A Novel Mu-Delta Opioid Agonist Demonstrates Enhanced Efficacy With Reduced Tolerance and Dependence in Mouse Neuropathic Pain Models

Abstract

Numerous studies have demonstrated a physiological interaction between the mu opioid receptor (MOR) and delta opioid receptor (DOR) systems. A few studies have shown that dual MOR-DOR agonists could be beneficial, with reduced tolerance and addiction liability, but are nearly untested in chronic pain models, particularly neuropathic pain. In this study, we tested the MOR-DOR agonist SRI-22141 in mice in the clinically relevant models of HIV Neuropathy and Chemotherapy-Induced Peripheral Neuropathy (CIPN). SRI-22141 was more potent than morphine in the tail flick pain test and had equal or enhanced efficacy versus morphine in both neuropathic pain models, with significantly reduced tolerance. SRI-22141 also produced no jumping behavior during naloxone-precipitated withdrawal in CIPN or naïve mice, suggesting that SRI-22141 produces little to no dependence. SRI-22141 also reduced tumor necrosis factor-α and cyclooxygenase-2 in CIPN in the spinal cord, suggesting an anti-inflammatory mechanism of action. The DOR-selective antagonist naltrindole strongly reduced CIPN efficacy and anti-inflammatory activity in the spinal cord, without affecting tail flick antinociception, suggesting the importance of DOR activity in these models. Overall, these results provide compelling evidence that MOR-DOR agonists could have strong efficacy with reduced side effects and an anti-inflammatory mechanism in the treatment of neuropathic pain. PERSPECTIVE: This study demonstrates that a MOR-DOR dual agonist given chronically in chronic neuropathic pain models has enhanced efficacy with strongly reduced tolerance and dependence, with a further anti-inflammatory effect in the spinal cord. This suggests that MOR-DOR dual agonists could be effective treatments for neuropathic pain with reduced side effects.

Keywords: HIV neuropathy; Mu opioid receptor; chemotherapy-induced neuropathy; delta opioid receptor; dependence; neuroinflammation; tolerance.

Figure 1:

Chemical Structure of SRI-22141

Figure 2:. Systemic SRI-22141 Produced Potent, Efficacious, and Long-Lasting Acute Anti-Nociception.

CD-1 mice were injected sc with 0.32-10 mg/kg SRI-22141 or 10 mg/kg morphine, and their tail flick latencies recorded over a 2 hour time course (52°C, 10 sec. cutoff). N = 7-13 mice per group, performed in 3 independent technical replicates by the same experimenter. Raw latencies in seconds reported as the mean ± SEM. A) Time courses shown for all doses. 3.2 mg/kg SRI-22141 produced a similar anti-nociceptive profile to 10 mg/kg morphine, while 10 mg/kg SRI-22141 produced maximal anti-nociception in all animals by 30 minutes that persisted for 2 hours. B) The data from A was transformed into % MPE, and graphed as a dose/response curve. The 3 doses in the linear range for SRI-22141 were fit by linear regression, which was used to calculate the potency (A₅₀ = 0.95 mg/kg; 0.64 – 1.31 95% Cl). C) Mice were tested for Rotarod latency to fall in seconds, measured as a baseline and 30 minutes after Vehicle, 10 mg/kg morphine, or 3.2-10 mg/kg SRI-22141 sc injection. N = 4 mice per group performed in 1 technical replicate, reported as the mean ± SEM. No drug treatment caused any significant change in Rotarod latency (p > 0.05).

Figure 3:. SRI-22141 Produced Efficacious Anti-Nociception with Reduced Tolerance in HIV Neuropathic Pain.

CD-1 mice had neuropathic pain induced by it injection of gpl20 protein over 3 days (see Methods). On day 15, morphine (10 mg/kg) or SRI-22141 (1-10 mg/kg) were injected sc twice daily, separated by 8 hours, for 3 days. Mechanical thresholds were measured using Von Frey filaments prior to and in a time course after each morning injection. Day 15 baselines were strongly reduced to near-zero levels from pre-gpl20 baselines, validating the pain state. Opioid treatment did not change the daily baselines (p > 0.05). N = 5-9 mice/group, performed in 2 technical replicates. Data reported as the mean ± SEM. A) Morphine vs. SRI-22141 time courses are shown for each day. Day 1 represents an acute injection and response without any chronic opioid treatment. 10 mg/kg Morphine and 10 mg/kg SRI-22141 had the same efficacy and time course on day 1, but morphine dropped off rapidly while SRI-22141 dropped more slowly over time. B) The data from A was transformed into %MPE and used to calculate the acute potency of SRI-22141 on day 1 as for the tail flick experiment above in Figure 2. A₅₀ = 2.3 mg/kg; 1.7 – 3.0 95% CI. C) AUC data were calculated from each time course curve for both drugs, and reported as the mean ± SEM over the 3 day period. Linear regression was performed, and the x-intercept with 95% CI for each drug curve used as the estimate of time in days to 0 efficacy. 10 mg/kg Morphine = 3.0 (2.7-3.6) days, SRI-22141 = 4.9 (3.6-15.2) days, a significant increase of 60%, demonstrating less tolerance by SRI-22141. Potency and tolerance values summarized in Table 2.

Figure 4:. SRI-22141 Produced Enhanced Efficacy with Reduced Tolerance in CIPN.

CD-1 mice had CIPN induced by ip injection of 2 mg/kg paclitaxel on days 1, 3, 5, and 7. On day 8, 0.32-10 mg/kg SRI-22141 or 10 mg/kg morphine was injected sc twice daily, separated by 8 hours, for 4 days. Mechanical thresholds were measured using Von Frey filaments prior to and in a time course after each morning injection. Day 8 baselines were strongly reduced to near-zero levels from pre-CIPN baselines, validating the pain state. Opioid treatment did not change the daily baselines (p > 0.05). N = 9-18 mice/group, performed in 2 or 4 independent technical replicates by the same experimenter. Data reported as the mean ± SEM. A-D) Morphine vs. SRI-22141 time courses are shown for each day. Day 1 represents an acute injection and response without any chronic opioid treatment. The 3.2 and 10 mg/kg doses of SRI-22141 demonstrated enhanced efficacy vs. morphine on day 1 (AUC increase of 165.2% for 3.2 mg/kg and 151.9% for 10 mg/kg of SRI-22141 over 10 mg/kg morphine). Morphine dropped to near-zero efficacy on day 2, while the 3.2 and 10 mg/kg doses of SRI-22141 fell more slowly, with 10 mg/kg SRI-22141 staying elevated over 3.2 mg/kg on days 2-4. E) The data from A-D was transformed into %MPE and used to calculate the acute potency of SRI-22141 on day 1 as for the tail flick experiment above in Figure 2. A₅₀ = 0.83 mg/kg; 0.70 – 0.97 95% CI. F) AUC data were calculated from each time course curve for both drugs and all doses, and reported as the mean ± SEM over the 4 day period. Linear regression was performed using dose and time points in the linear time-response range, and the x-intercept with 95% Cl for each drug curve used as the estimate of time in days to 0 efficacy. Morphine = 2.2 (1.9-3.3) days, 3.2 mg/kg SRI-22141 = 4.3 (3.8-5.2) days (95.5% increase), and 10 mg/kg SRI-22141 = 5.2 (4.3-9.4) days (136.4% increase); both doses are significantly increased vs. morphine, while the 0.32 and 1 mg/kg doses were not significantly different. Potency and tolerance values summarized in Table 2.

Figure 5:. SRI-22141 Produced Very Little Dependence and Withdrawal.

A) The CIPN mice from Figure 4 treated with 10 mg/kg morphine or 10 mg/kg SRI-22141 were injected with 30 mg/kg naloxone ip at the end of the behavioral time course, 3 hours after the final opioid injection. Withdrawal behaviors were observed and quantified for 20 minutes. Data reported as the mean ± SEM of N = 9-10 mice/group, performed in 2 independent technical replicates by the same experimenter. ** = p < 0.01 vs. morphine group by unpaired 2-tailed t test. SRI-22141 produced essentially no jumping behavior, but the same GI output as morphine. B) Pain-naïve CD-1 mice were injected twice daily with 10 mg/kg morphine or SRI-22141, 8 hours apart, for 4 days – the same injection regimen as in A except without the pain state. Four hours after the final injection on day 4, the mice were injected with naloxone and recorded as above. Data reported as the mean ± SEM of N = 9-10 mice/group, performed in 2 independent technical replicates by the same experimenter. ** = p < 0.01 vs. morphine group by unpaired 2-tailed t test. SRI-22141 produced essentially no jumping behavior, with a near-significant trend to produce less GI output than morphine (p = 0.07). C) As a control for the ability of 30 mg/kg naloxone to block 10 mg/kg SRI-22141, naïve CD-1 mice were injected with 30 mg/kg naloxone or vehicle ip, with a 10 minute treatment period, followed by 10 mg/kg SRI-22141 sc and a tail flick time course performed as in Figure 2. Data reported as the mean ± SEM of N = 5 mice/group performed in 1 technical replicate. **** = p < 0.0001 vs. same time point naloxone group by 2 Way ANOVA with Fisher’s Least Significant Difference post-hoc test.

Figure 6:. SRI-22141 Penetrates Into the CNS to Produce Anti-Nociception.

CD-1 mice were icv (A) or it (B) injected with 10 nmol naloxone or vehicle with a 10 minute treatment time, followed by 10 mg/kg SRI-22141 sc and a tail flick time course as in Figure 2. Data reported as the mean ± SEM with one technical replicate performed for each experiment. *, **** = p< 0.05, 0.0001 vs. same time point naloxone group by 2 Way ANOVA with Fisher’s Least Significant Difference post-hoc test. A) Icv injection group, N = 4-5 mice/group. Naloxone produced strong reversal of anti-nociception at early time points, but the effect progressively diminished over time and was gone after 60 minutes. B) It injection group, N = 5 mice/group. Naloxone produced essentially no blockade of anti-nociception. There was a significant difference at the 20 minute time point, but the effect size was very small – the naloxone group had an AUC of 95.4% of the vehicle group, only a 4.6% reduction.

Figure 7:. SRI-22141 Reduces Inflammatory Activation in the Spinal Cord in CIPN.

CD-1 mice were injected with paclitaxel or vehicle as in Figure 4 to produce CIPN (CIPN/ groups) or a sham pain state (Veh/Veh group). On day 8, mice were injected with vehicle, 10 mg/kg morphine, or 10 mg/kg SRI-22141 sc with a 4 hour treatment period, followed by spinal cord removal and analysis by qRT-PCR (see Methods). Inflammatory marker mRNA was normalized to GAPDH levels within each animal and further normalized to the Veh/Veh group. Data reported as the mean ± SEM of N = 5 mice/group performed in 1 technical replicate. * = p < 0.05 vs. Veh/Veh group; #, ## = p < 0.05, 0.01 vs. CIPN/Veh and CIPN/Morphine groups, both by 1 Way ANOVA with Fisher’s Least Significant Difference post-hoc test. A) Quantification of TNFα mRNA. CIPN induces TNFα mRNA levels over the Veh/Veh group. Morphine does not change the TNFα level in CIPN, but SRI-22141 reduces TNFα mRNA levels back to that seen in the Veh/Veh group. B) Quantification of COX-2 mRNA. CIPN/Veh and CIPN/Morphine have a trend to increase COX-2 levels over the Veh/Veh group in a similar pattern to that seen in A, but the effect is not significant. However, SRI-22141 in CIPN strongly decreases COX-2 mRNA vs. the CIPN/Veh and CIPN/Morphine groups (p < 0.01), demonstrating an anti-inflammatory effect of SRI-22141 in CIPN. The mean in the CIPN/22141 group is below that of the Veh/Veh group.

Figure 8:. SRI-22141 Acts Through the DOR to Promote Anti-Nociception and Anti-Inflammatory Activity in CIPN.

CD-1 mice used for all experiments, data reported as the mean ± SEM. A) CIPN was induced and measured with drug injected as in Figure 4. Naltrindole (10 mg/kg, ip) or Vehicle was injected 5 minutes prior to each SRI-22141 (3.2 mg/kg, sc) injection. N = 8-10 mice/group performed in 2 independent technical replicates by the same experimenter. Naltrindole treatment caused a strong reduction in SRI-22141 anti-nociception with the first acute treatment that persisted throughout the 4 day treatment period. *, **, ***, **** = p < 0.05, 0.01, 0.001, 0.0001 vs. same time point Naltrindole group by 2 Way ANOVA with Fisher’s Least Significant Difference post-hoc test. B) AUC data from A was used to create time-response curves, analyzed as in Figures 3–4, with x-intercept with 95% Cl used to estimate the time to zero efficacy. Vehicle = 3.0 (2.7-3.7) days; Naltrindole = 2.9 (2.4-4.7) days; not significantly different. C) CIPN or Sham was established, followed by Naltrindole (10 mg/kg, ip) or Vehicle for 5 minutes, then morphine or SRI-22141 (10 mg/kg, sc) for 4 hours, and TNFα mRNA measured in the spinal cord by qPCR, all as in Figure 7. N = 4-5 mice/group in 1 technical replicate. * = p < 0.05 vs. Veh/Veh group by 1 Way ANOVA with Fisher’s Least Significant Difference post-hoc test. Naltrindole treatment reverses the decrease in TNFα mRNA caused by SRI-22141. D) Naïve mice injected with Naltrindole (10 mg/kg, ip) or Vehicle followed by SRI-22141 (3.2 mg/kg, sc) and tail flick analysis performed as in Figure 2. N = 4-5 mice/group in 1 technical replicate. No differences between treatment groups (p > 0.05).