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

Ralvenius WT, Benke D, Acuña MA, Rudolph U, Zeilhofer HU - Nat Commun (2015)

Bottom Line: 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).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.These findings should foster the development of innovative subtype-selective BDZs for novel indications such as chronic pain.

View Article: PubMed Central - PubMed

Affiliation: 1] Institute of Pharmacology and Toxicology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland [2] Center for Neuroscience Zurich (ZNZ), Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.

ABSTRACT
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.

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Activity of DZP and MDZ at α1 and α2GABAARs, and PK/PD modelling.(a,b) Potentiation of recombinant α1β2γ2 and α2β3γ2 GABAAR currents by 1 μM DZP (a) and 1 μM MDZ (b). GABA concentration was EC5 (5 μM for α1β2γ2 and 2 μM for α2β3γ2). n=6 for both drugs and all concentrations. Scales bars, 200 pA, 2 s. (c,d) The H101R mutation prevented potentiation of α1β2γ2 GABAAR currents by MDZ (**P<0.01, n=5, unpaired t-test; c) and binding of MDZ to α1β2γ2 GABAARs (n=6 for all concentrations in wt and mutated receptors; d). (e–h) α1GABAAR-mediated sedation and α2GABAAR-mediated antihyperalgesia by DZP and MDZ. Sedation (black symbols) in HRRR mice was assessed in the open field test and expressed as percent maximum possible effect determined from the reduction in activity compared with vehicle-treated mice of the same genotype. Antihyperalgesia (red symbols) in RHRR mice was determined as the increase in mechanical withdrawal thresholds compared with pre-drug values 1 week after the CCI surgery. (e,f) Dose–response relationships. Data were fitted to Hill's equation with baseline fixed at 0. Sedation and antihyperalgesia were determined between 15 and 90 min after oral DZP (e) or between 15 and 60 min after oral MDZ (f). Mice were killed immediately afterwards (that is, at the time point of maximal effects), and brains and spinal cords were removed for further analyses. (g,h) Dependence on receptor occupancy (RO). (g) Sedation versus RO in brains of HRRR mice treated with DZP (0.03, 0.1, 0.3, 1, 3, 10 and 30 mg kg−1, n=6 mice for all doses). Antihyperalgesia versus RO in lumbar spinal cords of RHRR mice treated with 0.1, 1, 3, 10 or 30 mg kg−1 DZP, n=6 mice for all doses. (h) Same as g but MDZ. Sedation 0.1 (n=6 mice), 0.3 (7), 0.5 (7), 1.0 (7), 3 (6) and 10 (4) mg kg−1. Antihyperalgesia 0.1 (n=3), 0.3 (5), 1.0 (9), 3 (8), 5 (7) and 10 (7) mg kg−1 MDZ. Data were fitted to sigmoidal functions. Data shown in e,g (on DZP) and in f,h (on MDZ) are from the same two groups of mice. All data points are mean±s.e.m.
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f6: Activity of DZP and MDZ at α1 and α2GABAARs, and PK/PD modelling.(a,b) Potentiation of recombinant α1β2γ2 and α2β3γ2 GABAAR currents by 1 μM DZP (a) and 1 μM MDZ (b). GABA concentration was EC5 (5 μM for α1β2γ2 and 2 μM for α2β3γ2). n=6 for both drugs and all concentrations. Scales bars, 200 pA, 2 s. (c,d) The H101R mutation prevented potentiation of α1β2γ2 GABAAR currents by MDZ (**P<0.01, n=5, unpaired t-test; c) and binding of MDZ to α1β2γ2 GABAARs (n=6 for all concentrations in wt and mutated receptors; d). (e–h) α1GABAAR-mediated sedation and α2GABAAR-mediated antihyperalgesia by DZP and MDZ. Sedation (black symbols) in HRRR mice was assessed in the open field test and expressed as percent maximum possible effect determined from the reduction in activity compared with vehicle-treated mice of the same genotype. Antihyperalgesia (red symbols) in RHRR mice was determined as the increase in mechanical withdrawal thresholds compared with pre-drug values 1 week after the CCI surgery. (e,f) Dose–response relationships. Data were fitted to Hill's equation with baseline fixed at 0. Sedation and antihyperalgesia were determined between 15 and 90 min after oral DZP (e) or between 15 and 60 min after oral MDZ (f). Mice were killed immediately afterwards (that is, at the time point of maximal effects), and brains and spinal cords were removed for further analyses. (g,h) Dependence on receptor occupancy (RO). (g) Sedation versus RO in brains of HRRR mice treated with DZP (0.03, 0.1, 0.3, 1, 3, 10 and 30 mg kg−1, n=6 mice for all doses). Antihyperalgesia versus RO in lumbar spinal cords of RHRR mice treated with 0.1, 1, 3, 10 or 30 mg kg−1 DZP, n=6 mice for all doses. (h) Same as g but MDZ. Sedation 0.1 (n=6 mice), 0.3 (7), 0.5 (7), 1.0 (7), 3 (6) and 10 (4) mg kg−1. Antihyperalgesia 0.1 (n=3), 0.3 (5), 1.0 (9), 3 (8), 5 (7) and 10 (7) mg kg−1 MDZ. Data were fitted to sigmoidal functions. Data shown in e,g (on DZP) and in f,h (on MDZ) are from the same two groups of mice. All data points are mean±s.e.m.

Mentions: Our present results and previous pain studies employing subtype-selective BDZ site agonists in rodents52026272829 contrast with the lack of a clear analgesic or antihyperalgesic action of classical BDZs in human patients3031. Apart from species differences and differences between disease models and actual disease in human patients, we found one possible explanation particularly worth studying. The doses and the degrees of receptor activation required for a relevant effect might be significantly higher in case of antihyperalgesia than of sedation. As a consequence, antihyperalgesia would occur in patients only at doses already inducing strong sedation. The availability of the triple point-mutated mice allowed us to directly compare the doses and levels of receptor occupancy at the relevant GABAAR subtypes and sites. For these experiments, we chose midazolam (MDZ) as a second classical BDZ in addition to DZP. First, we determined for both drugs their α2/α1 selectivity profiles in electrophysiological experiments on recombinant GABAARs. As expected, we found that DZP potentiated α1/β2/γ2 and α2/β3/γ2 GABAARs with similar efficacy and potency (Fig. 6a and Table 3). By contrast, MDZ potentiated α1/β2/γ2 GABAARs more than twice as much as α2/β3/γ2 GABAARs (Fig. 6b and Table 3). We then verified that the H→R point mutation blocked not only DZP effects but also MDZ binding and GABAAR potentiation (Figs 6c,d; see also ref. 32). Next, we compared the dose dependency of DZP- and MDZ-induced antihyperalgesia in mice with only α2 BDZ-sensitive GABAARs (RHRR mice) with that of DZP- and MDZ-induced sedation in mice with only α1 BDZ-sensitive GABAARs (HRRR mice). We found that half maximal sedation occurred already at a doses of 0.33±0.05 and 0.52±0.11 mg kg−1 (mean±s.d.) for DZP and MDZ, respectively, while half maximal antihyperalgesia required 8±6 mg kg−1 (DZP) and 10.4±2.0 mg kg−1 (MDZ). A rightward shift of the response curve was also observed when the degrees of receptor occupancy required for antihyperalgesia and for sedation were compared. Half maximal sedation was reached when DZP had bound 47±6% brain α1GABAARs, while half maximal antihyperalgesia required 71±2% occupancy at spinal α2GABAARs. In case of MDZ, the required receptor occupancies were even further apart (24±2% of brain α1GABAARs and 71±2% of spinal α2GABAARs) consistent with the even less favourable α2/α1 selectivity ratio of MDZ. These data indicate that, when applied to wt mice, the DZP and MDZ doses needed for half maximal antihyperalgesia induce an almost complete (∼95%) reduction in locomotor activity, while at non-sedative doses both BDZs would not induce significant antihyperalgesia. Dose-limiting sedation is therefore the most likely reason for the absence of a relevant antihyperalgesic activity of classical, nonselective BDZs in human patients.


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

Ralvenius WT, Benke D, Acuña MA, Rudolph U, Zeilhofer HU - Nat Commun (2015)

Activity of DZP and MDZ at α1 and α2GABAARs, and PK/PD modelling.(a,b) Potentiation of recombinant α1β2γ2 and α2β3γ2 GABAAR currents by 1 μM DZP (a) and 1 μM MDZ (b). GABA concentration was EC5 (5 μM for α1β2γ2 and 2 μM for α2β3γ2). n=6 for both drugs and all concentrations. Scales bars, 200 pA, 2 s. (c,d) The H101R mutation prevented potentiation of α1β2γ2 GABAAR currents by MDZ (**P<0.01, n=5, unpaired t-test; c) and binding of MDZ to α1β2γ2 GABAARs (n=6 for all concentrations in wt and mutated receptors; d). (e–h) α1GABAAR-mediated sedation and α2GABAAR-mediated antihyperalgesia by DZP and MDZ. Sedation (black symbols) in HRRR mice was assessed in the open field test and expressed as percent maximum possible effect determined from the reduction in activity compared with vehicle-treated mice of the same genotype. Antihyperalgesia (red symbols) in RHRR mice was determined as the increase in mechanical withdrawal thresholds compared with pre-drug values 1 week after the CCI surgery. (e,f) Dose–response relationships. Data were fitted to Hill's equation with baseline fixed at 0. Sedation and antihyperalgesia were determined between 15 and 90 min after oral DZP (e) or between 15 and 60 min after oral MDZ (f). Mice were killed immediately afterwards (that is, at the time point of maximal effects), and brains and spinal cords were removed for further analyses. (g,h) Dependence on receptor occupancy (RO). (g) Sedation versus RO in brains of HRRR mice treated with DZP (0.03, 0.1, 0.3, 1, 3, 10 and 30 mg kg−1, n=6 mice for all doses). Antihyperalgesia versus RO in lumbar spinal cords of RHRR mice treated with 0.1, 1, 3, 10 or 30 mg kg−1 DZP, n=6 mice for all doses. (h) Same as g but MDZ. Sedation 0.1 (n=6 mice), 0.3 (7), 0.5 (7), 1.0 (7), 3 (6) and 10 (4) mg kg−1. Antihyperalgesia 0.1 (n=3), 0.3 (5), 1.0 (9), 3 (8), 5 (7) and 10 (7) mg kg−1 MDZ. Data were fitted to sigmoidal functions. Data shown in e,g (on DZP) and in f,h (on MDZ) are from the same two groups of mice. All data points are mean±s.e.m.
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f6: Activity of DZP and MDZ at α1 and α2GABAARs, and PK/PD modelling.(a,b) Potentiation of recombinant α1β2γ2 and α2β3γ2 GABAAR currents by 1 μM DZP (a) and 1 μM MDZ (b). GABA concentration was EC5 (5 μM for α1β2γ2 and 2 μM for α2β3γ2). n=6 for both drugs and all concentrations. Scales bars, 200 pA, 2 s. (c,d) The H101R mutation prevented potentiation of α1β2γ2 GABAAR currents by MDZ (**P<0.01, n=5, unpaired t-test; c) and binding of MDZ to α1β2γ2 GABAARs (n=6 for all concentrations in wt and mutated receptors; d). (e–h) α1GABAAR-mediated sedation and α2GABAAR-mediated antihyperalgesia by DZP and MDZ. Sedation (black symbols) in HRRR mice was assessed in the open field test and expressed as percent maximum possible effect determined from the reduction in activity compared with vehicle-treated mice of the same genotype. Antihyperalgesia (red symbols) in RHRR mice was determined as the increase in mechanical withdrawal thresholds compared with pre-drug values 1 week after the CCI surgery. (e,f) Dose–response relationships. Data were fitted to Hill's equation with baseline fixed at 0. Sedation and antihyperalgesia were determined between 15 and 90 min after oral DZP (e) or between 15 and 60 min after oral MDZ (f). Mice were killed immediately afterwards (that is, at the time point of maximal effects), and brains and spinal cords were removed for further analyses. (g,h) Dependence on receptor occupancy (RO). (g) Sedation versus RO in brains of HRRR mice treated with DZP (0.03, 0.1, 0.3, 1, 3, 10 and 30 mg kg−1, n=6 mice for all doses). Antihyperalgesia versus RO in lumbar spinal cords of RHRR mice treated with 0.1, 1, 3, 10 or 30 mg kg−1 DZP, n=6 mice for all doses. (h) Same as g but MDZ. Sedation 0.1 (n=6 mice), 0.3 (7), 0.5 (7), 1.0 (7), 3 (6) and 10 (4) mg kg−1. Antihyperalgesia 0.1 (n=3), 0.3 (5), 1.0 (9), 3 (8), 5 (7) and 10 (7) mg kg−1 MDZ. Data were fitted to sigmoidal functions. Data shown in e,g (on DZP) and in f,h (on MDZ) are from the same two groups of mice. All data points are mean±s.e.m.
Mentions: Our present results and previous pain studies employing subtype-selective BDZ site agonists in rodents52026272829 contrast with the lack of a clear analgesic or antihyperalgesic action of classical BDZs in human patients3031. Apart from species differences and differences between disease models and actual disease in human patients, we found one possible explanation particularly worth studying. The doses and the degrees of receptor activation required for a relevant effect might be significantly higher in case of antihyperalgesia than of sedation. As a consequence, antihyperalgesia would occur in patients only at doses already inducing strong sedation. The availability of the triple point-mutated mice allowed us to directly compare the doses and levels of receptor occupancy at the relevant GABAAR subtypes and sites. For these experiments, we chose midazolam (MDZ) as a second classical BDZ in addition to DZP. First, we determined for both drugs their α2/α1 selectivity profiles in electrophysiological experiments on recombinant GABAARs. As expected, we found that DZP potentiated α1/β2/γ2 and α2/β3/γ2 GABAARs with similar efficacy and potency (Fig. 6a and Table 3). By contrast, MDZ potentiated α1/β2/γ2 GABAARs more than twice as much as α2/β3/γ2 GABAARs (Fig. 6b and Table 3). We then verified that the H→R point mutation blocked not only DZP effects but also MDZ binding and GABAAR potentiation (Figs 6c,d; see also ref. 32). Next, we compared the dose dependency of DZP- and MDZ-induced antihyperalgesia in mice with only α2 BDZ-sensitive GABAARs (RHRR mice) with that of DZP- and MDZ-induced sedation in mice with only α1 BDZ-sensitive GABAARs (HRRR mice). We found that half maximal sedation occurred already at a doses of 0.33±0.05 and 0.52±0.11 mg kg−1 (mean±s.d.) for DZP and MDZ, respectively, while half maximal antihyperalgesia required 8±6 mg kg−1 (DZP) and 10.4±2.0 mg kg−1 (MDZ). A rightward shift of the response curve was also observed when the degrees of receptor occupancy required for antihyperalgesia and for sedation were compared. Half maximal sedation was reached when DZP had bound 47±6% brain α1GABAARs, while half maximal antihyperalgesia required 71±2% occupancy at spinal α2GABAARs. In case of MDZ, the required receptor occupancies were even further apart (24±2% of brain α1GABAARs and 71±2% of spinal α2GABAARs) consistent with the even less favourable α2/α1 selectivity ratio of MDZ. These data indicate that, when applied to wt mice, the DZP and MDZ doses needed for half maximal antihyperalgesia induce an almost complete (∼95%) reduction in locomotor activity, while at non-sedative doses both BDZs would not induce significant antihyperalgesia. Dose-limiting sedation is therefore the most likely reason for the absence of a relevant antihyperalgesic activity of classical, nonselective BDZs in human patients.

Bottom Line: 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).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.These findings should foster the development of innovative subtype-selective BDZs for novel indications such as chronic pain.

View Article: PubMed Central - PubMed

Affiliation: 1] Institute of Pharmacology and Toxicology, University of Zurich, Winterthurerstrasse 190, CH-8057 Zurich, Switzerland [2] Center for Neuroscience Zurich (ZNZ), Winterthurerstrasse 190, CH-8057 Zurich, Switzerland.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus