Methods for screening GABA-modulatory compounds for specified pharmacological activities

A method for screening compounds for cognitive enhancing activity, comprising:

• a) selecting compounds having a binding affinity less than 100 nM at any GABAA receptor;
• b) determining in vitro efficacy and EC50 values for each selected compound at an α1β2γ2 or α5β3γ2 GABAA subtype receptor;
• c) determining in vitro efficacy and EC50 values for each selected compound at a GABAA receptor comprised of an α2 or α3 subunit;
• and
• d) identifying as having cognitive enhancing activity any compound having an EC50 value determined in b) of less than 200 nM and an efficacy value measured in b) of less than −5%, and an efficacy value measured in c) of greater than 5%.

A method for screening a plurality of compounds for cognitive enhancing activity, comprising:

• a) determining in vitro efficacy and EC50 values for each compound at α1β2γ2 or α5β3γ2 GABAA receptors;
• b) determining in vitro efficacy for each compound at a GABAA receptor comprised of an α2 or α3 subunit;
• c) determining the in vivo effect of each compound in an animal model for measuring cognitive enhancement;
• d) determining the in vivo effects of each compound in an animal model for proconvulsant activity by measuring a seizure threshold in the presence of a seizure inducing compound or in an animal model that predicts anxiogenic effects;
• and
• e) identifying a cognitive enhancing compound as a compound having cognitive enhancing properties when the EC50 measured in step a) is less than 200 nM and the efficacy measured in step a) is less than −5% and the efficacy measured in step b) is greater than 5% and said compound produces a statistically significant (p<0.05) positive effect in the animal model indicative of cognitive enhancement and said compound does not produce an effect in the animal model predictive of proconvulsant activity of more than a 25% decrease in the seizure threshold in the presence of the seizure inducing drug, or does not produce a change that is statistically significant in said model, or the compound does not produce a statistically significant effect in the animal model that predicts anxiogenic effects.

A method for screening compounds for cognitive enhancing properties, comprising:

• a) selecting compounds having binding affinities of less than 100 nM at any GABAA receptor;
• b) measuring the in vitro efficacy of each compound at an α1β2γ2 or α5β3γ2 GABAA receptor;
• c) measuring the in vitro efficacy of each compound at a GABAA receptor comprised of an α2 or α3 subunit;
• d) measuring the in vivo effect of each compound in an animal model predictive of cognitive enhancement;
• e) measuring the in vivo side effects of each compound in an animal model that predicts proconvulsant activity by measuring a seizure threshold in the presence of a seizure inducing compound or measuring the in vivo side effects of each compound in an animal model that predicts anxiogenic effects;
• and
• f) identifying as a cognitive enhancing compound a particular compound for which the EC50 measured in step b) is less than 200 nM and the efficacy measured in step b) is less than −5% and the efficacy measured in step c) is greater than 5% and said particular compound produces a statistically significant (p<0.05) positive effect in the animal model indicative of cognitive enhancement and said particular compound does not produce an effect in the animal model predictive of proconvulsant activity of more than a 25% decrease in the seizure threshold in the presence of the seizure inducing drug, or does not produce a change that is statistically significant in said model, or said particular compound does not produce a statistically significant effect in the animal model that predicts anxiogenic effects.

A method for screening compounds for hypnotic activity, comprising:

• a) determining EC50 and in vitro efficacy of each compound at an α2β3γ2 GABAA subtype receptor or an α3β3γ2 GABAA subtype receptor;
• b) determining in vitro efficacy of each compound at a GABAA receptor comprised of an α1 or α5 subunit;
• and
• c) selecting a compound having an EC50 determined in a) of less than 200 nM, an in vitro efficacy determined in a) of greater than 10% for said α2β3γ2 GABAA subtype receptor or greater than 50% for said α3β3γ2 GABAA subtype receptor;
• and an in vitro efficacy value determined in b) of less than 50% for the GABAA receptor comprised of an α1 subunit or less than 45% for the GABAA receptor comprised of an α5 subunit.

A method for screening a plurality of compounds so as to identify at least one compound exhibiting hypnotic activity, comprising:

• a) selecting a plurality of compounds having a binding affinity of less than 100 nM at any GABAA receptor.
• b) determining EC50 and in vitro efficacy values for each selected compound at an α2β3γ2 GABAA subtype receptor or at an α3β3γ2 GABAA subtype receptor;
• c) determining in vitro efficacy values for each selected compound at a GABAA receptor comprised of an α1 or an α5 subunit;
• and
• d) identifying as exhibiting hypnotic activity each selected compound having an EC50 value determined in b) of less than 200 nM, an in vitro efficacy value measured in b) of greater than 10% for said α2β3γ2 GABAA subtype receptor or greater than 50% for said α3β3γ2 GABAA subtype receptor, and an in vitro efficacy value determined in c) of less than 50% for the GABAA receptor comprised of said α1 subunit or less than 45% for the GABAA receptor comprised of said α5 subunit.

A method for screening a plurality of compounds so as to identify compounds exhibiting hypnotic activity, comprising:

• a) measuring the EC50 and in vitro efficacy of each compound at an α2β3γ2 GABAA subtype receptor or an α3β3γ2 GABAA subtype receptor;
• b) measuring the in vitro efficacy of each compound at a GABAA receptor comprised of an α1 or α5 subunit;
• and
• c) measuring the in vivo effect of each compound in an animal model indicative of hypnotic effects;
• d) measuring the in vivo effect of each compound in an animal model indicative of cognitive impairment;
• and
• e) identifying a compound as having hypnotic activity when the EC50 measured in step a) is less than 200 nM, the in vitro efficacy measured in step a) is greater than 10% for said α2β3γ2 GABAA subtype receptor or greater than 50% for said α3β3γ2 GABAA subtype receptor, and the in vitro efficacy measured in step b) is less than 50% for the GABAA receptor comprised of said α1 subunit or less than 45% for the GABAA receptor comprised of said α5 subunit and said compound produces a statistically significant (p<0.05) positive effect in the animal model indicative of sedation and said compound does not produce a statistically significant effect in the animal model indicative of cognitive impairment.

A method for screening a plurality of compounds so as to identify at least one compound exhibiting hypnotic activity, comprising:

• a) selecting compounds having a binding affinity less than 100 nM at any GABAA receptor;
• b) measuring the EC50 and in vitro efficacy of each selected compound at an α2β3γ2 GABAA subtype receptor or an α3β3γ2 GABAA subtype receptor;
• c) measuring the in vitro efficacy of each selected compound at a GABAA receptor comprised of an α1 or α5 subunit;
• and
• d) measuring the in vivo effect of each selected compound in an animal model indicative of sedative effects;
• e) measuring the in vivo effect of each selected compound in an animal model indicative of cognitive impairment;
• and
• f) identifying as having hypnotic activity each selected compound for which the EC50 measured in step b) is less than 200 nM, the in vitro efficacy measured in step b) is greater than 10% for said α2β3γ2 GABAA subtype receptor or greater than 50% for said α3β3γ2 GABAA subtype receptor, and the in vitro efficacy measured in step c) is less than 50% for the GABAA receptor comprised of said α1 subunit or less than 45% for the GABAA receptor comprised of said α5 subunit and said compound produces a statistically significant (p<0.05) positive effect in the animal model indicative of hypnotic effects and said compound does not produce a statistically significant effect in the animal model indicative of cognitive impairment.

A method for screening a plurality of compounds so as to identify compounds exhibiting anxiolytic activity, comprising:

• a) determining in vitro efficacy and EC50 value for each compound at an α2β3γ2 GABAA subtype receptor or an α3β3γ2 GABAA subtype receptor;
• b) determining in vitro efficacy values for each compound at a GABAA receptor comprised of an α1 subunit or an α5 subunit;
• and
• c) identifying as exhibiting anxiolytic activity each compound having an EC50 value determined in a) of less than 200 nM and an efficacy value measured in a) greater than the efficacy measured in b).

A method for screening for compounds having anxiolytic activity, comprising:

• a) selecting a compound having a binding affinity less than 100 niM at any GABAA receptor;
• b) measuring in vitro efficacy and EC50 values for each compound at an α2β3γ2 or α3β3γ2 GABAA receptor;
• c) measuring in vitro efficacy values for each compound at a GABAA receptor comprised of an α1 or α5 subunit;
• and
• d) selecting a compound having an EC50 value measured in a) of less than 200 nM and an efficacy value measured in b) greater than the efficacy measured in c).

A method for screening compounds so as to select at least one compound having anxiolytic activity, comprising:

• a) measuring in vitro efficacy for each compound at an α2β3γ2 GABAA subtype receptor or an α3β3γ2 GABAA subtype receptor;
• b) measuring in vitro efficacy and EC50 values for each compound at a GABAA receptor comprised of an α1 or α5 subunit;
• c) measuring in vivo effects of each compound in an animal model indicative of anxiolytic activity;
• d) measuring in vivo effects of each compound in an animal model indicative of sedative effects;
• and
• e) selecting each compound having:
• an EC50 value measured in a) of less than 200 nM, an efficacy value measured in b) greater than the efficacy measured in step c), a statistically significant (p<0.05) positive effect in the animal model indicative of anxiolytic activity, and no statistically significant effect in the animal indicative of sedative effects.

A method for screening a plurality of compounds so as to identify at least one compound having anxiolytic activity, comprising:

• a) selecting a compound having a binding affinity less than 100 nM at any GABAA receptor;
• b) measuring in vitro efficacy and EC50 values for each selected compound at an α2β3γ2 or α3β3γ2 GABAA receptor;
• c) measuring in vitro efficacy for each selected compound at a GABAA receptor comprised of an α1 or α5 subunit;
• d) measuring in vivo effects of each selected compound in an animal model indicative of anxiolytic activity;
• e) measuring in vivo effect of each selected compound in an animal model indicative of sedative effects;
• and
• f) selecting a compound having:
• an EC50 value measured in b) of less than 200 nM, an efficacy measured in c) greater than the efficacy measured in d), a statistically significant (p<0.05) positive effect in the animal model indicative of anxiolytic activity, and no statistically significant effect in the animal indicative of sedative effects.

A method for screening a plurality of compounds so as to identify compounds exhibiting antidepressant activity, comprising:

• a) determining in vitro efficacy and EC50 values for each compound using an α2β3γ2 GABAA subtype receptor or an α3β3γ2 GABAA subtype receptor;
• b) determining in vitro efficacy values for each compound at a GABAA receptor comprised of an α1 or α5 subunit;
• and
• c) identifying as having antidepressant activity a compound having an EC50 value determined in a) of less than 200 nM and an efficacy value determined in a) of greater than the efficacy value determined in b).

A method for screening compounds for antidepressant activity, comprising:

• a) selecting compounds having a binding affinity less than 100 nM at any GABAA receptor;
• b) determining in vitro efficacy and EC50 values for the selected compounds using an α2β3γ2 GABAA subtype receptor or an α3β3γ2 GABAA subtype receptor;
• c) determining in vitro efficacy for the selected compounds using a GABAA receptor comprised of an α1 or an α5 subunit;
• and
• d) identifying as having antidepressant activity a compound having an EC50 as determined in b) of less than 200 nM and an efficacy value as determined in b) greater than the efficacy value determined in c).

A method for screening compounds for antidepressant activity, comprising:

• a) determining in vitro efficacy and EC50 values for each compound using an α2β3γ2 GABAA subtype receptor or α3β3γ2 GABAA subtype receptor;
• b) determining in vitro efficacy values for each compound at a GABAA receptor comprised of an α1 or an α5 subunit;
• c) determining in vivo effect of said compound in an animal model indicative of antidepressant activity;
• d) determining the in vivo effect of said compound in an animal model indicative of sedative effects;
• and
• e) identifying as an antidepressant a compound that produces an EC50 value as determined in a) of less than to 200 nM, and an efficacy value as determined in b) greater than the efficacy value from c), and (i) produces a statistically significant (p<0.05) positive effect in the animal model indicative of antidepressant activity and (ii) does not produce a statistically significant effect in the animal model indicative of sedative effects.

A method for screening compounds for antidepressant activity, comprising:

• a) selecting test compounds having a binding affinity less than 100 nM at any GABAA receptor;
• b) determining in vitro efficacy and EC50 value for each test compound using an α2β3γ2 GABAA subtype receptor or an α3β3γ2 GABAA subtype receptor;
• c) determining in vitro efficacy value for each test compound at a GABAA receptor comprised of an α1 subunit or an α5 subunit;
• d) determining the in vivo effect of each test compound in an animal model indicative of antidepressant activity;
• e) determining the in vivo effect of each test compound in an animal model indicative of sedative effects;
• and
• f) identifying as an antidepressant a compound that produces:
• an EC50 value as determined in b) of less than 200 nM, an efficacy value as determined in c) greater than the efficacy value from d), and (i) produces a statistically significant (p<0.05) positive effect in the animal model indicative of antidepressant activity and (ii) does not produce a statistically significant effect in the animal model indicative of sedative effects.