WO2009036768A2

WO2009036768A2 - Diagnosing potential weight gain in a subject

Info

Publication number: WO2009036768A2
Application number: PCT/DK2008/050227
Authority: WO
Inventors: Benny Bang-Andersen; John Nicholas Pearson
Original assignee: H. Lundbeck A/S
Priority date: 2007-09-19
Filing date: 2008-09-18
Publication date: 2009-03-26
Also published as: WO2009036768A3

Abstract

The present invention relates to a method for diagnosing or prognosing potential metabolic syndrome in a subject. The present invention further relates to a method for diagnosing or prognosing potential metabolic syndrome in a subject, which can be induced by medication. The present invention further relates to use of an agent in the manufacture of a medicament for treating a disorder or a disease in a subject. The present invention further relates to an agent for use in a method for treating a disorder or a disease in a subject. The present invention further relates to a method for treating a disorder or a disease in a subject comprising administering a therapeutically effective amount of an agent to a subject. The present invention further relates to a method for identifying an anti-psychotic compound.

Description

DIAGNOSING POTENTIAL WEIGHT GAIN IN A SUBJECT

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

Side effects such as weight gain can be a serious impediment to successful pharmacotherapy, and this has especially been true for the antipsychotic drug (APD)- treatment of for example schizophrenic patients.

Schizophrenia is distinguished from other psychotic disorders based on a characteristic cluster of symptoms, namely positive symptoms (i.e. delusion, hallucinations, disorganised thinking, disorganised behaviour and catatonia), negative symptoms (i.e. affective flattening, poverty of speech and an inability to initiate and persist in goal-directed activities), and cognitive symptoms (i.e. impairment of memory, executive function and attention).

The antipsychotic drugs are divided into classical and atypical APDs. The classical APDs were discovered in the 1950s with chlorpromazine as the first prominent example, whereas the atypical APDs were introduced into the treatment of schizophrenia during the 1990s with clozapine as the first example.

The term classical APD is linked to compounds that show effect in the treatment of positive symptoms (psychotic symptoms) at similar doses that induce extrapyramidal symptoms (EPS, i.e. parkinsonian symptoms, dystonia, akathisia and tardive dyskinesia). The classical APDs are also without effect on negative and cognitive symptoms, and it is generally agreed that these drugs may even worsen these symptoms. It has been argued that the worsening of negative and cognitive symptoms by classical APDs may be a consequence of their EPS, and the separation of the dose-response curves for antipsychotic effect and EPS is the foremost important property of the atypical APDs. The term atypical APD is linked to a diverse group of drugs having antipsychotic effect at doses not giving EPS.

All the APDs display some level of dopamine D₂ antagonism (besides from aripiprazole displaying D₂ partial agonism) but also agonism/antagonism/inverse agonism at a number of additional receptors/sites (e.g. 5-HT₂A, 5-HT₂c, H₁, etc) specific for each of the compounds.

For an extensive review of the receptor profile of APDs, see review by Roth et al. (Nature

Review Drug Discov. 2004, 4: 353-359). Consequently, APDs have their own compound specific advantages but also limitations, such as for example anticholinergic and antihistaminergic side effects, a tendency to prolong the QT interval and/or to increase weight. In particular, clozapine and olanzapine have been linked to weight gain and metabolic side effects, such as e.g. insulin resistance.

Olanzapine and clozapine have been associated with substantial weight gain in patients. Indeed, some patients have added over 30 kg after commencement of medication. In a recent extensive summary of the clinical data available for APDs, it was found that on average olanzapine and clozapine induced a 4 - 4.5 kg weight gain after 10 weeks medication. Other compounds such as quetiapine, chlorprothixene and thioridazine induced a weight gain of 2-3 kg over 10 weeks. Risperidone has been shown to induce a minor weight gain of up to 2 kg whilst no weight gain was observed for ziprasidone (Allison et al, 1999, Am J Psychiatry 156: 1686-1696). The weight gain associated with olanzapine has been attributed to increased food intake with an increase in peripheral fat (increased adiposity) (Eder et al., 2001, Am J Psychiatry 158:1719-1722).

Although much focus has centred on determining the mechanism behind APD-induced weight gain, the central mechanism remains elusive. It has been suggested that the extent of weight gain is mediated via receptors, such as for example 5-HT₂c and H₁. In support of the involvement of the Hi receptor, a recent study showed that AMP kinase was induced in the hypothalamus of mice treated with APDs (Kim et al, 2007, PNAS 104: 3456-3459). The authors showed that AMP kinase was upregulated, and it is known that AMP kinase is involved in appetite stimulation. The article also demonstrated that AMP kinase was not upregulated by APDs in mice lacking the Hi receptor. Deletion of the 5-HT₂C receptor in mice gives rise to obesity, which suggests that the 5-HT₂C receptor may be involved.

To date, no genetic markers have been identified that predict a patient's likelihood of increasing weight under drug treatment. Metabolonic (lipid serum) analysis of patients receiving APDs also failed to find a biomarker for drug-induced weight gain (Kaddurah- Daouk et al., 2007, Molecular Psychiatry, 12: 934-945).

Also associated with APDs, although not clealry linked to weight gain is the onset of Type 2 diabetes. Graham et al (Am J Psychiatry 2005,162:118-23) found that patients taking olanzapine had increased serum triglycerides and insulin levels, which is characteristic of newly onset type 2 diabetes. Farewell et al., (2004, J Gen Intern Med. 19: 1200-1205) showed that 8% of patients taking olanzapine developed diabetes but that this bore no relation to the extent of weight gain seen.

Engl et al., (2005, Molecular Psychiatry, 10:1089-1096) investigated the link between insulin resistance and olanzapine in rat muscle cells and found that olanzapine inhibited the insulin signaling cascade which resulted in the inhibition of glycogen synthesis. Olanzapine was shown to inhibit Insulin Receptor Substrate- 1 -associated PI3K activity and altered the extent of phosphorylation of AKT and GSK3 beta whilst increasing that of phosphorylated glycogen synthase (inactive form). The authors postulated that the diminished glycogen synthesis might lead to insulin resistance in patients receiving olanzapine.

Weight gain or diabetes induced by medication are of major concern for obvious health reasons. However, the threat of weight gain (obesity) or diabetes in patients suffering other conditions or disorders, such as hypertension and heart disease are of even greater concern. In these cases, the side effect can be life threatening.

While dietary education and exercise can be used as part of the treatment program, this may not be sufficient intervention to prevent weight gain. In subjects that are resistant to such lifestyle changes, for instance patients suffering a psychotic episode, drug intervention could be a possible solution. But such drugs also come with side effects. In extreme cases the patient is maybe forced to swap and change between various drugs where the antipsychotic therapy comes second to the weight issue.

Thus, there is a need for new measures for diagnosing a potential weight gain associated with medication, especially antipsychotic medication. Additionally, there is a need for the identification of sub-populations of patients that not to any substantial degree are prone to a potential weight gain associated with medication. Focus need to be aimed at understanding the underlying mechanism of APD induced weight gain. There is also a need to develop new methods for designing new drugs, such as new APDs that do not induce weight gain.

It has now been found that PBK delta (PIK3CD, EC 2.7.1.153 located on Chromosome 1 at Ip36.22) is inhibited by APDs, an inhibition that is correlated with the weight gain, which has been observed in patients taking these APDs.

SUMMARY OF THE INVENTION

In one aspect the present invention relates to a method for diagnosing or prognosing potential metabolic syndrome in a subject, comprising the step of analyzing a sample from said subject for PI3K delta activity.

In another aspect the present invention relates to a method for diagnosing or prognosing potential metabolic syndrome in a subject, which can be induced by medication, comprising the steps i) analyzing a sample from said subject for PI3K delta activity before said medication; ii) analyzing a sample from said subject for PI3K delta activity after medication.

In another aspect the present invention relates to use of an agent in the manufacture of a medicament for treating a disorder or a disease in a subject, characterized in that the PI3K delta activity in said subject has been measured to be about normal or higher than normal. In another aspect the present invention relates to an agent for use in a method for treating a disorder or disease in a subject, characterized in that the PBK delta activity in said subject has been measured to be about normal or higher than normal.

In another aspect the present invention relates to a method for treating a disorder or a disease in a subject comprising administering a therapeutically effective amount of an agent to a subject, characterized in that the PBK delta activity in said subject has been measured to be about normal or higher than normal.

In another aspect the present invention relates to a method for identifying an anti-psychotic compound comprising the steps of:

I) testing the compound in an assay predictive for an anti-psychotic potential and/or for interaction with a target relevant for psychosis, and II) testing the compound for inhibition of PBK delta activity.

BRIEF DESCRIPTION OF THE DRAWING

Figure 1. Glycogen depletion by 2-(6-Amino-purin-9-ylmethyl)-5-methyl-3-o-tolyl-3H- quinazolin-4-one. 2-(6-Amino-purin-9-ylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one a highly specific inhibitor of PBK delta, is shown here to inhibit the formation of glycogen in human skeletal muscle cells treated with various amounts of 2-(6-Amino-purin-9-ylmethyl)- 5-methyl-3-o-tolyl-3H-quinazolin-4-one 1 hour prior to 3 hours of insulin stimulation. Glycogen content was visualised by Periodic acid-Shiff staining followed by quantitation using Multi Gauge (FujiFilm). The inhibition of glycogen formation demonstrates that

PBKdelta is responsible for glycogen inhibition in muscle cells and that specific inhibitors of PBK delta will inhibit glycogen content of these cells in a similar manner to 2-(6-Amino- purin-9-ylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one. Y-axis: relative glycogen content. X-axis: concentration (μM). DESCRIPTION OF THE INVENTION

The present inventors have now found that weight gain associated with the use of antipsychotic medication can be ascribed to PBK delta activity. In particular it has been found that olanzapine, clozapine and other APDs selectively inhibit PBK delta to varying degrees but not any other isoform, such as alpha, beta or gamma. The degree of inhibition, together with the bio distribution, such as accumulation of the compound in the muscles, reflects the degree of glycogen synthesis inhibition.

In rat skeletal muscle cells, olanzapine disrupts glycogen synthesis and this was shown to disrupt insulin-signalling cascade that drives glycogen formation (Engl et al., 2005,

Molecular Psychiatry). The present inventors have found that APDs directly and specifically inhibit human delta isoform of phosphoinositide kinase (PBK delta or PIK3CD, EC 2.7.1.153), one of the first kinases in the insulin-signaling phosphorylation cascade but not any other isoform, such as alpha, beta or gamma. Under normal conditions, glycogen is formed from glucose when insulin stimulates the insulin receptor located on the plasma membrane of cells. A complex of proteins associated with the insulin receptor include PB kinases (isoforms alpha, beta, gamma and delta) and they, upon stimulation, phosphorylate the membrane bound phosphatidylinositol 3,4-triphosphate (PIP₂) to form phosphate- idylinositol 3,4,5-triphosphate (PIP₃). Once formed, PIP₃ recruits PDKl and PKB/AKTl to the membrane to form a complex that then phosphorylates GSK3 beta. Phospho-GSK3 beta is inactive and can no longer phosphorylate Glycogen Synthase enzyme (phosphor glycogen synthase is inactive and unable to synthesize glycogen) Thus, with insulin stimulation, glycogen synthesis is activated (see Cross et al., 1995 Nature 378: 785-789).

The APDs selectively inhibit PBK delta and not any of the other isoform, such as alpha, beta or gamma. The degree of inhibition varies from an IC₅₀ of 10-50 μM. PBK delta is heavily expressed in leukocytes (Chantry et al. 1997, J Biol Chem. 31:19236-19241), but also expressed in numerous other tissues including skeletal muscles (Seki et al. 1997, DNA Res. 4:355-358). The alpha isoform of PBK is the main PB kinase regulating glycogen formation in the liver (Carpenter et al., 1990, J. Biol. Chem. 265: 19704-19711 (1990). It has been shown that APDs are involved in glycogen depletion in muscles cells (Engl et al., 2005, Molecular Psychiatry, 10:1089-1096). Excess glucose from the muscles, normally destined for glycogen synthesis, is postulated to re-enter the blood stream where it is directed to the adipocytes and deposited as fat. Patients receiving APDs have been noted for their disregulation of serum glucose (Dwyer et al., 2001, Ann Clin Psych, 13:103-113).

APDs were recently shown to induce the expression of AMP -kinase in the brain and this is a vital enzyme involved in appetite regulation (Kim et al., 2007, PNAS, 104: 3456-3459). AMP-kinase, expressed in the muscles and brain, is usually associated with and recognizes glycogen reserves (Polekhina et al., 2005, Structure, 13:1453-1462). One of the functions of this kinase is to sense energy levels and it is directly involved in appetite regulation.

Therefore weight gain would seem to occur due to glycogen depletion stimulating AMPK activity and appetite.

Weight gain can be predicted by measuring PI3K delta activity (for method to measure PIK3 delta activity, see Gray et al., 2003 Anal. Biochem. 313:234-245) and the formation of glycogen. More specifically, this can be achieved by measuring the activity of the enzyme from tissue samples of medicated patients or the level of the products of PI3K delta. By direct measurement: activity of PI3K delta can be determined in blood samples or muscle biopsies by one of the following methods: the enzymatic formation of phosphatidylinositol 3,4,5-triphosphate (PIP3) or of a analogous product or PI3K delta inhibition can be determined by the cellular levels of protein PIP3 in cells relative to known control samples either via standard western blot analysis with antibodies directed specifically to PIP3 or via a fluorescently labelled marker of PIP3.

PI3K delta inhibition can also be measured indirectly by measuring the cellular levels of phosphorylated AKTl protein relative to known controls; likewise, PI3K delta inhibition can be measured by the cellular levels of phosphorylated GSK3 beta protein relative to known controls; PI3K delta inhibition can also be measured by the cellular levels of phosphorylated glycogen synthase protein relative to known controls.

PI3K delta inhibition can also be measured indirectly by the amounts of glycogen in blood or tissue to known controls. This can be measured either by elimination of free glucose followed by digestion of glycogen and subsequent measure of released glucose. Glycogen can also be quantitated in tissue with Periodic acid- S chiff staining or similar techniques.

PBK delta inhibition can also be predicted by comparing the exposure level in muscles of a specific compound with a known IC50 for PBK delta.

The distribution of compound in the muscles and elsewhere in the body can be determined either directly by taking biopsies and determining compound concentration using normal techniques or indirectly by supplying the patient with radio-labelled compound and following its movement through out the body. Calculation of compound concentration can be used to determine the likely extent of inhibition of PBK delta and probability of weight gain

Definitions

The term "biological sample" is intended to include tissues, cells, biological fluids, such as blood, and isolates thereof, isolated from a subject, as well as tissues, cells and fluids present within a subject.

Phosphoinositide kinase delta isoform refers to a human protein also known as phosphor- inositide-3-kinase, catalytic, delta polypeptide or a number of other synonyms (pi IOdelta, Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit delta isoform, PBK, PB- kinase pi 10 subunit delta, PtdIns-3 -kinase, pi 10). The reference DNA sequence is NM_005026 and the protein NP_005017.3.

The term "that the PBK delta activity in said subject is about normal" refers to the situation wherein the PBK delta activity in said subject is about the same in relation to expression and activity of PBK delta of the general population.

As used herein, the term "subject" refers to any warm-blooded species such as human and animal. The subject, such as a human, to be treated or diagnosed according to the present invention may in fact be any subject of the human population, male or female, which may be divided into children, adults, or elderly. Any one of these patient groups relates to an embodiment of the invention.

As used herein, the term "treating" or "treatment" refers to preventing or delaying the appearance of clinical symptoms of a disease or condition in a subject that may be afflicted with or predisposed to the disease or condition, but does not yet experience or display clinical or subclinical symptoms of the disease or condition. "Treating" or "treatment" also refers to inhibiting the disease or condition, i.e., arresting or reducing its development or at least one clinical or subclinical symptom thereof. "Treating" or "treatment" further refers to relieving the disease or condition, i.e., causing regression of the disease or condition or at least one of its clinical or subclinical symptoms. The benefit to a subject to be treated is either statistically significant or at least perceptible to the subject and/or the physician. Nonetheless, prophylactic (preventive) and therapeutic (curative) treatment are two separate embodiments of the invention.

The term "diagnosing" refers to identifying a disorder or a disease in a subject or the susceptibility of a subject to the disorder of the present invention (e.g., a predisposition to develop a disorder).

The term "metabolic syndrome" refers to a variety of disorders comprising glucose intolerance/insulin resistance, type 2 diabetes, arterial hypertension, dyslipidaemia and obesity.

As used herein, the term "pharmaceutically acceptable" refers to molecular entities and compositions that are "generally regarded as safe" - e.g., that are physiologically tolerable and do not typically produce an allergic or similar untoward reaction, such as gastric upset and the like, when administered to a human. In another embodiment, this term refers to molecular entities and compositions approved by a regulatory agency of the federal or a state government or listed in the U.S. Pharmacopeia or another generally recognized pharmacopeia for use in animals, and more particularly in humans.

The term "CNS-disorder" refers to a disorder of the central nervous system (CNS). The term "anti CNS-disorder agent" refers to a medicament approved for the treatment of CNS-disorder.

The term "effective amount" refers to the amount/dose of a compound or pharmaceutical composition that is sufficient to produce an effective response (i.e., a biological or medical response of a tissue, system, animal or human sought by a researcher, veterinarian, medical doctor or other clinician) upon administration to a subject. The "effective amount" will vary depending on inter alia the disease and its severity, and the age, weight, physical condition and responsiveness of the subject to be treated.

The term "agent" refers to a substance selected from the group that includes, but is not limited to, proteins, peptides or amino acids; antibodies or fragments thereof; nucleic acids such as DNA, such as full-length genes or fragments thereof derived from genomic, cDNA or artificial coding sequences, gene regulatory elements, RNA, including mRNA, tRNA, ribosomal RNA, ribozymes and antisense RNA, oligonucleotides and oligoribonucleotides, deoxyribonucleotides and ribonucleotides; carbohydrates; lipids; proteoglycans; organic molecules such as small molecules; inorganic molecules.

In a first aspect the present invention relates to a method for diagnosing or prognosing potential metabolic syndrome in a subject, comprising the step of analyzing a sample from said subject for PBK delta activity. In one embodiment of the first aspect of the invention the potential metabolic syndrome is potential weight gain. In another embodiment of the first aspect of the invention, said sample is a biological sample. In another embodiment of the first aspect of the invention the PBK delta activity is measured directly. In another em- bodiment of the first aspect of the invention the PBK delta activity is measured by enzymatic formation of PIP3 or of an analogous product, or by western blot analysis with antibodies directed specifically to PIP3, or with a fluorescently labelled marker of PIP3. In another embodiment of the first aspect of the invention PBK delta activity is measured indirectly. In another embodiment of the first aspect of the invention the PBK delta activity is measured by measuring cellular levels of phosphorylated AKTl or GSK3 beta, or by measuring cellular levels of phosphorylated glycogen synthase protein, or by measuring cellular levels of PBK delta mRNA or PBK delta protein, or by measuring the amounts of glycogen in blood or tissue. In another embodiment of the first aspect of the invention the subject is a human.

In a second aspect the present invention relates to a method for diagnosing or prognosing potential metabolic syndrome in a subject, which can be induced by medication, comprising the steps i) analyzing a sample from said subject for PBK delta activity before said medication; ii) analyzing a sample from said subject for PBK delta activity after medication. In one embodiment of the second aspect of the invention the potential metabolic syndrome is potential weight gain. In another embodiment of the second aspect of the invention step ii) is performed when medication has reached steady-state. In another embodiment of the second aspect of the invention step ii) is performed after a period of time equivalent to about 5-9 half lives of said medication, such as about 5, 6, 7, 8 or 9 half lives of said medication. In another embodiment of the second aspect of the invention said sample is a biological sample. In another embodiment of the second aspect of the invention the PBK delta activity is measured directly. In another embodiment of the second aspect of the invention the PBK delta activity is measured by enzymatic formation of PIP3 or of an analogous product, or by western blot analysis with antibodies directed specifically to PIP3, or with a fluorescently labelled marker of PIP3. In another embodiment of the second aspect of the invention PBK delta activity is measured indirectly. In another embodiment of the second aspect of the invention the PBK delta activity is measured by measuring cellular levels of phosphorylated AKTl or GSK3 beta, or by measuring cellular levels of phosphorylated glycogen synthase protein, or by measuring cellular levels of PBK delta mRNA or PBK delta protein, or by measuring the amounts of glycogen in blood or tissue. In another embodiment of the second aspect of the invention said medication is anti-psychotic medication. In another embodiment of the second aspect of the invention the anti-psychotic medication is selected from olanzapine, clozapine, quetiapine, ziprasidone or a pharmaceutically acceptable salt thereof. In another embodiment of the second aspect of the invention said medication is anti-depressant medication. In another embodiment of the second aspect of the invention the antidepressant medication is selected from amitriptyline or a pharmaceutically acceptable salt thereof. In another embodiment of the second aspect of the invention the subject is a human.

In a third aspect the present invention relates to use of an agent in the manufacture of a medicament for treating a disorder or a disease in a subject, characterized in that the PBK delta activity in said subject has been measured to be about normal or higher than normal. In one embodiment of the third aspect of the invention said agent is an anti CNS-disorder agent. In another embodiment of the third aspect of the invention said disorder is a CNS- disorder. In another embodiment of the invention the CNS-disorder is psychosis or schizo- phrenia and the anti CNS-disorder agent is an antipsychotic agent. In another embodiment of the third aspect of the invention the CNS-disorder is depression and the anti CNS- disorder agent is an anti-depressant agent. In another embodiment of the third aspect of the invention the anti-psychotic agent is selected from olanzapine, clozapine, quetiapine, ziprasidone or a pharmaceutically acceptable salt thereof. In another embodiment of the third aspect of the invention the anti-depressant agent is selected from amitriptyline or a pharmaceutically acceptable salt thereof. In another embodiment of the third aspect of the invention the PBK delta activity is determined by analyzing a biological sample from a subject. In another embodiment of the third aspect of the invention the PBK delta activity is measured directly. In another embodiment of the third aspect of the invention the PBK delta activity is measured by enzymatic formation of PIP3 or of an analogous product, or by western blot analysis with antibodies directed specifically to PIP3, or with a fluorescently labelled marker of PIP3. In another embodiment of the third aspect of the invention PBK delta activity is measured indirectly. In another embodiment of the third aspect of the invention the PBK delta activity is measured by measuring cellular levels of phosphorylated AKTl or GSK3 beta, or by measuring cellular levels of phosphorylated glycogen synthase protein, or by measuring cellular levels of PBK delta mRNA or PBK delta protein, or by measuring the amounts of glycogen in blood or tissue. In another embodiment of the third aspect of the invention the subject is a human.

In a fourth aspect the present invention relates to an agent for use in a method for treating a disorder or disease in a subject, characterized in that the PBK delta activity in said subject has been measured to be about normal or higher than normal. In one embodiment of the fourth aspect of the invention said agent is an anti CNS-disorder agent. In another embodiment of the fourth aspect of the invention said disorder is a CNS-disorder. In another em- bodiment of the fourth aspect of the invention the CNS-disorder is psychosis or schizophrenia and the anti CNS-disorder agent is an antipsychotic agent. In another embodiment of the fourth aspect of the invention the CNS-disorder is depression and the anti CNS- disorder agent is an anti-depressant agent. In another embodiment of the fourth aspect of the invention the anti-psychotic agent is selected from olanzapine, clozapine, quetiapine, ziprasidone or a pharmaceutically acceptable salt thereof. In another embodiment of the fourth aspect of the invention the anti-depressant agent is selected from amitriptyline or a pharmaceutically acceptable salt thereof. In another embodiment of the fourth aspect of the invention the PBK delta activity is determined by analyzing a biological sample from a subject. In another embodiment of the fourth aspect of the invention the PBK delta activity is measured directly. In another embodiment of the fourth aspect of the invention the PBK delta activity is measured by enzymatic formation of PIP3 or of an analogous product, or by western blot analysis with antibodies directed specifically to PIP3, or with a fluorescently labelled marker of PIP3. In another embodiment of the fourth aspect of the invention PBK delta activity is measured indirectly. In another embodiment of the fourth aspect of the invention the PBK delta activity is measured by measuring cellular levels of phosphorylated AKTl or GSK3 beta, or by measuring cellular levels of phosphorylated glycogen synthase protein, or by measuring cellular levels of PBK delta mRNA or PBK delta protein, or by measuring the amounts of glycogen in blood or tissue. In another embodiment of the fourth aspect of the invention the subject is a human.

In a fifth aspect the present invention relates to a method for treating a disorder or a disease in a subject comprising administering a therapeutically effective amount of an agent to a subject, characterized in that the PBK delta activity in said subject has been measured to be about normal or higher than normal. In one embodiment of the fifth aspect of the invention said agent is an anti CNS-disorder agent. In another embodiment of the fifth aspect of the invention said disorder is a CNS-disorder. In another embodiment of the fifth aspect of the invention the CNS-disorder is psychosis or schizophrenia and the anti CNS-disorder agent is an antipsychotic agent. In another embodiment of the fifth aspect of the invention the CNS- disorder is depression and the anti CNS-disorder agent is an anti-depressant agent. In another embodiment of the fifth aspect of the invention the anti-psychotic agent is selected from olanzapine, clozapine, quetiapine, ziprasidone or a pharmaceutically acceptable salt thereof. In another embodiment of the fifth aspect of the invention the anti-depressant agent is selected from amitriptyline or a pharmaceutically acceptable salt thereof. In another embodiment of the fifth aspect of the invention the PBK delta activity is determined by analyzing a biological sample from a subject. In another embodiment of the fifth aspect of the invention the PBK delta activity is measured directly. In another embodiment of the fifth aspect of the invention the PBK delta activity is measured by enzymatic formation of PIP3 or of an analogous product, or by western blot analysis with antibodies directed specifically to PIP3, or with a fluorescently labelled marker of PIP3. In another embodiment of the fifth aspect of the invention PI3K delta activity is measured indirectly. In another embodiment of the fifth aspect of the invention the PI3K delta activity is measured by measuring cellular levels of phosphorylated AKTl or GSK3 beta, or by measuring cellular levels of phosphorylated glycogen synthase protein, or by measuring cellular levels of PI3K delta mRNA or PI3K delta protein, or by measuring the amounts of glycogen in blood or tissue. In another embodiment of the fifth aspect of the invention the subject is a human.

In a sixth aspect the present invention relates to a method for identifying an anti-psychotic compound comprising the steps of:

I) testing the compound in an assay predictive for an anti-psychotic potential and/or for interaction with a target relevant for psychosis, and II) testing the compound for inhibition of PI3K delta activity.

In one embodiment of the sixth aspect of the invention said target by interaction with said compound will mediate an effect that can treat and/or prevent psychosis, or ameliorate the symptoms of psychosis. In another embodiment of the sixth aspect of the invention said compound when tested for inhibition of PI3K delta activity does not inhibit said PI3K delta activity. In another embodiment of the sixth aspect of the invention said method further comprises the step of measuring the bio-distribution of said compound.

In still another aspect the present invention relates to the marketing of a medicinal product comprising a compound for treating a disease or a disorder in a subject, said marketing comprising the public spreading of the information that the PI3K delta activity has an impact on potential metabolic syndrome of said subject. In one embodiment of this aspect of the invention said compound is an anti-psychotic compound for treating psychosis in a subject. In another embodiment of this aspect of the invention, said potential metabolic syndrome is a potential weight gain of said subject.

The disorders that can be treated and diagnosed according to the present invention are known according to established and accepted classifications, which can be found in various sources. For example, at present, the fourth edition of the Diagnostic and Statistical Manual of Mental Disorders (DSM-IV™) (2000, American Psychiatric Association, Washington, D. C), provides a diagnostic tool for identifying many of the disorders described herein. Also, the International Classification of Diseases, Tenth Revision, (ICD-IO) provides classifications for many of the disorders described herein. The skilled person in the art will recognize that there are alternative nomenclatures, nosologies, and classification systems for disorders described herein, including those as described in the DMS-IV and ICD-10, and that terminology and classification systems evolve with medical scientific progress. Moreover the scientific literature also gives definition on disorders.

The invention also provides the use as above wherein the medicament is for administration as a unit dose. In another embodiment of the invention, the unit dose is containing the active ingredient in an amount from about 10 μg/kg to 10mg/kg body weight, in another embodiment from about 25 μg/day/kg to 1.0 mg/day/kg, in yet another embodiment from about 0.1 mg/day/kg to 1.0 mg/day/kg body weight. In another embodiment, the unit dose is containing the active ingredient in an amount from 0.1 mg/day/kg to 1.0 mg/day/kg body weight.

According to the invention, the compounds mentioned above may be used as the base of the compound or as an acceptable acid addition salt thereof, such as a pharmaceutically acceptable acid addition salt thereof or as an anhydrate or hydrate of such salt. According to the invention, the compounds mentioned above or a pharmaceutically acceptable salt thereof may be administered in any suitable way e.g. orally or parenterally, and it may be presented in any suitable form for such administration, e.g. in the form of tablets, capsules, powders, syrups or solutions or dispersions for injection. In another embodiment, and in accordance with the purpose of the present invention, the compound of the invention is administered in the form of a solid pharmaceutical entity, suitably as a tablet or a capsule or in the form of a suspension, solution or dispersion for injection. The compound of the invention is most conveniently administered orally in unit dosage forms such as tablets or capsules, containing the active ingredient in an amount from about 10 μg/kg to 10mg/kg body weight, for example 25 μg/day/kg to 1.0 mg/day/kg. Methods for the preparation of solid or liquid pharmaceutical preparations are well known in the art. See, e.g., Remington: The Science and Practice of Pharmacy, 21st ed., Lippincott Williams & Wilkins (2005). Tablets may thus be prepared by mixing the active ingredients with an ordinary carrier, such as an adjuvant and/or diluent, and subsequently compressing the mixture in a tabletting machine. Non-limiting examples of adjuvants and/or diluents include: corn starch, lactose, talcum, magnesium stearate, gelatine, lactose, gums, and the like. Any other adjuvant or additive such as colourings, aroma, and preservatives may also be used provided that they are compatible with the active ingredients. The pharmaceutical compositions of the invention thus typically comprise an effective amount of a compound of the invention and a pharmaceutically acceptable carrier.

All non-patent references, patents, and patent applications cited and discussed in this specification are incorporated herein by reference in their entirety and to the same extent as if each was individually incorporated by reference.

EXAMPLES

The influence of APDs on the insulin-signaling cascade was investigated by kinase activity profiling. For PBK screening (beta, gamma and delta), the PIProfiler™ assay (see Gray et al., 2003 Ann. Biochem 313: 234-245) makes use of the specific, high affinity binding of the GRPl pleckstrin homology (PH) domain to PIP3, the product of the type I PI 3 -kinase acting on its physiological substrate PIP2. A sensor complex is formed involving a europium labeled anti-GST mAb, a GST tagged GRPl PH domain, biotinylated short chain PIP3 and Streptavidin-APC (allophycocyanin). This generates a stable time-resolved fluorescence resonance energy transfer (FRET) signal that can be displaced by competing PIP3 formed in the PI 3-kinase assay. For each compound, 0.5 μL of a 100% 50 x final concentration solution of compound was added to 14.5 μL reaction mix (5OmM Tris/HCl pH7.5, 30OmM NaCl, O.lmM EGTA, 0.03% Brij 35, 27OmM sucrose, 0.2mM PMSF, ImM benzamidine, 0.1% 2-mercaptoethanol plus 2 ng PI3 kinase). The reaction was started with 5 μL of 40 μM ATP and the reaction was incubated at room temperature for 30 min. The reaction was stopped with the addition of 5 μL stop solution (containing biotinylated-PIP3 and EDTA). 5μl of Detection Buffer containing Europium labelled anti-GST monoclonal antibody, GST tagged GRPl PH domain, and Streptavidin Allophycocyanin was then added and time -resolved fluorescence (Ex 380, Em 660) determined.

For each key compound an IC50 value was obtained from a concentration curve of inhibition over 10 concentrations.

For non-lipid kinases involved in the synthesis of glycogen, a radiometric assay is used (ref). The example shown here relates to GSK3 beta. In a final reaction volume of 25 μL, GSK3 beta (5-10 mU) is incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 20 μM YRRAAVPPSPSLSRHSSPHQS(p)EDEEE (phospho GS2 peptide), 10 mM MgAcetate and [γ-³³P-ATP] (specific activity approx. 500 cpm/pmol, concentration as required). The reaction is initiated by the addition of the MgATP mix. After incubation for 40 minutes at room temperature, the reaction is stopped by the addition of 5 μL of a 3% phosphoric acid solution. 10 μL of the reaction is then spotted onto a P30 filter mat and washed three times for 5 minutes in 50 mM phosphoric acid and once in methanol prior to drying and scintillation counting. Compound was added to the reaction at 10 and 100 μM final concentration and inhibition determined by reduced scintillation counts.

The results for a number of compounds are indicated below. Table 1 shows that none of the selected atypical APDs inhibit the selected kinases of the insulin-signaling cascade. However, Table 2 and 3 show that atypical APDs selectively inhibit the delta isoform of PI3K, with an IC50 down to 11 μM.

To demonstrate that PI3K delta is the enzyme responsible for controlling glycogen formation in muscles we obtained a highly specific PI3K delta inhibitor, 2-(6-Amino-purin- 9-ylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one. 2-(6-Amino-purin-9-ylmethyl)-5- methyl-3-o-tolyl-3H-quinazolin-4-one was prepared as described in US6667300 example D- 030. This compound was administered to human skeletal muscles cells (Lonza) cultured in 6-well plates and allowed to differentiate into myoblasts over an 5 day period once con- fluent. The myoblasts were then dosed with varying concentrations of reference compound for one hour before the cells were given insulin (100 ng/ml) to stimulate glycogen formation (as described for rat mucle cells by Engl et al., 2005, Molecular Psychiatry, 10:1089-1096). After 3 hours, the cells were washed with phosphate-buffered saline solution then fixed with an acid paraformaldehyde solution. The cells were then subjected to Periodic acid-Schiff staining (strictly according to manufacture's protocol - Sigma cat no. 395B-1KT). Upon complete development of colouring, the cells were photographed in black & white and the images digitized before quantification with Multi Gauge software (FujiFilm). The effect of 2-(6-Amino-purin-9-ylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one is shown in figure 1, where glycogen synthesis is almost completely inhibited by 10 nM of 2-(6-Amino-purin- 9-ylmethyl)-5-methyl-3-o-tolyl-3H-quinazolin-4-one.

Table 1 - Atypical APDs and non-lipid kinases involved in glycogen synthesis

Values are percentage and represent activity of enzyme in presence of compound. Values represent duplicate experiments carried out on separate days.

DYRK2 - Dual-specificity tyrosine phosphorylated and regulated protein kinase.

EEF-2K - Eukaryotic elongation factor-2 kinase.

GSK - Glycogen synthase kinase (alpha and beta isoforms). IR - Insulin receptor.

IRR - Insulin receptor-related receptor.

PDKl - Phosphoinositol (3,4,5) P3-dependent kinase.

PhKγ2 - Phosphorylating kinase gamma 2.

PKB/PKC - Phosphoralting kinase B/C. SGK - Serum- and glucocorticoid- induced kinase. Table 2 - Atypical APDs and PI3 kinases.

Values (%) represent activity of enzyme in presence of compound relative to controls. Values represent duplicate experiments carried out on separate days.

Table 3. IQsn profiles of key atypical APDs.

Values (%) represent activity of enzyme in presence of compound relative to controls.

Values represent duplicate experiments

^Clozapine quenched the fluorescent reaction and the values are considered inaccurate (and lower than recorded).

Claims

I . A method for diagnosing or prognosing potential metabolic syndrome in a subject, comprising the step of analyzing a sample from said subject for PBK delta activity. 2. The method of claim 1, wherein the potential metabolic syndrome is potential weight gain.

3. The method of any of the claims 1-2, wherein said sample is a biological sample.

4. The method of any of the claims 1-3 wherein the PI3K delta activity is measured directly. 5. The method of claim 4 wherein the PI3K delta activity is measured by enzymatic formation of PIP3 or of an analogous product, or by western blot analysis with antibodies directed specifically to PIP3, or with a fluorescently labelled marker of PIP3.

6. The method of any of the claims 1-3 wherein PI3K delta activity is measured indirectly.

7. The method of claim 6 wherein the PI3K delta activity is measured by measuring cellular levels of phosphorylated AKTl or GSK3 beta, or by measuring cellular levels of phosphorylated glycogen synthase protein, or by measuring cellular levels of PI3K delta mRNA or PI3K delta protein, or by measuring the amounts of glycogen in blood or tissue.

8. The method of any of the claims 1-7, wherein the subject is a human. 9. A method for diagnosing or prognosing potential metabolic syndrome in a subject, which can be induced by medication, comprising the steps i) analyzing a sample from said subject for PI3K delta activity before said medication; ii) analyzing a sample from said subject for PI3K delta activity after medication. 10. The method of claim 9, wherein the potential metabolic syndrome is potential weight gain.

I I. The method of any of the claims 9-10 wherein step ii) is performed when medication has reached steady-state.

12. The method of any of the claims 9-11 wherein step ii) is performed after a period of time equivalent to about 5-9 half lives of said medication, such as about 5, 6, 7, 8 or 9 half lives of said medication.

13. The method of any of the claims 9-12, wherein said sample is a biological sample.

14. The method of any of the claims 9-13 wherein the PI3K delta activity is measured directly.

15. The method of claim 14 wherein the PI3K delta activity is measured by enzymatic formation of PIP3 or of an analogous product, or by western blot analysis with antibodies directed specifically to PIP3, or with a fluorescently labelled marker of PIP3.

16. The method of any of the claims 9-13 wherein PI3K delta activity is measured indirectly.

17. The method of claim 16 wherein the PI3K delta activity is measured by measuring cellular levels of phosphorylated AKTl or GSK3 beta, or by measuring cellular levels of phosphorylated glycogen synthase protein, or by measuring cellular levels of PI3K delta mRNA or PI3K delta protein, or by measuring the amounts of glycogen in blood or tissue.

18. The method of any of the claims 9-17 wherein said medication is anti-psychotic medication. 19. The method of claim 18 wherein the anti-psychotic medication is selected from olanzapine, clozapine, quetiapine, ziprasidone or a pharmaceutically acceptable salt thereof. 20. The method of any of the claims 9-17 wherein said medication is anti-depressant medication. 21. The method of claim 20 wherein the anti-depressant medication is selected from amitriptyline or a pharmaceutically acceptable salt thereof.

22. The method according to any of the claims 9-21, wherein the subject is a human.

23. Use of an agent in the manufacture of a medicament for treating a disorder or a disease in a subject, characterized in that the PI3K delta activity in said subject has been measured to be about normal or higher than normal.

24. The use of claim 23, wherein said agent is an anti CNS-disorder agent.

25. The use of any of the claims 23-24, wherein said disorder is a CNS-disorder.

26. The use of claim 25, wherein said CNS-disorder is psychosis or schizophrenia and the anti CNS-disorder agent is an antipsychotic agent. 27. The use of claim 25, wherein said CNS-disorder is depression and the anti CNS- disorder agent is an anti-depressant agent.

28. The use of claim 26, wherein the anti-psychotic agent is selected from olanzapine, clozapine, quetiapine, ziprasidone or a pharmaceutically acceptable salt thereof.

29. The use claim 27, wherein the anti-depressant agent is selected from amitriptyline or a pharmaceutically acceptable salt thereof. 30. The use of any of the claims 23-29 wherein the PI3K delta activity is determined by analyzing a biological sample from a subject.

31. The use of any of the claims 23-30 wherein the PI3K delta activity is measured directly.

32. The use of claim 31 wherein the PI3K delta activity is measured by enzymatic formation of PIP3 or of an analogous product, or by western blot analysis with antibodies directed specifically to PIP3, or with a fiuorescently labelled marker of PIP3.

33. The use of any of the claims 23-30 wherein PI3K delta activity is measured indirectly.

34. The use of claim 33 wherein the PI3K delta activity is measured by measuring cellular levels of phosphorylated AKTl or GSK3 beta, or by measuring cellular levels of phosphorylated glycogen synthase protein, or by measuring cellular levels of PI3K delta mRNA or PI3K delta protein, or by measuring the amounts of glycogen in blood or tissue.

35. The use of any of the claims 23-34 wherein the subject is a human.

36. An agent for use in a method for treating a disorder or disease in a subject, characterized in that the PI3K delta activity in said subject has been measured to be about normal or higher than normal.

37. The agent of claim 36, wherein said agent is an anti CNS-disorder agent.

38. The agent of any of the claims 36-37, wherein said disorder is a CNS-disorder.

39. The agent of claim 38, wherein said CNS-disorder is psychosis or schizophrenia and the anti CNS-disorder agent is an antipsychotic agent. 40. The agent of claim 38, wherein said CNS-disorder is depression and the anti CNS- disorder agent is an anti-depressant agent.

41. The agent of claim 39, wherein the anti-psychotic agent is selected from olanzapine, clozapine, quetiapine, ziprasidone or a pharmaceutically acceptable salt thereof.

42. The agent claim 40, wherein the anti-depressant agent is selected from amitriptyline or a pharmaceutically acceptable salt thereof.

43. The agent of any of the claims 36-42 wherein the PI3K delta activity is determined by analyzing a biological sample from a subject.

44. The agent of any of the claims 36-43 wherein the PI3K delta activity is measured directly.

45. The agent of claim 44 wherein the PI3K delta activity is measured by enzymatic formation of PIP3 or of an analogous product, or by western blot analysis with antibodies directed specifically to PIP3, or with a fluorescently labelled marker of PIP3.

46. The agent of any of the claims 36-43 wherein PI3K delta activity is measured indirectly.

47. The agent of claim 46 wherein the PI3K delta activity is measured by measuring cellular levels of phosphorylated AKTl or GSK3 beta, or by measuring cellular levels of phosphorylated glycogen synthase protein, or by measuring cellular levels of PI3K delta mRNA or PI3K delta protein, or by measuring the amounts of glycogen in blood or tissue.

48. The agent of any of the claims 36-47 wherein the subject is a human.

49. A method for treating a disorder or a disease in a subject comprising administering a therapeutically effective amount of an agent to a subject, characterized in that the PI3K delta activity in said subject has been measured to be about normal or higher than normal.

50. The method of claim 49, wherein said agent is an anti CNS-disorder agent.

51. The method of any of the claims 48-49, wherein said disorder is a CNS-disorder.

52. The method of claim 51 , wherein said CNS-disorder is psychosis or schizophrenia and the anti CNS-disorder agent is an antipsychotic agent.

53. The method of claim 51, wherein said CNS-disorder is depression and the anti CNS- disorder agent is an anti-depressant agent.

54. The method of claim 52, wherein the anti-psychotic agent is selected from olanzapine, clozapine, quetiapine, ziprasidone or a pharmaceutically acceptable salt thereof. 55. The method claim 53, wherein the anti-depressant agent is selected from amitriptyline or a pharmaceutically acceptable salt thereof.

56. The method of any of the claims 49-55 wherein the PI3K delta activity is determined by analyzing a biological sample from a subject.

57. The method of any of the claims 49-56 wherein the PI3K delta activity is measured directly.

58. The method of claim 57 wherein the PBK delta activity is measured by enzymatic formation of PIP3 or of an analogous product, or by western blot analysis with antibodies directed specifically to PIP3, or with a fluorescently labelled marker of PIP3.

59. The method of any of the claims 49-56 wherein PI3K delta activity is measured indirectly.

60. The method of claim 59 wherein the PI3K delta activity is measured by measuring cellular levels of phosphorylated AKTl or GSK3 beta, or by measuring cellular levels of phosphorylated glycogen synthase protein, or by measuring cellular levels of PI3K delta mRNA or PI3K delta protein, or by measuring the amounts of glycogen in blood or tissue.

61. The method of any of the claims 49-60 wherein the subject is a human.

62. A method for identifying an anti-psychotic compound comprising the steps of:

63. The method of claim 62 wherein said target by interaction with said compound will mediate an effect that can treat and/or prevent psychosis, or ameliorate the symptoms of psychosis.

64. The method of claim 62, wherein said compound when tested for inhibition of PI3K delta activity does not inhibit said PI3K delta activity.

65. The method of any of the claims 62-64, wherein said method further comprises the step of measuring the bio-distribution of said compound.