BN-9, a chimeric peptide with mixed opioid and neuropeptide FF receptor agonistic properties, produces nontolerance-forming antinociception in mice

Abstract

Background and purpose: Neuropeptide FF (NPFF) behaves as an endogenous opioid-modulating peptide. In the present study, the opioid and NPFF pharmacophore-containing chimeric peptide BN-9 was synthesized and pharmacologically characterized.

Experimental approach: Agonist activities of BN-9 at opioid and NPFF receptors were characterized in in vitro cAMP assays. Antinociceptive activities of BN-9 were evaluated in the mouse tail-flick and formalin tests. Furthermore, its side effects were investigated in rotarod, antinociceptive tolerance, reward and gastrointestinal transit tests.

Key results: BN-9 acted as a novel multifunctional agonist at μ, δ, κ, NPFF1 and NPFF2 receptors in cAMP assays. In the tail-flick test, BN-9 produced dose-related antinociception and was approximately equipotent to morphine; this antinociception was blocked by μ and κ receptor antagonists, but not by the δ receptor antagonist. In the formalin test, supraspinal administration of BN-9 produced significant analgesia. Notably, repeated administration of BN-9 produced analgesia without loss of potency over 8 days. In contrast, repeated i.c.v. co-administration of BN-9 with the NPFF receptor antagonist RF9 produced significant antinociceptive tolerance. Furthermore, i.c.v. BN-9 induced conditioned place preference. When given by the same routes, BN-9 had a more than eightfold higher ED50 value for gastrointestinal transit inhibition compared with the ED50 values for antinociception.

Conclusions and implications: BN-9 produced a robust, nontolerance-forming analgesia with limited inhibition of gastrointestinal transit. As BN-9 is able to activate both opioid and NPFF systems, this provides an interesting approach for the development of novel analgesics with minimal side effects.

Figure 1

The chemical structure of the novel multi‐targeting peptide BN‐9 (Tyr‐D.Ala‐Gly‐Phe‐Gln‐Pro‐Gln‐Arg‐Phe‐NH₂). The amino acid sequence of BN‐9 contains overlapping opioid and NPFF motieties. The N‐terminus corresponds to that of the halfstructure of biphalin (Tyr‐D.Ala‐Gly‐Phe‐NH), and the C termini corresponds to NPFF(3–8) (NPFF = Phe‐Leu‐Phe‐Gln‐Pro‐Gln‐Arg‐Phe‐NH₂) respectively.

Figure 2

In vitro functional activities of BN‐9 and reference compounds in cAMP assays. Representative concentration‐response curves at μ, δ, κ, NPFF1 and NPFF2 receptors are presented. Data are mean of at least three individual experiments conducted in duplicate. Similar concentration‐response curves were used to generate pEC₅₀ and E_max (%) values for each test compound, which are summarized in Table 1.

Figure 3

Antinociceptive dose‐ and time‐response curves for supraspinal (i.c.v.) BN‐9 or morphine in the mouse tail‐flick test. Data points represent ± SEM from experiments conducted on eight mice. AUC calculated during 0–60 min from these data were statistically analysed and are presented in the text. *P < 0.05 versus saline according to one‐way ANOVA followed by the Dunnett's post hoc test performed on AUC data.

Figure 4

Antinociceptive dose‐ and time‐response curves for spinal (i.t.) BN‐9 or morphine in the mouse tail‐flick test. Data points represent means ± SEM from experiments conducted on at least seven mice. Final group sizes: (A) saline: n = 7, 0.125 nmol BN‐9: n = 7, 0.25 nmol BN‐9: n = 7, 0.5 nmol BN‐9: n = 7, 1 nmol BN‐9: n = 7, 2 nmol BN‐9: n = 8. (B) Saline: n = 7, 0.125 nmol morphine: n = 7, 0.25 nmol morphine: n = , 0.5 nmol morphine: n = 7, 1 nmol morphine: n = 7, 2 nmol morphine: n = 8. AUC calculated during 0–30 min from these data were statistically analysed and are presented in the text. *P < 0.05 versus saline according to one‐way ANOVA followed by the Dunnett's post hoc test performed on AUC data.

Figure 5

Antinociceptive dose‐ and time‐response curves for systemic (i.v.) BN‐9 or morphine in the mouse tail‐flick test. Data points represent means ± SEM from experiments conducted on seven mice. AUC calculated during 0–30 min from these data were statistically analysed and are presented in the text. *P < 0.05 versus saline according to one‐way ANOVA followed by the Dunnett's post hoc test performed on AUC data.

Figure 6

Antagonism of BN‐9‐induced supraspinal (i.c.v., A) or spinal (i.t., B) antinociception using various opioid antagonists. The effects of naloxone (5 nmol), β‐FNA (10 nmol), naltrindole (10 nmol) and nor‐BNI (10 nmol) on the central antinociception produced by BN‐9 were investigated in the mouse tail‐flick test. Data points represent means ± SEM from experiments conducted on eight mice. Effects of the NPFF receptor antagonist RF9 on the BN‐9‐induced supraspinal (i.c.v., C) or spinal (i.t., D) antinociceptionin in the mouse tail‐flick test. RF9 enhanced the central antinociception produced by BN‐9 after i.c.v. or i.t. injection. Data points represent means ± SEM from experiments conducted on at least seven mice. Final group sizes: (C) n = 8; (D) saline: n = 7, 10 nmol RF9 + saline: n = 7, saline +0.25 nmol BN‐9: n = 7, 10 nmol RF9 + 0.25 nmol BN‐9: n = 7, saline +0.5 nmol BN‐9: n = 8, 10 nmol RF9 + 0.5 nmol BN‐9: n = 8. AUC calculated during 0–60 or 0–30 min from these data are used for statistical analysis. *P < 0.05 versus Saline + BN‐9‐injected group, according to one‐way ANOVA followed by the Bonferroni's post hoc test performed on AUC data.

Figure 7

Antinociceptive effects of i.c.v. injection of BN‐9 or morphine in the mouse formalin test. (A) Time‐response cure for the antiniciception induced by 5 nmol BN‐9 and 10 nmol morphine after i.c.v. injection. The total time of licking and flinching /shaking the injected paw was recorded during the first 1 min of each interval after formalin injection. Data points represent means ± SEM from experiments conducted on eight mice. Antinociceptive dose–response curves for i.c.v. injection of BN‐9 or morphine in phase I (B) and phase II (C). The cumulative response time of licking and flinching/shaking the injected paw was measured during the period of 0–5 min (phase I) and 15–30 min (phase II). Data points represent means ± SEM from experiments conducted on at least seven mice. Final group sizes: (C) 0.078 nmol BN‐9: n = 9, 0.156 nmol BN‐9: n = 9, 0.313 nmol BN‐9: n = 8, 0.625 nmol BN‐9: n = 8, 1.25 nmol BN‐9: n = 8, 2.5 nmol BN‐9: n = 8, 5 nmol BN‐9: n = 8, 1.25 nmol morphine: n = 7, 2.5 nmol morphine: n = 8, 5 nmol morphine: n = 10, 10 nmol morphine: n = 8; (D) 0.625 nmol BN‐9: n = 8, 1.25 nmol BN‐9: n = 8, 2.5 nmol BN‐9: n = 8, 5 nmol BN‐9: n = 8, 1.25 nmol morphine: n = 7, 2.5 nmol morphine: n = 8, 5 nmol morphine: n = 10, 10 nmol morphine: n = 8. *P < .05 versus saline according to one‐way ANOVA followed by the Dunnett's post hoc test.

Figure 8

Effect of supraspinal (A, i.c.v.), spinal (B, i.t.) and systemic (C, i.v.) administrations of BN‐9 on the motor performance of mice in the rotarod test. The mean time (s) mice remained on the rotarod was measured 40 min after the administration of BN‐9 or vehicle. Data points represent means ± SEM from experiments conducted on at least eight mice. Final group sizes: (A) saline: n = 10, 1.25 nmol BN‐9: n = 9, 2.5 nmol BN‐9: n = 11, 5 nmol BN‐9: n = 11; (B) saline: n = 8, 0.6 nmol BN‐9: n = 10, 1 nmol BN‐9: n = 11, 2 nmol BN‐9: n = 11; (C) saline: n = 11, 224 nmol BN‐9: n = 11. *P < 0.05 versus saline according to one‐way ANOVA followed by the Dunnett's post hoc test.

Figure 9

Antinociceptive tolerance studies of BN‐9. Saline, morphine and BN‐9 were repeatedly administered daily i.c.v. (A) or i.t. (B) for 8 days in mice before the mouse tail‐flick test was performed. Ordinate values represent the daily analgesic response, expressed as MPE % [saline and BN‐9 at 15 min (i.c.v., A) or 10 min (i.t., B); morphine at 30 min (i.c.v., A) or 10 min (i.t., B)], after the injection. Data points represent mean ± SEM from experiments conducted on eight mice. Tolerance began to develop to the analgesic effects of morphine on day 4. *P < 0.05 versus effect of morphine on day 1, according to one‐way ANOVA followed by the Tukey's HSD post hoc test. (C) Effects of i.c.v. RF9 on the development of tolerance to antinociception of morphine or BN‐9. RF9 (10 nmol), a selective NPFF receptor antagonist, was i.c.v. co‐injected with morphine (4 nmol) or BN‐9 (1 nmol) for 5 days. On days 6–8, no RF9 was given. Ten nanomole RF9 was i.c.v. administered alone for 8 days as a control. Ordinatevalues represent the daily analgesic response, expressed as MPE % [saline and BN‐9 at 15 min; morphine at 30 min], after the injection. Data points represent mean ± SEM from experiments conducted on at least seven mice. Final group sizes: 10 nmol RF9: n = 7, 4 nmol morphine: n = 8, 4 nmol morphine + RF9 (Days 1–5): n = 8, 1 nmol BN‐9: n = 11, 1 nmol BN‐9 + RF9(Day 1–5): n = 11. * P < 0.05 versus effect on day 1, according to one‐way ANOVA followed by the Tukey HSD's post hoc test. ^& P < 0.05 versus effect of morphine, according to one‐way ANOVA followed by the Bonferroni's post hoc test. ^# P < 0.05 versus effect of BN‐9, according to one‐way ANOVA followed by the Bonferroni's post hoc test.

Figure 10

Evaluation of side effects of BN‐9. (A) The effects of i.c.v. administration of morphine or BN‐9 alone on place conditioning in mice. CPP score is expressed as time spent in the drug‐associated compartment on the post‐conditioning day minus time spent in the drug‐associated compartment during a period of 15 min on the pre‐conditioning day. Data are expressed as CPP score, that is, time spent in drug‐associated chamber on post‐conditioning day minus that on the pre‐conditioning day. Results are presented as mean ± SEM from experiments conducted on at least nine mice. Final group sizes: saline: n = 9, 4 nmol morphine: n = 9, 0.5 nmol BN‐9: n = 10, 1 nmol BN‐9: n = 10, 2 nmol BN‐9: n = 10, 4 nmol BN‐9: n = 0. *P < 0.05 versus saline according to one‐way ANOVA followed by the Tukey HSD test. The effects of supraspinal (B, i.c.v.), spinal (C, i.t.) and systemic (D, i.v.) administrations of morphine or BN‐9 on gastrointestinal transit in mice. Data points represent means ± SEM from experiments conducted on nine mice. *P < 0.05 versus saline according to one‐way ANOVA followed by the Dunnett's post hoc test.