Peripherally acting mu-opioid receptor agonist attenuates neuropathic pain in rats after L5 spinal nerve injury

Abstract

Studies in experimental models and controlled patient trials indicate that opioids are effective in managing neuropathic pain. However, side effects secondary to their central nervous system actions present barriers to their clinical use. Therefore, we examined whether activation of the peripheral mu-opioid receptors (MORs) could effectively alleviate neuropathic pain in rats after L5 spinal nerve ligation (SNL). Systemic loperamide hydrochloride (0.3-10 mg/kg, s.c.), a peripherally acting MOR-preferring agonist, dose-dependently reversed the mechanical allodynia at day 7 post-SNL. This anti-allodynic effect produced by systemic loperamide (1.5mg/kg, s.c.) was blocked by systemic pretreatment with either naloxone hydrochloride (10 mg/kg, i.p.) or methyl-naltrexone (5 mg/kg, i.p.), a peripherally acting MOR-preferring antagonist. It was also blocked by ipsilateral intraplantar pretreatment with methyl-naltrexone (43.5 microg/50 microl) and the highly selective MOR antagonist CTAP (5.5 microg/50 microl). However, this anti-allodynic effect of systemic loperamide was not blocked by intraplantar pretreatment with the delta-opioid receptor antagonist naltrindole hydrochloride (45.1 microg/50 microl). The anti-allodynic potency of systemic loperamide varied with time after nerve injury, with similar potency at days 7, 28, and 42 post-SNL, but reduced potency at day 14 post-SNL. Ipsilateral intraplantar injection of loperamide also dose-dependently (10-100 microg/50 microl) reversed mechanical allodynia on day 7 post-SNL. We suggest that loperamide can effectively attenuate neuropathic pain, primarily through activation of peripheral MORs in local tissue. Therefore, peripherally acting MOR agonists may represent a promising therapeutic approach for alleviating neuropathic pain.

Fig. 1. Systemic loperamide attenuated neuropathic pain after L5 spinal nerve ligation (SNL)

(A) L5 SNL induced a decrease in PWT on the hindpaw ipsilateral to the injured side that lasted for 56 days. *P < 0.05 versus pre-SNL baseline (n = 8–12). (B) Vehicle or different doses of loperamide (n = 7–10) were injected subcutaneously in different groups of rats on day 7 post-SNL. Loperamide dose-dependently reversed mechanical allodynia that developed on the hindpaw ipsilateral to the SNL. *, $, # P < 0.05 versus the respective pre-drug baseline. (C) MPE (%) for loperamide to attenuate mechanical allodynia was calculated at 30 min post-injection for each dose, and a dose-response function was established accordingly. PWT data are presented as median values, and MPE data are expressed as means ± SEM. ++P < 0.01 versus the vehicle-treated group. (D) Systemic loperamide (1.5 mg/kg, n = 6, s.c.) also attenuated heat hyperalgesia that developed in the hindpaw ipsilateral to the SNL in rats on day 7 post-SNL, but did not affect PWL on the contralateral side. *P < 0.05 versus pre-drug baseline, #P < 0.05 versus the ipsilateral hindpaw.

Fig. 2. The anti-allodynic effect of systemic loperamide varied with time after L5 SNL

(A) Loperamide (1.5 mg/kg, s.c) was given to different groups of animals on day 7 (n = 7), 14 (n = 8), 28 (n = 9), or 42 (n = 8) post-SNL. Note that the anti-allodynic effect of loperamide at 30 and 60 min after injection was substantially lower in the day-14 post-SNL group than in the day-7 post-SNL group. * (day 7, 28, 42), $ (day 7, 42), # (day 14) P < 0.05 from respective pre-drug baseline; + P < 0.05 from day 7 post-SNL group. (B) Systemic loperamide did not change PWT of the contralateral hindpaw at any post-SNL time point examined. (C) MPE (%) was calculated at 30 min post-injection and plotted against different post-SNL time points. +P < 0.05 versus day 7 post-SNL. (D) Intraperitoneal injection of loperamide (3.0 mg/kg, n = 6) produced a short-term attenuation of mechanical allodynia in the ipsilateral hindpaw on day 14 post-SNL. *P < 0.05 versus pre-drug baseline. PWT data are presented as medians. MPE data are presented as means ± SEM.

Fig. 3. Peripherally acting opioid receptor antagonists blocked systemic loperamide-induced reduction in allodynia

(A) Systemic pretreatment with methyl-naltrexone (5mg/kg, i.p., n = 10) and naloxone hydrochloride (10 mg/kg, i.p., n = 6) each significantly attenuated the anti-allodynic effect of loperamide (1.5 mg/kg, s.c.) on day 7 post-SNL, compared with saline pretreatment (n = 7). (B) PWT on the contralateral side did not change significantly after drug treatment. Data are expressed as medians. *P < 0.05 versus pre-drug baseline; #P < 0.05, ##P < 0.01 versus the saline-pretreated group.

Fig. 4. Intraplantar (i.pl.) injection of a mu-, but not a delta-, opioid receptor antagonist attenuated systemic loperamide-induced allodynia reduction

(A) On day 7 post-SNL, ipsilateral i.pl. pretreatment with either methyl-naltrexone (43.5 µg/50 µl, n = 7) or CTAP (5.5 µg/50 µl, n = 10) significantly attenuated systemic loperamide-induced allodynia reduction (1.5 mg/kg, s.c.), compared with saline pretreatment (50 µl, i.pl. n = 7). In contrast, pretreatment with a DOR antagonist, naltrindole hydrochloride (45.1 µg/50 µl, n = 7), was ineffective. (B) PWT on the contralateral side did not change significantly after any drug treatment. Data are expressed as medians. *P < 0.05, **P < 0.01 versus pre-drug baseline, +P < 0.05, ++P < 0.01, +++P < 0.001 versus the saline-pretreated group.

Fig. 5. Intraplantar administration of loperamide dose dependently reversed mechanical allodynia after L5 SNL

(A) Different groups of rats were given intraplantar injections of vehicle (n = 8) or loperamide (10 µg, n = 6; 50 µg, n = 6; 100 µg, n = 10) in a volume of 50 µl on day 7 post-SNL. Loperamide dose dependently reversed the mechanical allodynia when injected into the ipsilateral but not the contralateral (100 µg, n = 10) hindpaw. (B) No significant changes in the PWT of the contralateral hindpaw were observed in any group. (C) MPE (%) for ipsilateral intraplantar injection of loperamide to attenuate mechanical allodynia was calculated at 45 min post-injection, and dose-response function was established. PWT data are presented as medians, and MPE data are presented as means ± SEM. *P < 0.05, **P < 0.01 versus the respective pre-drug baseline; +P < 0.05 compared with the vehicle-treated group.

Fig. 6. The anti-allodynic effect of intraplantar loperamide did not change significantly at different time points after L5 SNL

(A) In a repetitive-drug treatment study, loperamide (100 µg/50 µl, n = 6) or vehicle (n = 4) was injected into the ipsilateral hindpaw at days 14, 28, 42, and 56 post-SNL. The day 7 post-SNL group (n = 10) from the single-drug treatment study was included for comparison. On days 7 to 56 post-SNL, mechanical allodynia was significantly attenuated at 15 and 45 min after loperamide administration. * (days 7, 14, 28, 42, 56 at 15 and 45 minutes after drug injection), # (day 7 at 90 minutes after drug injection), P < 0.05 from respective pre-drug baseline. (B) PWT on the contralateral side did not change significantly after loperamide injection. (C) The MPE values on days 14, 28, 42, and 56 post-SNL were not significantly different from that on day 7 post-SNL. PWT data are presented as medians, and MPE data are presented as means ± SEM.