(+)-naloxone, an opioid-inactive toll-like receptor 4 signaling inhibitor, reverses multiple models of chronic neuropathic pain in rats

Abstract

Previous work demonstrated that both the opioid antagonist (-)-naloxone and the non-opioid (+)-naloxone inhibit toll-like receptor 4 (TLR4) signaling and reverse neuropathic pain expressed shortly after chronic constriction injury. The present studies reveal that the TLR4 contributes to neuropathic pain in another major model (spinal nerve ligation) and to long established (2-4 months) neuropathic pain, not just to pain shortly after nerve damage. Additionally, analyses of plasma levels of (+)-naloxone after subcutaneous administration indicate that (+)-naloxone has comparable pharmacokinetics to (-)-naloxone with a relatively short half-life. This finding accounts for the rapid onset and short duration of allodynia reversal produced by subcutaneous (+)-naloxone. Given that toll-like receptor 2 (TLR2) has also recently been implicated in neuropathic pain, cell lines transfected with either TLR4 or TLR2, necessary co-signaling molecules, and a reporter gene were used to define whether (+)-naloxone effects could be accounted for by actions at TLR2 in addition to TLR4. (+)-Naloxone inhibited signaling by TLR4 but not TLR2. These studies provide evidence for broad involvement of TLR4 in neuropathic pain, both early after nerve damage and months later. Additional, they provide further support for the TLR4 inhibitor (+)-naloxone as a novel candidate for the treatment of neuropathic pain.

Perspective: These studies demonstrated that (+)-naloxone, a systemically available, blood-brain barrier permeable, small molecule TLR4 inhibitor can reverse neuropathic pain in rats, even months after nerve injury. These findings suggest that (+)-naloxone, or similar compounds, be considered as a candidate novel, first-in-class treatment for neuropathic pain.

Copyright © 2012 American Pain Society. All rights reserved.

Figures

Figure 1

(+)-Naloxone has a short plasma half-life when given s.c. (10 mg/kg) or p.o. (50 mg/kg) in rats (n=3 per group). The half-life values of (+) naloxone were 2.33±1.08 h and 1.57 ± 0.784 h for p.o. and s.c. administration. The AUC0→∞ (area under the curve) of p.o. (50 mg/kg) and s.c. (10 mg/kg administration) were 82.7 and 1825 hr*ng/mL, respectively. The relative bioavailability of (+)-naloxone (p.o. vs s.c.) was only 0.9%. The data are presented as mean ± S.D.

Figure 2

(−)-Naloxone significantly reversed SNL pain at a dose of 100 mg/kg, but not at lower doses. Data shown are from 1 hour following s.c. injection as all rats at each dose had returned to baseline 3 hours following s.c. (−)-naloxone injection. There were no baseline differences between groups and all rats developed significant allodynia in this within-subjects design (n=12). A star (*) indicates a significant (p

Figure 3

(+)-Naloxone reverses SNL pain 2 weeks following injury. There were no baseline differences between groups. All rats developed significant allodynia following SNL surgery which was significantly reversed 1 h, but not 3 h, following 100 mg/kg s.c. (+)-naloxone. (+)-Naloxone had no effect on response thresholds in sham operated rats, compared to saline (n=6). A star (*) indicates a significant difference (p

Figure 4

(+)-Naloxone reverses SNL pain 8 weeks following injury. Rats with SNL surgery (n=9) developed significant allodynia while rats which underwent a sham surgery (n=5) did not. SNL-induced allodynia was significantly reversed 1 h following a 100 mg/kg s.c. dose of (+)-naloxone, but saline vehicle did not significantly change SNL allodynia in this within-subjects design. Neither (+)-naloxone nor saline altered the response thresholds of the rats who received sham surgery. A star (*) indicates a significant difference (p

Figure 5

(+)-Naloxone reversed CCI pain of 4 months duration. There were no significant differences at baseline testing between groups. All rats developed chronic allodynia following CCI surgery. (+)-Naloxone (60 mg/kg, s.c., n=6) significantly reversed allodynia at 1 h, but not 3 h, following injection compared to saline injected rats (n=6). A star (*) indicates a significant difference (p

Figure 6

(+)-Naloxone antagonizes stimulated HEK-TLR4 cell SEAP expression, but not stimulated HEK-TLR2 cell SEAP expression. The increase in SEAP expression seen in LPS stimulated HEK-TLR4 cells was significantly blocked by coincubation with 1, 10 or 100 µM (+)-naloxone. The increase in SEAP expression caused by Pam in HEK-TLR2 cells was unaffected by coincubation with 1, 10 or 100 µM (+)-naloxone. A star (*) indicates a significant decrease (p