Antinociceptive Effects of Sinomenine Combined With Ligustrazine or Paracetamol in Animal Models of Incisional and Inflammatory Pain

Abstract

The management of postoperative and inflammatory pain has been a pressing challenge in clinical settings. Sinomenine (SN) is a morphinan derived alkaloid with remarkable analgesic properties in various kinds of pain models. The aim of the current study is to investigate if SN can enhance the effect of ligustrazine hydrochloride (LGZ) or paracetamol (PCM) in animal models of postoperative and inflammatory pain. And to determine if the combined therapeutic efficacies can be explained by pharmacokinetics changes. Pharmacological studies were performed using a rat model of incisional pain, and a mouse model of carrageenan induced inflammatory pain. Pharmacokinetic studies were performed using a microdialysis sampling and HPLC-MS/MS assay method to quantify SN, LGZ, and PCM levels in blood and extracellular fluid in brain. We found that SN plus LGZ or SN plus PCM produced marked synergistic analgesic effects. However, such synergy was subjected to pain modalities, and differed among pain models. Pharmacological discoveries could be partially linked to pharmacokinetic alterations in SN combinations. Though further evaluation is needed, our findings advocate the potential benefits of SN plus LGZ for postoperative pain management, and SN plus PCM for controlling inflammatory pain.

Keywords: carrageenan-induced inflammation; drug combinations; microdialysis; neuroimmune interaction; pharmacokinetics; post-operative pain.

Figure 1

The development of mechanical (A) and heat (B) hypersensitivities in rats after incision in their hand paws, and the development of mechanical (C) and heat (D) hypersensitivities in mice after carrageenan injection in their hand paws. N = 8 animals, data were presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, mechanical (A,C) or heat thresholds (B,D) from Day 1 to 7 in rats (A,B) or from Day 1 to 5 in mice (C,D) were compared with their baseline values, using Dunn's (A,C) or Dunnett's multiple comparisons test (B,D) following ANOVA. Gray shaded areas represent durations that rats or mice experiencing ongoing hypersensitivities.

Figure 2

Dose dependent effects of LGZ (A,B), SN (E,F), PCM (I,J); and SN combined with LGZ (C,D) or PCM (G,H) on mechanical and heat hypersensitivities in rats with incisional pain. N = 6–8 rats, data were presented as mean ± SEM. Black or Gray lines represent the average (mechanical / heat) thresholds, before or 1 day after incision, respectively. LGZ was applied at the dosages of 10, 20, and 80 mg/kg (A,B); SN was applied at the dosages of 10, 20, and 80 mg/kg (E,F); PCM was applied at the dosages 10, 30, and 100 mg/kg (I,J); SN (at 10 or 20 mg/kg) were applied 60 min before LGZ (at 10 or 20 mg/kg; C,D), or PCM (at 10 or 30 mg/kg; G,H) administrations. Mechanical/heat thresholds were measured for 240 min after completion of drug applications. Two Way ANOVA with repeated measures indicated overall significant differences (P < 0.05) between the groups (A–J). *P < 0.05, **P < 0.01, ***P < 0.001, post-drug thresholds were compared with pre-drug baselines at 0 min, using Dunn's (A,C,E,G,I) or Dunnett's (B,D,F,H,J) multiple comparisons test following ANOVA; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, post-drug effects were compared with vehicle treatments at each time points, using Bonferroni's multiple comparisons test following ANOVA.

Figure 3

Dose dependent effects of LGZ (A,B), SN (E,F), PCM (I,J); and SN combined with LGZ (C,D) or PCM (G,H) on mechanical and heat hypersensitivities in mice with carrageenan induced inflammatory pain. N = 6–8 mice, data were presented as mean ± SEM. Black or Gray lines represent the average (mechanical/heat) thresholds before or 1 day after carrageenan injection, respectively. LGZ was applied at the dosages of 10, 20, and 80 mg/kg (A,B); SN was applied at the dosages of 10, 20, and 80 mg/kg (E,F); PCM was applied at the dosages 10, 30, and 100 mg/kg (I,J); SN (at 10 or 20 mg/kg) were applied 60 min before LGZ (at 10 or 20 mg/kg; C,D), or PCM (at 10 or 30 mg/kg; G,H) administrations. Mechanical/heat thresholds were measured for 240 min after completion of drug applications. Two Way ANOVA with repeated measures indicated overall significant differences (P < 0.05) between the groups (A,B,E,F,G,H,J). *P < 0.05, **P < 0.01, ***P < 0.001, post-drug thresholds were compared with pre-drug baselines at 0 min, using Dunn's (A,C,E,G,I) or Dunnett's (B,D,F,H,J) multiple comparisons test following ANOVA; ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, post-drug effects were compared with controls (vehicle treatments) at each time points, using Bonferroni's multiple comparisons test following ANOVA.

Figure 4

Area Under Curve (AUC) normalized with control (vehicle animals) for the effects of single or combined therapies of SN, LGZ, and PCM at different dosages against mechanical and heat hypersensitivities in rats with incisional pain (A,B), or in mice with carrageenan induced inflammatory pain (C,D). N = 6–8 animals, data were presented as mean ± SEM. Blue, red, or green lines represent the average AUCs of mechanical/heat thresholds for SN 80 mg/kg, LGZ 80 mg/kg, or PCM 100 mg/kg treatment, respectively. Filled circles indicating AUCs of drug combinations were larger than the average AUCs of effective single drug treatments (SN 80 mg/kg and LGZ 80 mg/kg). AUCs of drug combinations were compared with SN (at 10 or 20 mg/kg, **P < 0.01, ***P < 0.001, significant differences with specific groups were illustrated by respective representing colors), LGZ (at 10 or 20 mg/kg, ^#P < 0.05, ^##P < 0.01, ^###P < 0.001, significant differences with specific groups were illustrated by respective representing colors); or PCM (at 10 or 30 mg/kg, ++P < 0.01, +++P < 0.001, illustrated by respective representing colors), using Bonferroni's multiple comparisons test following ANOVA. Gray shaded area marked the significant (and most effective) dosage combination for the combined drug therapies.

Figure 5

Investigation of pharmacokinetics changes of SN, LGZ, and PCM in combined formulas. Drug concentrations of SN, LGZ, and PCM in blood (A,C,E,G) and striatum of the brain (B,D,F,H) were monitored continuously in collected samples (using microdialysis), after intravenous injection of SN, LGZ, and PCM at 50 mg/kg or SN combine with LGZ/PCM at 50 or 25 mg/kg, in rats. N = 6 rats for each group. Data was presented as mean ± SD. Two Way ANOVA indicated significant differences between the groups (A–H). *P < 0.05, **P < 0.01, drug concentrations of different groups were compared at each time points using Bonferroni's multiple comparisons test following ANOVA (significant differences with specific groups were illustrated by respective representing colors).