Antinociceptive effects of two deltorphins analogs in the tail-immersion test in rats

Abstract

The antinociceptive effects of analogs of deltorphins: cyclo(Nδ,Nδ-carbonyl-D-Orn2, Orn4)deltorphin (DEL-6) and deltorphin II N-(ureidoethyl)amide (DK-4) after intracerebroventricular (i.c.v.) administration were investigated in the tail-immersion test in rats. Morphine, the most commonly used μ-opioid receptors (MOR) agonist, was employed as a reference compound. The contribution of the MOR, δ-(DOR) and κ-opioid receptors (KOR) in antinociceptive effects of the deltorphins analogs was studies using selective antagonists of these receptors. The results indicated that DK-4 (5, 10 and 20 nmol) and DEL-6 (5, 10 and 20 nmol) were the most effective in alleviating thermal pain at the dose of 20 nmol. The antinociceptive potency of DEL-6 at the dose of 20 nmol was approximately equal but DK-4 at the dose of 20 nmol was less effective than morphine at the dose of 13 nmol. DOR antagonist – naltrindole (NTI, 5 nmol) very strongly and, to the lower extent MOR antagonist – β-funaltrexamine (β-FNA, 5 nmol), inhibited antinociceptive effect of DK-4 (20 nmol). In turn, β-FNA was more potent than NTI in inhibition of the antinociceptive effects of DEL-6. Co-administration of DEL-6 and morphine at doses of 5 nmol, which do not produce measurable antinociception, generated additive antinociceptive effect. Chronic intraperitoneal (i.p.) injection of morphine (9 days) displayed a marked analgesic tolerance to the challenge dose of morphine and a slight cross-tolerance to challenge doses of DEL-6 and DK-4, given i.c.v. These findings indicate that the new deltorphin analogs recruit DOR and MOR to attenuate the nociceptive response to acute thermal stimuli.

1. Introduction

Opioid analgesics represent the front-line therapy for the clinical management of moderate to severe pain [35]. Three classes of opioid receptors, such as mu (MOR), delta (DOR) and kappa (KOR) mediate analgesia with distinct pharmacological profiles. The MOR agonists, such as morphine, remain the most powerful analgesics available to relieve post-operative and cancer pains. However, these compounds produce adverse effects, including development of tolerance and physical dependence, sedation, constipation, some respiratory depression, nausea, and vomiting [8,28,39]. Furthermore, MOR agonists are often ineffective under chronic pain conditions like neuropathic pain [12]. Selective KOR agonists produce analgesia without abuse potential, but their strong dysphoric effects may limit clinical utility of KOR agonists for pain treatment [41]. In turn, DOR agonists are of particular interest because they produce broad-spectrum antinociceptive effects in preclinical rodent models. Thus, they produce measurable antinociceptive effects in acute thermal nociception [2,16,32] and display high effectiveness in chronic inflammatory [9,11,29], neuropathic [9,15,20], and cancer pain [4,30]. Additionally, DOR agonists produce fewer undesirable side effects than MOR agonists [3,7,32]. Therefore, DOR remain potentially important therapeutic targets for the development of novel analgesic compounds with possible low abuse liability [9,36].

Deltorphins are linear heptapeptides, isolated from skin extracts of frogs belonging to Phyllomedusa genus, and have higher affinity and selectivity for DOR binding sites than any other endogenous compound known [10,23]. Two deltorphins with the sequences Tyr-D-Ala-Phe-Asp(or Glu)-Val-Val-Gly-NH₂ have been isolated from skin extracts of Phyllomedusa bicolor [10]. The deltorphins and their analogs are of considerable scientific interest because they have the potential to be used either as an effective therapeutic tool against acute and chronic pain, and/or in further elucidation of the structure–activity relationships of DOR agonists [19,24]. For example, modified deltorphin I analogs were prepared by introduction of D- or L-N-methylalanine (MeAla), D-or L-proline, α-aminoisobutyric acid (Aib), sarcosine or D-tertleucine (2-amino-3,3-dimethyl butyric acid) in place of D-Ala², or phenylalanine in place of Tyr¹. The D-MeAla²-analog was a slightly more potent DOR-agonist and showed two-fold higher antinociceptive potency in the tail-flick test in rats in comparison with the parent peptide. Substitution of Aib in the 2-position led to a sequence H-Tyr-Aib-Phe-Asp-Val-Val-Gly-NH₂, which displayed lower DOR-receptor affinity than deltorphin-I, but higher selectivity and, surprisingly, three times higher antinociceptive potency in the analgesic test [38]. In our study, two new analogs of deltorphins, such as cyclo(Nδ,Nδ-carbonyl-D-Orn2, Orn4) deltorphin (DEL-6) that contains an N-terminal cyclic structure and C-terminal sequence of native deltorphins and deltorphin II N-(ureidoethyl)amide (DK-4) – a linear peptide, were tested for their antinociceptive activity. We have conducted a comparison of the antinociceptive effects of these new analogs of deltorphins and the MOR agonist – morphine, following intracerebroventricular (i.c.v.) administration in rats. The antinociceptive potency of deltorphins analogs was investigated using an acute pain model based on the warm water tail withdrawal test (tail-immersion test). The functional activity of these two analogs toward DOR and MOR was initially determined in vitro using two bioassays, the guinea-pig ileum (GPI, a ‘MOR’ tissue) and the mouse vas deferens (MVD, a ‘DOR’ tissue) [21,42]. DEL-6 was reported to be 159 times more active in the MVD assay than in the GPI assay (IC₅₀ was 0.814 and 159 nmol, respectively) [42]. DK-4 was found to be about 685 times more active in the MVD assay than in the GPI (IC₅₀ 14.6 and >10,000 nmol, respectively) test [21]. To determine a respective contribution of MOR, DOR and KOR in the antinociceptive effects of deltorphins analogs in vivo, an influence of MOR, DOR and KOR selective antagonists on the antinociceptive effect of DEL-6 and DK-4 was estimated. Furthermore, other effects of both peptides, such as cross-tolerance with morphine and co-administration with non-effective doses of morphine were also assessed.

2. Materials and methods

2.1. Animals

The experiments were carried out according to the National Institute of Health Guidelines for the Care and Use of Laboratory Animals, the European Community Council Directive for Care and Use of Laboratory Animals, and approved by the Local Ethics Committee. Male Wistar rats (HZL, Warszawa, Poland), weighing 220 ± 20 g were used in all experiments. The animals were kept under a 12/12 h light–dark cycle and were adapted to the laboratory conditions for at least one week. The rats were handled once a day for 5 days before the beginning of the experiment. The animals were housed six per cage with standard food (Agropol-Motycz, Poland) and water ad libitum. All experiments were performed between 09:00 and 16:00 h.

2.2. Drugs and injection procedure

At least five days before the experiments, the rats were prepared for intracerebroventricular (i.c.v.) injections. Rats were anesthetized with pentobarbital (50 mg/kg, i.p., Vetbutal, Biowet, Pulawy, Poland) and placed in a stereotaxic instrument (Stoelting, Wood Dale, IL, USA). The animals were implanted with cannula (internal diameter 0.39 mm; outside diameter 0.71 mm; Milanowek, Poland). The coordinates for the i.c.v. injections were taken from bregma (1.5 mm lateral, 1.0 mm caudal, and 3.5 mm ventral), according to the atlas of Paxinos and Watson [31]. At the end of the experiments, position of the cannula was verified by injection of 5 μl of methylene blue. Results from the animals that did not exhibit a clear distribution of methylene blue in the ventricular system were excluded from further data evaluations.

Both analogs of deltorphins were synthesized in the Department of Chemistry, Warsaw University, Poland. Peptide chain of deltorphin II N-(ureidoethyl)amide (DK-4), was assembled on MBHA resin using the procedure described earlier [6]. Glutamic acid was incorporated using Boc-Glu(Bzl)-OH and tyrosine using Fmoc-Tyr(t-Bu)-OH. The peptide–resin was treated with 55% piperidine in DMF to remove the Fmoc group and treated with 55% TFA in methylene chloride to cleave the peptide from the resin. The products were purified using semipreparative reversed-phase high performance liquid chromatography (RP-HPLC). Homogeneous fractions were combined and lyophilized. The retention time (RT) of pure peptides was 13.5 min (Nucleosil 100 C18 column, 4 mm × 250 mm, 5 μm; solvent A: 0.05% TFA in water, solvent B: 60% CH₃CN in A; linear gradient 20–80% of B; flow rate 1 min/min). Structures were confirmed by ESI-MS (Finningan MAT 95S spectrometer, Bremen, Germany). Cyclo(Nδ,Nδ-carbonyl-D-Orn2, Orn4)deltorphin (DEL-6) is a cyclic deltorphin analog containing ornitine moieties in position 2 and 4. They were introduced into the ureid bridge by reaction of the amino group of the ornitine side chain with bis-(p-nitrophenyl)carbonate. Peptide was constructed on the 4-methylbenzhydrylamino solid support. Peptide were cleaved from the solid phase by use of the hydrogen fluoride and purified by means of RP-HPLC. The synthesis and purification of the DEL-6 was described in details by Zieleniak et al. [42] (as compound No. 6).

In the present study, DEL-6 and DK-4 were dissolved in physiological saline (0.9% NaCl) and injected i.c.v. at the doses of 5, 10, and 20 nmol in a volume of 5 μl. Morphine hydrochloride (Polfa, Kutno, Poland) was dissolved in saline and administered i.c.v. at the dose of 13 nmol (4.17 μg) (acute pain, tail-immersion test) or intraperitoneally (i.p.), at the dose of 10 mg/kg (cross-tolerance test). Naltrindole hydrochloride (NTI, 5 nmol), β-funaltrexamine hydrochloride (β-FNA, 5 nmol) and nor-binaltorphimine hydrochloride (nor-BNI, 10 nmol) were purchased from Tocris Cookson Ltd. (Bristol, UK). These opioid antagonists were freshly prepared as isotonic saline solutions prior to experiments, and given i.c.v. in the volume of 5 μl. All solutions were slowly injected over a period of 30 s. On the test day, following recording of the baseline values (response latency), the rats were assessed for antinociceptive response immediately after receiving the substances (morphine, DEL-6, DK-4) or saline.

2.3. Tail-immersion test

The tail-immersion test was carried out as described by Janssen et al. [18]. To determine the nociceptive reaction, the animals’ tails were placed in a water bath heated to 52 ºC, and the latency of response (in s; reflexive withdrawal of the distal half of the tail after its immersion in water) was measured before injections of the drugs (baseline latency response) and at 15 min intervals for the following 60 min, and then at 30 min intervals up to 180 min (post-treatment latency response) after drugs injection. The cut-off time of 20 s was set to prevent tail skin tissue damage. Morphine antinociception was induced by injection of morphine hydrochloride at the dose of 13 nmol, i.c.v. To examine an antinociceptive effect of DEL-6 and DK-4, the peptides were injected at the doses of 5, 10 or 20 nmol, i.c.v. Control group received saline instead of morphine or the peptide. To determine a respective contribution of MOR, DOR and KOR in the DEL-6 and DK-4 induced antinociception, the selective MOR antagonist – β-FNA (5 nmol, 24 h before peptides injection [14]), DOR antagonist – NTI (5 nmol, 5 min before peptides injection [34]), and KOR antagonist – nor-BNI (10 nmol, 1 h before peptides injection [40]) were given.

2.4. Combined administration of non-effective doses of morphine and DEL-6 on thermal nociception

DEL-6 (5 nmol, i.c.v.) and morphine (5 nmol, i.c.v.) were co-injected at non-effective doses and then the tail-immersion test was performed. Control groups received saline. The latency of response was measured before injections of the drugs (baseline latency response) and at 15 min intervals for the following 60 min, and then at 30 min intervals up to 180 min (post-treatment latency response) after drugs injection. The cut-off time was set to 20 s to avoid tissue damage.

2.5. Cross-tolerance studies

Tolerance to the antinociceptive effect of morphine (associative tolerance) was measured by the tail-immersion test as described previously [22]. At the beginning of the study, rats were randomly divided into two groups for pretreatment. For 9 days, the rats were taken to the experimental room each morning, and after at least 15 min of habituation, they were weighed. Next, one group of rats was injected with morphine (10 mg/kg, i.p.), once daily for 9 days and the other group was injected daily with vehicle (1 mg/kg, i.p.) and, 20 min later, the latency to withdraw the tail away from the warm water was measured (the tail-immersion test). On day 10 of the experiment, saline treated groups were challenged with saline, morphine (10 mg/kg, i.p.), DEL-6 or DK-4 at the doses of 5, 10 and 20 nmol, i.c.v. Rats tolerant to morphine were challenged with DEL-6, DK-4 or morphine at the same doses as saline treated group. The nociceptive threshold to heat was measured in the tail immersion test before and 20 min after the injections.

2.6. Statistical analysis

Data are given as means ± S.E.M. and are expressed as the percent maximum possible effect (%MPE) calculated as: $\text{MPE}(\%)=100\times [(\text{post}-\text{drug}\text{response}-\text{baseline}\text{response})/(\text{cut}-\text{off}-\text{response}-\text{baseline}\text{response})]$. Behavioral time course data were analyzed using a two-way ANOVA (followed by the Tukey–Kramer post hoc test), and a corresponding area under the curve (AUC) was analyzed using a one-way ANOVA and the Bonferroni post hoc test. The AUC data were calculated over the period 0–180 min. Cross-tolerance data were statistically compared by means of the one-way ANOVA, followed by the Bonferroni post hoc test. The value of P < 0.05 was considered statistically significant (GraphPad Prism 5.0, GraphPad Software, Inc., San Diego, CA, USA).

3. Results

3.1. Tail-immersion test

Injection of DEL-6 (5, 10 and 20 nmol, i.c.v.) dose-dependently produced increases in tail-immersion latencies, reaching maximal antinociceptive response at 45 min after the injection in rats (Fig. 1). The two-way ANOVA revealed significant effects for both dose [F(4, 387) = 73.31, P < 0.001] and time [F(8, 387) = 31.83, P < 0.001], as well as a statistically significant dose × time [F(32, 387) = 3.75, P < 0.001] interaction. From the results of AUC (0–180 min) (Fig. 1), it is clearly shown that DEL-6-induced antinociception at the dose of 20 nmol was almost equal to the morphine-induced antinociception at the dose of 13 nmol (AUC: 245.8 ± 18.78 vs. 254.6 ± 18.32, respectively). As shown in Fig. 2, the two-way ANOVA revealed significant effects for dose [F(4, 393) = 60.77, P < 0.001] and time [F(8, 393) = 25.95, P < 0.001], as well as a statistically significant dose × time [F(32, 393) = 3.04, P < 0.001] interaction. Co-injection of β-FNA (5 nmol, i.c.v., MOR antagonist) slightly more effectively than DOR antagonist, NTI (5 nmol, i.c.v.) blocked the antinociception induced by DEL-6 (20 nmol, i.c.v.). However, the KOR antagonist nor-BNI (10 nmol, i.c.v.) did not modify tail-immersion latency induced by i.c.v. administration of DEL-6 (20 nmol) (the two-way ANOVA: dose [F(1, 144) = 0.03, P = 0.858]; dose × time [F(8, 144) = 0.11, P = 0.99)].

Fig. 1.

Antinociceptive effect of DEL-6 (5, 10 and 20 nmol, i.c.v.) and morphine (13 nmol, i.c.v.) in the tail-immersion test in rats. Statistical analysis was performed using two-way ANOVA followed by the Tukey–Kramer post hoc test. The results are expressed as mean ± SEM (N = 8–10). AUC calculated using these data during 0–180 min were statistically analyzed (Bonferroni post hoc test after one-way ANOVA) and are presented in the inset. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. saline. MOR, morphine.

Fig. 2.

The influence of opioid antagonists: β-FNA (5 nmol, i.c.v., 24 h before test), naltrindole (5 nmol, i.c.v., 5 min before test), and nor-BNI (10 nmol, i.c.v., 1 h before test) on DEL-6 (20 nmol, i.c.v.) induced antinociception in the tail-immersion test in rats. Statistical analysis was performed using two-way ANOVA followed by the Tukey–Kramer post hoc test. AUC calculated using these data during 0–180 min were statistically analyzed (Bonferroni post hoc test after one-way ANOVA) and are presented in the inset. The results are expressed as a mean ± SEM (N = 6–10). ^**P < 0.01, ^***P < 0.001 vs. saline; ^#P < 0.05, ^##P < 0.001 vs. DEL-6.

As compared with vehicle-treated animals, i.c.v. injection of DK-4 (5, 10 and 20 nmol) dose-dependently produced significant increases in tail-immersion latencies, reaching maximal antinociceptive response at 45 min after the injection in rats (Fig. 3). The two-way ANOVA of these data revealed significant effects for both doses [F(4, 305) = 40.96, P < 0.0001] and time [F(8, 305) = 10.50, P < 0.0001]. From the results of AUC (0–180 min) it can be shown that DK-4 at the dose of 20 nmol was a somewhat less potent analgesic than morphine at the dose 13 nmol (AUC: 203.8 ± 20.85 vs. 254.6 ± 18.31) (Fig. 3). As shown in Fig. 4, the two-way ANOVA revealed significant effects for dose [F(4, 324) = 30.36, P < 0.001] and time [F(8, 393) = 12.06, P < 0.001]. Co-injection of NTI (5 nmol, i.c.v., DOR antagonist) slightly more effectively than MOR antagonist, β-FNA (5 nmol, i.c.v.) reduced the antinociception induced by DK-4 (20 nmol, i.c.v.). Similarly to the results with DEL-6, a KOR antagonist, nor-BNI (10 nmol, i.c.v.), did not change the tail-immersion latency induced by DK-4 (the two-way ANOVA: dose [F(1, 140) = 1.42, P = 0.235]; dose × time [F(8, 140) = 0.1, P = 0.999]).

Fig. 3.

Antinociceptive effect of DK-4 (5, 10 and 20 nmol, i.c.v.) and morphine (13 nmol, i.c.v.) in the tail-immersion test in rats. Statistical analysis was performed using two-way ANOVA followed by the Tukey–Kramer post hoc test. The results are expressed as mean ± SEM (N = 8–10). AUC calculated using these data during 0–180 min were statistically analyzed (Bonferroni post hoc test after one-way ANOVA) and are presented in the inset. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. saline. MOR, morphine.

Fig. 4.

The influence of opioid antagonists: β-FNA (5 nmol, i.c.v., 24 h before test), naltrindole (5 nmol, i.c.v., 5 min before test), and nor-BNI (10 nmol, i.c.v., 1 h before test) on DK-4 (20 nmol, i.c.v.) induced antinociception in the tail-immersion test in rats. Statistical analysis was performed using two-way ANOVA followed by the Tukey–Kramer post hoc test. The results are expressed as a mean ± SEM (N = 10–12). AUC calculated using these data during 0–180 min were statistically analyzed (Bonferroni post hoc test after one-way ANOVA) and are presented in the inset. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001 vs. saline; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. DK-4.

3.2. Combined administration of non-effective doses of morphine and DEL-6 on thermal nociception

Two-way ANOVA showed a significant difference among the groups of rats (dose [F(5, 335) = 33.95, P < 0.0001]; time [F(8, 335) = 7.15, P < 0.0001]; dose × time interaction [F(40, 335) = 2.15, P < 0.0001]). Post hoc test (Bonferroni) revealed that i.c.v. administration of DEL-6 or morphine at the dose of 5 nmol (i.c.v.) did not produce significant analgesia when given alone. However, an increasing effect of antinociception was observed following co-administration of the non-effective doses of morphine (5 nmol, i.c.v.) and DEL-6 (5 nmol, i.c.v.). Maximal antinociceptive effect was observed at 15, 30 and 45 min after co-injection of morphine and DEL-6 (Fig. 5). Thus, co-administration of DEL-6 and morphine increased morphine effect at the 15 min (17.44 ± 2.58 vs. 3.47 ± 1.22; P < 0.001), 30 min (16.31 ± 2.09 vs. 2.74 ± 1.21; P < 0.001) and 45 min (13.40 ± 2.23 vs. 1.076 ± 1.16; P < 0.01). However, the results of AUC (0–180 min) showed that co-administration of DEL-6 (5 nmol) (AUC: 22.65 ± 3.52 vs. saline 26.15 ± 1.9) and morphine (5 nmol) (AUC: 22.32 ± 5.09 vs. saline 26.15 ± 1.9) produced additive, rather than synergistic effect (57.59 ± 6.78 vs. DEL-6: 22.65 ± 3.52 or morphine: 22.32 ± 5.09; P < 0.001).

Fig. 5.

Combined administration of the non-effective doses of morphine (5 nmol, i.c.v.) and DEL-6 (5 nmol, i.c.v.) on thermal nociception in the tail-immersion test in rats. Statistical analysis was performed using two-way ANOVA followed by the Tukey–Kramer post hoc test. The results are expressed as mean ± SEM (N = 6–10). AUC calculated using these data during 0–180 min were statistically analyzed (Bonferroni post hoc test after one-way ANOVA) and are presented in the inset. ^#P < 0.05, ^##P < 0.01, ^###P < 0.001 vs. morphine or DEL-6.

3.3. Cross-tolerance between antinociceptive effects of morphine and DEL-6 or DK-4

One-way ANOVA showed a significant difference among the groups of rats [F(8, 99) = 16.83, P < 0.0001]. Post hoc test (Tukey–Kramer) revealed that daily i.p. administration of morphine for 9 days resulted in the development of tolerance to its antinociceptive action when rats were tested on day 10. Tolerance has been evidenced by the decrease in the antinociception for morphine group, as compared to the acute morphine (10 mg/kg, i.p.) injection on the day 10 of experiment. DEL-6 (5, 10 and 20 nmol) challenge indicated significant nociception at the dose of 10 and 20 nmol (P < 0.001) in the saline treated group but given to the morphine-tolerant rats (10 day of experiment) it induced less potent antinociceptive effect at the same doses (10 and 20 nmol; P < 0.05). These data suggest the existence of small cross-tolerance between morphine and DEL-6. However, the morphine-tolerant rats that were challenged with DEL-6 (10 and 20 nmol, P < 0.001) indicated higher antinociceptive effect than the morphine-tolerant rats challenged with morphine (Fig. 6).

Fig. 6.

Cross-tolerance between the antinociceptive effects of systemic morphine and i.c.v. DEL-6 administration in the tail-immersion test in rats. Challenge doses of DEL-6 (5, 10 and 20 nmol, i.c.v.) and morphine (10 mg/kg, i.p.) were given 20 min before the test (10th day of the experiment) to the saline-treated or morphine-tolerant (after 10 mg/kg, i.p., once daily for 9 days) rats. Statistical analysis was performed using one-way ANOVA followed by Bonferroni post hoc test. The results are expressed as mean ± SEM (N = 10–17). ^**P < 0.01, ^***P < 0.001 vs. saline treated rats; ^ˆˆP < 0.01, ^ˆˆˆP < 0.001 vs. morphine-tolerant rats; ⁺P < 0.05 vs. acute DEL-6 injection to saline treated rats; ^###P < 0.001 vs. acute morphine administration; ch, chronic (9 days) morphine/saline injection.

In a similar experiment involving DK-4, one-way ANOVA showed a significant difference among the groups of rats [F(8, 103) = 26.13, P < 0.0001]. On the day 10 of the experiment, DK-4 (5, 10 and 20 nmol, i.c.v.) challenge induced statistically significant antinociceptive effect at the doses of 10 and 20 nmol (P < 0.001) in the saline-treated groups (for 9 days), but in morphine tolerant rats a decrease in its antinociceptive effect was observed at the doses 10 and 20 nmol (P < 0.001) (Fig. 6). These results suggest that the cross-tolerance to antinociceptive effects of morphine and DK-4 exists indeed. However, the morphine-tolerant rats that were challenged with DK-4 (10 and 20 nmol, P < 0.001) indicated higher antinociceptive effect than the morphine-tolerant rats challenged with morphine (Fig. 7).

Fig. 7.

Cross-tolerance between the antinociceptive effects of systemic morphine and i.c.v. DK-4 administration in the tail-immersion test. Challenge doses of DK-4 (5, 10 and 20 nmol, i.c.v.) and morphine (10 mg/kg, i.p.) were given 20 min before the test (10th day of the experiment) to the saline-treated or morphine-tolerant (after 10 mg/kg, i.p., once daily for 9 days) rats. Statistical analysis was performed using one-way ANOVA followed by Bonferroni post hoc test. The results are expressed as a mean ± SEM (N = 9–19). ^**P < 0.01, ^***P < 0.001 vs. saline-treated rats; ^ˆˆˆP < 0.001 vs. morphine-tolerant rats; ⁺⁺⁺P < 0.001 vs. acute DK-4 injection to saline-treated rats; ^###P < 0.001 vs. acute morphine administration; ch, chronic (9 days) morphine/saline injection.

4. Discussion

Our results clearly indicated that the new deltorphins analogs, DEL-6 and DK-4, given supraspinally (i.c.v.), can mediate their antinociceptive effects in the tail-immersion test through MOR and DOR because both MOR selective antagonist, β-FNA and the DOR selective antagonist, NTI can attenuate their analgesic effects. The antinociceptive potency of both deltorphins analogs was generally weaker than morphine. Co-administration of DEL-6 and morphine at non-analgesic doses had an additive rather than synergistic analgesic effect. However, a slight cross-tolerance between morphine given systemically (i.p.) and both analogs of deltorphin given supraspinally (i.c.v.), was observed in morphine tolerant animals in the tail-immersion test.

Antinociceptive effects of opioids are derived from their interactions with MOR, DOR or KOR [8]. These receptors are found in the periphery at the presynaptic and postsynaptic level, in the spinal cord dorsal horn and in the brainstem, thalamus and cortex, where they represent an inhibitory system for the ascending and descending modulation of pain transmission [17]. In our study, both deltorphin analogs, DEL-6 and DK-4, given i.c.v., produced dose-dependent antinociception in the tail-immersion test in rats. They were the most effective analgesic at the dose of 20 nmol with the maximal peak effect at 45 min. Interestingly, these newly synthesized deltorphins analogs showed long-lasting antinociceptive effects that were comparable to morphine. These long-lasting effects may suggest that our modified deltorphins conferred higher resistance to enzymatic degradation. However, the analgesic potency of the deltorphins analogs was less than that of morphine. The antinociceptive effect of DEL-6 at the dose of 20 nmol was approximately equal to morphine (13 nmol) but DK-4 at the dose of 20 nmol was less effective than morphine at the dose of 13 nmol. In vitro (the GPI and MVD bioassays) functional activity of DEL-6 and DK-4 suggested their preferential activity at DOR [21,42]. However, this profile was only partially substantiated by our in vivo antagonist studies. Pretreatment with the selective MOR (β-FNA) or DOR (NTI) antagonists, given i.c.v., significantly attenuated but not fully antagonized antinociceptive actions of deltorphins analogs. DOR antagonist – NTI very strongly and, to the lower extent, MOR antagonist – β-FNA inhibited antinociceptive effect of DK-4. In turn, β-FNA was more potent than NTI in inhibition of the antinociceptive effects of DEL-6 nor-BNI was ineffective in blocking the antinociceptive effects of these peptides. Thus, our study suggests that DEL-6 and DK-4 are mixed MOR/DOR agonists in vivo because they exert their full antinociceptive activity involving both types of opioid receptors. DOR and MOR may be co-expressed in some neurons where they may form dimmers that could modulate G protein function (opioid receptors are functionally coupled to G-protein) differently from monomers [13,26]. Although recently Scherrer et al. [37] suggested a minimal possibility for such heterodimer formation in nociceptors, they do not exclude MOR-DOR interactions in the CNS neurons. Published data imply that ligands possessing dual agonistic activities at the DOR and MOR may allow for the effective treatment of pain with minimized MOR-mediated side effects [27].

Porreca et al. [33] provided evidence that the DOR agonists, such as DADLE and DPDPE at the dose that did not produce significant analgesia when given alone, significantly potentiated morphine response in mice when co-administered i.c.v. with morphine. Similar potentiation of morphine analgesia was observed after co-administration a synthetic enkephalin analog, FK33824 and morphine, both compounds at the doses that given alone in mice exhibited little or no analgesic activity [25]. In our study in rats, DEL-6 was co-injected with morphine, and both compounds were delivered at non-analgesic doses. Although combined administration of the non-effective doses of DEL-6 (5 nmol) and morphine (5 nmol) increased antinociceptive effect, the result of such co-injection seems to be rather additive but not synergistic (Fig. 5). Furthermore, as shown in Fig. 1, DEL-6 at the dose of 10 nmol produced equivalent antinociceptive effect to the combined treatment of DEL-6 (5 nmol) and morphine (5 nmol). Thus, we may suggest that opioid receptors (preferential MOR), on the same or distinct neurons, can interact to produce additive analgesic effect.

One approach used to distinguish analgesic activity at MOR or DOR involved evaluation of the possible cross-tolerance between selective agonist and morphine after induction of tolerance. The experiments performed on mice indicated that although the less selective DOR agonists (such as DSLET, DTLET and DADLE) produced analgesia, as do highly selective DOR ligands (DPDPE or DPLPE) after i.c.v. administration but, in contrast to highly selective ligands, they caused acute analgesic cross-tolerance to the MOR agonist – morphine, suggesting a common site of action with morphine [33]. Such effect was partially observed in our study in rats. Challenge doses of deltorphin analogs given i.c.v. to the morphine-tolerant rats indicated lower antinociceptive potency than that in saline treated animals. It suggests that the cross-tolerance was developed between both analogs of deltorphins and morphine. However, antinociception after challenge doses of deltorphins analogs was significantly higher than that in the morphine tolerant rats after challenge dose of morphine. It may suggest that deltorphins analogs induce their supraspinal antinociceptive potency also by the separate, MOR-independent mechanism(s), namely DOR. The data do not support the concept that thermal analgesia is necessarily the MOR-mediated event, as proposed previously [1,5], at least at supraspinal sites.

In summary, the present study found that DEL-6 and DK-4, given supraspinally, induced antinociceptive effects by their action via DOR and MOR binding sites in the tail-immersion test in rats. Co-administration of DEL-6 and morphine at the doses, which do not produce measurable antinociception, produced an additive antinociceptive effect in rats. Although DEL-6 and DK-4 indicated cross-tolerance with morphine, they partially preserved their DOR-dependent antinociceptive effects, thus suggesting that antinociceptive activity of deltorphins analogs is also mediated through the MOR-independent signaling pathway at supraspinal sites.