A METHOD FOR THE IDENTIFICATION OF COMPOUNDS WITH ANXIOLYTIC POTENTIAL

Abstract: The present invention relates to the use of a cloned GABAA receptor subtype composed of the alpha 2, beta 3 and gamma 2 subunits for the identification of benzodiazepine receptor ligands with selective anxiolytic properties (i.e. non-sedative anxiolytica). Affinity and/or efficacy of the ligands are measured at different cloned GABAA receptor subtypes.

Description: A METHOD FOR THE IDENTIFICATION OF COMPOUNDS WITH ANXIOLYTIC POTENTIAL TECHNICAL FIELD The present invention relates to the use of a cloned GABA A receptor subtype composed of the α 2 , β 3 and γ 2 subunits for the identification of benzodiazepine receptor ligands with selective anxiolytic properties (i.e. non-sedative anxiolytica). BACKGROUND ART Receptors for the major inhibitory neurotransmitter, γ-aminobutyric acid (GABA), are divided into two main classes, GABA A receptors and GABA B receptors. GABA A receptors are members of a ligand-gated ion channel family, and are the most abundant inhibitory receptors in the mammalian brain. Each GABA A receptor complex comprises a chloride ion channel that controls the chloride flux across the neuronal membrane, along with multiple recognition sites for small modulatory molecules such as benzodiazepines, barbiturates, picrotoxin, and certain steroids. When GABA interacts with its receptor, the ion channel is opened, the chloride flux is enhanced, and the cell becomes less responsive to excitatory stimuli. This GABA induced ion current can be regulated by various agents, including agents that interact with the benzodiazepine receptor or recognition site. Agents that bind or interact with the modulatory sites on the GABA A receptor complex, e.g. the benzodiazepine receptor, and have a positive modulatory effect on the action of GABA, are called benzodiazepine receptor agonists or partial agonists. Agonists generally produce muscle relaxant, hypnotic, sedative, anxiolytic, and/or anticonvulsant effects. Benzodiazepine receptor ligands with negative modulatory effect on the action of GABA are termed inverse agonists, while benzodiazepine receptor ligands with no intrinsic activity are termed antagonists. Numerous compounds belonging to different series of compounds having affinity for the benzodiazepine receptors have been synthesized during the last three decades. In particular it has been the aim of several groups to develop benzodiazepine receptor modulators having potent anxiolytic activity and devoid of sedative effects. However, although the benzodiazepine receptor sites are still considered as very attractive biological sites for interfering with the CNS to treat various disorders and diseases, then nearly all previously synthesized compounds acting at these receptor sites have failed during clinical development because of unacceptable side effects. The GABAA receptors are structurally constituted macromolecular heteropentameric assemblies, containing a combinations of , β, and γ/δ protein subunits. Several subunits of such GABA A receptors ( ι -6 , βι- 3 , δ and γ 1-3 ) have been characterized using techniques of modern molecular biology. Considering the number of individual mammalian GABA A receptor subunits, the number of pentameric subtypes with different subunit permutations that theoretically could exist, is overwhelming. It has therefore been a major challenge to delineate the subunit complements of native GABA receptors and to resolve their physiological role. It is believed that specific GABA A receptor subtypes are responsible for mediating the anxiolytic effect of benzodiazepines. But the specific subtypes and/or subunits involved in the patophysiology of anxiety disorders is not known at present. From anatomical/behavioral studies in rats it has been shown that the anti- conflict effect of the benzodiazepine Midazolam™ is mediated at the baso-lateral amygdaloid nucleus (European Journal of Pharmacology, 1982 82 115-116, and Neuroscience Letters, 1985 53 285-288). As mentioned above six subunits exists and benzodiazepines are believed to bind to α-subunits or at the interface between the α subunit and the β subunit. The αi subunit is distributed more or less uniformly throughout the rat brain, and is believed to be involved in the sedative affects of benzodiazepine receptor ligands. The affinity of benzodiazepines for 0 4 and β subunits is low and it is therefore highly unlikely that benzodiazepines mediate their effects via these subunits. From anatomical studies with in situ hybridization it has been shown that α-i, α 2 , 06 3 , 0( 4 and 0 5 subunits are present in the baso-lateral amygdaloid nucleus, 2 being the most abundant α-subunit (The Journal of Neuroscience, 1992 12 (3) 1040-1062). Additionally, It has been shown that the β 3 subunit is the most abundant β subunit in the baso-lateral amygdaloid nucleus and that the β 3 subunit seems predominantly to be expressed in combination with α 2 subunits (The Journal of Neuroscience, 1992 12 (3) 1040-1062, and Biochem. J. 1995 310 1 -9). Likewise the most abundant γ subunit in the baso-lateral amygdaloid nucleus is the γ 2 subunit. These findings suggests that the 0^ 3 7 2 GABA A receptor subtype may be the sole mediator of the anxiolytic effect of benzodiazepines. By measuring the affinity and efficacy of a novel benzodiazepine receptor agonist having potent anxiolytic properties and low sedative effects on cloned GABA A receptor subtypes it was found that this novel benzodiazepine receptor ligand is a full agonist to the α 2 β3T2 GABA A receptor subtype and a partial agonist the αιβ 2 γ 2 GABA A receptor subtype. Based on these findings a novel method for the identification of compounds with anxiolytic potential have been developed. SUMMARY OF THE INVENTION It is an object of the present invention to provide a method for the identification of benzodiazepine receptor ligands with selective anxiolytic properties (i.e. non-sedative) by measuring the affinity and/or efficacy of the test compounds to a cloned 0^ 3 7 2 GABAA receptor and comparing with the affinity and/or efficacy of the test compound to other GABA A receptor subtypes which are known not to mediate selective anxiolytic effects. The present invention provides a valuable tool for screening chemical libraries for compounds with selective anxiolytic activity and for designing drugs with selective anxiolytic activity. Accordingly, in its first aspect, the invention provides a method for the identification of chemical compounds having anxiolytic potential and no or only low sedative effects, which method comprises the steps of (i) measuring the affinity and/or efficacy of a chemical compound at a cloned α 2 β3T2 GABA A receptor; (ii) comparing the affinity and/or efficacy measured in step (i) with the affinity and/or efficacy of the same chemical compound at a different cloned GABA A receptor subtype; and (iii) selecting chemical compounds which are selective benzodiazepine receptor agonists at the α 2 β 3 Y 2 GABA A receptor subtype. In another aspect the invention provides a chemical compound having anxiolytic potential obtained by the method of the invention. In a third aspect the invention provides a chemical compound being a selective benzodiazepine receptor agonists at the oc2β3Y2 GABA A receptor subtype. In further aspects the invention relates to the use of a chemical compound of the invention for use as a medicament, and to pharmaceutical compositions comprising these compounds. Finally the invention relates to a method for the treatment of an individual suffering from anxiety comprising administering to said individual a therapeutically effective amount of a compound which is a selective benzodiazepine receptor agonist at the α 2 β 3 Y2 GABA receptor. DETAILED DISCLOSURE OF THE INVENTION The present invention provides for the identification of chemical compounds having potential as benzodiazepine receptor ligands with anxiolytic activity and no or only low sedative effects. The method of the invention comprises measuring the affinity and/or efficacy of a chemical compound (the test compound) for binding onto a cloned α 2 β 3 Y 2 GABAA receptor, and comparing the affinity and/or efficacy so determined with the affinity and/or efficacy of the same compound for binding onto another cloned GABA A receptor subtype. In this way compounds which are selective benzodiazepine receptor agonists at the α β 3 Y 2 GABA A receptor subtype can be identified. As defined herein, selective affinity for a GABA A receptor means that the affinity of a compound for the receptor is higher than the affinity of the compound to other GABAA receptors. Also, as defined herein, a selective agonist of a GABA A receptor is a positive modulator of a GABAA receptor which have a higher efficacy to this receptor that to other GABA A receptors. In a preferred embodiment, the method of the invention comprises (i) measuring the affinity of test compounds to cloned α 2 β 3 Y 2 GABA A receptors and to other cloned GABAA receptor subtypes; (ii) selecting compounds with selective affinity for the cloned oc 2 β 3 γ 2 GABA A receptors; (iii) measuring the efficacy of the selected compounds at the cloned α 2 β 3 γ 2 GABAA receptor; and (iv) selecting compounds which are selective agonist at the cloned GABA A receptor. In another preferred embodiment the method of the invention comprises (i) measuring the affinity of test compounds to a cloned α β 3 Y 2 GABA A receptor and to other cloned GABA A receptor subtypes; (ii) selecting compounds with affinity for the cloned α 2 β 3 Y 2 GABA A receptor; (iii) measuring the efficacy of the selected compounds at the cloned GABAA receptors; and (iv) selecting compounds which are selective agonists at the cloned α 2 β 3 Y 2 GABAA receptor. In the method of the invention, the affinity of the test compounds for the GABAA receptor subtypes may be determined by the ability of the test compounds to displace a radio labeled non-selective, high affinity benzodiazepine receptor ligand, such as tritium isotope radio labeled Flumazenil™ or Flunitrazepam™. Alternatively, the efficacy of the test compounds for the GABA A receptor subtypes may be determined by measuring the affinity of the compounds for the receptor in presence an in absence of GABA and calculating the GABA-ratio. In yet another embodiment, the efficacy of the test compounds for the GABAA receptor may be determined by measuring the chloride flux using patch clamp technique. In another preferred embodiment, the additional cloned GABA A receptor subtypes employed in the method of the invention, andnot being a cloned 0^ 3 7 2 GABAA receptor, are subtypes which are known not to mediate selective anxiolytic effects. More preferred this additional cloned GABAA receptor subtypes contain an αι, oc 3 or as GABA A receptor subunit. In a most preferred embodiment, this additional cloned GABA A receptor subtypes contain the subunit combination αιβ 3 γ 2 or α 3 β 2 γ 2 . In a particular embodiment of the invention, stabile CHO (Chinese hamster ovary) cells expressing cloned rat or human GABA receptor subunits is used to screen test compounds for their affinity to the α 2 β 3 γ 2 GABAA receptor subtype. The affinity of the test compounds to this receptor subtype is compared with the affinity of the compounds to GABA A receptor types which are believed not to mediate selective anxiolytic effects, e.g. GABAA receptor subtypes containing an αi receptor subunit. The affinity of the test compounds for the cloned receptor is determined by measuring the ability of the compounds to displace a non-selective radio labeled benzodiazepine receptor ligand, such as tritium isotope radio labeled Flumazenil™ or Flunitrazepam™. Inhibition of binding is measured by a conventional in vitro binding assays using membrane preparations of cells expressing cloned GABAA receptor subtypes. The efficacy of the test compounds, i.e. whether the compounds are agonists, inverse agonists or antagonists at the benzodiazepine receptor, can also be determined by calculating the GABA-ratio of the compounds for each of the receptor subtypes employed. GABA-ratio is the ratio between the affinity of a test compound measured in presence of GABA and the affinity of the compound measured in absence of GABA. Benzodiazepine receptor ligands which are full agonists generally have a GABA ratio between 1.8-3, partial agonists have a ratio between 1.0-1.8, antagonists have a ratio around 1 , whereas inverse agonist have a GABA ratio around 0.5 -1. Calculation of GABA-ratio therefore provides an indication of the efficacy of the test compound. The efficacy of the compound can also be measured in electrophysiological experiments using patch clamp technique. In the method of the invention any cell expressing GABAA receptor subunits may be employed. Preferred cells are CHO cells, or other rodent fibroblast cell lines such as BHK (baby kidney hamster cells) and mouse Ltk " cells, insects cells, such as Sf-9 cells, and HEK293 or HeLa cells. The GABA A subunits expressed by the cells may be of animal (rat or bovine) or human origin. In another aspect the invention provides a chemical compound having anxiolytic potential obtained by the method of the invention. In a third aspect the invention provides a chemical compound being a selective benzodiazepine receptor agonists at the α 2 β 3 Y 2 GABA A receptor subtype. In further aspects the invention relates to the use of a chemical compound of the invention for use as a medicament, and to pharmaceutical compositions comprising these compounds. Finally the invention relates to a method for the treatment of an individual suffering from anxiety comprising administering to said individual a therapeutically effective amount of a compound which is a selective benzodiazepine receptor agonist at the α 2 β3Y2 GABA receptor. EXAMPLES The invention is further illustrated with reference to the following example which is not intended to be in any way limiting to the scope of the invention as claimed. The affinity of 5-acetyl-1-(3-(3-pyridyl)phenyl)benzimidazole O-ethyl oxime for various GABAA receptor subtypes was studied using recombinant GABA A receptors expressed in insect cells. The ratio between affinity for the compound in the absence and presence of GABA (GABA ratio) depended on the subunit combination of the cloned receptors. The compound had a GABA ratio corresponding to a full agonist at the (* 2 β 3 γ 2 GABAA receptor subtype and a GABA ration corresponding to a partial agonist at the α-ιβ 2 γ 2 GABA A receptor subtype.

Claims: CLAIMS 1. A method for the identification of a chemical compound having anxiolytic potential and no or only low sedative effects, which method comprises the steps of (i) measuring the affinity and/or efficacy of a chemical compound at cloned 2 β3Y2 GABAA receptors; (ii) comparing the affinity and/or efficacy measured in step (i) with the affinity and/or efficacy of the same chemical compound at different cloned GABAA receptor subtypes; and (iii) selecting the chemical compound that is a selective benzodiazepine receptor agonist at the α 2 β3Y2 GABA A receptor subtypes. 2. The method according to claim 1 , comprising (i) measuring the affinity of the test compound to cloned 0^ 3 7 2 GABA A receptors and to different cloned GABA A receptor subtypes; (ii) selecting the compound which has selective affinity for the cloned α 2 β 3 Y 2 GABAA receptors; (iii) measuring the efficacy of the selected compound at the cloned α β 3 γ 2 GABAA receptors; and (iv) selecting the compound which is a selective agonist at the cloned GABAA receptors. 3. The method according to claim 1 , comprising (i) measuring the affinity of the test compound to cloned 2 β 3 Y 2 GABA A receptors and to different cloned GABAA receptor subtypes; (ii) selecting the compound which has affinity for the cloned α 2 β 3 γ 2 GABA A receptors; (iii) measuring the efficacy of the selected compound at the cloned GABAA receptors; and (iv) selecting the compound which is a selective agonist at the cloned α 2 β 3 Y 2 GABAA receptors. 4. The method according to any of claims 1-3, wherein the affinity of the test compound for the GABAA receptor subtypes is determined by measuring the ability of the test compound to displace a radio labeled non-selective, high affinity benzodiazepine receptor ligand, such as tritium isotope radio labeled Flumazenil™ or Flunitrazepam™. 5. The method according to any of claims 1-4, wherein the efficacy of the test compound for the GABA A receptor subtypes is determined by measuring the affinity of the compound for the receptors in the presence and in the absence of GABA, and calculating the GABA-ratio. 6. The method according to any of claims 1 -4, wherein the efficacy of the test compound for the GABAA receptor subtypes is determined by measuring the chloride flux using patch clamp technique. 7. The method according to any of claims 1-6, wherein the cloned GABA A receptor subtypes that is different from the cloned α β3Y2 GABAA receptors, are subtypes which are known not to mediate selective anxiolytic effects. 8. The method according to claim 1 , wherein the cloned GABA A receptor subtypes that is different from the cloned α 2 β3Y2 GABA A receptors, contain the αι, α 3 or 0 5 GABA A receptor subunits. 9. The method according to claim 1 , wherein the cloned GABA A receptor subtypes that is different from the cloned α 2 β3Y2 GABA A receptors, contain the subunit combination αι β 3 Y2 or 10. A chemical compound having anxiolytic potential obtained by the method according to any of claims 1 -9. 11. A chemical compound being a selective benzodiazepine receptor agonists at the αzβ GABAA receptor subtypes. 12. A pharmaceutical composition comprising the benzodiazepine receptor ligand according to either of claims 10-11. 13. The chemical compound according to either of claims 10-11 for use as a medicament. 14. Use of the chemical compound according to either of claims 10-11 for the manufacture of a medicament for the treatment of anxiety. 15. A method for the treatment of an individual suffering from anxiety comprising administering to said individual a therapeutically effective amount of a chemical compound which is a selective benzodiazepine receptor agonist at the 2 β 3 Y 2 GABA receptors.

Inventors: JENSEN LEIF HELTH, JOHANSEN TINA HOLM, JENSEN, LEIF, HELTH, JOHANSEN, TINA, HOLM
Applicants: JENSEN LEIF HELTH, JOHANSEN TINA HOLM, NEUROSEARCH AS, JENSEN, LEIF, HELTH, JOHANSEN, TINA, HOLM, NEUROSEARCH A/S
Application year: 1997
Main classification: G01N33
IPC codes: A61K45, A61P25, C12Q1, G01N33