We have examined the effects of cannabinoid agonists on hyperalgesia in a model of neuropathic pain in the rat and investigated the possible sites of action. The antihyperalgesic activity of the cannabinoids was compared with their ability to elicit behavioural effects characteristic of central cannabinoid activity. WIN55,212-2 (0.3-10 mg kg(-1)), CP-55,940 (0.03-1 mg kg(-1)) and HU-210 (0.001-0.03 mg kg(-1)) were all active in a 'tetrad' of tests consisting of tail-flick, catalepsy, rotarod and hypothermia following subcutaneous administration, with a rank order of potency in each of HU-210 > CP-55,940 > WIN55,212-2. The effects of WIN55,212-2 in each assay were blocked by the Cannabinoid1 (CB1) antagonist SR141716A. In the partial sciatic ligation model of neuropathic pain WIN55,212-2, CP-55,940 and HU-210 produced complete reversal of mechanical hyperalgesia within 3 h of subcutaneous administration with D50 values of 0.52, 0.08 and 0.005 mg kg(-1), respectively. In this model WIN55,212-2 was also effective against thermal hyperalgesia and mechanical allodynia. WIN55,212-2 produced pronounced reversal of mechanical hyperalgesia following intrathecal administration that was blocked by the CB1 antagonist SR141716A. Following intraplantar administration into the ipsilateral hindpaw, WIN55,212-2 produced up to 70% reversal of mechanical hyperalgesia, although activity was also observed at high doses following injection into the contralateral paw. The antihyperalgesic effect of WIN55,212-2 injected into the ipsilateral paw was blocked by subcutaneously administered SR141716A, but was not affected by intrathecally administered SR141716A. These data show that cannabinoids are highly potent and efficacious antihyperalgesic agents in a model of neuropathic pain. This activity is likely to be mediated via an action in both the CNS and in the periphery.

Vanilloid receptor type 1 (VR1) (TRPV1) is a ligand-gated ion channel expressed on sensory nerves that responds to noxious heat, protons, and chemical stimuli such as capsaicin. Herein, we have examined the activity of the VR1 antagonist capsazepine in models of inflammatory and neuropathic pain in the rat, mouse, and guinea pig. In naïve animals, subcutaneous administration of capsazepine (10 -100 mg/kg s.c.) did not affect withdrawal thresholds to noxious thermal or mechanical stimuli. However, pretreatment with capsazepine prevented the development of mechanical hyperalgesia induced by intraplantar injection of capsaicin, with a similar potency in all three species. Capsazepine (up to 100 mg/kg s.c.) did not affect mechanical hyperalgesia in the Freund's complete adjuvant (FCA)-inflamed hind paw of the rat or mouse. Strikingly, capsazepine (3-30 mg/kg s.c.) produced up to 44% reversal of FCA-induced mechanical hyperalgesia in the guinea pig. Capsazepine also produced significant reversal of carageenan-induced thermal hyperalgesia in the guinea pig at 30 mg/kg s.c., but was ineffective in the rat. Similarly, in the partial sciatic nerve ligation model of neuropathic pain, capsazepine was surprisingly effective in the guinea pig, producing up to 80% reversal of mechanical hyperalgesia (1-30 mg/kg s.c.) but had no effect in the rat or mouse. These data show that VR1 antagonists have antihyperalgesic activity in animal models of chronic inflammatory and neuropathic pain, and illustrate species differences in the in vivo pharmacology of VR1 that correlate with differences in pharmacology previously seen in vitro.

Double stranded, short interfering RNAs (siRNA) of 21-22 nt length initiate a sequence-specific, post-trancriptional gene silencing in animals and plants known as RNA interference (RNAi). Here we show that RNAi can block a pathophysiological pain response and provide relief from neuropathic pain in a rat disease model by down regulating an endogenous, neuronally expressed gene. Rats, intrathecally infused with a 21 nt siRNA perfectly complementary to the pain-related cation-channel P2X3, showed diminished pain responses compared to missense (MS) siRNA-treated and untreated controls in models of both agonist-evoked pain and chronic neuropathic pain. This form of delivery caused no adverse effects in any of the animals receiving P2X3 siRNA, MS siRNA or vehicle. Molecular analysis of tissues revealed that P2X3 mRNA expressed in dorsal root ganglia, and P2X3 protein translocated into the dorsal horn of the spinal cord, were significantly diminished. These observations open a path toward use of siRNA as a genetic tool for drug target validation in the mammalian central nervous system, as well as for proof of concept studies and as therapeutic agents in man.

Inflammatory processes occurring within the central nervous system (CNS) can produce 'illness induced behaviours' which include fever, sleep and the development of allodynia and hyperalgesia. Here we demonstrate the effects of the pro-inflammatory mediators, bacterial endotoxin, and rat recombinant interleukin 1 beta (rrIL-1 beta) or tumour necrosis factor-alpha (rrTNF alpha) on the integration of somatosensory information at the single neuronal level, via recordings from wide-dynamic range neurones in the dorsal horn of the spinal cord in anaesthetized rats. Intrathecal administration of E. coli lipopolysaccharide (LPS, 10 and 100 microg, i.t.) enhanced the activity of dorsal horn neurones, including facilitation of neuronal post-discharge. Intrathecal administration of IL-1 beta (5-5000 pg) or TNF-alpha (5-5000 pg) enhanced dorsal horn neuronal responses, including the acute responses to C-fibre stimulation, wind-up and post-discharge, however, the effects of IL-1 beta were more robust than those of TNF alpha. Intrathecal administration of IL-1 beta (1-1000 pg) also leads to the development of mechanical allodynia and hyperalgesia. On the other hand intrathecal application of TNF alpha did not produce changes in sensitivity to mechanical stimuli. Changes in the activity of spinal wide-dynamic range neurones induced by local inflammation may provide a pathomechanism for the clinical pathology of central pain syndrome, which can accompany CNS disease or acute CNS injury.

The excitation of nociceptive sensory neurons by ATP released in injured tissue is believed to be mediated partly by P2X3 receptors. Although an analysis of P2X3 knock-out mice has revealed some deficits in nociceptive signaling, detailed analysis of the role of these receptors is hampered by the lack of potent specific pharmacological tools. Here we have used antisense oligonucleotides (ASOs) to downregulate P2X3 receptors to examine their role in models of chronic pain in the rat. ASOs and control missense oligonucleotides (180 microg/d) were administered intrathecally to naive rats for up to 7 d via a lumbar indwelling cannula attached to an osmotic minipump. Functional downregulation of the receptors was confirmed by alphabeta-methylene ATP injection into the hindpaw, which evoked significantly less mechanical hyperalgesia as early as 2 d after treatment with ASOs relative to controls. At this time point, P2X3 protein levels were significantly downregulated in lumbar L4 and L5 dorsal root ganglia. After 7 d of ASO treatment, P2X3 protein levels were reduced in the primary afferent terminals in the lumbar dorsal horn of the spinal cord. In models of neuropathic (partial sciatic ligation) and inflammatory (complete Freund's adjuvant) pain, inhibition of the development of mechanical hyperalgesia as well as significant reversal of established hyperalgesia were observed within 2 d of ASO treatment. The time course of the reversal of hyperalgesia is consistent with downregulation of P2X3 receptor protein and function. This study demonstrates the utility of ASO approaches for validating gene targets in in vivo pain models and provides evidence for a role of P2X3 receptors in the pathophysiology of chronic pain.