An analog of [d-Trp]CJ-15,208 exhibits kappa opioid receptor antagonism following oral administration and prevents stress-induced reinstatement of extinguished morphine conditioned place preference

Abstract

Opioid use disorder (OUD) relapse rates are discouragingly high, underscoring the need for new treatment options. The macrocyclic tetrapeptide natural product CJ-15,208 and its stereoisomer [d-Trp]CJ-15,208 demonstrate kappa opioid receptor (KOR) antagonist activity upon oral administration which prevents stress-induced reinstatement of cocaine-seeking behavior. In order to further explore the structure-activity relationships and expand the potential therapeutic applications of KOR antagonism for the treatment of OUD, we screened a series of 24 analogs of [d-Trp]CJ-15,208 with the goal of enhancing KOR antagonist activity. From this screening, analog 22 arose as a compound of interest, demonstrating dose-dependent KOR antagonism after central and oral administration lasting at least 2.5 h. In further oral testing, analog 22 lacked respiratory, locomotor, or reinforcing effects, consistent with the absence of opioid agonism. Pretreatment with analog 22 (30 mg/kg, p.o.) prevented stress-induced reinstatement of extinguished morphine conditioned place preference and reduced some signs of naloxone-precipitated withdrawal in mice physically dependent on morphine. Collectively, these data support the therapeutic potential of KOR antagonists to support abstinence in OUD and ameliorate opioid withdrawal.

Keywords: Antagonist; Kappa opioid receptor; Macrocyclic peptide; Opioid use disorder; Reinstatement; Withdrawal.

Fig. 1.

Screening of the [d-Trp]CJ-15,208 analogs for antinociception and KOR antagonism following i.c.v. administration. A) The [d-Trp]CJ-15,208 analogs (10 nmol) were administered, and the mice tested 20 min later for antinociception in the 55 °C WWTW assay. Vehicle alone (50% DMSO/ 50% saline; left-most bar) was tested as a negative control, and morphine (30 nmol, i.c.v.) was tested as a positive control. B) Two and a half hours later the KOR antagonist U50,488 (10 mg/kg, i. p.) was administered, and mice were tested again in the 55 °C WWTW assay to assess [d-Trp]CJ-15,208 analog-mediated antagonism of U50,488-mediated antinociception. Points represent average % antinociception ± SEM from 8 mice for each bar. *significantly different from vehicle treatment group, † significantly different from U50,488 treatment group (p < 0.05; one-way ANOVA with Dunnett's multiple comparisons post hoc test).

Fig. 2.

Screening of [d-Trp]CJ-15,208 analogs 1–24 for antinociception and KOR antagonism following oral administration (30 mg/kg, p.o.). A) [d-Trp]CJ-15,208 analogs were administered, and the mice tested 30 min later for antinociception in the 55 °C WWTW assay. Vehicle alone (5% DMSO/15% Solutol/80% saline, p.o.; left-most bar) was tested as a negative control, and morphine (5 mg/kg, p.o.) was tested as a positive control. B) Two and a half hours later the KOR antagonist U50,488 (10 mg/kg, i.p.) was administered, and mice were tested again in the 55 °C WWTW assay to evaluate [d-Trp]CJ-15,208 analog-mediated antagonism of U50,488-mediated antinociception. Points represent average % antinociception ± SEM from 8 mice for each bar. *significantly different from vehicle treatment group, † significantly different from U50,488 treatment group (p < 0.05; one-way ANOVA with Dunnett's multiple comparisons post hoc test).

Fig. 3.

Antinociception and KOR antagonism in the 55 °C WWTW assay following i.c.v. and p.o. administration of [d-Trp]CJ-15,208 analogs A) 1 and B) 21, or analog 22 after C) i.c.v. administration or D) p.o. administration. Points and bars represent average % antinociception ± SEM from 8 mice. *significantly different from U50,488, (p < 0.05; one-way ANOVA Dunnett's multiple comparisons post hoc test).

Fig. 4.

Characterization of analog 22 opioid partial agonist and antagonist activity in the 55 °C warm-water tail-withdrawal assay. A) Opioid receptor involvement in antinociception induced by 22 30 min after i.c.v. administration of a 30 nmol dose to wildtype (C57BL/6J), MOR KO, KOR KO, or DOR KO mice. B) A 2.5-hour pretreatment with analog 22 (30 mg/kg, p.o.) prevented U50,488-induced antinociception (10 mg/kg, i.p.), but not that of morphine (10 mg/kg, i.p.) or SNC-80 (100 nmol, i.c.v.). C) Time-dependent KOR antagonism by analog 22. The antinociceptive effect of U50,488 (10 mg/kg, i.p.) was determined in mice pretreated for 2.5, 6 or 24 h with analog 22 (30 mg/kg, p.o.). Points represent average % antinociception ± SEM from 8 to 17 mice for each bar. *significantly different from response of agonist alone (p < 0.05); two-way ANOVA with Sidak's multiple comparison or Dunnett's post hoc tests.

Fig. 5.

Effects of analog 22 on respiration and ambulation tested in the CLAMS/Oxymax system. A) Respiration and B) ambulation were monitored after oral administration (30 mg/kg, p.o.) of 22 or morphine. Points represented as % vehicle response ± SEM; breaths per minute, BPM, or ambulation, XAMB, from 12 mice (8 for morphine). *significantly different from vehicle treated mice (p < 0.05); two-way RM ANOVA with Sidak's multiple comparison post hoc test.

Fig. 6.

Evaluation of potential rewarding or aversive properties of analog 22. Analog 22 alone did not produce conditioned place preference or aversion. After determination of initial preconditioning preferences, C57BL/6 J mice were place conditioned daily for 2 days with morphine (10 mg/kg, i.p.), U50,488 (30 mg/kg, i.p.), or 22 (30 mg/kg, p.o.) with a counterbalanced design as described in the Methods. Data is presented as mean difference in time spent in the drug-paired side ± SEM (n = 17–26 mice). *significantly different from matching preconditioning preference (p < 0.05), two-way ANOVA with Sidak's multiple comparison post hoc test.

Fig. 7.

Prevention of stress-induced reinstatement of extinguished morphine-CPP by analog 22. A) Reinstatement paradigm schematic. B) Following 4 days of morphine administration (10 mg/kg, i.p. daily), mice exhibited significant preference for the morphine-paired environment, with extinction occurring 9 weeks later. Mice were then exposed to forced swim stress or another round of morphine place conditioning, reinstating preference. Pretreatment with 22 (30 mg/kg, p.o.) prevented stress-induced reinstatement of place preference when administered either 5 min or 2.5 h prior to FSS exposure; however, it had no significant effect on drug-seeking behavior when administered 5 min prior to an additional round of morphine place conditioning. Bars represent means of n = 16–29 mice (with 7 vehicle-treated, FSS-exposed mice); morphine place conditioning data in the left panel represents combined responses of all 99 mice. *significantly different from pre-conditioning place preference response; †significantly different from post-conditioning place preference response; ‡significantly different from vehicle pretreatment reinstatement of place preference response (p < 0.05); one-way ANOVA followed by Tukey's multiple comparison post hoc test.

Fig. 8.

Frequency of jumping behavior following administration of naloxone or analog 22. Mice that underwent twice daily injections of escalating doses of morphine (10–75 mg/kg, i.p.) for five days demonstrated significant elevations of jumping behavior upon administration of the opioid antagonist naloxone (10 mg/kg, s.c.), a behavior characteristic of precipitated withdrawal. Mice that underwent five days of morphine injections but received analog 22 (30 mg/kg, p.o.) 1 h prior to naloxone demonstrated significantly reduced instances of jumping behavior, as did mice that only received chronic saline prior to naloxone on day five. Data is presented as mean jumping frequency ± SEM (n = 10–12 mice). *significantly different from 5d morphine treatment group (p < 0.05), †significantly different from 5d-saline treatment (p < 0.05); two-way ANOVA with Tukey's multiple comparison post hoc test. # = value of 0 ± 0 for this measure.