Tolerance develops to the antiallodynic effects of the peripherally acting opioid loperamide hydrochloride in nerve-injured rats

Abstract

Peripherally acting opioids are potentially attractive drugs for the clinical management of certain chronic pain states due to the lack of centrally mediated adverse effects. However, it remains unclear whether tolerance develops to peripheral opioid analgesic effects under neuropathic pain conditions. We subjected rats to L5 spinal nerve ligation (SNL) and examined the analgesic effects of repetitive systemic and local administration of loperamide hydrochloride, a peripherally acting opioid agonist. We found that the inhibition of mechanical hypersensitivity, an important manifestation of neuropathic pain, by systemic loperamide (1.5mg/kg subcutaneously) decreased after repetitive drug treatment (tolerance-inducing dose: 0.75 to 6.0mg/kg subcutaneously). Similarly, repeated intraplantar injection of loperamide (150 μg/50 μL intraplantarly) and D-Ala(2)-MePhe(4)-Glyol(5) enkephalin (300 μg/50 μL), a highly selective mu-opioid receptor (MOR) agonist, also resulted in decreased inhibition of mechanical hypersensitivity. Pretreatment with naltrexone hydrochloride (5mg/kg intraperitoneally) and MK-801 (0.2mg/kg intraperitoneally) attenuated systemic loperamide tolerance. Western blot analysis showed that repetitive systemic administration of morphine (3mg/kg subcutaneously), but not loperamide (3mg/kg subcutaneously) or saline, significantly increased MOR phosphorylation in the spinal cord of SNL rats. In cultured rat dorsal root ganglion neurons, loperamide dose-dependently inhibited KCl-induced increases in [Ca(2+)]i. However, this drug effect significantly decreased in cells pretreated with loperamide (3 μM, 72 hours). Intriguingly, in loperamide-tolerant cells, the delta-opioid receptor antagonist naltrindole restored loperamide's inhibition of KCl-elicited [Ca(2+)]i increase. Our findings indicate that animals with neuropathic pain may develop acute tolerance to the antiallodynic effects of peripherally acting opioids after repetitive systemic and local drug administration.

Keywords: Nerve injury; Neuropathic pain; Peripheral opioid receptor; Rats; Tolerance.

Fig. 1

Repetitive drug administration induced acute tolerance to the antiallodynic effect of systemic loperamide in nerve-injured rats. (A) The diagram shows the tolerance-inducing protocol. (B) In rats on day 5 after an L5 spinal nerve injury (SNL), systemic administration of loperamide (L, 1.5 mg/kg subcutaneously in the back) reversed the decrease in paw withdrawal threshold (PWT) of the hind paw ipsilateral to the injured (left) side. The inhibition of mechanical hypersensitivity by systemic loperamide (1.5 mg/kg subcutaneously) decreased after repetitive systemic drug treatment at various tolerance-inducing doses (0.75 to 6.0 mg/kg subcutaneously, n = 8 to 9/group). The inhibition of mechanical hypersensitivity from a centrally penetrating opioid, morphine sulfate (M, 3.0 mg/kg subcutaneously), was not decreased in loperamide-tolerant rats. (C) After tolerance induction, the PWT measured after a 1.5-mg/kg loperamide injection was normalized to the preinjection baseline and plotted according to tolerance-inducing dose. (D) The increase in PWT induced by 1.5 mg/kg (n = 8) and 3.0 mg/kg (n = 8) systemic loperamide gradually decreased after repetitive injections. PWT was measured at 30 minutes postinjection (peak drug effect). Data are expressed as mean ± SEM. *P < .05, **P < .01, ***P < .001 versus preinjection PWT in (B) and (D), and versus vehicle in (C). ^# P < .05 versus postinjection PWT under pretolerance conditions.

Fig. 2

Repeated local administration of loperamide into hind paw tissue also resulted in tolerance to the drug’s inhibitory effects on neuropathic mechanical hypersensitivity. (A) The antiallodynic effects of intraplantar injections of loperamide (150 µg/50 µL, n = 6) and DAMGO (300 µg/50 µL, n = 7) diminished after repetitive local drug administration. The antiallodynic effects of intraplantar loperamide (150 µg/50 µL n = 6) largely remained intact in rats treated with saline. (B) The antiallodynic effect of intraplantar loperamide (150 µg/50 µL) decreased in rats that had received repetitive systemic (3 mg/kg subcutaneously, n = 6) or repetitive local (300 µg/30 µL, intraplantarly, n = 8) injections of loperamide, compared to the effect observed before tolerance induction. However, the antiallodynic effect of systemic loperamide (1.5 mg/kg subcutaneously) was still present in rats that had become tolerant to locally administered loperamide. The paw withdrawal threshold was measured 30 minutes after injection. Data are expressed as mean ± SEM. **P < .01, ***P < .001 versus predrug paw withdrawal threshold.

Fig. 3

Effects of pretreatment with opioid receptor antagonists and N-methyl-d-aspartate receptor antagonists on systemic loperamide tolerance. (A) Systemic loperamide (1.5 mg/kg subcutaneously) increased the ipsilateral paw withdrawal threshold (PWT) in spinal nerve ligation (SNL) rats under pretolerance conditions. Pretreatment with the long-lasting opioid receptor antagonist naltrexone HCl (5 mg/kg intraperitoneally), but not saline, reduced loperamide tolerance in SNL rats. However, pretreatment with naloxone methiodide (5 mg/kg intraperitoneally), an opioid receptor antagonist that does not cross the blood-brain barrier, or naloxone HCl (10 mg/kg intraperitoneally), a relatively short-acting antagonist, did not block the development of tolerance. Antagonist or saline was administered 10 minutes before each tolerance-inducing injection of loperamide (6.0 mg/kg subcutaneously). (B) Rats were administered MDL 105,519 (1 mg/kg intraperitoneally, n = 6), MK-801 (0.2 mg/kg, intraperitoneally n = 6), or saline (n = 3) 10 minutes before each tolerance-inducing injection of loperamide (6.0 mg/kg subcutaneously). After the tolerance-inducing protocol, systemic loperamide did not increase PWT in MDL 105,519- or vehicle-treated groups, but did increase PWT in the MK-801–treated group from preinjection baseline. Data are expressed as mean ± SEM. **P < .01, ***P < .001 versus preinjection PWT. ^#P < .05 versus preinjection PWT under pretolerance conditions.

Fig. 4

Pretreatment with locally administered naloxone methiodide and MK-801 did not prevent the development of tolerance to intraplantar loperamide. Intraplantar injection of loperamide (150 µg/50 µL) significantly increased the ipsilateral paw withdrawal threshold (PWT) of spinal nerve ligation (SNL) rats (i.e., reversed mechanical allodynia) under pretolerance conditions. During induction of tolerance, rats were administered an intraplantar injection of MK-801 (10 µg/20 µL, n = 6), naloxone methiodide (100 µg/20 µL, n = 6), or saline (n = 4) 10 minutes before each injection of loperamide (300 µg/30 µL intraplantarly). After tolerance induction, the inhibitory effect of local loperamide (150 µg/50 µL intraplantarly) diminished in all 3 groups. However, systemic loperamide (1.5 mg/kg subcutaneously) significantly increased the PWT in local loperamide-tolerant animals. Data are expressed as mean ± SEM. **P < .01, ***P < .001 versus preinjection PWT.

Fig. 5

Repetitive systemic administration of morphine, but not loperamide, increased MOR phosphorylation in spinal cord of nerve-injured rats. (A) Representative immunoblots (IB) show that repetitive systemic administration of morphine sulfate (M, 3 mg/kg subcutaneously, n = 3), but not loperamide (L, 3 mg/kg subcutaneously, n = 3) or saline (S, n = 3), significantly increased the level of p-MOR-Ser³⁷⁵ in the L5 spinal cord segment of SNL rats. The total MOR protein levels in SNL groups were all significantly lower than that in the sham-operated group. (B) Treatment with morphine, but not loperamide or saline, also induced a significant increase in MOR phosphorylation in SNL rats at the L4 spinal cord. MOR protein level at the L4 spinal segment was not significantly different between groups. Data are expressed as mean + SEM. *P < .05, ***P < .001 versus SNL+saline; ⁺P < .05, ⁺⁺P < .01 versus sham; ^#P < .05, ^##P < .01 versus SNL+morphine.

Fig. 6

Tolerance developed to loperamide inhibition of KCl-induced [Ca²⁺]_i transients in cultured DRG neurons. (A) Using a fluorescent plate reader, KCl-induced [Ca²⁺]_i transients in adult DRG neurons were measured in a 96-well Fluo-4AM assay. Inset: The dose dependence of KCl on [Ca²⁺]_i transient size is shown by increasing area under the curve (AUC) with increasing KCl concentration. (B) A 5-minute pretreatment with loperamide (0.1 to 10 µM) dose-dependently reduced KCl (30 µM)-induced [Ca²⁺]_i transients. Inset: Dose-dependent loperamide inhibition on KCl-induced [Ca²⁺]_i transients. (C) To model loperamide tolerance in vitro, cells were incubated with loperamide (3 µM) for 72 hours. At 24 hours posttreatment, loperamide (0.1 to 10 µM) inhibition of KCl-induced [Ca²⁺]_i transients was significantly decreased across all doses, as compared to that of loperamide-naïve neurons. (D) However, a 5-minute exposure to the delta opioid receptor antagonist naltrindole (1 µM) before application of loperamide (3 µM) restored loperamide inhibition of KCl-induced [Ca²⁺]_i transients in loperamide-tolerant cells, suggesting a reversal of tolerance. (E) Bar graph showing naltrindole alone had no effect on KCl-induced [Ca²⁺]_i transients, and loperamide (3 µM) inhibited KCl-induced [Ca²⁺]_i transients in nontolerant cells, but not in tolerant cells. Pretreatment with naltrindole partially restored the diminished drug effect in tolerant cells. Data are expressed as mean + SEM. *P < .05 versus naltrindole group.