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

Reversal of chronic pain by activation of single GABAA receptor subtypes

SUMMARY The ability to perceive pain is a basic property of humans and of all other higher order animals. The primary role of pain sensation and nociception (that is, the neuronal activity encoding pain) is to protect against potentially harmful threats arriving from the environment or the body interior. Pain can however also become dysfunctional and persist for extended periods of time without an apparent benefit, instead becoming a major burden that requires medical attention. Chronic pain is a major socio-economic challenge, which – despite scientific advances in the understanding of its causes – remains poorly responsive to the drugs available on the market today. Recent insights into the mechanisms of chronic pain states suggest that a common factor for many kinds of persistent pain states is a loss of inhibition in spinal cord circuits that normally control nociceptive input to the brain. The so- called benzodiazepines – first marketed by Hoffmann-La Roche in the 1960s – are drugs that facilitate synaptic inhibition throughout the CNS and have as such the potential to reverse pathological disinhibition. Benzodiazepines facilitate inhibition by increasing the activity of γ-aminobutyric acid (GABA) at its receptor, a heteropentameric anion permeable ion channel. Although rodent studies have shown that pathologically increased pain sensitivity can be normalized by intrathecal (spinal) injection of benzodiazepines, these drugs do not exert clinically relevant analgesia in human patients, at least not after systemic application. In this thesis, I have tested the hypothesis that benzodiazepines reverse pathological pain after systemic application if their action is restricted to well-defined subtypes of GABAA receptors. Using GABAA receptor point-mutated mice, I was able to demonstrate that selective targeting of GABAA receptors that contain the α2 subunit (α2-GABAA) receptors evoke pronounced pain relief in the absence of confounding and undesired sedation. I could also confirm previous findings that had proposed that activation of the same GABAA receptors induces anxiolysis and muscle relaxation. Importantly, selective targeting of α2- GABAA receptors avoided several unwanted effects of classical non-selective benzodiazepines including sedation, impairment of motor coordination, and the progressive loss of therapeutic efficacy over time. Using mice in which the action of classical benzodiazepine agonists was restricted to only a single GABAA receptor subtype, I could also propose a new hypothesis explaining why classical benzodiazepines lack clinically relevant analgesic properties. For two clinically used benzodiazepines (diazepam and midazolam), I could demonstrate that strong α1-GABAA receptor-mediated sedation occurs already at doses

Reversal of chronic pain by activation of single GABA_A receptor subtypes

SUMMARY The ability to perceive pain is a basic property of humans and of all other higher order animals. The primary role of pain sensation and nociception (that is, the neuronal activity encoding pain) is to protect against potentially harmful threats arriving from the environment or the body interior. Pain can however also become dysfunctional and persist for extended periods of time without an apparent benefit, instead becoming a major burden that requires medical attention. Chronic pain is a major socio-economic challenge, which – despite scientific advances in the understanding of its causes – remains poorly responsive to the drugs available on the market today. Recent insights into the mechanisms of chronic pain states suggest that a common factor for many kinds of persistent pain states is a loss of inhibition in spinal cord circuits that normally control nociceptive input to the brain. The so- called benzodiazepines – first marketed by Hoffmann-La Roche in the 1960s – are drugs that facilitate synaptic inhibition throughout the CNS and have as such the potential to reverse pathological disinhibition. Benzodiazepines facilitate inhibition by increasing the activity of γ-aminobutyric acid (GABA) at its receptor, a heteropentameric anion permeable ion channel. Although rodent studies have shown that pathologically increased pain sensitivity can be normalized by intrathecal (spinal) injection of benzodiazepines, these drugs do not exert clinically relevant analgesia in human patients, at least not after systemic application. In this thesis, I have tested the hypothesis that benzodiazepines reverse pathological pain after systemic application if their action is restricted to well-defined subtypes of GABAA receptors. Using GABAA receptor point-mutated mice, I was able to demonstrate that selective targeting of GABAA receptors that contain the α2 subunit (α2-GABAA) receptors evoke pronounced pain relief in the absence of confounding and undesired sedation. I could also confirm previous findings that had proposed that activation of the same GABAA receptors induces anxiolysis and muscle relaxation. Importantly, selective targeting of α2- GABAA receptors avoided several unwanted effects of classical non-selective benzodiazepines including sedation, impairment of motor coordination, and the progressive loss of therapeutic efficacy over time. Using mice in which the action of classical benzodiazepine agonists was restricted to only a single GABAA receptor subtype, I could also propose a new hypothesis explaining why classical benzodiazepines lack clinically relevant analgesic properties. For two clinically used benzodiazepines (diazepam and midazolam), I could demonstrate that strong α1-GABAA receptor-mediated sedation occurs already at doses

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

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

TP003 is a non-selective benzodiazepine site agonist that induces anxiolysis via α2GABA receptors

Benzodiazepines (BDZ), which potentiate the action of GABA at four subtypes of GABA receptors (α1, α2, α3, and α5GABARs), are highly effective against anxiety disorders, but also cause severe side effects greatly limiting their clinical application. Both, preclinical studies in genetically engineered mice, and preclinical and clinical trials with subtype-selective compounds indicate that undesired effects can in principle be avoided by targeting specific GABAR subtypes. While there is general consensus that activity at α1GABARs should be avoided, controversy exists as to whether α2 or α3GABARs need to be targeted for anxiolysis. While previous experiments in GABAR point-mutated mice demonstrated a critical role of α2GABARs, studies solely relying on pharmacological approaches suggested a dominant contribution of α3GABARs. As most α1GABAR-sparing BDZ site agonists discriminate little between α2 and α3GABARs, these claims rest almost exclusively on a single compound, TP003, that has been reported to be a selective α3GABAR modulator. Here, we have revisited the in vitro pharmacological profile of TP003 and, in addition, tested TP003 in GABAR triple point-mutated mice, in which only either α1, α2, or α3GABARs were left BDZ sensitive. These experiments revealed that TP003 behaves as a partial, rather non-selective BDZ site agonist in vitro that acts in vivo through α1, α2, and α3GABARs (α5GABAR-mediated effects were not tested). With respect to anxiolysis, our results support a critical contribution of α2GABARs, but not of α3GABARs. TP003 should therefore not be considered an α3GABAR selective agent. Previously published studies using TP003 should be interpreted with caution

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

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

Localization and production of peptide endocannabinoids in the rodent CNS and adrenal medulla.

The endocannabinoid system (ECS) comprises the cannabinoid receptors CB1 and CB2 and their endogenous arachidonic acid-derived agonists 2-arachidonoyl glycerol and anandamide, which play important neuromodulatory roles. Recently, a novel class of negative allosteric CB1 receptor peptide ligands, hemopressin-like peptides derived from alpha hemoglobin, has been described, with yet unknown origin and function in the CNS. Using monoclonal antibodies we now identified the localization of RVD-hemopressin (pepcan-12) and N-terminally extended peptide endocannabinoids (pepcans) in the CNS and determined their neuronal origin. Immunohistochemical analyses in rodents revealed distinctive and specific staining in major groups of noradrenergic neurons, including the locus coeruleus (LC), A1, A5 and A7 neurons, which appear to be major sites of production/release in the CNS. No staining was detected in dopaminergic neurons. Peptidergic axons were seen throughout the brain (notably hippocampus and cerebral cortex) and spinal cord, indicative of anterograde axonal transport of pepcans. Intriguingly, the chromaffin cells in the adrenal medulla were also strongly stained for pepcans. We found specific co-expression of pepcans with galanin, both in the LC and adrenal gland. Using LC-MS/MS, pepcan-12 was only detected in non-perfused brain (∼40 pmol/g), suggesting that in the CNS it is secreted and present in extracellular compartments. In adrenal glands, significantly more pepcan-12 (400-700 pmol/g) was measured in both non-perfused and perfused tissue. Thus, chromaffin cells may be a major production site of pepcan-12 found in blood. These data uncover important areas of peptide endocannabinoid occurrence with exclusive noradrenergic immunohistochemical staining, opening new doors to investigate their potential physiological function in the ECS. This article is part of a Special Issue entitled 'Fluorescent Neuro-Ligands'

Localization and production of peptide endocannabinoids in the rodent CNS and adrenal medulla

The endocannabinoid system (ECS) comprises the cannabinoid receptors CB1 and CB2 and their endogenous arachidonic acid-derived agonists 2-arachidonoyl glycerol and anandamide, which play important neuromodulatory roles. Recently, a novel class of negative allosteric CB1 receptor peptide ligands, hemopressin-like peptides derived from alpha hemoglobin, has been described, with yet unknown origin and function in the CNS. Using monoclonal antibodies we now identified the localization of RVD-hemopressin (pepcan-12) and N-terminally extended peptide endocannabinoids (pepcans) in the CNS and determined their neuronal origin. Immunohistochemical analyses in rodents revealed distinctive and specific staining in major groups of noradrenergic neurons, including the locus coeruleus (LC), A1, A5 and A7 neurons, which appear to be major sites of production/release in the CNS. No staining was detected in dopaminergic neurons. Peptidergic axons were seen throughout the brain (notably hippocampus and cerebral cortex) and spinal cord, indicative of anterograde axonal transport of pepcans. Intriguingly, the chromaffin cells in the adrenal medulla were also strongly stained for pepcans. We found specific co-expression of pepcans with galanin, both in the LC and adrenal gland. Using LC-MS/MS, pepcan-12 was only detected in non-perfused brain (∼40 pmol/g), suggesting that in the CNS it is secreted and present in extracellular compartments. In adrenal glands, significantly more pepcan-12 (400-700 pmol/g) was measured in both non-perfused and perfused tissues. Thus, chromaffin cells may be a major production site of pepcan-12 found in blood. These data uncover important areas of peptide endocannabinoid occurrence with exclusive noradrenergic immunohistochemical staining, opening new doors to investigate their potential physiological function in the ECS. This article is part of a Special Issue entitled 'Fluorescent Neuro-Ligands'