Antihyperalgesia by α2-GABAA receptors occurs via a genuine spinal action and does not involve supraspinal sites.

Drugs that enhance GABAergic inhibition alleviate inflammatory and neuropathic pain after spinal application. This antihyperalgesia occurs mainly through GABAA receptors (GABAARs) containing α2 subunits (α2-GABAARs). Previous work indicates that potentiation of these receptors in the spinal cord evokes profound antihyperalgesia also after systemic administration, but possible synergistic or antagonistic actions of supraspinal α2-GABAARs on spinal antihyperalgesia have not yet been addressed. Here we generated two lines of GABAAR-mutated mice, which either lack α2-GABAARs specifically from the spinal cord, or, which express only benzodiazepine-insensitive α2-GABAARs at this site. We analyzed the consequences of these mutations for antihyperalgesia evoked by systemic treatment with the novel non-sedative benzodiazepine site agonist HZ166 in neuropathic and inflammatory pain. Wild-type mice and both types of mutated mice had similar baseline nociceptive sensitivities and developed similar hyperalgesia. However, antihyperalgesia by systemic HZ166 was reduced in both mutated mouse lines by about 60% and was virtually indistinguishable from that of global point-mutated mice, in which all α2-GABAARs were benzodiazepine insensitive. The major (α2-dependent) component of GABAAR-mediated antihyperalgesia was therefore exclusively of spinal origin, whereas supraspinal α2-GABAARs had neither synergistic nor antagonistic effects on antihyperalgesia. Our results thus indicate that drugs that specifically target α2-GABAARs exert their antihyperalgesic effect through enhanced spinal nociceptive control. Such drugs may therefore be well-suited for the systemic treatment of different chronic pain conditions.

Presynaptic alpha2-GABAA receptors in primary afferent depolarization and spinal pain control.

Spinal dorsal horn GABA(A) receptors are found both postsynaptically on central neurons and presynaptically on axons and/or terminals of primary sensory neurons, where they mediate primary afferent depolarization (PAD) and presynaptic inhibition. Both phenomena have been studied extensively on a cellular level, but their role in sensory processing in vivo has remained elusive, due to inherent difficulties to selectively interfere with presynaptic receptors. Here, we address the contribution of a major subpopulation of GABA(A) receptors (those containing the α2 subunit) to spinal pain control in mice lacking α2-GABA(A) receptors specifically in primary nociceptors (sns-α2(-/-) mice). sns-α2(-/-) mice exhibited GABA(A) receptor currents and dorsal root potentials of normal amplitude in vitro, and normal response thresholds to thermal and mechanical stimulation in vivo, and developed normal inflammatory and neuropathic pain sensitization. However, the positive allosteric GABA(A) receptor modulator diazepam (DZP) had almost completely lost its potentiating effect on PAD and presynaptic inhibition in vitro and a major part of its spinal antihyperalgesic action against inflammatory hyperalgesia in vivo. Our results thus show that part of the antihyperalgesic action of spinally applied DZP occurs through facilitated activation of GABA(A) receptors residing on primary nociceptors.

A virus-like particle-based anti-nerve growth factor vaccine reduces inflammatory hyperalgesia: potential long-term therapy for chronic pain.

Chronic pain resulting from inflammatory and neuropathic disorders causes considerable economic and social burden. For a substantial proportion of patients, conventional drug treatments do not provide adequate pain relief. Consequently, novel approaches to pain management, involving alternative targets and new therapeutic modalities compatible with chronic use, are being sought. Nerve growth factor (NGF) is a major mediator of chronic pain. Clinical testing of NGF antagonists is ongoing, and clinical proof of concept has been established with a neutralizing mAb. Active immunization, with the goal of inducing therapeutically effective neutralizing autoreactive Abs, is recognized as a potential treatment option for chronic diseases. We have sought to determine if such a strategy could be applied to chronic pain by targeting NGF with a virus-like particle (VLP)-based vaccine. A vaccine comprising recombinant murine NGF conjugated to VLPs from the bacteriophage Qß (NGFQß) was produced. Immunization of mice with NGFQß induced anti-NGF-specific IgG Abs capable of neutralizing NGF. Titers could be sustained over 1 y by periodic immunization but declined in the absence of boosting. Vaccination with NGFQß substantially reduced hyperalgesia in collagen-induced arthritis or postinjection of zymosan A, two models of inflammatory pain. Long-term NGFQß immunization did not change sensory or sympathetic innervation patterns or induce cholinergic deficits in the forebrain, nor did it interfere with blood-brain barrier integrity. Thus, autovaccination targeting NGF using a VLP-based approach may represent a novel modality for the treatment of chronic pain.

Hoxb8-Cre mice: A tool for brain-sparing conditional gene deletion.

The spinal cord is the first site of temporal and spatial integration of nociceptive signals in the pain pathway. Neuroplastic changes occurring at this site contribute critically to various chronic pain syndromes. Gene targeting in mice has generated important insights into these processes. However, the analysis of constitutive (global) gene-deficient mice is often hampered by confounding effects arising from supraspinal sites. Here, we describe a novel Cre mouse line that expresses the Cre recombinase under the transcriptional control of the Hoxb8 gene. Within the neural axis of these mice, Hoxb8-Cre expression is found in spinal cord neurons and glial cells, and in virtually all neurons of the dorsal root ganglia, but spares the brain apart from a few cells in the spinal trigeminal nucleus. The Hoxb8-Cre mouse line should be a valuable new tool for the in vivo analysis of peripheral and spinal gene functions in pain pathways.

Pharmacokinetics and pharmacodynamics of nasally delivered midazolam.

AIMS: To investigate the pharmacokinetics and pharmacodynamics of nasal formulations containing midazolam (5-30 mg ml(-1)) complexed with cyclodextrin. METHODS: An open-label sequential trial was conducted in eight healthy subjects receiving single doses of 1 mg and 3 mg intranasally and 1 mg midazolam intravenously. Pharmacokinetic parameters were obtained by non-compartmental and two-compartmental models. Pharmacodynamic effects of midazolam were assessed using VAS and a reaction time test. RESULTS: Mean bioavailability of midazolam after nasal administration ranged from 76 +/- 12% to 92 +/- 15%. With formulations delivering 1 mg midazolam, mean C(max) values between 28.1 +/- 9.1 and 30.1 +/- 6.6 ng ml(-1) were reached after 9.4 +/- 3.2-11.3 +/- 4.4 min. With formulations delivering 3 mg midazolam, mean C(max) values were between 68.9 +/- 19.8 and 80.6 +/- 15.2 ng ml(-1) after 7.2 +/- 0.7-13.0 +/- 4.3 min. Chitosan significantly increased C(max) and reduced t(max) of midazolam in the high-dose formulation. Mean ratios of dose-adjusted AUC after intranasal and intravenous application for 1'-hydroxymidazolam were between 0.97 +/- 0.15 and 1.06 +/- 0.24, excluding relevant gastrointestinal absorption of intranasal midazolam. The pharmacodynamic effects after the low-dose nasal formulations were comparable with those after 1 mg intravenous midazolam. The maximum increase in reaction time by the chitosan-containing formulation delivering 3 mg midazolam was greater compared with 1 mg midazolam i.v. (95 +/- 78 ms and 19 +/- 22 ms, mean difference 75.5 ms, 95% CI 15.5, 135.5, P < 0.01). Intranasal midazolam was well tolerated but caused reversible irritation of the nasal mucosa. CONCLUSIONS: Effective midazolam serum concentrations were reached within less than 10 min after nasal application of a highly concentrated midazolam formulation containing an equimolar amount of the solubilizer RMbetaCD combined with the absorption enhancer chitosan.

Spinal endocannabinoids and CB1 receptors mediate C-fiber-induced heterosynaptic pain sensitization.

Diminished synaptic inhibition in the spinal dorsal horn is a major contributor to chronic pain. Pathways that reduce synaptic inhibition in inflammatory and neuropathic pain states have been identified, but central hyperalgesia and diminished dorsal horn synaptic inhibition also occur in the absence of inflammation or neuropathy, solely triggered by intense nociceptive (C-fiber) input to the spinal dorsal horn. We found that endocannabinoids, produced upon strong nociceptive stimulation, activated type 1 cannabinoid (CB1) receptors on inhibitory dorsal horn neurons to reduce the synaptic release of gamma-aminobutyric acid and glycine and thus rendered nociceptive neurons excitable by nonpainful stimuli. Our results suggest that spinal endocannabinoids and CB1 receptors on inhibitory dorsal horn interneurons act as mediators of heterosynaptic pain sensitization and play an unexpected role in dorsal horn pain-controlling circuits.

Subtype-selective GABAA receptor mimetics--novel antihyperalgesic agents?

Agonists at the benzodiazepine-binding site of ionotropic gamma-aminobutyric acid (GABA(A)) receptors are in clinical use as hypnotics, anxiolytics, and anticonvulsants since the early 1960. Analgesic effects of classical benzodiazepines have occasionally been reported in certain subgroups of patients suffering from chronic pain or after spinal delivery through intrathecal catheters. However, these drugs are generally not considered as analgesics but should in fact be avoided in patients with chronic pain. Recent evidence from genetically modified mice now indicates that agents targeting only a subset of benzodiazepine (GABA(A)) receptors should provide pronounced antihyperalgesic activity against inflammatory and neuropathic pain. Several such compounds have been developed recently, which exhibit significant antihyperalgesia in mice and rats and appear to be devoid of the typical side-effects of classical benzodiazepines.

Reversal of pathological pain through specific spinal GABAA receptor subtypes.

Inflammatory diseases and neuropathic insults are frequently accompanied by severe and debilitating pain, which can become chronic and often unresponsive to conventional analgesic treatment. A loss of synaptic inhibition in the spinal dorsal horn is considered to contribute significantly to this pain pathology. Facilitation of spinal gamma-aminobutyric acid (GABA)ergic neurotransmission through modulation of GABA(A) receptors should be able to compensate for this loss. With the use of GABA(A)-receptor point-mutated knock-in mice in which specific GABA(A) receptor subtypes have been selectively rendered insensitive to benzodiazepine-site ligands, we show here that pronounced analgesia can be achieved by specifically targeting spinal GABA(A) receptors containing the alpha2 and/or alpha3 subunits. We show that their selective activation by the non-sedative ('alpha1-sparing') benzodiazepine-site ligand L-838,417 (ref. 13) is highly effective against inflammatory and neuropathic pain yet devoid of unwanted sedation, motor impairment and tolerance development. L-838,417 not only diminished the nociceptive input to the brain but also reduced the activity of brain areas related to the associative-emotional components of pain, as shown by functional magnetic resonance imaging in rats. These results provide a rational basis for the development of subtype-selective GABAergic drugs for the treatment of chronic pain, which is often refractory to classical analgesics.