Efficacy-Based Perspective to Overcome Reduced Opioid Analgesia of Advanced Painful Diabetic Neuropathy in Rats

Abstract

Reduction of the opioid analgesia in diabetic neuropathic pain (DNP) results from μ-opioid receptor (MOR) reserve reduction. Herein, we examined the antinociceptive and antiallodynic actions of a novel opioid agonist 14-O-methymorphine-6-O-sulfate (14-O-MeM6SU), fentanyl and morphine in rats with streptozocin-evoked DNP of 9-12 weeks following their systemic administration. The antinociceptive dose-response curve of morphine but not of 14-O-MeM6SU or fentanyl showed a significant right-shift in diabetic compared to non-diabetic rats. Only 14-O-MeM6SU produced antiallodynic effects in doses matching antinociceptive doses obtained in non-diabetic rats. Co-administered naloxone methiodide (NAL-M), a peripherally acting opioid receptor antagonist failed to alter the antiallodynic effect of test compounds, indicating the contribution of central opioid receptors. Reduction in spinal MOR binding sites and loss in MOR immunoreactivity of nerve terminals in the spinal cord and dorsal root ganglia in diabetic rats were observed. G-protein coupling assay revealed low efficacy character for morphine and high efficacy character for 14-O-MeM6SU or fentanyl at spinal or supraspinal levels (E _max values). Furthermore, at the spinal level only 14-O-MeM6SU showed equal efficacy in G-protein activation in tissues of diabetic- and non-diabetic animals. Altogether, the reduction of spinal opioid receptors concomitant with reduced analgesic effect of morphine may be circumvented by using high efficacy opioids, which provide superior analgesia over morphine. In conclusion, the reduction in the analgesic action of opioids in DNP might be a consequence of MOR reduction, particularly in the spinal cord. Therefore, developing opioids of high efficacy might provide analgesia exceeding that of currently available opioids.

Keywords: 14-O-methylmorphine-6-O-sulfate; diabetes; fentanyl; morphine; neuropathic pain; opioid efficacy.

FIGURE 1

Representative figure illustrating the experimental protocol. For each experiment different animals were used. For in vitro studies different groups were used.

FIGURE 2

The changes in blood glucose levels in mmol/ml (A), and hind paw withdrawal thresholds (B) in grams prior to and after STZ- or vehicle treatments. Animals not matching predefined criteria for diabetic neuropathy were excluded from experiments (at least 14 mmol/ml blood glucose level and at least 20% decrease in PPT compared to weight matched animals). Figure represents data from one series of experiments. Each value represents the mean ± SEM. ^∗p < 0.05 vs. the signed groups (one way ANOVA followed by Newman–Keuls post hoc test).

FIGURE 3

Dose-response curves of 14-O-MeM6SU (A), fentanyl (B) and morphine (C) in diabetic and non-diabetic animals obtained with DPA. Data are represented as mean ± SEM (n = 5–10).

FIGURE 4

The systemic antinociceptive effect of 14-O-MeM6SU (A), fentanyl (B) and morphine (C) in STZ treated diabetic rats with neuropathy on DPA test following systemic (s.c.) administration at 9th week after STZ injection. Data were obtained 60 min after the injection of 14-O-MeM6SU, 10 min after fentanyl injection and 30 min in the case of morphine injection (time of peak effect). Each value represents the mean in grams ± SEM. ^∗p < 0.05 vs. diabetic baseline and saline treated group. #p < 0.05 vs. weight match control group (one way ANOVA followed by Newman–Keuls post hoc test).

FIGURE 5

The antagonist effect of s.c. co-administered NAL-M (10.6 μmol/kg) on the analgesic effect of s.c. 14-O-MeM6SU (A) and morphine (B) in STZ treated neuropathic animals in doses that reversed the allodynia and elevated PPT on diabetic and non-diabetic animals. Data were obtained 60 min after the injection of 14-O-MeM6SU and 30 min in the case of morphine injection. Each value represents the mean in grams ± SEM. ^∗p < 0.05 vs. diabetic baseline and saline treated group. #p < 0.05 vs. weight match control group (one way ANOVA followed by Newman–Keuls post hoc test).

FIGURE 6

The immunohistological assay shows reduction in MOR number in DRG (A,B) and spinal cord dorsal horn tissues (A,C) of STZ treated diabetic rats in comparison with non-diabetic animals (n = 5). Scale bar = 20 μm for DRG and 40 μm for spinal cord section. Each value represents the mean ± SEM. ^∗p < 0.05 Student t-test.

FIGURE 7

[³H]DAMGO binding in membrane tissues from dorsal spinal cord of diabetic and non-diabetic rats (n = 3–5). ^∗p < 0.05; vs. non-diabetic control group (^∗∗p < 0.01). (Two-way ANOVA followed by Fisher’s LSD post hoc test.)

FIGURE 8

Agonist activity of 14-O-MeMSU (A,D) compared to fentanyl (B,E) and morphine (C,F) in rat whole spinal cord membrane homogenates treated with vehicle or STZ for 9 (A–C) or 12 weeks (D–F) after treatment in [³⁵S]GTPγS binding assays. Figures represents the specific binding of [³⁵S]GTPγS in the presence of increasing concentrations (0.1 nM–10 μM) of the indicated ligands. Points represent means ± S.E.M. for at least three experiments performed in triplicate. “Basal” on the x-axis indicates the basal activity of the monitored G-protein, which is measured in the absence of the compounds and also represents the total specific binding of [³⁵S]GTPγS. The level of basal activity was defined as 100% (indicated by dotted line). The calculated E_max and EC₅₀ ± S.E.M. values are presented in Table 1. ^∗p < 0.05 diabetic vs. non-diabetic samples (^∗∗p < 0.01; Two-way ANOVA, Fisher’s LSD post hoc test).

FIGURE 9

Agonist activity of 14-O-MeMSU (A,D) compared to fentanyl (B,E) and morphine (C,F) in rat whole brain membrane homogenates treated with vehicle or STZ 9 weeks (A–C) or 12 weeks (D–F) after treatment in [³⁵S]GTPγS binding assays. Figures represents the specific binding of [³⁵S]GTPγS in the presence of increasing concentrations (0.1 nM–10 μM) of the indicated ligands. Points represent means ± S.E.M. for at least three experiments performed in triplicate. “Basal” on the x-axis indicates the basal activity of the monitored G-protein, which is measured in the absence of the compounds and also represents the total specific binding of [³⁵S]GTPγS. The level of basal activity was defined as 100% (indicated by dotted line). The calculated E_max and EC₅₀ ± S.E.M. values are presented in Table 2.