WO2013139700A1

WO2013139700A1 - Memory genes

Info

Publication number: WO2013139700A1
Application number: PCT/EP2013/055374
Authority: WO
Inventors: Dominique De Quervain; Andreas Papassotiropoulos
Original assignee: Ac Immune S.A.; Universität Basel
Priority date: 2012-03-21
Filing date: 2013-03-15
Publication date: 2013-09-26

Abstract

A method for identifying targets of therapeutic compounds with medical indications other than memory that can be used for memory-modulation in a subject is provided as well as a method for identifying candidate compounds for their potential of modulating said target. Further provided are pharmaceutical compositions for use in memory modulation in a subject and a method of modulating aversive memory processes and enhancing working memory in a subject in need of such a modulation.

Description

Memory Genes

The present invention relates to a method for identifying targets of therapeutic compounds with medical indications other than memory that can be used for memory-modulation in a subject, to a method for screening candidate compounds for their potential of modulating said target and to pharmaceutical compositions for use in memory modulation in a subject. The invention further relates to a method of modulating working and aversive memory processes in a subject in need of such a modulation.

Searching for molecules related to human emotional and cognitive function is instrumental for understanding the biological mechanisms related to such complex traits as memory capacity and for identifying pathways possibly amenable to pharmacological interventions. From a genetic standpoint, memory can be defined as a genetically complex behavioral trait with substantial heritability estimates (i.e., genetic factors account for a significant proportion of this phenotype's variance). Several twin studies report heritability values between 30% and 60% (Alarcon, 1998; Bouchard, 1990;Finkel, 1995; Johansson, 1999; McClearn, 1997; Swan, 1999; Volk, 2006), indicating that naturally occurring genetic variations have an important impact on memory capacity. Recent advances in the development of high-density genotyping platforms allow for high-resolution genome-wide association studies (GWAS) of genetically complex traits and have already led to a substantial increase in knowledge of the genetic underpinnings of physiological and pathological conditions of human emotional and cognitive functions (De Quervain, 2003; De Quervain, 2007; Papassotiropoulos, 2011 ).

Emotional experiences are typically well remembered, but there is a large, partly genetically controlled, variability for this phenomenon (De Quervain, 2007). On the one hand, enhanced memory for emotionally arousing events can be seen as an adaptive mechanism, which helps us to remember important information (McGaugh, 2003). On the other hand, strong memory of aversive events may contribute to the development and symptoms of psychiatric disorders, such as anxiety disorders, particularly posttraumatic stress disorder (PTSD) or phobias (Green, 2008; Papassotiropoulos, 201 1 ; Papassotiropoulos, 201 1), and depression.

Working memory refers to the ability of maintaining task-relevant information while performing a specific cognitive task (Shah, 1999). Working memory can be defined as a limited-capacity system for remembering information for a short time and performing mental operations on this information (Gazzaniga, 2009). This cognitive capacity is related to the proper function of a variety of brain regions, such as the prefrontal cortex and the basal ganglia, and is impaired in several neuropsychiatric disorders such as schizophrenia, depression, bipolar disease, neurodegenerative disorders (e.g. Alzheimer's and Parkinson's disease), attention deficit / hyperactivity disorder, autism, multiple sclerosis, epilepsy, anxiety, post-traumatic stress disorder, and other diseases affecting working memory-related brain regions. A promising therapeutic approach for the treatment of pathological conditions characterized by working memory deficits is to pharmacologically enhance working memory performance.

There was therefore a need for identifying genes or gene clusters that are involved in the regulation of these adaptive mechanisms and which would thus qualify as targets that can be used in memory modulation in patients, which are, for example, in danger of developing anxiety disorders or loss of cognitive capacities including working memory.

This need could be satisfied within the scope of the present invention by providing the methods and means, which are further defined in the claims.

The present invention thus relates in certain embodiments to a method of identifying targets of therapeutic compounds with medical indications other than memory that can be used in memory-modulation in a subject comprising

a. carrying out a genome-wide association study (GWAS) filtered for known targets of therapeutic compounds with medical indications other than memory in a sample, particularly a genomic DNA (gDNA) sample, of healthy subjects characterized for memory performance, particularly emotional memory and/or working memory performance;

b. identifying genes or gene clusters of known targets of therapeutic compounds with medical indications other than memory significantly associated with memory performance, particularly emotional memory and/or working memory performance.

In one embodiment, the GWAS in step a) is complemented by tagging SNPs, particularly equal to or below the significance threshold (P) of 0.001 , and/or a gene set enrichment analysis (GSEA). In one embodiment, the GWAS in step a) is performed after correction for minor allele frequency (MAF), particularly excluding tagging SNPs with MAF < 1 %.

In one embodiment, the GWAS in step a) is performed after correction for deviation from Hardy-Weinberg equilibrium (HWE), particularly excluding tagging SNPs with PHWE < 0.01.

In one embodiment, the GSEA analysis is done in a genome-wide manner, particularly with genome-wide false discovery rate (FDR) correction with Ρ set to equal to or below 0.05, particularly with the "mask x MHC" option activated.

In one embodiment, the number of genes in a pathway is set to 20 - 200, particularly 20-100, particularly 20-50.

In one embodiment of the invention, the sample mentioned in step a) above is processed on a Genome-Wide Human SNP Array, particularly an Affymetrix Genome-Wide Human SNP Array, comprising between 500 and 2500, particularly between 700 and 2300, particularly between 900 and 2100, particularly between 1000 and 2000 DNA samples. In a specific embodiment of the invention, 1800 DNA samples are used.

In one embodiment of the invention, determination of genetic associations as mentioned above in step b) is complemented by an analysis under the additive and dominant genetic model.

In one embodiment, genes or gene clusters are identified in step b) which are significantly associated with negative and/or positive emotional memory.

In certain embodiments of the invention, the method according to the invention as described herein comprises additional pre-analytical steps including, without being limited thereto, standard array quality control (QC), and/or control for population stratification.

In one embodiment, the method according to the invention as described herein comprises the analysis of genes or gene clusters or of the expression products thereof, which are known targets of therapeutic compounds with medical indications other than memory. In one embodiment of the invention, the target gene or gene cluster identified in the method according to the invention as described herein is a gene encoding a G- protein coupled plasma membrane receptor, particularly a gene encoding a G- protein coupled plasma membrane receptor from the family of histamine receptors and genes encoding receptor interacting molecules, respectively. In one embodiment of the invention, the receptor from the family of histamine receptors identified in the method according to the invention as described herein is a histamine H1 receptor or a histamine H1 receptor interacting molecule.

In one embodiment of the invention, the target gene or gene cluster identified in the method according to the invention as described herein is a gene from the family of angiotensin receptor genes and genes encoding receptor interacting molecules, respectively.

In one embodiment of the invention, the receptor from the family of angiotensin receptors identified in the method according to the invention as described herein is an angiotensin II receptor or a angiotensin II receptor interacting molecule. In one embodiment of the invention, the target gene or gene cluster identified in the method according to the invention as described herein is a gene related to episodic memory, particularly a gene encoding the beta-2-adrenergic receptor (ADRB2R), or a gene encoding a molecule which directly or indirectly interacts with said beta-2-adrenergic receptor (ADRB2R). In one embodiment of the invention, the target gene or gene cluster identified in the method according to the invention as described herein is a gene related to episodic memory, particularly a gene encoding plasminogen (PLG), or a gene encoding a molecule which directly or indirectly interacts with plasminogen. In one embodiment of the invention, the target gene or gene cluster identified in the method according to the invention as described herein is a gene encoding the calcium-sensing receptor (CASR) and genes encoding CASR interacting molecules, respectively.

In one embodiment of the invention, the target gene or gene cluster identified in the method according to the invention as described herein is a gene encoding the neuronal amiloride-sensitive cation channel 1 (ACCN1 ) and genes encoding ACCN1 interacting molecules, respectively.

In one embodiment of the invention, the target gene or gene cluster identified in the method according to the invention as described herein is a gene encoding at least one of the members of the family of specific voltage-gated potassium channels in the CNS, or a gene encoding a molecule directly or indirectly interacting with at least one of said family members.

In certain embodiments, the present invention relates to a method for screening candidate compounds with medical indications other than memory for their potential of modulating a target gene or gene cluster, or the expression product thereof significantly associated with memory performance, particularly emotional or working memory performance, comprising administering to a subject a candidate compound suspected to be able to induce the desired modulation effect in the target gene or gene cluster, or the expression product thereof, and determining the effect of said compound on memory performance, particularly emotional or working memory performance, in said subject. The effect on memory performance, particularly emotional or working memory performance may be determined in a clinical trial.

In one embodiment, the effect on negative and/or positive emotional memory is determined, particularly in a test system selected from the group consisting of picture memory task or working memory task.

In one embodiment, the target gene or gene cluster to be used in the screening method of the invention as described herein, is a gene or gene cluster or the expression products thereof, which are known targets of therapeutic compounds with medical indications other than memory.

In certain embodiments of the invention, said gene or gene cluster encodes a G- protein coupled plasma membrane receptor, particularly a G-protein coupled plasma membrane receptor of the family of histamine receptors or a molecule directly or indirectly interacting with said receptor.

In one embodiment of the invention, the receptor from the family of histamine receptors is a histamine H1 receptor.

In certain embodiments of the invention, said gene or gene cluster encodes the family of angiotensin receptors or a molecule directly or indirectly interacting with said receptors.

In one embodiment of the invention, the receptor from the family of angiotensin receptors is an angiotensin II receptor.

In certain embodiments of the invention, said gene or gene cluster is related to episodic memory, encoding the beta-2-adrenergic receptor (ADRB2R) and plasminogen (PLG), respectively.

In certain embodiments of the invention, said gene or gene cluster encodes the calcium-sensing receptor (CASR), or a molecule directly or indirectly interacting with said receptor.

In certain embodiments of the invention, said gene or gene cluster encodes neuronal amiloride-sensitive cation channel 1 (ACCN1 ), or a molecule directly or indirectly interacting with said receptor.

In certain embodiments, the present invention relates to a method for screening therapeutic candidate compounds with a medical indication other than memory, for their potential of modulating a target gene or gene cluster, or the expression product thereof significantly associated with memory performance comprising

a. ) contacting a candidate compound with cells in culture; and b. ) determining the modulation of target gene expression within the cells, and/or

c. ) determining the modulation of the activity of the expression product within the cells.

In one embodiment, the modulation of target gene expression is determined on transcript level.

In another embodiment, the modulation of target gene expression is determined on protein level.

In one embodiment, the endogenous target gene expression within the cells is quantified, by specific nucleic acid quantification methods, such as quantitative RT-PCR, micro array, or by Northern blot.

In one embodiment, the cultured cells are transfected cells comprising an expression cassette comprising a reporter gene under the control of the target gene promoter, particularly a reporter gene selected from the group consisting of a luciferase, a fluorescent protein, beta-galactosidase, chloramphenicol acetyltransferase, beta-glucuronidase, alkaline phosphatase, a resistance- conferring gene, and a gene for growth selection.

In one embodiment, the cultured cells are transfected cells comprising an expression cassette comprising a cDNA of the target gene, particularly a cDNA comprising 3' and/or 5' untranslated regions (UTR), under the control of an exogenous promoter.

In one embodiment, the cultured cells are transfected cells comprising an expression cassette comprising a nucleic acid encoding a fusion protein under control of a target gene promoter or an exogenous promoter.

In one embodiment, the modulation of target gene expression is quantified by determining the expression level of said cDNA or of the fusion protein. In one embodiment, the modulation of target gene expression is determined by quantifying the endogenous level of target protein in the cultured cell using a protein quantification method selected from the group consisting of ELISA, Western blot, or immunoprecipitation.

In one embodiment, the cells are of a cultured cell line, particularly a cell line that naturally expresses the target gene, especially CNS derived cell lines.

In one embodiment, said target gene used in the method according to the invention and as described herein is a gene encoding i) a receptor from the family of histamine and / or angiotensin receptor proteins or a molecule directly or indirectly interacting with said receptor; or ii) plasminogen (PLG), or a molecule directly or indirectly interacting with plasminogen; or iii) the beta-2-adrenergic receptor (ADRB2R) or a molecule directly or indirectly interacting with said receptor; or iv) the neuronal amiloride-sensitive cation channel 1 (ACCN1 ) or a molecule directly or indirectly interacting with ACCN1 ; or. v) a member of the family of specific voltage-gated potassium channels or a molecule directly or indirectly interacting with said member; or vi) a calcium-sensing receptor (CASR) or a molecule directly or indirectly interacting with said receptor.

To screen for compounds that are capable of modulating expression or activity of target gene or gene cluster, or the expression product thereof significantly associated with memory performance such as histamine H1 receptor, angiotensin II receptor, beta-2-adrenergic receptor (ADRB2R) and plasminogen (PLG), respectively, calcium-sensing receptor (CASR), and/or neuronal amiloride- sensitive cation channel 1 (ACCN1 ), a cultured cell line may be used, particularly cell lines that naturally express histamine H1 receptor, angiotensin II receptor, beta-2-adrenergic receptor (ADRB2R) and plasminogen (PLG), respectively, calcium-sensing receptor (CASR), and/or neuronal amiloride-sensitive cation channel 1 (ACCN1 ), such as a CNS derived cell including neural cells, e.g. hippocampal cells, as the screen addresses neuronal processes. Target gene expression can be measured on the gene expression level (amount of mRNA) by methods known in the art such as RT-PCR (e.g. Roche LightCycler®), micro array technology (e.g. Affymetrix GeneChip®) or Northern-blotting methods, using probes complementary to the histamine H1 receptor, angiotensin II receptor, beta-2-adrenergic receptor (ADRB2R) and plasminogen (PLG) cDNA sequence, respectively, calcium-sensing receptor (CASR), and/or neuronal amiloride- sensitive cation channel 1 (ACCN1 ), .

Advantageously, micro array technologies allow comparisons of target gene levels to many other genes, and thus conclusions as to the specificity of the effects caused by a test compound.

Alternatively, alteration of target gene expression can also be assessed by using cells transfected by a reporter gene construct, wherein the reporter gene is under the control of the promoter of the target gene. Suitable reporter genes are e.g.. without being limited thereto: luminescence causing enzymes (such as firefly luciferase, renilla luciferase, or bacterial luciferase operon (Photorhabdus luminescens luxCDABE)), fluorescent proteins (such as green fluorescent protein (Aequorea victoria) and derivatives thereof (e.g . EYFP), or red fluorescent protein (Discosoma sp.)), substrate converting enzymes (such as beta-galactosidase, chloramphenicol, acetyltransferase, beta-glucuronidase, or alkaline phosphatase), or resistance-conferring genes or else genes for growth selection. Protein expression can be determined e.g. within an antibody based ELISA assay quantifying the amount or concentration of individual proteins. The expression level assessed according to the present invention is influenced by the synthesis rate as well as by the degradation rate or the stability of the transcript and the protein. In addition the cultured cells can be transfected with an expression construct before being contacted with the candidate compound. The expression construct may comprise full length cDNA of the target gene, including 3' and/or 5' untranslated regions (UTR), under the control of an exogenous promoter. The quantification of the expression level of said full length cDNA reflects the alteration of its stability due to the contacting of the cells with the candidate compound.

Further details and alternatives to the here described methods and tools for use in the screening process according to the invention can be found in WO 2011095363, the disclosure of which is incorporated herewith. In one embodiment, the invention relates to an expression cassette comprising a reporter gene under the control of a promoter of a target gene significantly associated with memory performance.

In one embodiment, the invention relates to an expression cassette comprising a cDNA of a target gene significantly associated with memory performance, particularly a cDNA comprising 3' and/or 5' untranslated regions (UTR), under the control of an exogenous promoter.

In one embodiment, the invention relates to an expression cassette comprising a nucleic acid encoding a fusion protein under control of a promoter of a target gene significantly associated with memory performance or of an exogenous promoter. In one embodiment, the expression cassette of the invention and as described herein comprises a target gene, which is a gene encoding i) a receptor from the family of histamine and / or angiotensin receptor proteins or a molecule directly or indirectly interacting with said receptor; or ii) plasminogen (PLG), or a molecule directly or indirectly interacting with plasminogen; or iii) the beta-2-adrenergic receptor (ADRB2R) or a molecule directly or indirectly interacting with said receptor; or iv) the neuronal amiloride-sensitive cation channel 1 (ACCN 1 ) or a molecule directly or indirectly interacting with ACCN1 ; or v) a member of the family of specific voltage-gated potassium channels or a molecule directly or indirectly interacting with said member; or vi) a calcium-sensing receptor (CASR) or a molecule directly or indirectly interacting with said receptor.

In another embodiment, the invention relates to a transgenic animal comprising an expression cassette according to the invention and as described herein.

In certain embodiments, the present invention relates to a pharmaceutical composition comprising a compound which modulates a target identified in a method according to the invention as described herein, particularly for use in modulation of emotional memory and/or working memory performance.

In certain embodiments, the present invention relates to a pharmaceutical composition comprising a compound which modulates a G-protein coupled plasma membrane receptor, particularly a G-protein coupled plasma membrane receptor of the histamine and/or angiotensin receptor family or a molecule directly or indirectly interacting with said receptor for use in memory-modulation, particularly for use in modulation of emotional memory, particularly negative emotional memory. In particular, said modulating compound may be an agonist or an antagonist for the receptor or for the molecule directly or indirectly interacting with said receptor.

In one embodiment, the pharmaceutical composition according to the invention as described herein, comprising a compound which modulates a G-protein coupled plasma membrane receptor, particularly a G-protein coupled plasma membrane receptor of the histamine and/or angiotensin receptor family or a molecule directly or indirectly interacting with said receptor for use in memory-modulation, is used for suppressing emotional memory or for enhancing working memory performance.

In certain embodiments, the present invention relates to a pharmaceutical composition comprising a compound, which modulates a G-protein coupled plasma membrane receptor, particularly a G-protein coupled plasma membrane receptor of the histamine and/or angiotensin receptor family or a molecule directly or indirectly interacting with said receptor, for use in the treatment of psychiatric disorders, particularly anxiety disorders selected from the group consisting of posttraumatic stress disorder (PTSD), and phobias.

In particular, said modulating compound may be an agonist or an antagonist for the receptor or for the molecule directly or indirectly interacting with said receptor.

In one embodiment, the present invention relates to a pharmaceutical composition comprising a compound, which modulates a G-protein coupled plasma membrane receptor, particularly a G-protein coupled plasma membrane receptor of the histamine and/or angiotensin receptor family or a molecule directly or indirectly interacting with said receptor, for use in the treatment of posttraumatic stress disorder (PTSD) in heavily traumatized subjects.

In one embodiment of the invention, said treatment leads to the reduction of long- term recall of negative pictures.

In one embodiment of the invention, said treatment leads to the reduction of long- term recall of positive pictures.

In one embodiment, the invention relates to a pharmaceutical composition as described herein, comprising a compound, which modulates a G-protein coupled plasma membrane receptor of the histamine receptor family or a molecule directly or indirectly interacting with said receptor family, wherein said receptor from the family of histamine receptors is histamine H1 receptor.

In one embodiment, the invention relates to a pharmaceutical composition as described herein, wherein said compound is an antagonist of histamine H1 receptor is diphenyl-hydramine. In one embodiment, the invention relates to a pharmaceutical composition as described herein, comprising a compound, which modulates a G-protein coupled plasma membrane receptor of the family of angiotensin receptors or a molecule directly or indirectly interacting with said receptor family, wherein said receptor from the family of angiotensin receptors is angiotensin II receptor.

In one embodiment, the invention relates to a pharmaceutical composition as described herein, wherein said compound is an antagonist of angiotensin II receptor is losartan.

In certain embodiments, the present invention relates to a pharmaceutical composition comprising a compound which modulates a calcium-sensing receptor (CASR) or a molecule directly or indirectly interacting with said receptor, for use in memory-modulation, particularly for use in working memory modulation.

In particular, said modulating compound may be an agonist or an antagonist for the receptor or for the molecule directly or indirectly interacting with said receptor. In one embodiment, the pharmaceutical composition according to the invention as described herein comprising a compound which modulates a calcium-sensing receptor (CASR) or a molecule directly or indirectly interacting with said receptor, is used for enhancing working memory performance.

In certain embodiments, the present invention relates to a pharmaceutical composition comprising a compound which modulates a calcium-sensing receptor (CASR) or a molecule directly or indirectly interacting with said receptor, for use in the treatment of pathological conditions characterized by working memory deficits, particularly pathological condition selected from the group consisting of schizophrenia, depression, bipolar disease, neurodegenerative disorders (e.g. Alzheimer's and Parkinson's disease), attention deficit / hyperactivity disorder, autism, multiple sclerosis, epilepsy, anxiety, post-traumatic stress disorder, and other diseases affecting working memory-related brain regions.

Said modulating compound may be an agonist or an antagonist for the receptor or for the molecule directly or indirectly interacting with said receptor. In particular, said molecule directly or indirectly interacting with the calcium- sensing receptor (CASR) is a molecule as shown in Figure 3, 6a and 6b.

In one embodiment, the invention relates to a pharmaceutical composition as described herein, wherein said compound modulating a calcium-sensing receptor (CASR) or a molecule directly or indirectly interacting with said receptor is a calcimimetic agent that in-creases the sensitivity of the calcium-sensing receptor (CASR) to activation by extracellular calcium.

In one embodiment, the invention relates to a pharmaceutical composition as described herein, wherein said calcium-sensing receptor (CASR) modulating compound is Cinacalcet.

In certain embodiments, the present invention relates to a pharmaceutical composition comprising a compound which modulates a neuronal amiloride- sensitive cation channel 1 (ACCN1 ) or a molecule directly or indirectly interacting with ACCN1 , for use in the treatment of pathological conditions characterized by working memory deficits, particularly pathological condition selected from the group consisting of schizophrenia, depression, bipolar disease, neurodegenerative disorders (e.g. Alzheimer's and Parkinson's disease), attention deficit / hyperactivity disorder, autism, multiple sclerosis, epilepsy, anxiety, post-traumatic stress disorder, and other diseases affecting working memory-related brain regions.

In particular, said modulating compound may be an agonist or an antagonist for ACCN1 or for the molecule directly or indirectly interacting with ACCN1.

In one embodiment of the invention, said molecule directly or indirectly interacting with ACCN1 is a molecule as shown in Figure 7a, 7b. In one embodiment, the invention relates to a pharmaceutical composition as described herein, wherein said compound modulating a neuronal amiloride- sensitive cation channel 1 (ACCN1 ) or a molecule directly or indirectly interacting with ACCN1 is a molecule that has working memory enhancing properties.

In one embodiment, the invention relates to a pharmaceutical composition as described herein, wherein said ACCN1 modulating compound is amiloride.

In certain embodiments, the present invention relates to a pharmaceutical composition comprising a compound which modulates at least one of the members of the family of specific voltage-gated potassium channels in the CNS, or a molecule directly or indirectly interacting with at least one of said family members, for use in memory-modulation in a subject, particularly for use in

(i) modulation of emotional memory, particularly negative emotional memory;

(ii) modulation of working memory, particularly for enhancing working memory performance; or

(iii) the treatment of psychiatric disorders, particularly anxiety disorders selected from the group consisting of posttraumatic stress disorder (PTSD), or phobias.

In another specific embodiment, the invention relates to a pharmaceutical composition as disclosed herein comprising a compound which modulates at least one of the members of the family of specific voltage-gated potassium channels in the CNS, or a molecule directly or indirectly interacting with at least one of said family members, for use in the treatment of pathological conditions characterized by working memory deficits, particularly pathological condition selected from the group consisting of schizophrenia, depression, bipolar disease, neurodegenerative disorders (e.g. Alzheimer's and Parkinson's disease), attention deficit / hyperactivity disorder, autism, multiple sclerosis, epilepsy, anxiety, post-traumatic stress disorder, and other diseases affecting working memory-related brain regions.

In one embodiment of the invention, said modulating compound may be an agonist or an antagonist, for at least one of the members of the family of specific voltage- gated potassium channels in the CNS, or for a molecule directly or indirectly interacting with at least one of said family members.

In particular, said modulating compound is an agonist of at least one member of the family of specific voltage-gated potassium channels or a molecule directly or indirectly interacting with at least one of said family members. In a specific embodiment the invention relates to a pharmaceutical composition as described herein comprising a compound which modulates at least one of the members of the family of specific voltage-gated potassium channels in the CNS, or a molecule directly or indirectly interacting with at least one of said family members, which comprises Ezogabine (syn. Retigabine).

In particular, the Ezogabine compound affects the expression of the target genes KCNQ2, KCNQ3, KCNQ4, KCNQ5, GABRA1 , GABRA5, GABRB1 , GABRB2, GABRB3, GABRG2, GABRG3 and is capable of passing the blood-brain barrier. In one embodiment, the invention relates to a pharmaceutical composition according to the invention as described herein, which, upon administration to a subject, does not cause side-effects, particularly side effects selected from the group consisting of attenuation of positive recall, verbal recall, figure recall, attention and working memory.

In certain embodiments, the present invention relates to a pharmaceutical composition comprising a compound which modulates the beta-2-adrenergic receptor or plasminogen, or a molecule directly or indirectly interacting with the beta-2-adrenergic receptor or plasminogen, for use in memory-modulation in a subject, particularly for use in

(i) modulation of episodic memory;

(iii) the treatment of psychiatric disorders, particularly psychotic, mood and anxiety disorders including depression, anxiety, post-traumatic stress disorder, schizophrenia.

In another specific embodiment, the invention relates to a pharmaceutical composition as disclosed herein comprising a compound which modulates the beta-2-adrenergic receptor or plasminogen, or a molecule directly or indirectly interacting with the beta-2-adrenergic receptor or plasminogen, for use in the treatment of pathological conditions characterized by episodic and/or working memory deficits, particularly pathological condition selected from the group consisting of depression, anxiety, post-traumatic stress disorder, schizophrenia, and other diseases affecting episodic and/or working memory-related brain regions.

In one embodiment of the invention, said modulating compound may be an agonist or an antagonist, for the beta-2-adrenergic receptor or plasminogen, or for a molecule directly or indirectly interacting with the beta-2-adrenergic receptor or plasminogen.

In a specific embodiment the invention relates to a pharmaceutical composition as described herein comprising a compound which modulates the beta-2-adrenergic receptor or a molecule directly or indirectly interacting with the beta-2-adrenergic receptor, which compound is salbutamol (targeting the beta-2-adrenergic receptor).

In a specific embodiment the invention relates to a pharmaceutical composition as described herein comprising a compound which modulates plasminogen or a molecule directly or indirectly interacting with plasminogen, which compound is tranexamic acid (targeting plasminogen).

In certain embodiments, the present invention relates to a method for use in memory-modulation, particularly for use in modulation of emotional memory and/or working memory performance, comprising administering to said subject a compound identified in a method according to the invention and as described in any one of the preceding embodiments or a pharmaceutical composition according to the invention as described herein.

In certain embodiments, the present invention relates to a method for use in the treatment of anxiety disorders, particularly anxiety disorders selected from the group consisting of posttraumatic stress disorder (PTSD), and phobias, comprising administering to said subject a compound identified in a method according to the invention and as described in any one of the preceding embodiments or a pharmaceutical composition according to the invention as described herein, particularly a composition comprising a compound, which modulates a G-protein coupled plasma membrane receptor, particularly a G-protein coupled plasma membrane receptor of the histamine and/or angiotensin receptor family or a molecule directly or indirectly interacting with said receptor.

In particular, said modulating compound as used in the method for memory- modulation as described herein, may be an agonist or an antagonist for the receptor or for the molecule directly or indirectly interacting with said receptor.

In certain embodiments, the present invention relates to a method for modulating aversive memory processes in a subject in need of such a modulation comprising administering to said subject a compound identified in a method according to the invention and as described in any one of the preceding embodiments or a pharmaceutical composition according to the invention as described herein, particularly a composition comprising a compound, which modulates a G-protein coupled plasma membrane receptor, particularly a G-protein coupled plasma membrane receptor of the histamine and/or angiotensin receptor family or a molecule directly or indirectly interacting with said receptor.

In particular, said modulating compound as used in the method for memory- modulation as described herein, may be an agonist or an antagonist for the receptor or for the molecule directly or indirectly interacting with said receptor. In one embodiment of the invention, said method for memory-modulation as described herein, is used for the treatment of psychiatric disorders, particularly anxiety disorders selected from the group consisting of posttraumatic stress disorder (PTSD), and/or phobias.

In one embodiment, said method for memory-modulation as described herein, is used for the treatment of posttraumatic stress disorder (PTSD) in heavily traumatized subjects.

In one embodiment, the said method for memory-modulation as described herein, leads to the reduction of long-term recall of negative pictures.

In one embodiment, the said method for memory-modulation as described herein, leads to the reduction of long-term recall of positive pictures.

In one embodiment, the compound used in the method for memory-modulation according to the invention as described herein is an antagonist of receptors from the family of histamine and/or angiotensin receptors, particularly an antagonist of histamine H1 receptor or of angiotensin II receptor.

In one embodiment said antagonist is diphenyl-hydramine.

In one embodiment said antagonist is losartan.

In certain embodiments, the present invention relates to a method for use in working memory modulation comprising administering to said subject a compound identified in a method according to the invention and as described in any one of the preceding embodiments or a pharmaceutical composition according to the invention, particularly a pharmaceutical composition comprising a compound which modulates a calcium-sensing receptor (CASR) or a molecule directly or indirectly interacting with said receptor. In particular, said method for working memory modulation may be used for the treatment of pathological condition selected from the group consisting of schizophrenia, depression, bipolar disease, neurodegenerative disorders (e.g. Alzheimer's and Parkinson's disease), attention deficit / hyperactivity disorder, autism, multiple sclerosis, epilepsy, anxiety, post-traumatic stress disorder, and other diseases affecting working memory-related brain regions.

In one embodiment said calcium-sensing receptor (CASR) modulating compound is Cinacalcet. In certain embodiments, the present invention relates to a method for use in working memory modulation comprising administering to said subject a compound identified in a method according to the invention and as described in any one of the preceding embodiments or a pharmaceutical composition according to the invention comprising a compound which modulates a neuronal amiloride-sensitive cation channel 1 (ACCN1 ) or a molecule directly or indirectly interacting with ACCN1.

In particular, said method for working memory modulation may be used for the treatment of pathological condition selected from the group consisting of schizophrenia, depression, bipolar disease, neurodegenerative disorders (e.g. Alzheimer's and Parkinson's disease), attention deficit / hyperactivity disorder, autism, multiple sclerosis, epilepsy, anxiety, post-traumatic stress disorder, and other diseases affecting working memory-related brain regions.

In one embodiment, said compound as used in the method for working memory modulation according to the invention as described herein, modulating a neuronal amiloride-sensitive cation channel 1 (ACCN1 ) or a molecule directly or indirectly interacting with ACCN1 is a molecule that has working memory enhancing properties.

In one embodiment, said ACCN1 modulating compound is amiloride.

In certain embodiments, the present invention relates to a method for memory- modulation in a subject, comprising administering to said subject a compound identified in a method according to the invention and as described in any one of the preceding embodiments or a pharmaceutical composition according to the invention, which modulates at least one of the members of the family of specific voltage-gated potassium channels in the CNS, or a molecule directly or indirectly interacting with at least one of said family members, for use in

(i) modulation of emotional memory, particularly negative emotional memory;

In another specific embodiment, the invention relates to a method as disclosed herein, for the treatment of pathological conditions characterized by working memory deficits, particularly pathological condition selected from the group consisting of schizophrenia, depression, bipolar disease, neurodegenerative disorders (e.g. Alzheimer's and Parkinson's disease), attention deficit / hyperactivity disorder, autism, multiple sclerosis, epilepsy, anxiety, post-traumatic stress disorder, and other diseases affecting working memory-related brain regions.

In one embodiment, said compound as used in the method according to the invention as described herein for the treatment of pathological conditions characterized by working memory deficits, is a modulator of at least one member of the family of specific voltage-gated potassium channels, or a molecule directly or indirectly interacting with at least one of said family members.

In a specific embodiment, said compound as used in the method according to the invention as described herein, is an agonist of at least one member of the family of specific voltage-gated potassium channels, or a molecule directly or indirectly interacting with at least one of said family members, particularly said agonist is Ezogabine.

In particular, the Ezogabine compound affects the expression of KCNQ2, KCNQ3, KCNQ4, KCNQ5, GABRA1 , GABRA5, GABRB1 , GABRB2, GABRB3, GABRG2, GABRG3 and is capable of passing the blood-brain barrier.ln one embodiment, administration of a compound identified in a method according to the invention and as described in any one of the preceding embodiments or a pharmaceutical composition according to the invention as described herein does not cause side- effects, particularly side-effects selected from the group consisting of attenuation of positive recall, verbal recall, figure recall, attention and working memory.

In certain embodiments, the present invention relates to a method for use in episodic and/or working memory modulation comprising administering to said subject a compound identified in a method according to the invention and as described in any one of the preceding embodiments or a pharmaceutical composition according to the invention, particularly a pharmaceutical composition comprising a compound which modulates the beta-2-adrenergic receptor or plasminogen, or a molecule directly or indirectly interacting with the beta-adrenergic receptor or plasminogen.

In particular, said method for episodic and/or working memory modulation may be used for the treatment of pathological condition selected from the group consisting of depression, anxiety, post-traumatic stress disorder, schizophrenia, and other diseases affecting episodic and/or working memory-related brain regions.

In a specific embodiment, said modulating compound may be an agonist or an antagonist for the beta-2-adrenergic receptor, or for a molecule directly or indirectly interacting with the beta-2-adrenergic receptor.

In a specific embodimentr, said modulating compound may be an agonist or an antagonist of plasminogen or a molecule directly or indirectly interacting with plasminogen.

In one embodiment said compound is salbutamol (targeting the beta-2-adrenergic receptor).

In another specific embodiment, said compound is tranexamic acid (targeting plasminogen).

Brief Description of Figures

Figure 1 shows genetic association results (Y-axis, -logioP) of the HRH1 locus. Black dots represent P values of association with negative emotional memory. Chromosomal positions were retrieved from the March 2006 UCSC genome browser assembly. Four known HRH1 transcripts are visualized in the lower part of the figure.

Figure 2 shows genetic association results (Y-axis, -log-ιοΡ) of the AGTR1 locus. Black dots represent P values of association with negative emotional memory. Chromosomal positions were retrieved from the March 2006 UCSC genome browser assembly. Five known AGTR1 transcripts are visualized in the lower part of the figure.

Figure 3 shows genetic association results (Y-axis, -log-|₀P) of the CASR locus. Black dots represent P values of association with working memory (i.e. 2-back - 0- back). Chromosomal positions were retrieved from the March 2006 UCSC genome browser assembly. Two known CASR transcripts are visualized in the lower part of the figure.

Figure 4 shows genetic association results (Y-axis, -logi₀P) of the ACCN1 locus. Black dots represent P values of association with working memory (i.e. 2-back - 0- back). Chromosomal positions were retrieved from the March 2006 UCSC genome browser assembly. Two known ACCN1 transcripts are visualized in the lower part of the figure.

Figure 5a shows the result of an Ingenuity Pathway Analysis (I PA®) revealed following network of AGTR1 -interacting molecules

Figure 5b shows molecules directly interacting with AGTR1.

These molecules/molecule groups are in addition to AGTR1 candidate drug targets for modulation of aversive memory capacity. Based on the genetic data there is an expectation that either AGTR1/AGTR1 network agonists or AGTR1/AGTR1 network antagonists have aversive memory-suppressing properties.

Figure 6a shows the result of an Ingenuity Pathway Analysis (IPA®) revealed following network of CASR-interacting molecules.

Figure 6b shows molecules directly interacting with CASR.

Figure 7a shows the result of an Ingenuity Pathway Analysis (IPA®) revealed following network of ACCN1 -interacting molecules.

Figure 7b shows molecules directly interacting with ACCN1.

These molecules/molecule groups are in addition to ACCN1 candidate drug targets for modulation of working memory capacity, with ACCN1 inhibitor amiloride being a first-priority candidate drug. Based on the genetic data it may be expected that either ACCN1/ACCN1 network agonists or ACCN1/ACCN1 network antagonists (such as amiloride) have working memory-enhancing properties. Definitions:

A "modulating compound" refers to a compound as described herein, which may either up-regulate (e.g., activate or stimulate), down-regulate (e.g., inhibit or suppress) or otherwise change a functional property or biological activity of a target molecule or gene. A modulating compound may act to modulate a target molecule or a gene encoding said target molecule either directly or indirectly. In certain embodiments, a modulating compound may be an activating compound or an-inhibiting compound.

The term "expression" is meant to refer to both, gene expression as well as protein expression, unless indicated otherwise.

"Inhibitors" or "antagonists" are compounds that, e.g., bind to, partially or totally block activity, decrease, prevent, delay activation, inactivate, desensitize, or downregulate the activity or expression of the target molecule or gene, particularly a target molecule or gene associated with memory performance.

"Activators" or "agonists" are compounds that increase, open, activate, facilitate, enhance activation, sensitize, agonize, or upregulate activity of the target molecule or gene, particularly a target molecule or gene associated with memory performance.

As used herein, the term "downregulate" refers to any statistically significant decrease in a biological activity and/or expression of a target molecule or gene, , particularly a target molecule or gene associated with memory performance, including full blocking of the activity (i.e., complete inhibition) and/or expression. For example, "downregulation" can refer to a decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% activity and/or expression of a target molecule or gene, particularly a target molecule or gene associated with memory performance.

As used herein, the term "inhibit" or "inhibiting" refers to any statistically significant decrease in a biological activity and/or expression of a target molecule or gene, , particularly a target molecule or gene associated with memory performance, including full blocking of the activity and/or expression. For example, "inhibition" can refer to a decrease of about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% in activity and/or expression of a target molecule or gene, particularly a target molecule or gene associated with memory performance.

"Memory modulation" refers to the adjusting of the adaptive mechanisms which allow one to remember important information. In some cases, "Memory modulation" refers to abating memory of aversive events. This adjustment should be such that the abating of memory of aversive events is not accompanied by an attenuation of other cognitive functions such as, for example, positive recall, verbal recall, figure recall, attention and working memory. In some cases, "Memory modulation" refers to enhancing working memory.

The terms "treatment", "treating" and the like are used herein to generally mean obtaining a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of partially or completely curing a disease and/or adverse effect attributed to the disease. The term "treatment" as used herein covers any treatment of a disease in a subject and includes: (a) preventing a disease related to an undesired immune response from occurring in a subject which may be predisposed to the disease; (b) inhibiting the disease, i.e. arresting its development; or (c) relieving the disease, i.e. causing regression of the disease.

A "patient" or "subject" for the purposes of the present invention is used interchangeably and meant to include both humans and other animals, particularly mammals, and other organisms. Thus, the methods are applicable to both human therapy and veterinary applications. In the preferred embodiment the patient or subject is a mammal, and in the most preferred embodiment the patient or subject is a human. The expressions "pharmaceutical composition" and "therapeutical composition" are used herein interchangeably in the widest sense. They are meant to refer, for the purposes of the present invention, to a therapeutically effective amount of the modulating compound, optionally, together with a pharmaceutically acceptable carrier or diluent.

It embraces compositions that are suitable for the curative treatment, the control, the amelioration, an improvement of the condition or the prevention of a disease or disorder in a human being or a non-human animal. Thus, it embraces pharmaceutical compositions for the use in the area of human or veterinary medicine. Such a "therapeutic composition" is characterized in that it embraces at least one modulating compound, and optionally a carrier or excipient whereby the salt and the carrier and excipient are tolerated by the target organism that is treated therewith.

A "therapeutically effective amount" refers to that amount which provides a therapeutic effect for a given condition and administration regimen. In particular, "therapeutically effective amount" means an amount that is effective to prevent, alleviate or ameliorate symptoms of the disease or prolong the survival of the subject being treated, which may be a human or non-human animal. Determination of a therapeutically effective amount is within the skill of the person skilled in the art.

The therapeutically effective amount or dosage of a compound according to this invention can vary within wide limits and may be determined in a manner known in the relevant art. The dosage can vary within wide limits and will, of course, have to be adjusted to the individual requirements in each particular case.

While it is possible for the compounds of the present invention to be administered alone, it is preferable to formulate them into a pharmaceutical composition in accordance with standard pharmaceutical practice. Thus, the invention also provides a pharmaceutical composition which comprises a therapeutically effective amount of a compound of the invention as described herein in admixture with a pharmaceutically acceptable carrier or excipient.

Pharmaceutically acceptable carrier or excipients are well known in the pharmaceutical art, and are described, for example, in Remington's Pharmaceutical Sciences, 15^th Ed., Mack Publishing Co., New Jersey (1991 ). The pharmaceutical excipient can be selected with regard to the intended route of administration and standard pharmaceutical practice. The excipient must be acceptable in the sense of being not deleterious to the recipient thereof.

Pharmaceutically useful excipients that may be used in the formulation of the pharmaceutical composition of the present invention may comprise, for example, carriers, vehicles, diluents, solvents such as monohydric alcohols such as ethanol, isopropanol and polyhydric alcohols such as glycols and edible oils such as soybean oil, coconut oil, olive oil, safflower oil cottonseed oil, oily esters such as ethyl oleate, isopropyl myristate, binders, adjuvants, solubilizers, thickening agents, stabilizers, disintegrants, glidants, lubricating agents, buffering agents, emulsifiers, wetting agents, suspending agents, sweetening agents, colorants, flavors, coating agents, preservatives, antioxidants, processing agents, drug delivery modifiers and enhancers such as calcium phosphate, magnesium state, talc, monosaccharides, disaccharides, starch, gelatine, cellulose, methylcellulose, sodium carboxymethyl cellulose, dextrose, hydroxypropyl-B-cyclodextrin, polyvinylpyrrolidone, low melting waxes, and ion exchange resins.

The routes for administration (delivery) of the compounds of the invention include, but are not limited to, one or more of: oral (e. g. as a tablet, capsule, or as an solution), topical, mucosal (e. g. as a nasal spray or aerosol for inhalation), nasal, parenteral (e. g. by an injectable form), gastrointestinal, intraspinal, intraperitoneal, intramuscular, intravenous, intrauterine, intraocular, intradermal, intracranial, intratracheal, intravaginal, intracerebroventricular, intracerebral, subcutaneous, ophthalmic (including intravitreal or intracameral), transdermal, rectal, buccal, epidural and sublingual.

For example, the compounds can be administered orally in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavoring or coloring agents, for immediate-, delayed-, modified-, sustained-, pulsed- or controlled-release applications.

The tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included. Solid compositions of a similar type may also be employed as fillers in gelatin capsules. Preferred excipients in this regard include lactose, starch, a cellulose, milk sugar or high molecular weight polyethylene glycols. For aqueous suspensions and/or elixirs, the agent may be combined with various sweetening or flavoring agents, coloring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.

If the compounds of the present invention are administered parenterally, then examples of such administration include one or more of: intravenously, intraarterial^, intraperitoneally, intrathecal^, intraventricularly, intraurethrally, intrasternally, intracranially, intramuscularly or subcutaneously administering the compounds; and/or by using infusion techniques. For parenteral administration, the compounds are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary. The preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

As indicated, the compounds of the present invention can be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurized container, pump, spray or nebulizer with the use of a suitable propellant, e. g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, a hydrofluoroalkane such as 1 ,1 ,1 ,2-tetrafluoroethane (HFA134AT) or 1 ,1 ,1 ,2,3,3,3- heptafluoropropane (HFA 227EA), carbon dioxide or other suitable gas. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. The pressurized container, pump, spray or nebulizer may contain a solution or suspension of the active compound, e. g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e. g. sorbitan trioleate. Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of the compound and a suitable powder base such as lactose or starch.

Alternatively, the compounds of the present invention can be administered in the form of a suppository or pessary, or it may be applied topically in the form of a gel, hydrogel, lotion, solution, cream, ointment or dusting powder. The compounds of the present invention may also be dermally or transdermally administered, for example, by the use of a skin patch.

They may also be administered by the pulmonary or rectal routes. They may also be administered by the ocular route. For ophthalmic use, the compounds can be formulated as micronized suspensions in isotonic, pH adjusted, sterile saline, or, preferably, as solutions in isotonic, pH adjusted, sterile saline, optionally in combination with a preservative such as a benzylalkonium chloride. Alternatively, they may be formulated in an ointment such as petrolatum.

For application topically to the skin, the compounds of the present invention can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, emulsifying wax and water. Alternatively, they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, cetyl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.

Typically, a physician will determine the actual dosage which will be most suitable for an individual subject. The specific dose level and frequency of dosage for any particular individual may be varied and will depend upon a variety of factors including the activity of the specific compound employed, the metabolic stability and length of action of that compound, the age, body weight, general health, sex, diet, mode and time of administration, rate of excretion, drug combination, the severity of the particular condition, and the individual undergoing therapy.

A proposed dose of the compounds according to the present invention for administration to a human (of approximately 70 kg body weight) is 0.1 mg to 1 g, preferably 1 mg to 500 mg of the active ingredient per unit dose. The unit dose may be administered, for example, 1 to 4 times per day. The dose will depend on the route of administration. It will be appreciated that it may be necessary to make routine variations to the dosage depending on the age and weight of the patient as well as the severity of the condition to be treated. The precise dose and route of administration will ultimately be at the discretion of the attendant physician or veterinarian.

The compounds of the invention may also be used in combination with other therapeutic agents. When a compound of the invention is used in combination with a second therapeutic agent active against the same disease the dose of each compound may differ from that when the compound is used alone.

The combinations referred to above may conveniently be presented for use in the form of a pharmaceutical formulation. The individual components of such combinations may be administered either sequentially or simultaneously in separate or combined pharmaceutical formulations by any convenient route. When administration is sequential, either the compound of the invention or the second therapeutic agent may be administered first. When administration is simultaneous, the combination may be administered either in the same or different pharmaceutical composition. When combined in the same formulation it will be appreciated that the two compounds must be stable and compatible with each other and the other components of the formulation. When formulated separately they may be provided in any convenient formulation, conveniently in such manner as are known for such compounds in the art.

The pharmaceutical compositions of the invention can be produced in a manner known per se to the skilled person as described, for example, in Remington's Pharmaceutical Sciences, 15^th Ed., Mack Publishing Co., New Jersey (1991 ).

Examples A Genetic studies

1 - High-resolution genome-wide association studies (GWAS)

Samples were processed as described in the Genome-Wide Human SNP Nsp/Sty 6.0 User Guide (Affymetrix). Briefly, genomic DNA concentration was determined by fluorometry (Qubit dsDNA BR Assay Kit, Invitrogen) in a Qubit 1.0 fluorometer and adjusted to 50ng/pl in water. 250ng of DNA was digested in parallel with 10 units of Sty I and Nsp I restriction enzymes (New England Biolabs, Beverly, MA) for 2 hours at 37°C. Enzyme specific adaptor oligonucleotides were then ligated onto the digested ends with T4 DNA Ligase for 3 hours at 16°C. After adjustment to 100μΙ with water, 10μΙ of the diluted ligation reactions were subjected to PCR. Three PCR reactions of 100μΙ were performed for Sty digested products and four PCR reactions for Nsp. PCR was performed with Titanium Taq DNA Polymerase (Clontech, Mountain View, CA) in the presence of 4.5 μΜ PCR primer 002 (Affymetrix), 350 μΜ each dNTP (Clontech), 1 M G-C Melt (Clontech), and 1X Titanium Taq PCR Buffer (Clontech). Cycling parameters were as follows: initial denaturation at 94°C for 3 minutes, amplification at 94°C for 30 seconds, 60°C for 45 seconds and extension at 68°C for 15 seconds repeated a total of 30 times, final extension at 68°C for 7 minutes. Reactions were then verified to migrate at an average size between 200-1 100 bps using 2% TBE gel electrophoresis. PCR products were combined and purified with the Filter Bottom Plate (Millipore, P/N MDRLN0410) using Agencourt AMPure XP Beads (Beckman Coulter, Fuillerton, CA). Purified PCR products were quantified on a Zenith 200rt microplate reader (Anthos-Labtec, Cambridge, UK). 4 to 5pg/Ml were obtained on average for each sample. From this stage on, the SNP Nsp/Sty 5.0/6.0 Assay Kit (Affymetrix) was used. Around 250 pg of purified PCR products were fragmented using 0.5 units of DNAse I at 37°C for 35 minutes. Fragmentation of the products to an average size less than 180 bps was verified using 4% TBE gel electrophoresis. Following fragmentation, the DNA was end labeled with 105 units of terminal deoxynucleotidyl transferase at 37°C for 4 hours. The labeled DNA was then hybridized onto Genome-Wide Human SNP 6.0 Array at 50°C for 18 hours at 60 rpm. The hybridized array was washed, stained, and scanned according to the manufacturer's (Affymetrix) instructions using Affymetrix GeneChip Command Console (AGCC, version 3.2.0.1515).

Generation of SNP calls and Array quality control were performed using the command line programs of the Affymetrix Power Tools package (version: apt- 1.14). According to the manufacturer's recommendation, Contrast QC was chosen as QC metric, using the default value of greater or equal than 0.4. Mean Call Rate for all samples averaged >98.5%. All samples passing QC criteria were subsequently genotyped using the Birdseed (v2) algorithm. GWAS were performed with 1802 DNA samples from healthy young adults characterized for emotional and working memory performance. All samples were processed on the Affymetrix® Genome-Wide Human SNP Array 6.0. Pre-analytical steps for this particular study included standard array quality control (QC), correction for minor allele frequency (SNPs with MAF < 1 % were excluded), correction for deviation from Hardy-Weinberg equilibrium (SNPs with PHWE < 0.01 were excluded), and control for population stratification. Genetic association analyses were done under the additive and dominant genetic model. In order to identify gene clusters and pathways related to the core phenotypes (i.e. emotional memory and working memory) the following selection criteria were used:

A) Tagging SNPs (i.e. plus/minus 20 kb adjacent to the corresponding transcript) significantly associated (i.e. P < 0.001 ) with the target phenotype, or

B) Gene set enrichment analysis (GSEA) and the open platform i- GSEA4GWAS (http://gsea4qwas.psvch.ac.cn/).

Pathway analysis was performed on the /^"-GSEA4GWAS (improved GSEA for GWAS) web server. Briefly, the implemented algorithm performs a gene-set enrichment analysis (GSEA) by calculating a gene-set enrichment score (ES) based on the Kolmogorov-Smirnov statistic using genes' max -log(P value). For increased sensitivity, the test statistic is weighted by the proportion of the number of significant genes in a gene set. SNP label permutation is used to analyze SNP P values and to correct for gene and gene set variation. k/K is multiplied to the ES to obtain the significance proportion based enrichment score (SPES), where k is the proportion of significant genes in the gene set and K is the proportion of significant genes in the GWAS. False-discovery rate (FDR) is used for multiple testing correction. The utilized gene sets are extracted and curated from the MSigDB v2.5 database (http://www.broadinstitute.org/gsea/msigdb), which includes pathways from different online databases (KEGG, Gene Ontology GO and BioCarta.com). We used a gene set size ranging between 20 and 200 genes to avoid both too narrow and too broad functional pathway categories. Gene and SNP annotations were derived from the Ensemble Biomart database (Release 56- 15 September 2009, http://www.ensempl.org/biomart/martview), and each gene was represented by the maximum SNP P value within ±0kb of the annotated gene (i.e. we used intragenic SNPs only). The GSEA analysis was done in a genome-wide manner with the "mask x MHC" option activated. Furthermore, networks with genome-wide FDR corrected P values of < 0.05 were considered significant.

In addition, a third criterion C was used, where the genetic analysis was restricted to genes with known and approved (in Switzerland) therapeutic compounds with medical indications other than memory. This criterion was applied subsequent to A and/or B.

Criteria A) and B) revealed a number of different genes and pathways thereof to be significantly associated with negative or positive emotional memory (i.e. ratio of freely recalled negative over neutral pictures; ratio of freely recalled positive over neutral pictures, respectively) or with working memory (i.e. mean accuracy of performance in the 2-back - 0-back task). Tagging SNPs of the genes encoding the histamine receptor H1 (HRH1) and the angiotensin II receptor, type 1 (AGTR1) were significantly associated with negative emotional memory (see Fig. 1 and Fig. 2, respectively). Tagging SNPs of the genes encoding the calcium-sensing receptor (CASR) and the neuronal amiloride-sensitive cation channel 1 (ACCN1) were significantly associated with working memory (see Fig. 3 and Fig. 4, respectively). Results HRH1 results:

HRH1 was identified by applying criterion B.

The most significant HRH1 SNP was SNP_A-8656444 (dbSNP ID: rs1 1915050) followed by SNP_A-8318149, SNP_A-8704733, and SNP_A- 8704733. Homozygous major allele (A) carriers of SNP_A-8656444 had increased negative emotional memory performance than carriers of the minor allele (G) (P=0.0006, dominant model of inheritance). Statistical significance of SNP_A-8656444 also survived gene-wide Bonferroni correction. From these genetic association results it can be concluded that HRH1 is a candidate drug target for modulation of aversive (negative) memory capacity. AGTR1 results: AGTR1 was identified by applying criterion B.

The most significant AGTR1 SNP was SNP_A-2104975 (dbSNP ID: rs275643) adjacent to the 3'UTR of AGTR1. Homozygous major allele (A) carriers of SNP_A-2104975 had decreased negative emotional memory performance compared to heterozygous or homozygous carriers of the minor allele (G) (PO.05, additive model of inheritance). It can be concluded that also AGTR1 is a candidate drug target for modulation of aversive (negative) memory capacity. Ingenuity Pathway Analysis (IPA®) revealed a network of AGTR1- interacting molecules as shown in Figures 5a and 5b, respectively.

2.3 CASR results:

CASR was identified by applying criterion A.

The most significant CASR SNP was SNP_A-4224830 (dbSNP ID: rs17252533) adjacent to the 3'UTR of CASR. Minor allele (A) carriers of SNP_A-4224830 had better working memory performance than homozygous carriers of the major allele (C) (P=0.001 , additive model of inheritance). It can be concluded that CASR is a candidate drug target for modulation of working memory capacity.

Ingenuity Pathway Analysis (IPA®) revealed a network of CASR-interacting molecules as shown in Figure 6a and 6b, respectively.

The molecules/molecule groups identified in Figure 6a and 6b may also qualify as drug targets for modulation of working memory capacity. Based on the genetic data it can be concluded that either CASR/CASR network agonists (such as cinacalcet) or CASR/CASR network antagonists (such as NPS-2143) have working memory-enhancing properties.

2.4 ACCN1 results:

ACCN1 was identified by applying criterion A.

The most significant ACCN1 SNP was the intronic SNP_A-2180700 (dbSNP ID: rs8067435). Minor allele (C) carriers of SNP_A-2180700 had better working memory performance than homozygous carriers of the major allele (A) (P=0.00005, additive model of inheritance). It can be concluded that ACCN1 is a candidate drug target for modulation of working memory capacity.

Ingenuity Pathway Analysis (IPA®) revealed a network of ACCN1 -interacting molecules as shown in Figures 7a and 7b, respectively. These molecules/molecule groups are in addition to ACCN1 candidate drug targets for modulation of working memory capacity, with ACCN1 inhibitor amiloride being a first-priority candidate drug. Based on the genetic data it can be expected that either ACCN1/ACCN1 network agonists or ACCN1/ACCN1 network antagonists (such as amiloride) have working memory-enhancing properties.

Phase II clinical trials

HRH1 antagonist: Diphenhydramine

To investigate if the HRH1 antagonist diphenhydramine can modulate emotional memory processes in healthy humans, a phase II clinical study was conducted (see table 1 for study specification summary).

Table : Study specification summary

Study medication

Diphenhydramine 50mg (histamine H1 receptor antagonist, Nardyl® Schlaftablette 50mg manufactured by Vifor SA, Villars-sur-Glane formulated for oral administration). Diphenhydramine is an antihistamine originally used for treating allergic reactions. Antihistamines compete with histamine for histamine H1 receptors. Diphenhydramine is approved in Switzerland as a sedative. Diphenhydramine (Tashiro, Duan et al. 2008) is known to cross the blood-brain barrier and is therefore able to influence intracerebral his-tamine H1 receptors (Hill, Ganellin et al. 1997). For Placebo, mannitol 50mg formulated for oral administration was used. Subjects received a single oral administration of diphenhydramine or placebo in a cross-over design. Diphenhydramine and the placebo were encapsulated in identically looking capsules. The preparation of study medication, blinding and randomization list was performed by the Pharmacy of the University Hospital Basel according to GMP and GCP. Randomization was stratified in a counterbalanced way according to treatment, gender and order of medication (i.e. either diphenhydramine or placebo first).

Screening Procedures

During the screening visits the investigator explained to the subject the aims of the study, the study procedures, the drug under investigation and potential risks. Written informed consent was obtained from all participants. Subjects, who were candidates for enrolment into the study, were evaluated for eligibility during the screening visits by the investigator (inclusion, exclusion criteria). Screening visits took place within 4 weeks and at least one day before first test visit and consist of assessment of personal and family history, psychosocial assessment, assessment of medication history and physical examination.

The following questionnaires were used to assess psychiatric or somatic disorders: sociodemographic self assessment questionnaire, self assessment questionnaire covering mental health. Furthermore, there was medical examination (including physical examination and ECG).

Procedure on test days

After the administration of the study medication (i.e. active drug or placebo), there was a 3h-waiting period allowing the medication to reach maximal plasma concentrations. After this time period, emotional and cognitive functions were tested. After 8 + 3 days, subjects returned for a second test day, this time with the study medication they did not receive the first time (i.e. active drug or placebo).

Cognitive and emotional task

Picture memory task (Rasch, 2009): Subjects were presented with 24 neutral, 24 positive and 24 negative photographs in a random order. The photographs were taken from the international affective picture system (IAPS) and were presented for 2.5 s each. Immediately following the presentation of each photograph, subjects were asked to rate it for valence and arousal using the IAPS rating scales. Free recall was tested 5 min (short delay) and 90 min (long delay) after presentation of all photographs. To document recall performance, subjects had to describe in writing each picture with a few words. Two trained investigators rated the description independently. Scores was calculated by summing the correctly remembered photographs per valence. A parallel version was used for the second test day.

Verbal memory task (De Quervain, 2003): Participants viewed six series of five semantically unrelated nouns presented at a rate of one word per second with the instruction to learn the words for immediate free recall after each series. The number of correctly recalled words (hits) was the relevant output. Total score was calculated by summing the number of correctly recalled words. Delayed recall of all 30 words was tested 15 min (short delay) and 1 15 min (long delay) after presentation. A parallel version was used for the second test day. 4.3 Figural memory task, Rey (Rey, 1958): Free recall of visual material was assessed with the Rey-15-figures test. The figures had to be drawn 5 min and 1 h after learning. Total score was calculated by summing the number of correctly recalled figures. A parallel version was used for the second test day. 4.4 d2 cancellation test (Brickenkamp, 1998): Selective attention and concentration was assessed with the d2 cancellation test. Performance is defined by the number of correctly crossed signs minus false positives during 5 minutes.

4.5 Digit span task: Working memory was assessed with the digit span task, a subtest of the Wechsler intelligence inventory for adults. Total score was calculated as described in the manual.

4.6 Mood state was assessed with the self-rating instrument MDBF (Steyer, 1997) consisting of 12 items to be rated in a 5 scale mode. The total score is calculated by summing the answers in each of the 3 dimensions good/bad mood, alertness/sleepiness, rest /restlessness with 4 items in each dimension.

Version A and B for test days 1 and 2.

4.7 Montgomery-Asberg Depression Scale (MADRS, Schmidtke, 1988):

Depressive symptoms were assessed with the self-rating questionnaire MADRS. This scale consists of 9 items assessing subjects' mood, feelings of unease, sleep, appetite, ability to concentrate, initiative, emotional involvement, pessimism and zest for life. Each item is scored between 0 and 3, with three intermediate levels (0.5, 1.5, 2.5). The total score is calculated by summing the answers of the nine items, ranging between 0 and 27 (higher scores indicate increased impairment).

4.8 Anxiety was measured with the self-rating instrument STAI-G form X1 (state) (Laux, 1981 ). This instrument consists of 20 items that is scored between 1-4. The total score is calculated by summing the answers, ranging between 20 to 80.

4.9 Sleepiness was rated by the subject using a visual analogue scale (VAS). Subjects specify their level of agreement to the statement by indicating a position along a continuous line (10 cm) between the two end-points "wide awake" and "extremely sleepy". The score ranges between 0 and 10.

Matrix reasoning. The Bochumer Matrizentest (BOMAT, Hossiep 2001 ) was administered to measure fluid intelligence (Gf) consisting of 29 items. A parallel version was used for the second visit. A time-limited version was used according to Jaeggi (Jaeggi, 2008). The total score was calculated by summing the correct solutions, ranging between 0 to 29.

Saliva sampling for DNA analysis

A saliva sample (2ml) was obtained from each person using oragene DNA Self-Collection Kit OG-500, manufactured by DNA Genotek Inc, Canada. DNA was extracted from saliva samples by using standard procedures.

Statistics

Paired t-tests were used to analyze treatment effects (i.e. diphenhydramine vs. placebo) on emotional memory outcome measures. Sex, age, sleepiness, weight and treatment order were considered as covariates. A significance level of p < 0.05 (two-sided) was considered as significant.

Results of Clinical Trial

The study population consisted of 40 healthy human subjects (21 males) with European ancestry with a mean age of 23 years (SE: 0.8) and with a mean weight of 70.1 kg (SE: 1.9). As expected, diphenhydramine significantly increased sleepiness (as measured with a visual analog scale during the test phase, see table 2). However, there were no significant effects of diphenhydramine on anxiety, attention or fluid intelligence (see table 2). Table 2

Diphen: Diphenhydramine

Effects of diphenhydramine on sleepiness (measured with visual analog scale), anxiety (measured with STAI state), attention (measured with d2 test) and fluid intelligence (measured with BOMAT).

Picture memory task

Diphenhydramine did not affect valence or arousal ratings of the pictures (P > 0.2).

A significant effect of diphenhydramine was found on free recall performance of negative and neutral information after a short delay (5 min after picture presentation), see table 3

Table 3

Data are statistically corrected for sleepiness. Diphen: Diphenhydramine

Further, a significant effect of diphenhydramine on free recall performance of negative pictures was found after a long delay (90 min after picture presentation), see table 4. The effect of Diphenhydramine on free recall of negative pictures (90-min-delay) was observed at a medium effect size (Cohen's d = 0.46).

Table 4

Data are statistically corrected for sleepiness. Diphen: Diphenhydramine

The effect of Diphenhydramine on 90 min delayed free recall of negative pictures was also significant after correcting additionally for treatment order: P = 0.008, sex: P = 0.008, age: P = 0.007 or body weight: P = 0.007. Also without any correction the drug effect was significant: P = 0.028. For SNP_A- 8656444. A significant SNP x drug treatment effect (P < 0.05, repeated measurements ANOVA) was also observed.

Diphenhydramine did not affect significantly verbal recall, figural recall, working memory, depression scores or side effects (P > 0.05). Conclusion

In conclusion, the results from this clinical trial with diphenhydramine provide a proof of concept for the use of human genetic data to discover memory- modulating drugs. Diphenhydramine reduces long-term (90 min) memory recall of negative pictorial information and might therefore be suited for the treatment of anxiety disorders. It can be concluded that HRH1 SNPs (e.g.

SNP_A-8656444) can be used for pharmacogenetic purposes to identify individuals with high probability for optimal treatment effects. Angiotensin II receptor antagonist: Losartan

To investigate if the angiotensin II receptor antagonist losartan can modulate emotional memory processes in healthy humans, a phase II clinical study was conducted (see table 5 for study specification summary). Table 5: Study specification summary

Study medication

Losartan (angiotensin II receptor antagonist), Cosaar® tablet 50 mg (manufactured by Merck Sharp&Dohme-Chibret AG, Schaffhauserstrasse 136, 8152 Opfikon-Glattbrugg, Switzerland) formulated for oral administration. Angiotensin binds to angiotensin receptors found in many tissues. Angiotensin's binding to these receptors in smooth muscle cells causes an increase in blood pressure. Losartan (more specifically, the active metabolite EXP3174 converted in the liver) is a selective angiotensin II receptor blocker. Losartan was approved in Switzerland for treatment of hypertension. EXP3174 is known to cross the blood-brain barrier (Polidori, Ciccocioppo et al. 1996) and is therefore able to influence intracerebral angiotensin II receptors (Phillips and de Oliveira 2008). For Placebo mannitol 50mg formulated for oral administration was used. Subjects received a single oral administration of losartan or placebo in a cross-over design. Losartan and the placebo were encapsulated in identically looking capsules. The preparation of study medication, blinding and randomization list was performed by the Pharmacy of the University Hospital Basel according to GMP and GCP. Randomization was stratified in a counterbalanced way according to treatment, gender and order of medication (i.e. either losartan or placebo first). Screening Procedures

During the screening visits the investigator explained to the subject the aims of the study, the study procedures, the drug under investigation and potential risks. Written informed consent was obtained from all participants. Subjects, who were candidates for enrolment into the study, were evaluated for eligibility during the screening visits by the investigator (inclusion, exclusion criteria).

Screening visits took place within 4 weeks and at least one day before first test visit and consist of assessment of personal and family history, psychosocial assessment, assessment of medication history and physical examination.

The following questionnaires were used to assess psychiatric or somatic disorders: sociodemographic self assessment questionnaire, self assessment questionnaire covering mental health. Furthermore, there was medical examination (including physical examination and ECG). Procedure on test days

After the administration of the study medication, there was a 3h-waiting period allowing the medication to reach maximal plasma concentrations. After this time period, emotional and cognitive functions were tested. After 8 + 3 days, subjects returned for a second test day, this time with the study medication they did not receive the first time (placebo/losartan). Cognitive and emotional tasks

Picture memory task [Rasch, 2009): Subjects were presented with 24 neutral, 24 positive and 24 negative photographs in a random order. The photographs were taken from the international affective picture system (I APS) and were presented for 2.5 s each. Immediately following the presentation of each photograph, subjects were asked to rate it for valence (i.e. the intrinsic attractiveness (positive valence) or aversiveness (negative valence) of a photograph) and arousal using the IAPS rating scales. Free recall was tested 5 min (short delay) and 90 min (long delay) after presentation of all photographs. To document recall performance, subjects had to describe in writing each picture with a few words. Two trained investigators rated the description independently. Scores was calculated by summing the correctly remembered photographs per valence. A parallel version was used for the second test day. Verbal memory task (De Quervain, 2003): Participants viewed six series of five semantically unrelated nouns presented at a rate of one word per second with the instruction to learn the words for immediate free recall after each series. The number of correctly recalled words (hits) was the relevant output. Total score was calculated by summing the number of correctly recalled words. Delayed recall of all 30 words was tested 15 min (short delay) and 115 min (long delay) after presentation. A parallel version was used for the second test day.

Figural memory task, Rey (Rey, 1958): Free recall of visual material was assessed with the Rey-15-figures test. The figures had to be drawn 5 min and 1 h after learning. Total score was calculated by summing the number of correctly recalled figures. A parallel version was used for the second test day. d2 cancellation test (Brickenkamp, 1998): Selective attention and concentration was assessed with the d2 cancellation test. Performance is defined by the number of correctly crossed signs minus false positives during 5 minutes.

Digit span task: Working memory was assessed with the digit span task, a subtest of the Wechsler intelligence inventory for adults. Total score was calculated as described in the manual.

Mood state was assessed with the self-rating instrument MDBF{Steyer, 1997 #468} consisting of 12 items to be rated in a 5 scale mode. The total score is calculated by summing the answers in each of the 3 dimensions good/bad mood, alertness/sleepiness, rest /restlessness with 4 items in each dimension. Version A and B for test days 1 and 2.

Montgomery-Asberg Depression Scale (MADRS, Schmidtke, 1988): Depressive symptoms were assessed with the self-rating questionnaire MADRS. This scale consists of 9 items assessing subjects' mood, feelings of unease, sleep, appetite, ability to concentrate, initiative, emotional involvement, pessimism and zest for life. Each item is scored between 0 and 3, with three intermediate levels (0.5, 1.5, 2.5). The total score is calculated by summing the answers of the nine items, ranging between 0 and 27 (higher scores indicate increased impairment).

Anxiety was measured with the self-rating instrument STAI-G form X1 (state){Laux, 1981 #126}. This instrument consists of 20 items that is scored between 1-4. The total score is calculated by summing the answers, ranging between 20 to 80. 4.9 Sleepiness was rated by the subject using a visual analogue scale (VAS). Subjects specify their level of agreement to the statement by indicating a position along a continuous line (10 cm) between the two end-points "wide awake" and "extremely sleepy". The score ranges between 0 and 10.

4.10 Matrix reasoning. The Bochumer Matrizentest (BOMAT, Hossiep 2001 ) was administered to measure fluid intelligence (Gf) consisting of 29 items. A parallel version was used for the second visit. A time-limited version according to Jaeggi (Jaeggi, 2008) was used. The total score was calculated by summing the correct solutions, ranging between 0 to 29. 5. Saliva sampling for DNA analysis

Statistics

Paired t-tests were used to analyze treatment effects (i.e. losartan vs. placebo) on emotional memory outcome measures. Sex, age, sleepiness, weight and treatment order were considered as covariates. A significance level of p < 0.05 (two-sided) was considered as significant.

7. Results of Clinical Trials 7.1 The study population consisted of 38 healthy human subjects (19 males) with European ancestry with a mean age of 23.5 years (SE: 0.9) and with a mean weight of 68.3 kg (SE: 1.7). Losartan significantly increased sleepiness (as measured with a visual analog scale during the test phase, see table 6). Further, losartan lowered diastolic blood pressure (measured 3 h after medication). There were no significant effects of losartan on anxiety, attention or fluid intelligence (see table 6).

Table 6: Effects of losartan on sleepiness (measured with visual analog scale), anxiety (measured with STAI state), attention (measured with d2 test), fluid intelligence (measured with BOMAT) and on systolic blood pressure (BP syst) and diastolic blood pressure (BP diast).

Picture memory task: _Losartan did not affect valence or arousal ratings of the pictures (P > 0.2). No significant effect of losartan on short-delay free recall performance 5 min after picture presentation (P > 0.05) was found. However, losartan significantly affected free recall performance of positive pictures after a long delay (90 min after picture presentation), see table 7. The effect of losartan on free recall of positive pictures (90-min-delay) was observed at a small to medium effect size (Cohen's d = 0.36).

Table 7

Data are statistically corrected for sleepiness.

The effect of losartan on free recall of positive pictures was also significant when correcting additionally for treatment order: P = 0.037, sex: P = 0.037, age: P = 0.035 or body weight: P = 0.037. Also without any correction the drug effect was significant: P = 0.039. Losartan did not affect significantly verbal recall, figural recall, working memory or depression scores (P > 0.05). There were significantly (P = 0.04) more side effects 3 h after losartan ingestion as compared to placebo. These side effects were mild and consisted of dizziness, headache and feeling cold.

8. Conclusion

In conclusion, the results from this clinical trial with losartan provide further evidence for the use of human genetic data to discover memory-modulating drugs. Losartan reduces long-term (90 min) memory recall of emotional pictorial information (positive information). Because compounds interfering with the AGTR1 pathway might also affect memory recall of emotional pictorial information, including negative information, compounds interfering with the AGTR1 pathway might be suited for the treatment of anxiety disorders. III. Cinacalcet

To investigate if the calcimimetic agent cinacalcet can modulate cognitive processes (in particular attention, working memory and episodic memory functions) in healthy humans, a phase II clinical study (see table 8 for study specification summary) is conducted. Table 8: Study specification summary

Healthy, normotensive male or female, aged between 18 and 40, native or fluent German-speaking, Caucasian (European ancestry), BMI between 19 and 27 kg/m2, able

Inclusion Criteria and willing to give written informed consent and comply with the requirements of the study protocol, willing to donate saliva sample for DNA-analysis and blood sample for calcium analysis. Females: willing to perform a pregnancy test at the beginning of the two test visits.

Acute or chronic psychiatric or somatic disorder, pathological ECG, low serum calcium, known hypersensitivity to the IMP under investigation (cinacalcet), pregnancy, breast-feeding, long-term medication within last 3 months (oral contraceptives are

Exclusion Criteria

disregarded), smoking (>3 cigarettes per day), seizures in participants and first degree relatives, concurrent participation in another study, participation in one of our previous studies using the same memory tests, inability to read and understand the participant's information.

Investigational

The calcimimetic agent cinacalcet (Mimpara®) 90mg Product

Attention, working memory and episodic memory

Primary Endpoints

functions

Secondary Mood, depression, anxiety, attention, fluid intelligence, Endpoints motivation, nausea.

Statistical

Paired t-tests

Methodology Study medication

Active study medication consists of

• Cinacalcet Mimpara®, film coated tablet 90mg (manufactured by Amgen Switzerland AG, 6301 Zug) formulated for oral administration. Cinacalcet is a calcimimetic agent that in-creases the sensitivity of the calcium- sensing receptor (CaSR) to activation by extracellular calcium.

• Placebo lactose 90mg formulated for oral administration.

Subjects receive a single oral administration of cinacalcet and placebo in a cross-over design. The study includes a total of 40 healthy subjects. After the administration, there is a 3.5h period allowing the medications to reach maximal plasma concentrations. After this time period, the cognitive test battery (see section I. 4 and II. 4 above) is administered. 2. Saliva sampling for DNA analysis

A saliva sample (2ml) is obtained from each person using oragene DNA Self- Collection Kit OG-500, manufactured by DNA Genotek Inc, Canada. DNA is extracted from saliva samples by using standard procedures. 3. Statistics

Paired t-tests are used to analyze treatment effects (i.e. cinacalcet vs. placebo) on emotional memory outcome measures. Sex, age, sleepiness, weight and treatment order are considered as covariates. A significance level of p < 0.05 (two-sided) is considered as significant.

The effect of Cinacalcet on performance in attention, working memory and episodic memory in healthy humans is determined.

IV Ezogabine

The gene pathway analysis for aversive memory performed independently in the two sub-samples comprising the entire sample identified the anticonvulsant Ezogabine (syn. Retigabine) as a high-priority drug, because it was significant in both independent pathway analyses. Ezogabine is used as a treatment for partial epilepsies, can be administered orally and is also registered in Switzerland. Ezogabine reaches maximum plasma concentrations 1-2 hours after oral administration and readily passes the blood-brain barrier. It acts primarily by opening specific voltage-gated potassium channels in the CNS. Thus ezogabine is a high-priority drug candidate for suppressing memory, especially aversive (negative) memory.

To investigate if ezogabine can modulate cognitive and emotional processes (in particular aversive memory and working memory) in healthy humans, a phase II clinical study (see table 9 for study specification summary) is conducted. Table 9: Study specification summary

Study medication

Active study medication consists of ^• Ezogabine Trobalt®, film coated tablet 200mg (manufactured by GlaxoSmithKline AG, 3053 Munchenbuchsee, Switzerland) formulated for oral administration. Ezogabine is an opener of voltage-gated potassium channels.

Placebo consists of lactose 200mg formulated for oral administration.

After the administration of the study medication, there is a 1.5 hour waiting period allowing the medication to reach maximal plasma concentrations. After this time period, the cognitive/emotional test battery (see section I. 4 and II. 4 above) is administered. Saliva sampling for DNA analysis

A saliva sample (2ml) is obtained from each person using oragene DNA Self- Collection Kit OG-500, manufactured by DNA Genotek Inc, Canada. DNA is extracted from saliva samples by using standard procedures. Statistics

Paired t-tests are used to analyze treatment effects (i.e. Ezogabine vs. placebo) on emotional memory outcome measures. Sex, age, sleepiness, weight and treatment order are considered as covariates. A significance level of p < 0.05 (two-sided) is considered as significant. Salbutamol

By applying the gene set enrichment analysis (criterion B) to episodic memory (delayed recall of words or pictures), and by applying criterion C (restriction of the genetic analysis to genes with known and approved (in Switzerland) therapeutic compounds with medical indications other than memory), a gene related to episodic memory was identified: ADRB2R encoding the beta-adrenergic receptor.

For the proof of concept a clinical trial is conducted with salbutamol (targeting the beta-2-adrenergic receptor), which is available in Switzerland.

To investigate if Salbutamol can modulate cognitive processes (in particular episodic memory) in healthy humans, a phase II clinical study (see table 10 for study specification summary) is conducted.

Table 10: Study specification summary

Study medication

Active study medication consists of

•Salbutamol (Ventolin® Sirup) 2-8 mg (manufactured by GlaxoSmithKline AG, 3053 Munchenbuchsee, Switzerland) formulated for oral administration. Salbutamol is beta 2-adrenergic receptor agonist used for the relief of bronchospasm in conditions such as asthma and chronic obstructive pulmonary disease.

Placebo consists of sweetened (with saccharinum) syrup formulated for oral administration. After the administration of the study medication, there is a 2- hour waiting period allowing the medication to reach maximal plasma concentrations. After this time period, the cognitive/emotional test battery is administered. Saliva samplinp for DNA analysis

Paired t-tests are used to analyze treatment effects (i.e. salbutamol vs. placebo) on cognitive and emotional outcome measures. Sex, age, sleepiness, weight and treatment order are considered as covariates. A significance level of p < 0.05 (two-sided) is considered as significant. Tranexamic acid

By applying the gene set enrichment analysis (criterion B) to episodic memory (delayed recall of words or pictures), and by applying criterion C (restriction of the genetic analysis to genes with known and approved (in Switzerland) therapeutic compounds with medical indications other than memory), a second gene related to episodic memory was identified: PLG, encoding plasminogen.

For the proof of concept a clinical trial is conducted with tranexamic acid (targeting plasminogen), which is available in Switzerland.

To investigate if Tranexamic acid can modulate cognitive processes (in particular episodic memory) in healthy humans, a phase II clinical study (see table 11 for study specification summary) is conducted.

Table 11 : Study specification summary

Statistical

Paired t-tests

Methodology

Study medication

Active study medication consists of

Tranexamic acid (Cyklokapron®) 500-2000 mg (tablets a 500 mg) (manufactured by MEDA Pharma GmbH, 8602 Wangen-Briittisellen, Switzerland) formulated for oral administration. Tranexamic acid is a synthetic derivative of the amino acid lysine. It is used to treat or prevent excessive blood loss during surgery and in various other medical conditions. It is an antifibrinolytic that competitively inhibits the activation of plasminogen to plasmin, by binding to specific sites of both plasminogen and plasmin.

Placebo consists of lactose 1000 mg (2 tablets a 500 mg) formulated for oral administration. After the administration of the study medication, there is a 2- hour waiting period allowing the medication to reach maximal plasma concentrations. After this time period, the cognitive/emotional test battery is administered. Saliva sampling for DNA analysis

Paired t-tests are used to analyze treatment effects (i.e. salbutamol vs. placebo) on cognitive and emotional outcome measures. Sex, age, sleepiness, weight and treatment order are considered as covariates. A significance level of p < 0.05 (two-sided) is considered as significant. Additional potential drug target candidates identified by GWAS drug discovery approach

In addition to the above shown detailed results, the high-resolution genome- wide association studies (GWAS) based drug discovery approach as described herein has unveiled further drug target candidates with effects on aversive memory-related gene pathways. In order to facilitate the subsequent clinical trials the following priority criteria were applied to these compounds with respect to their suitability in the context of a clinical trial: 1. Existing registration as therapeutic compound by the Swiss Agency for Therapeutic Products (Swissmedic) and availability on the Swiss market, 2. Acceptable side-effects profile (i.e. no major side effects such as nephrotoxicity, neurotoxicity or myelotoxicity (e.g. anti-cancer drugs were not considered)), and 3. Favorable application mode (e.g. oral administration was prioritized over intravenous medication). Application of these priority criteria to the list of existing compounds (Table 13) resulted in 19 compounds/compound groups (corresponding to 36 genes) with potentially aversive memory-modulating properties (Table 12).

Table 12: Compounds/compound groups with potentially aversive memory- modulating properties identified by GWAS-based drug discovery approach.

Calcitonin CALCR

Cyclopyrrolones (e.g. eszopiclone) GABRA1, GABRA5, GABRB1,

GABRB2, GABRB3, GABRG2, GABRG3, GABRP

Ergot alkaloids (e.g. ergotamine) DRD3, HTR1E, HTR2A

Ezogabine KCNQ2, KCNQ3, KCNQ4, KCNQ5,

GABRA1 , GABRA5, GABRB1 , GABRB2, GABRB3, GABRG2, GABRG3

Fasoracetam GRM1, GRM3, GRM4, GRM5,

GRM7, GRM8

Isoniazid GABRA1, GABRA5, GABRB1,

GABRB2, GABRB3, GABRG2, GABRG3, GABRP

Memantine GRIN2A, GRIN2B, GRIN3A

Neuroleptics CHRM2, CHRM3, CHRM5, DRD3,

GABRA1, GABRA5, GABRB1, GABRB2, GABRB3, GABRG2, GABRG3, GRIN2A, HRH1, HTR2A

Nitrates (e.g. nitrogylcerin) GUCY1A2, GUCY1A3, GUCY1B3

Opioids (e.g. morphine) GRIN2A, GRIN2B, GRIN3A

Thyroid hormones THRB

Triptans HTR1E

Xanthine derivatives (e.g. ADORA3, DRD3, HTR1E, HTR2A theophylline)

13: Further therapeutic compounds/compound groups with potential aversive memory-modulating properties identified by GWAS-based drug discovery approach.

Clofarabine ADORA3

Xanthine derivatives (e.g. ADORA3 theophylline)

AVP and AVP analogs AVPR1A

Conivaptan AVPR1A

Anatibant BDKRB2 lcatibant BDKRB2

Vemurafenib BRAF

Sorafenib BRAF

Clevidipine CACNA1A

Calcitonin CALCR

Antimuscarinics (e.g. atropine, CHRM2 fesoterodine, cyclopentolate,

trihexyphenidyl)

ABT-089 CHRM2

Diphenhydramine CHRM2

Quinidine CHRM2

Olanzapine CHRM2

Antimuscarinics (e.g. atropine, CHRM3 fesoterodine, cyclopentolate,

trihexyphenidyl)

ABT-089 CHRM3

Diphenhydramine CHRM3

Quinidine CHRM3

Olanzapine CHRM3

Antimuscarinics (e.g. atropine, CHRM5 fesoterodine, cyclopentolate,

trihexyphenidyl)

ABT-089 CHRM5 Diphenhydramine CHRM5

Quinidine CHRM5

Olanzapine CHRM5

Dopamine DRD3

Neuroleptics DRD3

Pergolide DRD3

Dihydroergotamine DRD3

Apomorphine DRD3

FLT1 inhibitors (e.g. sunitinib) FLT1

CEP 7055 FLT1

Menotropins FSHR

Barbiturates (e.g. methohexital) GABRA1

Cyclopyrrolones (e.g. pagoclone) GABRA1

SEP 174559 GABRA1

Anesthetics (e.g. isoflurane) GABRA1

Gaboxadol GABRA1

Isoniazid GABRA 1

Felbamate GABRA1

Muscimol GABRA1

Neuroleptics GABRA1

Benzodiazepines GABRA1

Ezogabine GABRA 1

Pregnenolone GABRA1

Barbiturates (e.g. methohexital) GABRA5

Cyclopyrrolones (e.g. pagoclone) GABRA5

SEP 174559 GABRA5 Anesthetics (e.g. isoflurane) GABRA5

Gaboxadol GABRA5

Isoniazid GABRA5

Felbamate GABRA5

Muscimol GABRA5

Neuroleptics GABRA5

Benzodiazepines GABRA5

Ezogabine GABRA5

Pregnenolone GABRA5

Barbiturates (e.g. methohexital) GABRB1

Cyclopyrrolones (e.g. pagoclone) GABRB1

Alphadolone GABRB1

Anesthetics (e.g. isoflurane) GABRB1

Gaboxadol GABRB1

Isoniazid GABRB1

Felbamate GABRB1

Muscimol GABRB1

Neuroleptics GABRB1

Benzodiazepines GABRB1

Ezogabine GABRB1

Pregnenolone GABRB1

Barbiturates (e.g. methohexital) GABRB2

Cyclopyrrolones (e.g. pagoclone) GABRB2

Alphadolone GABRB2

Anesthetics (e.g. isoflurane) GABRB2

Gaboxadol GABRB2 Isoniazid GABRB2

Felbamate GABRB2

Muscimol GABRB2

Neuroleptics GABRB2

Benzodiazepines GABRB2

Ezogabine GABRB2

Pregnenolone GABRB2

Barbiturates (e.g. methohexital) GABRB3

Cyclopyrrolones (e.g. pagoclone) GABRB3

Alphadolone GABRB3

Anesthetics (e.g. isoflurane) GABRB3

Gaboxadol GABRB3

Isoniazid GABRB3

Felbamate GABRB3

Muscimol GABRB3

Neuroleptics GABRB3

Benzodiazepines GABRB3

Ezogabine GABRB3

Pregnenolone GABRB3

Barbiturates (e.g. phenobarbital) GABRG2

Cyclopyrrolones (e.g. pagoclone) GABRG2

Alphadolone GABRG2

SEP 174559 GABRG2

Tracazolate GABRG2

Anesthetics (e.g. isoflurane) GABRG2

Gaboxadol GABRG2 Isoniazid GABRG2

Felbamate GABRG2

Muscimol GABRG2

Neuroleptics GABRG2

Benzodiazepines GABRG2

Ezogabine GABRG2

Pregnenolone GABRG2

Barbiturates (e.g. phenobarbital) GABRG3

Cyclopyrrolones (e.g. pagoclone) GABRG3

Alphadolone GABRG3

SEP 174559 GABRG3

Tracazolate GABRG3

Anesthetics (e.g. isoflurane) GABRG3

Gaboxadol GABRG3

Isoniazid GABRG3

Felbamate GABRG3

Muscimol GABRG3

Neuroleptics GABRG3

Benzodiazepines GABRG3

Ezogabine GABRG3

Pregnenolone GABRG3

Cyclopyrrolones (e.g. eszopiclone) GABRP

Alphadolone GABRP

Anesthetics (e.g. isoflurane) GABRP

Isoniazid GABRP

Felbamate GABRP Neuroleptics GABRP

Benzodiazepines GABRP

Barbiturates (e.g. phenobarbital) GABRP

Pegvisomant GHR

Growth hormone / Somatrem GHR

Tesamorelin GHRH

Sermorelin GHRH

Liraglutide GLP1R

T-0632 GLP1R

GLP-1 (7-36) amide GLP1R

Exenatide GLP1R

Talampanel GRIA1

Org 24448 GRIA1

LY451395 GRIA1

Tezampanel GRIA1

Talampanel GRIA2

Org 24448 GRIA2

LY451395 GRIA2

Tezampanel GRIA2

Talampanel GRIA4

Org 24448 GRIA4

LY451395 GRIA4

Tezampanel GRIA4

Opioids (e.g. morphine) GRIN2A

Neramexane GRIN2A

Bicifadine GRIN2A Delucemine GRIN2A

CR 2249 (Nebostinel) GRIN2A

Besonprodil GRIN2A

UK-240455 GRIN2A

Ketamine GRIN2A

Felbamate GRIN2A

Memantine GRIN2A

Orphenadrine GRIN2A

Cycloserine GRIN2A

N-(2-indanyl)glycinamide GRIN2A

1 -aminocyclopropane-1 -carboxylic GRIN2A acid

Opioids (e.g. morphine) GRIN2B

Neramexane GRIN2B

Bicifadine GRIN2B

Delucemine GRIN2B

CR 2249 (Nebostinel) GRIN2B

Besonprodil GRIN2B

UK-240455 GRIN2B

Ketamine GRIN2B

Felbamate GRIN2B

Memantine GRIN2B

Orphenadrine GRIN2B

Cycloserine GRIN2B

N-(2-indanyl)glycinamide GRIN2B

1 -aminocyclopropane-1 -carboxylic GRIN2B acid Opioids (e.g. morphine) GRIN3A

Neramexane GRIN3A

Bicifadine GRIN3A

Delucemine GRIN3A

CR 2249 (Nebostinel) GRIN3A

Besonprodil GRIN3A

UK-240455 GRIN3A

Ketamine GRIN3A

Felbamate GRIN3A

Memantine GRIN3A

Orphenadrine GRIN3A

Cycloserine GRIN3A

N-(2-indanyl)glycinamide GRIN3A

1 -aminocyclopropane-1 -carboxylic GRIN3A acid

Fasoracetam GRM1

Fasoracetam GRM3

Fasoracetam GRM4

Fasoracetam GRM5

Fasoracetam GRM7

Fasoracetam GRM8

Nitrates (e.g. 68itroglycerin) GUCY1A2

Nitrates (e.g. 68itroglycerin) GUCY1A3

Nitrates (e.g. 68itroglycerin) GUCY1B3

Antihistamines (e.g. HRH1 diphenhydramine)

Neuroleptics HRH1 Tesmilifene HRH4

Antihistamines (e.g. buclizine) HRH4

Ergot alkaloids (e.g. ergotamine) HTR1E

Triptans HTR1E

Fenfluramine HTR1E

Atypical neuroleptics HTR2A

Ergot alkaloids (e.g. ergotamine) HTR2A

Eplivanserin HTR2A

Psilocybine HTR2A

APD125 HTR2A

Antidepressants (e.g. mirtazapine) HTR2A

Cyproheptadine HTR2A

Epinastine HTR2A

Apomorphine HTR2A

Azatadine HTR2A

IGF1 IGF1R

OSI-906 IGF1R

Cixutumumab IGF1R

IL1-trap IL1A

IL1-trap IL1B

Canakinumab IL1B

Anakinra IL1R1

ORG 41841 LHCGR

Menotropins LHCGR

SCIO-469 MAPK14

RO-3201195 MAPK14 Ramelteon MTNR1A

GW 273629 NOS1

Omega-N-methylarginine NOS1

GW 273629 NOS3

Omega-N-methylarginine NOS3

Contulakin-G NTSR1

SF 1126 PIK3CA

PX-866 PIK3CA

NVP-BEZ235 PIK3CA

GDC-0941 PIK3CA

BKM120 PIK3CA

XL 147 PIK3CA

Varespladib PLA2G2A

Varespladib PLA2G5

L-threo-safingol PRKCA

Prostaglandin E1 PTGER3

Tafluprost PTGFR

Travoprost PTGFR

Isopropyl unoprostone PTGFR

Bimatoprost PTGFR

Latanoprost PTGFR

Teriparatide PTH2R

Vemurafenib RAF1

Sorafenib RAF1

Dantrolene RYR1

MK-0517 TACR1 AV608 TACR1

Aprepitant TACR1

3,5-diiodothyropropionic acid THRB

Amiodarone THRB

Levothyroxine THRB

L-triiodothyronine THRB

Capsaicin TRPV1

SB-705498 TRPV1

ORG 41841 TSHR REPLICATION STUDY

In an additional population of 781 Swiss healthy young adults, who underwent the identical phenotyping and genome-wide genotyping procedure as the first sample, we also performed gene set enrichment pathway analysis. Pathway analysis replicated (at the identical genome-wide FDR < 0.05 level) 2 of the 4 pathways of the first sample, namely the neuroactive ligand receptor interaction pathway and the long-term depression pathway (see Materials and Methods). A total of 20 replicated genes (Table 16) of the two pathways were significantly associated with aversive memory in both the hypothesis-testing (Table 14) and the replication (Table 15) samples. These genes served as input for the subsequent selection of suitable drugs. Study design

Behavioral genetic study, replication sample

N=781 healthy, young Swiss university students or age-matched employees/trainees (484 females, 297 males). Age was 22.4 ± 0.1 years (mean ± standard error). After complete description of the study to the subjects, written informed consent was obtained. The local ethics committee approved the study protocol. Participants of this sample performed the identical picture task as those of the hypothesis-testing sample. As in the hypothesis-testing sample, aversive memory performance (i.e. enhanced free recall performance for previously shown pictures with negative emotional valence) was the trait of interest and was analyzed as a continuous variable. Replication procedure

In the replication sample, pathway analysis was also performed on the /^'- GSEA4GWAS web server(24) with the identical settings as those used in the hypothesis-testing sample. In particular, only intragenic SNPs were included and the FDR<0.05 threshold was used to define a pathway as being significant. Gene set size ranged between 20 and 200 genes, and each gene was represented by the maximum SNP P value within ±0kb of the annotated gene. Because for a number of gene sets in the pathway databases (including MSigDB), the proportion of between-set overlapping genes is highly significant, a gene set of the hypothesis-testing sample was considered replicated, if the particular set or at least one highly overlapping set (P₀veria_P <

10^"8, as defined by MSigDB) also surpassed the FDR<0.05 threshold in the replication sample. This was the case for the neuroactive ligand receptor interaction pathway (significant gene sets in the replication sample: cellular cation homeostasis, cation homeostasis, substrate-specific channel activity, ion channel activity, gated channel activity, homeostatic process, hsa04020 calcium signaling pathway) and for the long-term depression pathway (significant gene set in the replication sample: hsa04020 calcium signaling pathway) (see Table 15 for a list of significant, non-significant, and unmapped genes of the replicated pathways in the replication sample). This replication criterion was not fulfilled by either the VEGF or the IL-1 R pathway. At the gene level, replication was defined as occurrence of a gene in the significant gene category in the pathways of the hypothesis-testing sample and the replicated pathways in the replication sample (Table 16). Table 14: Pathway genes (testing sample)

Table 14: Pathway genes (testing sample), cont'd

Table 14: Pathway genes (testing sample), cont'd

Table 14: Pathway genes (testing sample), cont'd

Table 15: Pathway genes (replication sample)

Table 15: Pathway genes (replication sample), cont'd

Table 15: Pathway genes (replication sample), cont'd

Table 15: Pathway genes (replication sample), cont'd

Table 15: Pathwa genes (replication sample), cont'd

Table 15: Pathway genes (replication sample), cont'd

Table 15: Pathway genes (replication sample), cont'd

Table 16: Replicated pathway genes

List of References

Alarcon, M., Plomin, R., Fulker, D.W., Corley, R. & DeFries, J.C.

Multivariate path analysis of specific cognitive abilities data at 12 years of age in the Colorado Adoption Project. Behavior genetics 28, 255-64 (1998).

Bouchard, T.J., Jr., Segal, N.L. & Lykken, D.T. Genetic and environmental influences on special mental abilities in a sample of twins reared apart. Acta geneticae medicae et gemellologiae 39, 193-206 (1990).

Finkel, D., Pedersen, N. & McGue, M. Genetic influences on memory performance in adulthood: comparison of Minnesota and Swedish twin data. Psychology and aging 10, 437-46 (1995).

Johansson, B. ef al. Origins of individual differences in episodic memory in the oldest-old: a population-based study of identical and same-sex fraternal twins aged 80 and older. The journals of gerontology. Series B, Psychological sciences and social sciences 54, P173-9 (1999).

McClearn, G.E. et al. Substantial genetic influence on cognitive abilities in twins 80 or more years old. Science 276, 1560-1563 (1997).

Swan, G.E. et al. Differential genetic influence for components of memory in aging adult twins. Archives of neurology 56, 1127-32 (1999).

Volk, H.E., McDermott, K.B., Roediger, H.L., 3rd & Todd, R.D. Genetic influences on free and cued recall in long-term memory tasks. Twin research and human genetics : the official journal of the International Society for Twin Studies 9, 623-31 (2006).

De Quervain, D.J. et al. A functional genetic variation of the 5-HT2a receptor affects human memory. Nat.Neurosci. (2003).

De Quervain, D.J. et al. A deletion variant of the alpha2b-adrenoceptor is related to emotional memory in Europeans and Africans. Nat.Neurosci. 10, 1 137-1 139 (2007).

Papassotiropoulos, A. & de Quervain, D.J. Genetics of human episodic memory: dealing with complexity. Trends in cognitive sciences 15, 381 -7 (2011 ). 11. Green, A.E. et al. Using genetic data in cognitive neuroscience: from growing pains to genuine insights. Nat Rev Neurosci (2008).

12. Papassotiropoulos, A. et al. A genome-wide survey and functional brain imaging study identify CTNNBL1 as a memory-related gene. Molecular psychiatry (201 1 ).

13. McGaugh, J.L. The Making of Lasting Memory, in Memory and Emotion:

(Weidenfeld and Nicolson, London, 2003).

14. Phelps, E.A. & LeDoux, J.E. Contributions of the amygdala to emotion processing: from animal models to human behavior. Neuron 48, 175-87 (2005).

15. Pitman, R.K. Post-traumatic stress disorder, hormones, and memory.

Biol. Psychiatry 26, 221 -223 (1989).

16. American Psychiatric, A. Diagnostic and statistical manual of mental disorders, fourth edition (DSM-IV), Washington, DC, 1994, (Washington, DC, 1994).

17. Brewin, C.R. & Holmes, E.A. Psychological theories of posttraumatic

stress disorder. Clin Psychol Rev 23, 339-76 (2003).

18. Rasch, B. et al. A genetic variation of the noradrenergic system is related to differential amygdala activation during encoding of emotional memories. Proc Natl Acad Sci U S A 106, 19191-6 (2009).

19. Rey, A. L'examen clinique en psychologie, (Presses universitaires de France, Paris, 1958).

20. Brickenkamp, R. & Zillmer, E. The d2 Test of Attention, (Hogrefe & Huber Publishers, Seattle, Washington, 1998).

21. Steyer, R., Schwenkmezger, P., Notz, P. & Eid, M. Mehrdimensionaler Befindlichkeitsfragebogen (MDBF) [Multidimensional Mood

Questionnaire], (Hogrefe, G"ttingen, 1997).

22. Laux, L., Glanzmann, P., Schaffner, P. & Spielberger, CD. Das State- Trait Angstinventar. Theoretische Grundlagen und Handanweisung, (Beltz, Weinheim, 1981 ).

23. Jaeggi, S.M., Buschkuehl, M., Jonides, J. & Perrig, W.J. Improving fluid intelligence with training on working memory. Proceedings of the National Academy of Sciences of the United States of America 105, 6829-33 (2008).

24. Wechsler, D. Wechsler Memory Scale-Revised Manual, (Psychological Corp, New York, NY, 1987).

25. Cooper, J.A., Sagar, H.J., Jordan, N., Harvey, N.S. & Sullivan, E.V.

Cognitive impairment in early, untreated Parkinson's disease and its relationship to motor disability. Brain 114 ( Pt 5), 2095-122 (1991 ).

26. Benton, A.L. Differential behavioural effects in frontal lobe disease.

Neuropsychologia 6, 53-60 (1968).

27. Lezak, M.D. Neuropsychological assessment, (Oxford University Press, New York, Oxford, 1983).

28. Price, A.L. et al. Principal components analysis corrects for stratification in genome-wide association studies. Nat Genet 38, 904-9 (2006).

29. Devlin, B. & Roeder, K. Genomic control for association studies.

Biometrics 55, 997-1004 (1999).

30. Marchini, J., Howie, B., Myers, S., McVean, G. & Donnelly, P. A new

multipoint method for genome-wide association studies by imputation of genotypes. Nat Genet 39, 906-13 (2007).

Claims

1. A pharmaceutical composition comprising a memory modulator identified in a method comprising

a. carrying out a genome-wide association study (GW AS) filtered for targets of therapeutic compounds with medical indications other than memory in a sample of healthy subjects characterized for memory performance;

b. identifying genes or gene clusters of targets of therapeutic compounds with medical indications other than memory significantly associated with memory performance.

2. The pharmaceutical composition of claim 1 comprising a compound which modulates i) a receptor from the family of histamine and / or angiotensin receptor proteins or a molecule directly or indirectly interacting with said receptor; or ii) plasminogen (PLG), or a molecule directly or indirectly interacting with plasminogen; or iii) the beta-2-adrenergic receptor (ADRB2R) or a molecule directly or indirectly interacting with said receptor; or iv) the neuronal amiloride-sensitive cation channel 1 (ACCN1 ) or a molecule directly or indirectly interacting with ACCN1 ; or. v) a member of the family of specific voltage-gated potassium channels or a molecule directly or indirectly interacting with said member; or vi) a calcium-sensing receptor (CASR) or a molecule directly or indirectly interacting with said receptor.

3. The pharmaceutical composition of claim 2, wherein said modulating compound is an antagonist.

4. The pharmaceutical composition of claim 2, wherein said modulating compound is an agonist.

5. The pharmaceutical composition of claim 2, wherein said receptor is a receptor from the families of histamine and /or angiotensin.

6. The pharmaceutical composition of claims 5, wherein said receptor from the families of histamine and /or angiotensin receptors is histamine H1 receptor or angiotensin II receptor.

7. The pharmaceutical composition of claims 6, wherein said compound is an histamine H1 receptor antagonist, particularly diphenylhydramine.

8. The pharmaceutical composition of claims 6, wherein said compound is an angiotensin II receptor antagonist, particularly losartan.

9. The pharmaceutical composition of claim 2, wherein said compound modulates plasminogen or a molecule directly or indirectly interacting with plasminogen.

10. The pharmaceutical composition of claims 9, wherein said compound is tranexamic acid.

11. The pharmaceutical composition of claim 2, wherein said compound modulates the beta-2-adrenergic receptor (ADRB2R) or a molecule directly or indirectly interacting with the receptor.

12. The pharmaceutical composition of claims 11 , wherein said compound is salbutamol.

13. The pharmaceutical composition of claim 2, wherein said compound modulates a neuronal amiloride-sensitive cation channel 1 (ACCN1 ) or a molecule directly or indirectly interacting with ACCN1.

14. The pharmaceutical composition of claims 13, wherein said compound is amiloride.

15. The pharmaceutical composition of claim 2, wherein said compound modulates the activity of at least one member of the family of specific voltage-gated potassium channels or a protein directly interacting with at least one member of the family of specific voltage-gated potassium channels.

16. The pharmaceutical composition of claims 15, wherein said compound is Ezogabine.

17. The pharmaceutical composition of claim 2, wherein said compound modulates a calcium-sensing receptor (CASR) or a molecule directly or indirectly interacting with said receptor, for use in memory-modulation, particularly for use in working memory modulation.

18. The pharmaceutical composition of claims 17, wherein said compound is Cinacalcet.

19. The pharmaceutical composition of any one of claims 1-18 for use in modulation of emotional memory, particularly negative emotional memory.

20. The pharmaceutical composition of any one of claims 1 - 18 for use in modulation of working memory, particularly for enhancing working memory performance.

21. The pharmaceutical composition of claim 19 for reducing emotional memory, particularly negative emotional memory.

22. The pharmaceutical composition of any of any of claims 1-18 for use in the treatment of psychiatric disorders, particularly anxiety disorders selected from the group consisting of posttraumatic stress disorder (PTSD), or phobias.

23. The pharmaceutical composition of claim 22 for use in the treatment of posttraumatic stress disorder (PTSD) in heavily traumatized subjects.

24. The pharmaceutical composition of any of claims 1-18 for use in the treatment of pathological conditions characterized by working memory deficits.

25. The pharmaceutical composition of claim 24, wherein said pathological conditions are selected from the group consisting of schizophrenia, depression, bipolar disease, neurodegenerative disorders (e.g. Alzheimer's and Parkinson's disease), attention deficit / hyperactivity disorder, autism, multiple sclerosis, epilepsy, anxiety, post-traumatic stress disorder, and other diseases affecting working memory-related brain regions.

26. The pharmaceutical composition of any one of claims 1 - 25, which does not cause side-effects, particularly side effects selected from the group consisting of attenuation of positive recall, verbal recall, figure recall, attention and working memory.

27. The pharmaceutical composition of any one of claims 1 - 26, which comprises a pharmaceutically acceptable carrier or excipient.

28. The pharmaceutical composition of any one of claims 1-27, which comprises the modulating compound in a therapeutically effective amount.

29. A method of identifying targets of therapeutic compounds with medical indications other than memory that can be used in memory-modulation in a subject comprising

a. carrying out a genome-wide association study (GWAS) filtered for targets of therapeutic compounds with medical indications other than memory in a sample of healthy subjects characterized for memory performance;

30. The method of claim 29, wherein the GWAS in step a) is complemented by tagging SNPs equal to or below the significance threshold (P) of 0.001 , and/or a gene set enrichment analysis (GSEA) with genome-wide false discovery rate (FDR) correction, with P set to equal to or below 0.05, particularly with the "mask x MHC" option activated.

31. The method of claims 29-30, wherein the sample mentioned in step a) above is processed on a Genome-Wide Human SNP Array comprising between 500 and 2500 DNA samples.

32. The method of claims 29-31 , wherein genes or gene clusters are identified in step b) which are significantly associated with negative and/or positive emotional memory.

33. The method of claims 29-31 , wherein genes or gene clusters are identified in step b) which are significantly associated with working memory.

34. The method of claims 29-31 , wherein the gene identified in step b) is a gene encoding a receptor from the family of histamine and / or angiotensin receptor proteins or a protein directly interacting with said receptor.

35. The method of claim 34, wherein said receptor protein is a histamine H1 receptor or an angiotensin II receptor.

36. The method of anyone of claims 29-31 , wherein said gene identified in step b) is a gene encoding plasminogen (PLG).

37. The method of anyone of claims 29-31 , wherein said gene identified in step b) is a gene encoding plasminogen (PLG).

38. The method of claims 29-31 , wherein the gene identified in step b) is a gene encoding the calcium-sensing receptor (CASR) or a molecule directly or indirectly interacting with said receptor.

39. The method of claims 29-31 , wherein said gene identified in step b) is a gene encoding the neuronal amiloride-sensitive cation channel 1 (ACCN1 ) or a molecule directly or indirectly interacting with ACCN1 .

40. The method of claims 29-31 , wherein said gene identified in step b) is a gene encoding for a member of the family of specific voltage-gated potassium channels in the CNS or a molecule directly or indirectly interacting with at least one member of the family of specific voltage-gated potassium channels in the CNS.

41 . A method for screening therapeutic candidate compounds with a medical indication other than memory, for their potential of modulating a target gene or gene cluster, or the expression product thereof significantly associated with memory performance, comprising administering to a subject a candidate compound suspected to be able to induce the desired modulation effect in the target gene or gene cluster, or the expression product thereof, and determining the effect of said compound on memory performance.

42. The method of claim 41 , wherein the effect of the candidate compound is determined in a test system selected from the group consisting of picture memory task.

43. The method of claim 41 , wherein the effect of the candidate compound is determined in a test system selected from the group consisting of working memory task.

44. The method of anyone of claims 41 - 43, wherein the genes or gene clusters are genes or gene clusters identified in a method according to any one of claims 29 - 40.

45. The method of anyone of claims 41 - 43, wherein the genes or gene clusters or the expression products thereof are known targets of therapeutic compounds with medical indications other than memory.

46. The method of anyone of claims 41 - 45, wherein said gene or gene cluster encodes a receptor from the families of histamine and /or angiotensin receptors or a protein directly interacting with said receptor.

47. The method of claim 46, wherein the histamine and / or angiotensin receptor is histamine H1 receptor or angiotensin II receptor.

48. The method of anyone of claims 41 - 45, wherein said gene or gene cluster encodes plasminogen (PLG)

49. The method of anyone of claims 41 - 45, wherein said gene or gene cluster encodes the beta-2-adrenergic receptor (ADRB2R).

50. The method of anyone of claims 41 - 45, wherein said gene or gene cluster encodes the calcium-sensing receptor (CASR), or a molecule directly or indirectly interacting with said receptor.

51. The method of anyone of claims 41 - 45, wherein said gene or gene cluster encodes the neuronal amiloride-sensitive cation channel 1 (ACCN1 ) or a molecule directly or indirectly interacting with ACCN1.

52. The method of anyone of claims 41 - 45, wherein said gene or gene cluster encodes for a member of the family of specific voltage-gated potassium channels in the CNS or a molecule directly or indirectly interacting with at least one member of the family of specific voltage-gated potassium channels in the CNS.

53. A method for screening therapeutic candidate compounds with a medical indication other than memory, for their potential of modulating a target gene or gene cluster, or the expression product thereof significantly associated with memory performance comprising

a. contacting a candidate compound with cells in culture; and b. determining the modulation of target gene expression within the cells, and/or

c. determining the modulation of the activity of the expression product within the cells.

54. The method of claim 53, wherein the modulation of target gene expression is determined on transcript level.

55. The method of claim 53, wherein the modulation of target gene expression is determined on protein level.

56. The method of claim 54, wherein the endogenous target gene expression within the cells is quantified, by specific nucleic acid quantification methods, such as quantitative RT-PCR, micro array, or by Northern blot.

57. The method of claim 53, wherein the cultured cells are transfected cells comprising an expression cassette comprising a reporter gene under the control of the target gene promoter.

58. The method of claim 57, wherein the reporter gene is selected from the group consisting of a luciferase, a fluorescent protein, beta-galactosidase, chloramphenicol acetyltransferase, beta-glucuronidase, alkaline phosphatase, a resistance-conferring gene, and a gene for growth selection.

59. The method of claim 53, wherein the cultured cells are transfected cells comprising an expression cassette comprising a target cDNA, particularly a cDNA comprising 3' and/or 5' untranslated regions (UTR), under the control of an exogenous promoter.

60. The method of claim 53, wherein the cultured cells are transfected cells comprising an expression cassette comprising a nucleic acid encoding a fusion protein under control of a target gene promoter or an exogenous promoter.

61. The method of claim 59 or 60, wherein modulation of target gene expression is quantified by determining the expression level of said cDNA or of the fusion protein.

62. The method of claim 55, wherein the modulation of target gene expression is determined by quantifying the endogenous level of target protein in the cultured cell using a protein quantification method selected from the group consisting of ELISA, Western blot, or immunoprecipitation.

63. The method of any of claims 53, wherein the cells are of a cultured cell line, particularly a cell line that naturally expresses the target gene, especially CNS derived cell lines.

64. The method according to any one of claims 53 - 63, wherein said target gene is a gene encoding i) a receptor from the family of histamine and / or angiotensin receptor proteins or a molecule directly or indirectly interacting with said receptor; or ii) plasminogen (PLG), or a molecule directly or indirectly interacting with plasminogen; or iii) the beta-2-adrenergic receptor (ADRB2R) or a molecule directly or indirectly interacting with said receptor; or iv) the neuronal amiloride-sensitive cation channel 1 (ACCN1 ) or a molecule directly or indirectly interacting with ACCN1 ; or. v) a member of the family of specific voltage-gated potassium channels or a molecule directly or indirectly interacting with said member; or vi) a calcium-sensing receptor (CASR) or a molecule directly or indirectly interacting with said receptor.

65. An expression cassette comprising a reporter gene under the control of a promoter of a target gene significantly associated with memory performance.

66. An expression cassette comprising a cDNA of a target gene significantly associated with memory performance, particularly a cDNA comprising 3' and/or 5' untranslated regions (UTR), under the control of an exogenous promoter.

67. An expression cassette comprising a nucleic acid encoding a fusion protein under control of a promoter of a target gene significantly associated with memory performance or of an exogenous promoter.

68. An expression cassette of any one of claims 65 - 67, wherein said target gene is a gene encoding i) a receptor from the family of histamine and / or angiotensin receptor proteins or a molecule directly or indirectly interacting with said receptor; or ii) plasminogen (PLG), or a molecule directly or indirectly interacting with plasminogen; or iii) the beta-2-adrenergic receptor (ADRB2R) or a molecule directly or indirectly interacting with said receptor; or iv) the neuronal amiloride-sensitive cation channel 1 (ACCN1 ) or a molecule directly or indirectly interacting with ACCN1 ; or. v) a member of the family of specific voltage-gated potassium channels or a molecule directly or indirectly interacting with said member; or vi) a calcium-sensing receptor (CASR) or a molecule directly or indirectly interacting with said receptor.

69. A transgenic animal comprising an expression cassette according to any one of claims 65 - 68.

70. A diagnostic method for determining the probability for an individual for responding to a treatment with a compound identified in a method according to any one of claims 41 - 64 comprising a. obtaining a sample of said individual; b. determining presence or absence in said sample of a genetic variant of a gene significantly associated with memory performance, c. calculating the probability of said individual for responding to a treatment with a compound identified in a method according to any one of claims 41 - 64.

71. The method of claim 70, wherein said compound is diphenhydramine and the genetic variant is a variant of the HRH1 gene.

72. The method of claim 71 , wherein said genetic variant is SNP_A-8656444.

73. The method of claim 70, wherein said compound is losartan and the genetic variant is a variant of the gene encoding the angiotensin II receptor.

74. The method of claim 70, wherein said compound is tranexamic acid and the genetic variant is a variant of the gene encoding plasminogen (PLG).

75. The method of claim 70, wherein said compound is salbutamol and the genetic variant is a variant of the gene encoding the beta-2-adrenergic receptor (ADRB2R).

76. The method of claim 70, wherein said compound is Cinacalcet and the genetic variant is a variant of the gene encoding the calcium-sensing receptor (CASR).

77. The method of claim 70, wherein said compound is amiloride and the genetic variant is a variant of the gene encoding the neuronal amiloride- sensitive cation channel 1 (ACCN1 ).

78. The method of any one of claims 73 - 77, wherein said genetic variants are all Affymetrix 6.0 array SNPs within 20 kb 5' and 3" of the respective gene.

79. A pharmaceutical composition comprising a memory modulator identified in a method according to any one of claims 29-64.