Evaluation of Analgesia, Tolerance, and the Mechanism of Action of Morphine-6-O-Sulfate Across Multiple Pain Modalities in Sprague-Dawley Rats

Abstract

Background: Morphine-6-O-sulfate (M6S) is a mixed μ/δ-opioid receptor (OR) agonist and potential alternative to morphine for treatment of chronic multimodal pain.

Methods: To provide more support for this hypothesis, the antinociceptive effects of M6S and morphine were compared in tests that access a range of pain modalities, including hot plate threshold (HPT), pinprick sensitivity threshold (PST) and paw pressure threshold tests.

Results: Acutely, M6S was 2- to 3-fold more potent than morphine in HPT and PST tests, specifically, derived from best-fit analysis of dose-response relationships of morphine/M6S half-effective dose (ED50) ratios (lower, upper 95% confidence interval [CI]) were 2.8 (2.0-5.8) in HPT and 2.2 (2.1, 2.4) in PST tests. No differences in analgesic drug potencies were detected in the PPT test (morphine/M6S ED50 ratio 1.2 (95% CI, 0.8-1.4). After 7 to 9 days of chronic treatment, tolerance developed to the antinociceptive effects of morphine, but not to M6S, in all 3 pain tests. Morphine-tolerant rats were not crosstolerant to M6S. The antinociceptive effects of M6S were not sensitive to κ-OR antagonists. However, the δ-OR antagonist, naltrindole, blocked M6S-induced antinociception by 55% ± 4% (95% CI, 39-75) in the HPT test, 94% ± 4% (95% CI, 84-105) in the PST test, and 5% ± 17% (95% CI, -47 to 59) or 51% ± 14% (95% CI, 14-84; 6 rats per each group) in the paw pressure threshold test when examined acutely or after 7 days of chronic treatment, respectively.

Conclusions: Activity via δ-ORs thus appears to be an important determinant of M6S action. M6S also exhibited favorable antinociceptive and tolerance profiles compared with morphine in 3 different antinociceptive assays, indicating that M6S may serve as a useful alternative for rotation in morphine-tolerant subjects.

Fig. 1. Time profiles, dose response relationships and total antinociception scores of morphine and M6S in the HPT assay (acute treatment)

A – Time course of analgesic effects of morphine (open circles and dashed lines), M6S (closed circles and solid lines) in the HPT assay at a dose of 5.6 mg/kg. Repeated testing of vehicle-treated rats (solid triangles and solid line) did not effect HPT thresholds (One way RM ANOVA, F(4,29) = 0.995; p=0.433). A two-way RM ANOVA demonstrated statistically significant (p < 0.01) main effects of both drug treatment (F(1,30) = 12.63) and time (F(3,30) = 93.1, n = 6 rats per group). However, a significant interaction between factors was also detected. Nonetheless, the Bonferroni post hoc test confirmed that the antinociceptive effect of M6S was greater than that of morphine at 1, 3, and 5 hrs post-injection (asterisks, p<0.01). Mean morphine/M6S effect differences were, in °C, −0.57 (95%CI: 1.1, 2.1), −0.8 (95%CI: 1.3, 2.3), −0.75 (95%CI: 0.2, 1.2) and 0.2 (95%CI: −0.3, 0.7) °C for 1, 3, 5 and 7 hr time points, respectively). B - In each set of experiments data were corrected for HPT changes observed in vehicle-treated rats and normalized to respective maximum effect. A two-way ANOVA does not reveal a significant interaction or overall effect of treatment (morphine vs. M6S; F(1,10) = 3.05; p = 0.111); however at the 5 hr time point morphine and M6S mean effect data are different at p < 0.05 by Bonferroni post-test (asterisk; n=6 rats per group; drug effect differences, in %, were: 0.01 (95%CI: −0.28, 0.31), −0.17 (95%CI: −0.47, 0.13), −0.45 (95%CI: −0.75, −0.15) and 0.14 (95%CI: −0.16, 0.44) % for 1, 3, 5 and 7 hr time points, respectively). C - Dose-response relationships for the analgesic effects of M6S (solid circles and lines) and morphine (open circles and dashed lines) in the HPT assay. Sigmoidal curves for both the drugs were fitted using best curve fit procedures and a Boltzman sigmoidal function (note: logarithmic dose scale). The best-fit parameters for M6S are ED₅₀ = 0.35 ± 0.07 mg/kg (95%CI: 0.16, 0.54 mg/kg) and efficacy = 1.4 ± 0.1 °C (95%CI: 1.07, 1.70 °C; fit adjusted R² = 0.902). The best-fit parameters for morphine are ED₅₀ =0.99 ± 0.03 mg/kg (95%CI: 0.92, 1.08 mg/kg) and efficacy = 0.98 ± 0.01 °C (95%CI: 0.94, 1.07 °C; adjusted R² = 0.998). Horizontal dashed lines represent the baseline (set to zero across the tests) and maximum effect possible in the test. Arrow indicates effect of 10 mg/kg of morphine and M6S, data points excluded from the fit procedures above (n = 6 to 12 rats per group; the legend as in panel B; Note Y-axis break at 2.5 °C ΔHPT value). D - Total antinociceptive score (area under the curve, AUC) for morphine (light grey bars) and M6S (dark grey bars) across 0.5–5.6 mg/kg drug dose ranges in HPT pain assays; Asterisks indicate statistical significant difference of adjacent data sets (two-way RM ANOVA, main effect of treatment, F(1, 30) = 33.4; p.< 0.01; main effect of dose, F(3, 30) = 10.4; p <0.01; no significant interaction between the factors was observed; 6 rats per group; drug effect differences (CI of difference) were 3.64 (95%CI: 1.04, 6.25), 3.04 (95%CI: 0.44, 5.64), 3.01 (95%CI: 0.41, 5.61) and 3.51 (95%CI: 0.90, 6.11) AUC units for 0.5, 1.5, 3 and 5.6 mg/kg drug dose, respectively (Bonferroni test).

Fig. 2. Chronic treatment effects of morphine and M6S; and effects of δ- and κ-OR-specific antagonists on M6S antinociception in the HPT assay

A: Baseline thresholds are expressed as percentages of respective first day baseline HPT values in rats chronically treated with vehicle, morphine and M6S (open, light and dark grey bars, respectively). Two way RM ANOVA detected statistically significant (p<0.05) main effects of treatment, time and interaction between main effects. However, Bonferoni post-hoc test detected a statistically significant difference between day 1 vs. day 3, 7 or 9 baseline HPT in M6S-treated rats only (p< 0.01; 6 rats per group). Compared to the effect of M6S produced on day 1, effects measured on day 3, 7 and 9 were weaker by −0.98 (95%CI: −1.90, −0.07), −1.05 (95%CI−1.96, −0.14) and −1.27 (95%−2.18, −0.35) °C of the HPT change, respectively. In morphine- or vehicle- treated rats differences of baseline HPT on different days of the study never exceeded 0.38°C (95%CI −1.49, 0.74; day 1 vs. day 3 baseline HPT difference observed in vehicle-treated rats) B: Relative (% of the 1^st day) analgesic effects of M6S (solid circles and solid lines) and morphine (open circles and dotted lines) on HPT during chronic daily treatment protocol. Two way RM ANOVA detected statistically significant (p<0.05) main effects of treatment, time and interaction between main effects. However, statistically significant (p<0.01, asterisks) development of tolerance to the antinociceptive effect was observed in morphine- but not M6S- treated rats (Bonferroni post hoc test; mean differences of effects of morhine on day 1 vs day 3, 7 and 9 were (in %) −43.85 (95%CI: −109.1, 21.4), −85.1 (95%CI: −150.3, −19.80) and −82.2 (95%CI: −147.5, −16.9). For M6S, the largest difference in the drug effects on HPT was observed between days 1 and day 9 of treatment (17.3% with 95%CI: −48.0, 82.5%). Treatment of morphine-tolerant rats with 1.5 mg/kg M6S (dark grey bar) resulted an antinociceptive effect of M6S that was indistinguishable from that observed in either acute or chronic M6S-HPT studies (one-way ANOVA, F(2,17) = 0.0306, p = 0.969; 6 rats per group). No differences in M6S effects on HPT (in °C of HPT change) were detected when morphine/M6S-replacement and acute M6S treatment groups of animals were compared (95%CI: −0.61, 0.61) and this difference constituted just 0.05 °C (95%CI: −0.66, 0.56) for morphine/M6S-replacement and chronic M6S treatment groups (Tukey’s multiple comparison post hoc test). C. Percent maximum effect on HPT for morphine and M6S alone or after pre-treatment with δ- or κ-OR-specific antagonists naltrindole (NLD, 1mg/kg, i.p.) or norbinaltorphimine (nor-BNI, 5mg/kg, i.p.). Asterisks indicates statistically significant difference between M6S and M6S +NLD rat groups (one-way ANOVA, F(3,23) = 9.353; p = 0.0005) The mean difference of relative effects of M6S given alone or after pre-treatment with NLD was 57% (95%CI: 23.9, 91.0%; p < 0.01) the same value for morphine was 10% (95%CI: −24.0, 43.1%; p >0.05; post hoc Tukey test; 6 rats per group).

Fig. 3. Time profiles, dose response relationships and total antinociception scores of morphine and M6S in the PPT assay (acute treatment)

A – Time course of analgesic effects of morphine, open circles and dashed lines), morphine-6-O-sulfate (M6S, closed circles and solid lines) in the PPT assay at a dose of 5.6 mg/kg. Repeated testing of vehicle-treated rats (solid triangles and solid line) resulted in a steady decline in PPT (RM ANOVA, F(5,29) = 4.392; p=0.0027) with PPT measured at 180 and 270 min time points being statistically lower than baseline PPT (p<0.05; Tukey test). A two-way RM ANOVA detected significant effect of time (F(6,60) = 34.34; p <0.01) but no effect of treatment or time x treatment interactions. The largest difference in the effects of morphine and M6S on PPT (22.3 g greater effect in M6S vs morphine-treated rats was observed at the 2-hr time point, but was not statistaically significantly different from zero (95%CI: −28.3, 73.0; Bonferroi post-test; 6 rats per group). B – Comparison of M6S and morphine analgesic time-profiles after the data presented in panel A were corrected for PPT changes observed in vehicle-treated rats and normalized to respective maximum effect. C - Dose-response relationships for the analgesic effects of M6S (solid circles and lines) and morphine (open circles and dashed lines) in the PPT assay. Best fit parameters of sigmoidal functions describing the dose-response data were similar for both drugs: for morphine ED₅₀ = 1.4 ± 0.1 mg/kg (95%CI 0.8, 2.1 mg/kg) and E_max = 72 ± 7 g (95%CI 43, 102g; adjusted R² = 0.963 and for M6S ED₅₀ =1.2 ± 0.1 mg/kg (95%CI 0.9, 1.5 mg/kg) and E_max = 70 ± 6 g (95%CI 53, 88 g; adjusted R² = 0.951). Horizontal dashed lines represent the baseline (set to zero across the tests) and maximum effect possible on the test. An arrow indicates effect of 10 mg/kg of morphine and M6S, data points excluded from the fit procedures above (n = 6–12 rats per group). D - Total antinocicieptive score (area under the curve, AUC) for morphine (light grey bars) and M6S (dark grey bars) did not differ across various dose ranges in the PPT assay. A two-way ANOVA detected a significant main effect of drug dose, F (3, 40) = 23.58; p < 0.01, but not of treatment and no treatment x drug dose interactions (6–12 rats per group). The largest difference in effects of morphine and M6S on PPT was observed at the 1.5 mg/kg dose, but this was not statistically significantly different from zero (95%CI: −14830, 2333; Bonferroi post hoc post-test).

Fig. 4. Chronic treatment effects of morphine and M6S, and effect of δ- and κ-OR- specific antagonists on M6S antinociception in the PPT assay

A: Baseline thresholds expressed as percentages of respective first day baseline PPT values in rats chronically treated with vehicle, morphine or M6S (open, light and dark grey bars, respectively). Two-way RM ANOVA detected main effect of time; F(3, 66) = 5.44, P = 0.002), but no interaction between the factors or the significant effects of treatment were observed. Nonetheless the baseline PPT in rats chronically treated with morphine and M6S was statistically lower on day 7 than on day 1(asterisks, Bonferroni post hoc test; p< 0.01; 6 rats per group). This difference constituted −15.5% (95%CI −32.7, 0.86) in morphine-treated rats and −18.4% (95%CI: −34.7, −2.1) in M6S-treated rats. B: Relative (% of the 1^st day value) analgesic effects of M6S (solid circles and solid lines) and morphine (open circles and dotted lines) on PPT during chronic daily treatment protocol. A two-way RM ANOVA revealed a significant (p < 0.05) effect of time and time x treatment interaction. A Bonferroni post hoc test detected decreased antinociception on days 5 and 7 (compared to day 1 respective mean differences were −34.2% (95%CI: −63, −5.2) and −38.9% (95%CI: −67.9, −9.9), asterisks; p <0.05) in rats treated with morphine, but not with M6S. In the latter group, the largest relative decline in the antinociceptive action of M6S was observed on day 3 of experiment, but did not reach statistical significance: −23.0% (95%CI −52.0, 5.9). Treatment of morphine-tolerant rats with 1.5 mg/kg M6S (dark grey bar) resulted in weakened (50–60% of observed in experiments with either acute or day 7 chronic treatment) antinociceptive effect of M6S. This decrease however was not statistically significant; Kruskal-Wallis test, followed by Dunn’s multiple comparison post hoc test; 12 rats per group). C. Percent maximum effect on PPT for M6S alone or after pre-treatment with δ- or κ-OR-specific antagonists: naltrindole (NLD, 1mg/kg, i.p.) or norbinaltorphimine (nor-BNI, 5mg/kg, i.p.). Asterisks indicates statistically significant difference between M6S and M6S +NLD rat groups (one-way ANOVA, F(3,23) = 9.353; p = 0.0005; post hoc Tukey; p < 0.001; 6 rats per group). Pre-treatment with NLD decreased antinociceptive effect of M6S in PPT test by 33.6 g (95%CI: 1.78, 65.4).

Fig. 5. Comparison of time profiles, dose response relationships and total antinociception scores of morphine and M6S in the PST assay (acute treatment)

A – Time course of analgesic effects of morphine (open circles and dashed lines), M6S (closed circles and solid lines) in the PST assay at a dose of 5.6 mg/kg. Repeated testing of vehicle-treated rats (solid triangles and solid lines) did not effect PST. The antinociceptive effect of M6S was significantly stronger than that of morphine (6 rats per each group). Furthermore, an acute phase of hyperalgesia developed after morphine, but not M6S treatment. A two-way (treatment x time) RM ANOVA confirmed a statistical significance of time and treatment as well as time x treatment interaction. The Bonferroni post hoc test for multiple comparisons confirmed the effects of M6S treatment being significantly different from those of morphine treatment at 30, 45, 60 and 120 min of experimentation (p<0.05; asterisks). Mean morphine/M6S effect differences at these time points were (in grams of PST change), respectively: −33.9 (95%CI: −40.7, −27.0), −36.5 (95%CI: −43.3, −30.0), −37.3 (95%CI: −44.1, −30.5) and −10.0 (95%CI: −16.9, −3.2). At other time points studied, differences never exceeded −5.2 g PST change value (95%CI −12.1, 1.6; 15 min post-treatment). B - In each set of experiments, data were corrected for PST changes observed in vehicle-treated rats and normalized to the respective maximum effect. The Bonferroni post hoc test of normalized profiles detected significant differences at all time points studied except for the 4- and 5-hr time points (asterisks, at 4- and 5-hr time points mean drug effect differences were −0.31 g (95%CI: −0.77, 0.15) and −0.14 g (95%CI: −0.60, 0.32)) C - Dose-response relationships for the analgesic effects of M6S (solid circles and lines) and morphine (open circles and dashed lines) in the PST assay. The best-fit parameters for M6S are ED₅₀ = 2.3 ± 0.1 mg/kg (95%CI: 1.8, 2.7 mg/kg) and efficacy = 40 ± 1 g (95%CI: 36, 44 g; fit adjusted R² = 0.991). The best-fit parameters for morphine were ED₅₀ =5.1 ± 0.4 mg/kg (95%CI: 3.8, 6.4 mg/kg) and efficacy = 24 ± 2 g (95%CI: 19, 30 g; adjusted R² = 0.982). Horizontal dashed lines represent the baseline (set to zero across the tests) and maximum effect possible on the test (n = 6 rats per group). D – Dose-response relationships for total antinocicieptive score (area under the curve, AUC) for M6S (closed circles) and AUC for antinociceptive (morphine analgesia, opened circles) and pro-nociceptive phases (morphine hyperalgesia, open squares) of morphine’s acute effects on PST. The ED₅₀ and E_max of M6S action were 2.3 ± 0.1 mg/kg and 3336 ± 134 g*hr (best fit of sigmoidal function parameters; adjusted R² = 0.998). Best fit of dose-response relationships for antinociceptive and hyperalgesic effects of morphine predicted very similar slopes of fitted lines: 81 ±11 g*hr (95%CI 47, 115 g*hr; adjusted R² = 0.933) and 101 ± 10 g*hr (95%CI 69, 133 g*hr; adjusted R² = 0.961). Intercept was fixed to zero in both cases.

Fig. 6. Chronic treatment effects of morphine, and M6S, and the effect of δ- and κ-OR- specific antagonist on M6S antinociception in the PST assay

A: Baseline thresholds expressed as percentages of respective first day baseline PST values in rats chronically treated with vehicle, morphine and M6S (open, light and dark grey bars, respectively). Asterisks indicate a statistically significant difference of day 1 vs. day 3 and 7 baseline PST in M6S- and morphine-treated rats (two way RM ANOVA, main effect of time; F (2, 30) = 8.54, P = 0.001); followed by Bonferroni test; p< 0.05; 6 rats per group). Day 3 vs day 1 drug baseline PST differences constituted −1.93 g (morphine; 95%CI: −3.92, 0.06) and −1.37 g (M6S; 95%CI: −3.36, 0.56) and day 7 vs day 1 differenses were −1.90 g (morphine; 95%CI: −3.89, 0.09) and −2.25 g (M6S; 95%CI: −4.24, −0.26). B: Absolute (g of PST change) analgesic effects of 1.5 mg/kg M6S (solid circles and solid lines) and morphine (open circles and dotted lines) on PST during chronic daily treatment protocol. Antinociceptive efficacy of M6S is not different betweeen 1^st, 3^rd and 7^th days of chronic treatment (one way RM ANOVA, F(2,17) = 1.861; P = 0.2804). By Tukey post hoc test day 1 vs 3 and day 1 vs 7 mean differences of the effect of M6S on PST were not different from zero: −4.2 g (95%CI: −10.3, 1.8) and −2.8 (95%CI: −8.9, 3.3), respectively. The antinociceptive effect of M6S administered to rats chronically treated with morphine is was not statistically different from its effects produced on any of the days studied in chronic M6S treatment experiments (dark grey bar, compare to data presented by filled circles; one-way RM ANOVA; F( 3,23) = 2.539; P = 0.096; mean effect differences never exceeded 4.3 g (95%CI: −1.1, 9.8), the value observed when action of M6S in morphine-tolerant rats was compared to its action on day 3 of chronic treatment). C. Percent maximum effect on PST for morphine and M6S alone or after pre-treatment with δ- or κ-OR-specific antagonists: naltrindole (NLD, 1mg/kg, i.p.) or norbinaltorphimine (nor-BNI, 5mg/kg, i.p.). Asterisks indicates statistically significant difference between M6S and M6S +NLD rat groups (one way ANOVA F(2,17) = 56.55; P < 0.01; post hoc Tukey test p < 0.01; 6 rats per group). Mean effect differences from control were 95.8 % (95%CI: 70.4, 121.3) for M6S+NLD and 12.5% (95%CI: −13.0, 38.0) for M6S+NBNI group of rats.