment time, an antagonist dose-response curve was generated using NAQ.

Fig. 3

NAQ locomotor study with acute morphine (32 mg/kg, s.c.) administration at doses of 1.0 mg/kg (A), 3.2 mg/kg (B), and 10 mg/kg (C). Morphine/saline (s.c.), and NAQ/saline (i.v.) were injected at t = 30, 60 min, respectively.

Fig. 3

NAQ locomotor study with acute morphine (32 mg/kg, s.c.) administration at doses of 1.0 mg/kg (A), 3.2 mg/kg (B), and 10 mg/kg (C). Morphine/saline (s.c.), and NAQ/saline (i.v.) were injected at t = 30, 60 min, respectively.

Fig. 4

NAQ acute dependence study evaluating vertical jumps (A), weight of feces and urines (B), and weight loss (C).

Fig. 4

NAQ acute dependence study evaluating vertical jumps (A), weight of feces and urines (B), and weight loss (C).

Fig. 5

NAQ chronic dependence study evaluating vertical jumps (A), weight of feces and urines (B), and weight loss (C).

Fig. 5

NAQ chronic dependence study evaluating vertical jumps (A), weight of feces and urines (B), and weight loss (C).