Alanine analogues of [D-Trp]CJ-15,208: novel opioid activity profiles and prevention of drug- and stress-induced reinstatement of cocaine-seeking behaviour

Abstract

Background and purpose: The novel macrocyclic peptide cyclo[Phe-D-Pro-Phe-D-Trp] ([D-Trp]CJ-15,208) exhibits κ opioid (KOP) receptor antagonist activity in both in vitro and in vivo assays. The four alanine analogues of this peptide were synthesized and characterized both in vitro and in vivo to assess the contribution of different amino acid residues to the activity of [D-Trp]CJ-15,208.

Experimental approach: The peptides were synthesized by a combination of solid phase peptide synthesis and cyclization in solution. The analogues were evaluated in vitro in receptor binding and functional assays, and in vivo with mice using a tail-withdrawal assay for antinociceptive and opioid antagonist activity. Mice demonstrating extinction of cocaine conditioned-place preference (CPP) were pretreated with selected analogues to evaluate prevention of stress or cocaine-induced reinstatement of CPP.

Key results: The alanine analogues displayed pharmacological profiles in vivo distinctly different from [D-Trp]CJ-15,208. While the analogues exhibited varying opioid receptor affinities and κ and μ opioid receptor antagonist activity in vitro, they produced potent opioid receptor-mediated antinociception (ED50 = 0.28-4.19 nmol, i.c.v.) in vivo. Three of the analogues also displayed KOP receptor antagonist activity in vivo. Pretreatment with an analogue exhibiting both KOP receptor agonist and antagonist activity in vivo prevented both cocaine- and stress-induced reinstatement of cocaine-seeking behaviour in the CPP assay in a time-dependent manner.

Conclusions and implications: These unusual macrocyclic peptides exhibit in vivo opioid activity profiles different from the parent compound and represent novel compounds for potential development as therapeutics for drug abuse and possibly as analgesics.

Keywords: CJ-15,208; analgesic; cocaine reinstatement; macrocyclic peptide; κ opioid receptor antagonist.

Figure 1

Structures of (A) [D-Trp]CJ-15,208 (1) and (B) alanine analogues 2–5. The residues are numbered 1–4, arbitrarily starting with the Phe C-terminal to the D-Trp residue.

Figure 2

Antinociceptive activity of [D-Trp]CJ-15,208 (1) and the alanine analogues 2–5 in vivo following i.c.v. administration in the 55°C warm-water tail-withdrawal assay in C57BL/6J mice. Data shown are mean % antinociception ± SEM. (A) Dose–response curves at peak response, which was 30 min for 1 or 20 min for analogues 2–5. (B) Time course for antinociceptive activity for 1 (30 nmol), 2 (10 nmol), 3 (30 nmol), 4 (3 nmol) and 5 (1 nmol).

Figure 3

Opioid receptor selectivity of the antinociceptive activity produced by alanine analogues of [D-Trp]CJ-15,208 in the mouse 55°C warm-water tail-withdrawal assay in C57BL/6J mice. The antinociceptive activity of the analogues was determined at the indicated doses after i.c.v. administration alone (solid bars), 24 h after administration of β-FNA or nor-BNI, and 15 min after pretreatment with naltrindole. Tail withdrawal latencies were measured 30 min after analogue administration. Data shown are mean % antinociception ± SEM.*P < 0.05, significantly different from response of matching administered analogue alone; one-way anova followed by Tukey's post hoc test.

Figure 4

Further analysis of opioid receptor mediation of the antinociceptive activity of analogue 2. The antinociceptive dose–response of analogue 2 in the mouse 55°C warm-water tail-withdrawal assay was determined in MOP KO mice, KOP KO mice and C57BL/6J WT mice pretreated 15 min with naltrindole (20 mg·kg⁻¹, i.p.) prior to administration of 2. Tail withdrawal latencies were measured 20 min after analogue administration, except in KOP KO mice where the tail withdrawal latencies were measured 50 min after analogue administration. Data shown are mean % antinociception ± SEM.

Figure 5

Dose-dependent antagonism of U50,488-induced antinociception by pretreatment with the alanine analogues 2, 4 and 5 compared with [D-Trp]CJ-15,208 [1; from Ross et al. (2012)] in the mouse 55°C warm-water tail-withdrawal assay. Mice were pretreated i.c.v. with the [D-Trp]CJ-15,208 or one of the analogues 2–5, 140 min prior to administration of the KOP receptor-selective agonist U50,488, and antinociceptive activity determined 40 min later. Alanine analogue 3 did not antagonize U50,488-induced antinociception at doses up to 30 nmol. Data shown are mean % antinociception ± SEM. *P < 0.05, significantly different from response of U50,488 administered alone; one-way anova followed by Tukey's post hoc test.

Figure 6

Duration of macrocyclic tetrapeptide-mediated antagonism of U50,488-induced antinociception in the mouse 55°C warm-water tail-withdrawal assay. Mice were pretreated i.c.v. with [D-Trp]CJ-15,208 [1, 3 nmol; from Ross et al. (2012)] or one of the analogues displaying KOP receptor antagonism (2, 1 nmol; 4 or 5, 30 nmol each), and the antinociception induced by U50,488 was determined 3–24 h later. Tail withdrawal latencies were determined 40 min after U50,488 administration. Data shown are mean % antinociception ± SEM. *P < 0.05, significantly different from response of U50,488 administered alone; one-way anova followed by Tukey's post hoc test.

Figure 7

Opioid receptor selectivity of antagonism by the alanine analogues of [D-Trp]CJ-15,208 in the mouse 55°C warm-water tail-withdrawal assay. Antinociception induced by morphine (left group) or SNC-80 (right group) was not significantly decreased by a 3 h pretreatment with 1 [3 nmol; from Ross et al. (2012)], 2 (10 nmol), 3 (30 nmol), 4 (30 nmol) or 5 (10 nmol), in contrast to the antinociceptive effect of U50,488 (centre group), which was significantly antagonized by pretreatment with 1, 2, 4 and 5. Tail withdrawal latencies were determined 40 min after selective agonist administration. Data shown are mean % antinociception ± SEM. *P < 0.05, significantly different from response of U50,488 administered alone; one-way anova followed by Tukey's post hoc test.

Figure 8

Time-dependent prevention of reinstatement of extinguished cocaine–CPP following pretreatment with analogues 2 and 5. (A) Schematic showing timing of the extinction, treatment and reinstatement protocol. Vehicle (50% DMSO) or analogues 2 or 5 (10 or 30 nmol, i.c.v., respectively) was administered on days 28 and 29, 2 h prior to initial exposure to forced swim stress; for cocaine place conditioning the mice were treated on days 28 and 29 with vehicle or peptide, followed by cocaine place conditioning 5 min to 2 h after the injection (square) on day 29. (B) Mice exhibited significant preference for the cocaine (10 mg kg⁻¹, s.c. daily for 4 days)-paired compartment, with extinction occurring over the next 3 weeks (left bars). Mice were then exposed to forced swim stress (centre bars) or an additional round of cocaine place conditioning (right bars), resulting in the reinstatement of place preference in vehicle-treated mice. Pretreatment for 2 h with either alanine analogue 2 or 5 prevented stress-induced, but not cocaine-primed, reinstatement of cocaine-seeking behaviour in the CPP assay. (C) Analogue 2 also prevented cocaine-primed reinstatement of cocaine CPP when administered 5 or 30 min, but not 1 or 2 h, prior to cocaine. Pretreatment with analogue 2 without cocaine (rightmost bar) did not induce reinstatement by itself. Data shown are mean (± SEM) difference in time spent on the drug-paired side. *P < 0.05, significantly different from preconditioning place preference response (leftmost bar); †P < 0.05, significantly different from post-CPP response (leftmost solid black bar); ‡P < 0.05, significantly different from vehicle-treated, stress-induced or cocaine-primed reinstatement of place preference response; anova followed by Tukey's post hoc test.