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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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Related in: MedlinePlus

Sedation, muscle relaxation and motor coordination in GABAAR-mutated mice.Effects of DZP (10 mg kg−1, p.o.) on locomotor activity in the open field test (a), on muscle relaxation in the horizontal wire test (b), and on motor coordination in the rotarod test (c). ***P<0.001, *P<0.05 significant versus vehicle-treated wt (HHHH) mice (ANOVA followed by Dunnett's post hoc test). Statistics: locomotor activity F(6,177)=79.5 (n=106, 26, 8, 8, 9, 13 and 13 mice, for vehicle and DZP-treated wt mice, and DZP-treated HRRR, RHRR, RRHR, RRRH and RRRR mice, respectively). Horizontal wire F(6,165)=44.0 (n=109, 14, 8, 8, 9, 13 and 10 mice). Rotarod F(6,41)=11.5 (n=8, 5, 6, 7, 6, 8 and 8 mice). (d) Effects of DZP on locomotor activity and horizontal wire performance in quadruple GABAAR point-mutated (RRRR) mice. ***P<0.001; **P<0.05 significant versus vehicle (ANOVA followed by Dunnett's post hoc test) F(3,33)=13.4 (locomotor activity), n=9, 13, 8 and 7 mice, for vehicle, and 10, 30 and 100 mg kg−1 DZP; F(3,30)=0.44; P>0.60 (horizontal wire test), n=9, 10, 8 and 7 mice, for vehicle, and 10, 30 and 100 mg kg−1 DZP. All data points are mean±s.e.m.
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f4: Sedation, muscle relaxation and motor coordination in GABAAR-mutated mice.Effects of DZP (10 mg kg−1, p.o.) on locomotor activity in the open field test (a), on muscle relaxation in the horizontal wire test (b), and on motor coordination in the rotarod test (c). ***P<0.001, *P<0.05 significant versus vehicle-treated wt (HHHH) mice (ANOVA followed by Dunnett's post hoc test). Statistics: locomotor activity F(6,177)=79.5 (n=106, 26, 8, 8, 9, 13 and 13 mice, for vehicle and DZP-treated wt mice, and DZP-treated HRRR, RHRR, RRHR, RRRH and RRRR mice, respectively). Horizontal wire F(6,165)=44.0 (n=109, 14, 8, 8, 9, 13 and 10 mice). Rotarod F(6,41)=11.5 (n=8, 5, 6, 7, 6, 8 and 8 mice). (d) Effects of DZP on locomotor activity and horizontal wire performance in quadruple GABAAR point-mutated (RRRR) mice. ***P<0.001; **P<0.05 significant versus vehicle (ANOVA followed by Dunnett's post hoc test) F(3,33)=13.4 (locomotor activity), n=9, 13, 8 and 7 mice, for vehicle, and 10, 30 and 100 mg kg−1 DZP; F(3,30)=0.44; P>0.60 (horizontal wire test), n=9, 10, 8 and 7 mice, for vehicle, and 10, 30 and 100 mg kg−1 DZP. All data points are mean±s.e.m.

Mentions: We next used the same triple point-mutated mouse approach to investigate non-pain-related effects. In these experiments, we focused on diminished locomotor activity (as a surrogate parameter of sedation), muscle strength and motor coordination. No significant differences were observed in the behaviour of drug-naive wt and point-mutated mice (Table 2). The DZP treatment strongly reduced locomotor activity in wt mice and in mice with BDZ-sensitive GABAARs of only the α1 subtype (HRRR mice; Fig. 4a). No sedative effects were observed in any of the other triple point-mutated mice. Mice with only α2 BDZ-sensitive GABAARs (RHRR mice) even showed a strong increase in locomotor activity, which may originate from the anxiolytic effect of DZP occurring through α2GABAARs (ref. 12). Impairment of muscle strength was assessed in the horizontal wire test. Significant muscle relaxation was detected in wt mice and in mice with BDZ-sensitive GABAARs of either only the α2 (RHRR mice) or α3 subtype (RRHR mice; Fig. 4b). Motor coordination, tested in the rotarod test, was significantly impaired by DZP in mice with only α1 (HRRR mice) and with only α3 BDZ-sensitive GABAARs (RRHR mice; Fig. 4c). Quadruple point-mutated (RRRR) mice were completely protected from DZP-induced muscle relaxation and motor impairment. They did, however, show a trend towards reduced locomotor activity (compare Fig. 4a). We therefore assessed changes in locomotion also after higher DZP doses in the quadruple point-mutated mice and found significant and dose-dependent impairment starting at 30 mg kg−1 (Fig. 4d). Unlike α1GABAAR-mediated sedation, impairment of muscle strength was absent in the quadruple point-mutated mice even at doses ⩾30 mg kg−1. The sedative action of DZP remaining in the quadruple point-mutated mice may be attributed to a low-affinity BDZ-binding site at α1GABAARs described earlier2122.


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)

Sedation, muscle relaxation and motor coordination in GABAAR-mutated mice.Effects of DZP (10 mg kg−1, p.o.) on locomotor activity in the open field test (a), on muscle relaxation in the horizontal wire test (b), and on motor coordination in the rotarod test (c). ***P<0.001, *P<0.05 significant versus vehicle-treated wt (HHHH) mice (ANOVA followed by Dunnett's post hoc test). Statistics: locomotor activity F(6,177)=79.5 (n=106, 26, 8, 8, 9, 13 and 13 mice, for vehicle and DZP-treated wt mice, and DZP-treated HRRR, RHRR, RRHR, RRRH and RRRR mice, respectively). Horizontal wire F(6,165)=44.0 (n=109, 14, 8, 8, 9, 13 and 10 mice). Rotarod F(6,41)=11.5 (n=8, 5, 6, 7, 6, 8 and 8 mice). (d) Effects of DZP on locomotor activity and horizontal wire performance in quadruple GABAAR point-mutated (RRRR) mice. ***P<0.001; **P<0.05 significant versus vehicle (ANOVA followed by Dunnett's post hoc test) F(3,33)=13.4 (locomotor activity), n=9, 13, 8 and 7 mice, for vehicle, and 10, 30 and 100 mg kg−1 DZP; F(3,30)=0.44; P>0.60 (horizontal wire test), n=9, 10, 8 and 7 mice, for vehicle, and 10, 30 and 100 mg kg−1 DZP. All data points are mean±s.e.m.
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f4: Sedation, muscle relaxation and motor coordination in GABAAR-mutated mice.Effects of DZP (10 mg kg−1, p.o.) on locomotor activity in the open field test (a), on muscle relaxation in the horizontal wire test (b), and on motor coordination in the rotarod test (c). ***P<0.001, *P<0.05 significant versus vehicle-treated wt (HHHH) mice (ANOVA followed by Dunnett's post hoc test). Statistics: locomotor activity F(6,177)=79.5 (n=106, 26, 8, 8, 9, 13 and 13 mice, for vehicle and DZP-treated wt mice, and DZP-treated HRRR, RHRR, RRHR, RRRH and RRRR mice, respectively). Horizontal wire F(6,165)=44.0 (n=109, 14, 8, 8, 9, 13 and 10 mice). Rotarod F(6,41)=11.5 (n=8, 5, 6, 7, 6, 8 and 8 mice). (d) Effects of DZP on locomotor activity and horizontal wire performance in quadruple GABAAR point-mutated (RRRR) mice. ***P<0.001; **P<0.05 significant versus vehicle (ANOVA followed by Dunnett's post hoc test) F(3,33)=13.4 (locomotor activity), n=9, 13, 8 and 7 mice, for vehicle, and 10, 30 and 100 mg kg−1 DZP; F(3,30)=0.44; P>0.60 (horizontal wire test), n=9, 10, 8 and 7 mice, for vehicle, and 10, 30 and 100 mg kg−1 DZP. All data points are mean±s.e.m.
Mentions: We next used the same triple point-mutated mouse approach to investigate non-pain-related effects. In these experiments, we focused on diminished locomotor activity (as a surrogate parameter of sedation), muscle strength and motor coordination. No significant differences were observed in the behaviour of drug-naive wt and point-mutated mice (Table 2). The DZP treatment strongly reduced locomotor activity in wt mice and in mice with BDZ-sensitive GABAARs of only the α1 subtype (HRRR mice; Fig. 4a). No sedative effects were observed in any of the other triple point-mutated mice. Mice with only α2 BDZ-sensitive GABAARs (RHRR mice) even showed a strong increase in locomotor activity, which may originate from the anxiolytic effect of DZP occurring through α2GABAARs (ref. 12). Impairment of muscle strength was assessed in the horizontal wire test. Significant muscle relaxation was detected in wt mice and in mice with BDZ-sensitive GABAARs of either only the α2 (RHRR mice) or α3 subtype (RRHR mice; Fig. 4b). Motor coordination, tested in the rotarod test, was significantly impaired by DZP in mice with only α1 (HRRR mice) and with only α3 BDZ-sensitive GABAARs (RRHR mice; Fig. 4c). Quadruple point-mutated (RRRR) mice were completely protected from DZP-induced muscle relaxation and motor impairment. They did, however, show a trend towards reduced locomotor activity (compare Fig. 4a). We therefore assessed changes in locomotion also after higher DZP doses in the quadruple point-mutated mice and found significant and dose-dependent impairment starting at 30 mg kg−1 (Fig. 4d). Unlike α1GABAAR-mediated sedation, impairment of muscle strength was absent in the quadruple point-mutated mice even at doses ⩾30 mg kg−1. The sedative action of DZP remaining in the quadruple point-mutated mice may be attributed to a low-affinity BDZ-binding site at α1GABAARs described earlier2122.

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