William T. Ralvenius

Targeted Ablation, Silencing, and Activation Establish Glycinergic Dorsal Horn Neurons as Key Components of a Spinal Gate for Pain and Itch

Summary The gate control theory of pain proposes that inhibitory neurons of the spinal dorsal horn exert critical control over the relay of nociceptive signals to higher brain areas. Here we investigated how the glycinergic subpopulation of these neurons contributes to modality-specific pain and itch processing. We generated a GlyT2::Cre transgenic mouse line suitable for virus-mediated retrograde tracing studies and for spatially precise ablation, silencing, and activation of glycinergic neurons. We found that these neurons receive sensory input mainly from myelinated primary sensory neurons and that their local toxin-mediated ablation or silencing induces localized mechanical, heat, and cold hyperalgesia; spontaneous flinching behavior; and excessive licking and biting directed toward the corresponding skin territory. Conversely, local pharmacogenetic activation of the same neurons alleviated neuropathic hyperalgesia and chloroquine- and histamine-induced itch. These results establish glycinergic neurons of the spinal dorsal horn as key elements of an inhibitory pain and itch control circuit.

Analgesia and unwanted benzodiazepine effects in point-mutated mice expressing only one benzodiazepine-sensitive GABAA receptor subtype

Agonists at the benzodiazepine-binding site of GABAA receptors (BDZs) enhance synaptic inhibition through four subtypes (α1, α2, α3 and α5) of GABAA receptors (GABAAR). When applied to the spinal cord, they alleviate pathological pain; however, insufficient efficacy after systemic administration and undesired effects preclude their use in routine pain therapy. Previous work suggested that subtype-selective drugs might allow separating desired antihyperalgesia from unwanted effects, but the lack of selective agents has hitherto prevented systematic analyses. Here we use four lines of triple GABAAR point-mutated mice, which express only one benzodiazepine-sensitive GABAAR subtype at a time, to show that targeting only α2GABAARs achieves strong antihyperalgesia and reduced side effects (that is, no sedation, motor impairment and tolerance development). Additional pharmacokinetic and pharmacodynamic analyses in these mice explain why clinically relevant antihyperalgesia cannot be achieved with nonselective BDZs. These findings should foster the development of innovative subtype-selective BDZs for novel indications such as chronic pain.

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.

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 Qβ (NGFQβ) was produced. Immunization of mice with NGFQβ 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 NGFQβ substantially reduced hyperalgesia in collagen-induced arthritis or postinjection of zymosan A, two models of inflammatory pain. Long-term NGFQβ 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.

Chapter Four – Restoring the Spinal Pain Gate: GABAA Receptors as Targets for Novel Analgesics

GABAA receptors (GABA(A)Rs) and glycine receptors are key elements of the spinal control of nociception and pain. Compromised functioning of these two transmitter systems contributes to chronic pain states. Restoring their proper function through positive allosteric modulators should constitute a rational approach to the treatment of chronic pain syndromes involving diminished inhibitory spinal pain control. Although classical benzodiazepines (i.e., full agonists at the benzodiazepine binding site of GABA(A)Rs) potentiate synaptic inhibition in spinal pain controlling circuits, they lack clinically relevant analgesic activity in humans. Recent data obtained from experiments in GABA(A)R point-mutated mice suggests dose-limiting sedative effects of classical nonspecific benzodiazepines as the underlying cause. Experiments in genetically engineered mice resistant to the sedative effects of classical benzodiazepines and studies with novel less sedating benzodiazepines have indeed shown that profound antihyperalgesia can be obtained at least in preclinical pain models. Present evidence suggests that compounds with high intrinsic activity at α2-GABA(A)R and minimal agonistic activity at α1-GABA(A)R should possess relevant antihyperalgesic activity without causing unwanted sedation. On-going preclinical studies in genetically engineered mice and clinical trials with more selective benzodiazepine site agonists should soon provide additional insights into this emerging topic.

Restoring the spinal pain gate: GABA(A) receptors as targets for novel analgesics.

GABAA receptors (GABA(A)Rs) and glycine receptors are key elements of the spinal control of nociception and pain. Compromised functioning of these two transmitter systems contributes to chronic pain states. Restoring their proper function through positive allosteric modulators should constitute a rational approach to the treatment of chronic pain syndromes involving diminished inhibitory spinal pain control. Although classical benzodiazepines (i.e., full agonists at the benzodiazepine binding site of GABA(A)Rs) potentiate synaptic inhibition in spinal pain controlling circuits, they lack clinically relevant analgesic activity in humans. Recent data obtained from experiments in GABA(A)R point-mutated mice suggests dose-limiting sedative effects of classical nonspecific benzodiazepines as the underlying cause. Experiments in genetically engineered mice resistant to the sedative effects of classical benzodiazepines and studies with novel less sedating benzodiazepines have indeed shown that profound antihyperalgesia can be obtained at least in preclinical pain models. Present evidence suggests that compounds with high intrinsic activity at α2-GABA(A)R and minimal agonistic activity at α1-GABA(A)R should possess relevant antihyperalgesic activity without causing unwanted sedation. On-going preclinical studies in genetically engineered mice and clinical trials with more selective benzodiazepine site agonists should soon provide additional insights into this emerging topic.

Phosphorylation state–dependent modulation of spinal glycine receptors alleviates inflammatory pain

Diminished inhibitory neurotransmission in the superficial dorsal horn of the spinal cord is thought to contribute to chronic pain. In inflammatory pain, reductions in synaptic inhibition occur partially through prostaglandin E2- (PGE2-) and PKA-dependent phosphorylation of a specific subtype of glycine receptors (GlyRs) that contain α3 subunits. Here, we demonstrated that 2,6-di-tert-butylphenol (2,6-DTBP), a nonanesthetic propofol derivative, reverses inflammation-mediated disinhibition through a specific interaction with heteromeric αβGlyRs containing phosphorylated α3 subunits. We expressed mutant GlyRs in HEK293T cells, and electrophysiological analyses of these receptors showed that 2,6-DTBP interacted with a conserved phenylalanine residue in the membrane-associated stretch between transmembrane regions 3 and 4 of the GlyR α3 subunit. In native murine spinal cord tissue, 2,6-DTBP modulated synaptic, presumably αβ heteromeric, GlyRs only after priming with PGE2. This observation is consistent with results obtained from molecular modeling of the α-β subunit interface and suggests that in α3βGlyRs, the binding site is accessible to 2,6-DTBP only after PKA-dependent phosphorylation. In murine models of inflammatory pain, 2,6-DTBP reduced inflammatory hyperalgesia in an α3GlyR-dependent manner. Together, our data thus establish that selective potentiation of GlyR function is a promising strategy against chronic inflammatory pain and that, to our knowledge, 2,6-DTBP has a unique pharmacological profile that favors an interaction with GlyRs that have been primed by peripheral inflammation.

The positive allosteric GABAB receptor modulator rac-BHFF enhances baclofen-mediated analgesia in neuropathic mice

Neuropathic pain is associated with impaired inhibitory control of spinal dorsal horn neurons, which are involved in processing pain signals. The metabotropic GABAB receptor is an important component of the inhibitory system and is highly expressed in primary nociceptors and intrinsic dorsal horn neurons to control their excitability. Activation of GABAB receptors with the orthosteric agonist baclofen effectively reliefs neuropathic pain but is associated with severe side effects that prevent its widespread application. The recently developed positive allosteric GABAB receptor modulators lack most of these side effects and are therefore promising drugs for the treatment of pain. Here we tested the high affinity positive allosteric modulator rac-BHFF for its ability to relief neuropathic pain induced by chronic constriction of the sciatic nerve in mice. rac-BHFF significantly increased the paw withdrawal threshold to mechanical stimulation in healthy mice, indicating an endogenous GABABergic tone regulating the sensitivity to mechanical stimuli. Surprisingly, rac-BHFF displayed no analgesic activity in neuropathic mice although GABAB receptor expression was not affected in the dorsal horn as shown by quantitative receptor autoradiography. However, activation of spinal GABAB receptors by intrathecal injection of baclofen reduced hyperalgesia and its analgesic effect was considerably potentiated by co-application of rac-BHFF. These results indicate that under conditions of neuropathic pain the GABAergic tone is too low to provide a basis for allosteric modulation of GABAB receptors. However, allosteric modulators would be well suited as an add-on to reduce the dose of baclofen required to achieve analgesia.

The clobazam metabolite N-desmethyl clobazam is an α2 preferring benzodiazepine with an improved therapeutic window for antihyperalgesia

Data from genetically modified mice suggest that benzodiazepine (BDZ)-site agonists with improved selectivity for α2-subtype GABAA receptors (α2GABAAR) are potentially useful for the treatment of neuropathic pain. Subtype-selective compounds available for preclinical tests in rodents support this concept but have not been approved for human use, hindering proof-of-concept studies in patients. We recently proposed that N-desmethyl clobazam (NDMC), the main metabolite of the licensed BDZ clobazam (CBZ), is responsible for most of the antihyperalgesia observed in mice after CBZ administration. In order to assess a potentially favorable pharmacological profile of NDMC, we analyzed differences in the GABAAR subtype specificity of CBZ, NDMC and diazepam (DZP) in recombinant receptors. DZP and CBZ potentiated sedating α1GABAARs and antihyperalgesic α2GABAARs with similar efficacies, whereas NDMC preferred α2GABAARs over α1GABAARs across a wide concentration range. In vivo, DZP and NDMC reduced neuropathic pain at doses between 3 and 30 mg/kg. At these doses, DZP had strong locomotor sedating effects while NDMC caused no or only weak sedation. Sedative effects of NDMC became apparent when the action of NDMC was restricted to α1GABAARs. However, when GABAAR point-mutated mice were studied that allow the analysis of antihyperalgesia and sedation in isolation, we found that, compared to DZP, NDMC had a significantly improved therapeutic window, consistent with its more favorable α2/α1 in vitro activity ratio. Given that NDMC should share the safety profile of its parent compound CBZ, it should be well-suited for proof-of-concept studies in human volunteers or patients.

GABAA receptor subtypes in the mouse brain: Regional mapping and diazepam receptor occupancy by in vivo [18F]flumazenil PET

Abstract Classical benzodiazepines, which are widely used as sedatives, anxiolytics and anticonvulsants, exert their therapeutic effects through interactions with heteropentameric GABAA receptors composed of two &agr;, two &bgr; and one &ggr;2 subunit. Their high affinity binding site is located at the interface between the &ggr;2 and the adjacent &agr; subunit. The &agr;‐subunit gene family consists of six members and receptors can be homomeric or mixed with respect to the &agr;‐subunits. Previous work has suggested that benzodiazepine binding site ligands with selectivity for individual GABAA receptor subtypes, as defined by the benzodiazepine‐binding &agr; subunit, may have fewer side effects and may even be effective in diseases, such as schizophrenia, autism or chronic pain, that do not respond well to classical benzodiazepines. The distributions of the individual &agr; subunits across the CNS have been extensively characterized. However, as GABAA receptors may contain two different &agr; subunits, the distribution of the subunits does not necessarily reflect the distribution of receptor subtypes with respect to benzodiazepine pharmacology. In the present study, we have used in vivo [18F]flumazenil PET and in vitro [3H]flumazenil autoradiography in combination with GABAA receptor point‐mutated mice to characterize the distribution of the two most prevalent GABAA receptor subtypes (&agr;1 and &agr;2) throughout the mouse brain. The results were in agreement with published in vitro data. High levels of &agr;2‐containing receptors were found in brain regions of the neuronal network of anxiety. The &agr;1/&agr;2 subunit combinations were predictable from the individual subunit levels. In additional experiments, we explored in vivo [18F]flumazenil PET to determine the degree of receptor occupancy at GABAA receptor subtypes following oral administration of diazepam. The dose to occupy 50% of sensitive receptors, independent of the receptor subtype(s), was 1–2 mg/kg, in agreement with published data from ex vivo studies with wild type mice. In conclusion, we have resolved the quantitative distribution of &agr;1‐ and &agr;2‐containing homomeric and mixed GABAA receptors in vivo at the millimeter scale and demonstrate that the regional drug receptor occupancy in vivo at these GABAA receptor subtypes can be determined by [18F]flumazenil PET. Such information should be valuable for drug development programs aiming for subtype‐selective benzodiazepine site ligands for new therapeutic indications. Graphical abstract Figure. No Caption available. Highlights[18F]Flumazenil PET with point‐mutated mice to predict GABAA receptor pharmacology.Mapping of homomeric and mixed &agr;1 and &agr;2 GABAA receptors in vivo in mouse brain.In vivo regional GABAA receptor occupancy in mouse brain by [18F]flumazenil PET.