WO2006032859A2

WO2006032859A2 - Treatment of schizophrenia or suicidal behaviour with a 5-ht2c receptor antagonist

Info

Publication number: WO2006032859A2
Application number: PCT/GB2005/003603
Authority: WO
Inventors: Anne T. Bruinvels
Original assignee: Curidium Limited
Priority date: 2004-09-20
Filing date: 2005-09-20
Publication date: 2006-03-30
Also published as: GB0420873D0; WO2006032859A3

Abstract

Methods for determining whether a patient diagnosed with schizophrenia, or suffering from suicidal behaviour or from mild cognitive impairment, is suitable for treatment with a 5-HT2C receptor antagonist, and associated uses of 5-HT2C receptor antagonists.

Description

Methods of Diagnosis

Field of the Invention

The present invention describes a method for determining, within a group of diagnosed patients, those patients most susceptible to treatment with a 5HT2C receptor antagonist.

Background to the Invention

Schizophrenia, a devastating mental disorder, is a chronic disease characterised by severe psychological symptoms such as perception (hallucinations), ideation, reality testing (delusions), thought processes (loose associations), feeling (flatness, inappropriate effect), behaviour (catatonia, disorganisation) , attention, concentration, motivation (avolition, impaired intentions and planning) and judgement. For a full listing of the symptoms associated with schizophrenia, see for example the Diagnostic and Statistical Manual of Mental Disorders IV, American Psychiatric Association.

In general the schizophrenia disease symptoms are divided into positive and negative symptoms, with hallucinations and delusions being positive features and features such as flatness, poverty of speech and impaired executive functions representing negative symptoms. Clinical rating scales such as the Positive and Negative Syndrome Scale (PANSS) (Kay et al., 1991 Compr Psychiatry, 32 (4) : 355-61) and the Scale for the Assessment of Negative Symptoms (SANS) (Andreasen et al., 1982, Arch Gen Psychiatry 39(7) :784-8) provide criteria to differentiate between, and rate, positive and negative symptoms . Frequently included in the description of negative symptoms are the cognitive deficits schizophrenic and schizotypical patients suffer from. These include impairment in attention, verbal fluency, executive functions such as planning, working memory and visual and verbal learning and memory. These types of cognitive dysfunction can be measured with a variety of tests, such as Visual Search (Portnoff et al., 1981, Percept Mot Skills 53 (2) : 411-8; Kurachi et al . , 1994, Schizophr Res. 12 (1) : 75-80), Verbal Fluency, Wisconsin Card Sorting, Trail Making - Part B (see Goldberg et al . , 1988, Int J Neurosci. 42 (1-2) : 51-8) , Symbol Digit, Hopkins Verbal Learning, Digit Span, Stroop-Color-Word and Attentional Capacity (see Mahurin et al . , 1998, Psychiatry Res. 79(2) : 139-49) . Importantly, it has been found that cognitive measures predict work function and overall outcome as assessed by the Global Assessment Scale and Quality of Life Scale (see Meltzer et al . , 1996, Brit. J. Psychiatry 168: 23- 31) . Several studies have now demonstrated that neuropsychological functions, reflecting several negative and cognitive symptoms of the disease, may be more impaired in male schizophrenic patients when compared to female patients (see Goldstein et al . 1998, Am J Psychiatry. 155 (10) : 1358-64; Goldstein et al . , 1994, J. Psychiatr. Res. 28 ( 6) : 483-98; Goldstein and Link, 1988, J. Psychiatr. Res. 22 (2) : 141-55) . Further, there are a number of other psychiatric diseases such as schizotypical and schizoaffective disorder, other acute- and chronic psychoses and bipolar disorder which have an overlapping symptomatology with schizophrenia.

Suicide is the major cause of premature death in patients with schizophrenia, with 40% of patients reporting suicidal thoughts, 2 to 40% making unsuccessful suicide attempts and 9% to 13% ending their lives in suicide (Siris, 2001, J. Psychopharmacol. 15 (2) : 127-35; Meltzer, 1998, J. Clin. Psychiatry, 59 Suppl 3:15-20) . Although the pathophysiology of suicide and suicidality remains unclear, the 5-HT2A receptor has been associated with suicide (Du et al, 2001, Crisis 22 (2) :54-60; Pandey et al, 1997, Ann. N Y Acad. Sci. 836:182-200) . A recent report (Niswender et al . , 2001, Neuropsychopharmacology, 24 (5) : 478-91) describes altered levels of an edited form of 5-HT2C receptor messenger RNA in suicide victims.

Mild cognitive impairment is a term given to patients whose clinical state presents as memory impaired, but who are otherwise functioning well and do not meet the clinical criteria for dementia. Mild cognitive impairment represents a transitional state of cognitive impairment between normal aging and early Alzheimer disease. Diagnostic criteria typically include memory complaint (preferably corroborated) , objective memory impairment, normal general cognitive function, intact activities of daily living but no dementia. Mild cognitive impairment may also be referred to as incipient dementia, questionable dementia, age-associated cognitive decline and isolated memory impairment and the present invention embraces all these, and other commonly used, synonyms for mild cognitive impairment. Numerous studies have been performed on mild cognitive impairment and the reviews of these studies have indicated that individuals with mild cognitive impairment are at an increased risk of developing Alzheimer disease and, in most cases, convert to dementia and/or Alzheimer disease within several years.

PCT/GB2004/001225 describes that a 5-HT2C receptor antagonist may be a particularly useful medicament for the treatment of cognitive dysfunction in and/or negative symptoms of schizophrenia, in the treatment of refractory schizophrenia or in the treatment of suicidality or mild cognitive impairment.

Although the 5-HT2C receptor has been investigated for a role in schizophrenia many conflicting results have been reported. Recent reviews however, which discuss the putative role of 5-HT2C receptor antagonists in antipsychotic treatments, have primarily concluded that 5-HT2C receptor antagonism 1) does not contribute to antipsychotic activity (Leysen et al . , 1993, Psychopharmacol . 110: 27-36), 2) leads to weight gain (Leysen, 2000, in Atypical Antipsychotics, Birkhauser Verlag, Basel-Boston-Berlin) and 3) that it functionally opposes 5- HT2A receptor antagonism (Meltzer, 1999,

Neuropsychopharmacology, 21(2 Suppl) :106S-115S) .

The 5-HT2C receptor is a heavily regulated receptor, its activity not only affected by genetic variations of the gene itself, but also by post-translational processes such as pre- messenger RNA editing (Burns et al., 1997, Nature, 387:303- 308), regulation by receptor phosphorylation by G-protein receptor kinases (Berg et al . , 2001, J. Pharmacol. Exp. Ther. 299:593-602), by MAP kinase (Hurley et al., 2003, BMC Neuroscience, 4:10) and regulation by various transcription factors. Also several mRNA splice variants exist resulting in both active and inactive receptors (Canton et al., 1996, MoI. Pharmacol. 50 (4) :799-807) .

Summary of the Invention

The present invention provides for the first time evidence that determination of the activity of a specific receptor can enable a delineation of a patient group most likely to benefit from a treatment directed at that receptor. The present invention is concerned with a method for determining whether a schizophrenic or a suicidal patient or a patient with mild cognitive impairment is susceptible to treatment with a 5-HT2C receptor antagonist.

Accordingly the invention provides a method for determining whether a patient diagnosed with schizophrenia, or suffering from suicidal behaviour or from mild cognitive impairment, is suitable for treatment with a 5-HT2C receptor antagonist, which comprises:

(a) determining ex vivo the level of 5-HT2C receptor activity in a schizophrenic patient, or an individual suffering from suicidal behaviour or mild cognitive impairment; and

(b) if the activity of the 5-HT2C receptor falls within the range of 5-HT2C receptor activity which is more prevalent in the patient population than the normal population concluding that said patient or individual is suitable for treatment with a 5-HT2C receptor antagonist.

The invention additionally provides:

- use of a 5HT2C receptor antagonist in the manufacture of a medicament for the treatment of a patient diagnosed with schizophrenia, or an individual suffering from suicidal behaviour or from mild cognitive impairment, wherein said patient has been determined to be suitable for treatment with a 5HT2C receptor antagonist by a method comprising determining from the level of 5-HT2C receptor activity in said schizophrenic patient, or an individual suffering from suicidal behaviour or mild cognitive impairment, whether the activity of the 5-HT2C receptor falls within the range of 5-HT2C receptor activity which is more prevalent in the patient population than the normal population;

- use of a VNTR motif listed in Table 1 or an SNP listed in Table 2 for the assessment of a genotype predictive of 5HT2C receptor activity in a patient diagnosed with schizophrenia, or suffering from suicidal behaviour or from mild cognitive impairment;

- use of a VNTR sequence motif listed in Table 1 or an SNP listed in Table 2 for the generation of a nucleic acid probe or primer for the assessment of a genotype predictive of 5HT2C receptor activity in a patient diagnosed with schizophrenia, or suffering from suicidal behaviour or from mild cognitive impairment;

- a nucleic acid probe or primer which is suitable for detecting a VNTR motif listed in Table 1 or an SNP listed in Table 2;

- a nucleic acid probe or primer of the invention for use in medicine;

- use of a probe or primer of the invention for the in vitro assessment of a genotype predictive of 5HT2C receptor activity in a sample from a patient diagnosed with schizophrenia, or suffering from suicidal behaviour or from mild cognitive impairment; and

- a kit for determining suitability for treatment with a 5- HT2C receptor antagonist of a patient diagnosed with schizophrenia, or suffering from suicidal behaviour or from mild cognitive impairment, which kit comprises a probe or primer of the invention.

Brief Description of the Figures Figure 1 : Table 1 - a list of variable number tandem repeat (VNTR) motifs useful in the design of primer and probe sets for use as (part of) a genetic test.

Figure 2 : Table 2 - a list of single nucleotide polymorphisms (SNPs) useful in the design of primer and probe sets for use as (part of) a genetic test.

Detailed Description

In its first aspect, the present invention provides a method for determining whether a patient diagnosed with schizophrenia, suffering from suicidal behaviour or from mild cognitive impairment is suitable for treatment with a 5-HT2C receptor antagonist which comprises:

(a) determining the level of 5-HT2C receptor activity in a schizophrenic patient, an individual suffering from suicidal behaviour or mild cognitive impairment; and

(b) if the activity of the 5-HT2C receptor falls within the range of 5-HT2C receptor activity which is more prevalent in the patient population than the normal population concluding that said patient is suitable for treatment with a 5-HT2C receptor antagonist.

In a second aspect, the present invention provides the use of a 5HT2C receptor antagonist in the manufacture of a medicament for the treatment of a patient diagnosed with schizophrenia, suffering from suicidal behaviour or from mild cognitive impairment, wherein said patient has been determined to be suitable for treatment with a 5HT2C receptor antagonist by a method comprising determining from the level of 5-HT2C receptor activity in said schizophrenic patient, or an individual suffering from suicidal behaviour or mild cognitive impairment, whether the activity of the 5-HT2C receptor falls within the range of 5-HT2C receptor activity which is more prevalent in the patient population than the normal population.

The diagnostic test of the present invention therefore allows a more accurate determination of the patient group most susceptible to successful treatment with a 5HT2C receptor antagonist .

In one embodiment the methods of the invention may further comprise the step of treating the patient or individual with a suitable 5HT2C receptor antagonist.

In the methods of the present invention, the level of 5-HT2C receptor activity may be determined ex vivo. Measurements of 5HT2C receptor activity may be made in vitro, such as on a sample from a patient or individual. Any appropriate sample may be used, typically of bodily fluid or tissue. Samples are preferably taken from blood, including lymphocytes, lymphoblastoid cells, fibroblasts, platelets, mononuclear cells or other blood cells, from saliva, cerebrospinal fluid, liver, kidney, pancreas or heart, urine or from any other tissue, fluid, cell or cell line derived from the human body.

In the methods of the present invention, the patient whose suitability for treatment with a 5HT2C receptor antagonist is being determined has previously been diagnosed with schizophrenia according to the symptoms set out in DSM IV ) or other similar diagnostic criteria) and/or is suffering from suicidal behaviour or mild cognitive impairment. Performing a diagnosis of these conditions is not embraced by the present invention. In the methods of the present invention, the activity level of the 5HT2C receptor is measured to determine whether the activity of that receptor falls within the range of 5HT2C activity which is more prevalent in the patient population than in the normal population. By "normal population" in this respect is meant a group of individuals neither diagnosed with, nor demonstrating the symptoms of, schizophrenia, suicidality or mild cognitive impairment. By "patient population" is meant a group of individuals diagnosed with schizophrenia, according to the requirements of DSM IV or other similar diagnostic criteria, or suffering from suicidal behaviour or mild cognitive impairment.

In order for a determination to be made of the suitability of the patient for treatment with a 5HT2C receptor antagonist, the activity of the 5HT2C receptor must fall within the range of activity which is more prevalent in the patient population than in the normal population. By "more prevalent" in this respect is meant that a larger number of patients in the patient population have this range of activity than do individuals in the normal population. Typically, 10% or more of patients in the patient population will have this range of activity, preferably 20% or more and more preferable 25% or more.

It has been discovered that the activity of the 5HT2C receptor in a patient diagnosed with schizophrenia or suffering from suicidal behaviour or mild cognitive impairment will typically be enhanced or increased. An increase in activity of the 5HT2C receptor can be defined as being a level of activity in the patient population which is higher than the level of activity determined from a normal population. The precise amount of the deviation of the level of activity from the norm is not essential to the present invention, although it is anticipated that the greater the deviation from the norm, the greater the susceptibility of the patient to treatment with a 5HT2C receptor antagonist.

In the present invention, therefore, the level of activity of the 5HT2C receptor in the patient will be at least 10% greater than that in the normal population, preferably at least 30% greater and more preferably from 50-100% greater.

Although previous studies had demonstrated that 5-HT receptor function, may be increased in patients who benefit from treatment with the atypical antispcyhotic clozapine relative to patients who fail to improve on this drug (Kahn et al . Am J Psychiatry, 1993, 150 (9) : 1337-42) , the present invention is the first to suggest that psychiatric patients with increased sensitivity of the 5-HT2C receptor, measured directly or indirectly as 5-HT2C receptor activity, may benefit from treatment with a 5-HT2C receptor antagonist.

5HT2C receptor activity may be measured either directly or indirectly. The precise method for measurement of this activity is not essential to the present invention. Of more import is the comparison between the levels of activity in a patient and the levels found in a normal population.

5-HT2C receptor activity may be measured by direct assessment involving measurement of 5-HT2C receptor activity in any human tissue sample, such as lymphocytes, blood or liver cells or any other cell or cell line derived from a patient. Methods that may be used include: measurement of phospholipase C activity (Cussac et al . , 2002, Naunyn Schmiederbergs Arch. Pharmacol. 365 (3) :242-52; Cussac et al., 2000, Naunyn Schmiederbergs Arch. Pharmacol. 361 (2) :221-3; Berg et al . , 1998, MoI. Pharmacol. 54 (1) : 94-104; Bany et al . , 1995, Arch. Immunol. Ther. Exp. 43(l) :51-4; Bacon et al. , 1994, Blood, 81 (2) : 430-6) ; measurement of calcium mobilisation (Tong et al., 2004, Transplantation, 77(2) :259- 67; Laskowska-Bozek et al . , 1996, Neuroimmunomodulation, 3 (4) :247-53) ; measurement of GTPgammaS incorporation (Gonsiorek et al . , 2003, J. Immunol. Methods. 273 (1-2) : 15-27; Pietruck et al . , 1998, Diabetologia, 41 (1) : 94-100; Siffert et al., 1995, J. Clin. Invest. 96 (2) :759-66) , measurement of phospholipase A2-mediated arachidonic acid release (Berg et al., 1998, MoI. Pharmacol. 54 (1) : 94-104) , in basal conditions and in the presence of a 5-HT2C receptor agonist.

5-HT2C receptor activity may also be measured by conducting an in vivo scanning method, such as positron emission topography (PET) , spectrophotometry emission chromatography (SPECT), functional magnetic resonance image (MRI) scanning of the brain or another organ with functional 5-HT2C receptors .

Indirect assessment of 5-HT2C receptor activity may be measured for example by:

1. the measurement of 5HT2C messenger RNA levels in any human tissue sample, such as lymphocytes, blood or liver cells or any other cell or cell line derived from a patient (Marazziti et al. Neuropsychobiology, 2001, 43:123-6; WO 00/05409; Castensson et al . , 2002, Biol Psychiatry, 54(11) :1212-21) ;

2. measurement of 5-HT2C receptor expression by radioligand binding (Lopez-Gimenez et al . , J Neurosci Res. 2002, 1;67(1) : 69-85) or by antibody binding (Abramowski et al. Neuropharmacol., 1995, 12:1635-1645);

3. measurement of 5-HT2C RNA editing products in any human tissue sample, such as lymphocytes, blood or liver cells or any other cell or cell line derived from a patient (see Flomen et al., Nucleic Acids Res. 2004 Apr 15 32 (7) :2113-22; Dracheva et al, J Neurochem. , 2003 Dec; 87 (6) : 1402-12; Schmauss, Neuroscientist, 2003, 9(4) :237-42; Niswender et al, Ann N Y Acad Sci., 1998 Dec 15; 861 : 38-48; Burns et al, Nature 1997, 387:303-8; Fitzgerald et al . , Neuropsychopharmacology,

1999, Aug 21(2 Suppl) : 82S-90S; Wang et al, J Neurochem.,

2000, 74 (3) :1290-300; Sodhi et al . , MoI Psychiatry., 2001, 6(4) :373-9) ;

4. by the use of a genetic test measuring genotypes of the 5-HT2C receptor gene and/or of genes influencing 5-HT2C receptor activity, as described by Lappalainen et al.,1995, Genomics, 27(2) :274-9; Ebstein et al . , 1997, Am. J. Med., Genet. 74(1) : 65-72; Segman et al., 1997, Psychiatr Genet., 7:75-8; Song et al . , MoI Genet Metab., 1999, 66(3) :224-7; Meyer et al . 2002, J. Neural. Transm. 109 (5-6) : 939-46 Zhang et al., 2002, Zhonghua Yi Xue Za Zhi. 82 (16) : 1097-101; Mottagui-Tabar et al., 2004, Alcohol Alcohol.. 39(5) :380-5 or by detecting genetic variances as listed in Table 1 and Table 2;

5. by determination of the prevalence or presence of specific gene alleles which can influence the activity of 5- HT2C receptor activity, such as, for example, the nucleotide 68 polymorphism resulting in either a cysteine or a serine at position 23 of the receptor protein (Lappalainen et al . , supra) , or the polymorphic variance as described by Song et al . , supra; or

6. by elucidation of the presence of 5-HT2C-Ser genotype (hemizygotes in male or homozygotes in female; Segman et al. supra) .

It is known that 5-HT2C receptor activity is highly regulated by several other factors such as adenosine deaminases (ADARs, Yang et al., 2004, Brain Res. MoI. Brain Res. 124 (1) : 70-8) , the G-protein G13 (Berg et al . supra) , MAP kinase (Hurley et al. supra), G-protein kinase (Berg et al . supra) and the dopamine D4 receptor (Ebstein et al. supra) .

Various ways to express 5-HT2C receptor activity may be used. An indirect measure of 5-HT2C receptor activity is the measurement of levels of mRNA in human tissues. It is expected that those individuals with increased activity of the 5-HT2C receptor have decreased levels of 5-HT2C mRNA. Therefore patients with low levels of mRNA (that is, for example from 20 to 40%, preferably less than 30%, of the range of mRNA expression in the control population) may be selected for treatment with a 5-HT2C receptor antagonist.

Although any of the above methods may be used to measure 5- HT2C receptor activity, or any other method available from the art, it is preferred to measure 5-HT2C receptor activity either by any of the direct methods discussed hereinabove and/or by determination of mRNA in lymphocytes or by genetic tests or by measurement of 5-HT2C receptor activity in lymphocytes . Preferred mRNA expression tests include, for example, the use of primer and probe sets to assess the level of expression of mRNA of different (splice or editing) variants of the 5-HT2C receptor. Such tests are preferably carried out in lymphocytes and the results preferably demonstrate a low expression (i.e. from 20 to 40%, preferably less than 30%, of the range of mRNA expression in the control population) in lymphocytes .

This test may be performed alone or in combination with any of the other tests mentioned herein, and, in particular in combination with genetic tests, which include for example: the use of probes/primers for variable number tandem repeats (VNTRs) and/or single nucleotide polymorphisms (SNPs) , such as those listed in Tables 1 and 2 respectively; the presence of the G/C-68 polymorphism: 5-HT2C-Ser allele; the assessment of the genotype the mRNA editing enzyme ADAR, the G-protein G13, MAP kinase, G-protein kinase.

The most preferred such combined test is the determination of low levels of 5-HT2C mRNA in lymphocytes (from 20 to 40%, preferably less than 30% of the range of mRNA expression in the control population) together with the presence of one (male) or two (female) alleles of the 5HT2C Ser gene.

VNTR and SNP variances in this chromosomal location which may be used in the genetic tests of the invention have been identified. VNTR motifs and surrounding sequences which may be used in the design of primers and/or probes are illustrated in Table 1. SNPs which may be used in the design of primers and/or probes are illustrated in Table 2. The VNTRs and SNPs listed in Tables 1 and 2 may be used in the assessment of a genotype associated with or predictive of 5HT2C receptor activity. The testing will also assess genetic marker haplotypes in the region which may have an influence on expression of the gene.

In accordance with the above, the present invention therefore also provides the use of a VNTR motif as listed in Table 1 herein in the assessment of (a genotype predictive of) 5-HT2C receptor activity in a schizophrenic, suicidal or mild cognitively impaired patient.

The invention also provides nucleic acid probes and/or primers which are suitable for detection of a VNTR motif listed in Table 1. For example, a VNTR motif listed in Table 1 may be used in the generation of such a probe/primer. In particular, in this aspect, the present invention provides the use of a VNTR motif of Table 1 or a nucleic acid surrounding said motif in the generation of a probe and/or primer for the assessment of 5-HT2C receptor activity, in particular the assessment of a genotype predictive of 5-HT2C receptor activity, in a schizophrenic, suicidal or mild cognitively impaired patient. The sequence surrounding the motif may be referred to as the flanking sequence and may extend from 1 to 250 bases upstream or downstream of the motif.

Probes or primers for detection of a VNTR, such as those generated using the VNTRs in Table 1 will be designed to measure the number of repeats that an individual has . The sequence around the VNTR, or flanking sequence (500bp in total) may be used to design selective and sensitive primers/probes which can be used to detect the motif and determine the number of repeats . A nucleic acid probe or primer suitable for detection of a VNTR, such as one generated using a VNTR motif in Table 1, may in one aspect comprise or consist (essentially) of: (a) a fragment of the flanking sequence of the VNTR motif; or (b) a nucleic acid sequence complementary to (a); wherein the VNTR motif is one listed in Table 1 and wherein the flanking sequence is the sequence from 1 to 250 bases upstream or downstream of the motif.

Furthermore the present invention provides the use of an SNP listed in Table 2 herein in the assessment of (a genotype predictive of) 5-HT2C receptor activity in a schizophrenic, suicidal or mild cognitively impaired patient.

The invention also provides nucleic acid probes and/or primers which are suitable for detection of an SNP listed in Table 2. For example, an SNP listed in Table 2 may be used in the generation of such a probe/primer. In particular, in this aspect, the present invention provides the use of an SNP of Table 2 in the generation of a probe and/or primer for the assessment of 5-HT2C receptor activity, in particular the assessment of a genotype predictive of 5-HT2C receptor activity, in a schizophrenic, suicidal or mild cognitively impaired patient.

A nucleic acid probe or primer suitable for detection of an SNP, such as one generated using an SNP in Table 2 typically comprises the SNP. The probe/primer in general comprises sequence surrounding the SNP. In one embodiment the SNP may^¬ be the central base of the probe. Typically a probe or primer has a length of from 15 to 60, such as from 20 to 50, 20 to 40, 15 to 30 or 15 to 40 bases. For example, a probe/primer may be 15, 16, 17, 18, 19, 20, 22, 25, 27 or 30 bases in length, typically 20 bases.

As far as the sequences of the VNTRs and SNPs are not previously known, the present invention also provides a VNTR or SNP having a sequence as listed in Tables 1 and 2, respectively.

The probes or primers of the invention, for example, those generated as described herein, are useful in determining 5HT2C receptor activity in a schizophrenic, suicidal or mild cognitively impaired patient. In particular, the probes/primers may be used to determine a genotype predictive of 5HT2C receptor activity. Thus the invention also provides a nucleic acid probe or primer of the invention for use in medicine. Also provided is the use of a probe or primer of the invention for the in vitro determination of a genotype predictive of 5HT2C receptor activity in a sample from a patient diagnosed with schizophrenia or suffering from suicidal behaviour or from mild cognitive impairment.

Further provided is a kit for determining suitability for treatment with a 5HT2C receptor antagonist of a patient diagnosed with schizophrenia, or suffering from suicidal behaviour or from mild cognitive impairment, which kit comprises a probe or primer of the invention. The kit may be used to assess 5HT2C receptor activity in subject such as a schizophrenic, suicidal or mild cognitively impaired patient, for example by means of a genetic test carried out on a sample taken from the patient. Typically the kit is suitable for use in a method for determining whether a schizophrenic, suicidal or mild cognitively impaired patient is suitable for treatment with a 5HT2C receptor antagonist as described herein. Such a kit may additionally comprise, suitable nucleic acid labelling and/or detection means, reaction buffer, suitable enzymes and/or instructions for use.

In its second aspect, the present invention provides the use of a 5HT2C receptor antagonist in the manufacture of a medicament for the treatment of a patient diagnosed with schizophrenia, suffering from suicidal behaviour or from mild cognitive impairment, wherein said patient has been determined to be suitable for treatment with a 5HT2C receptor antagonist by a method comprising determining from the level of 5-HT2C receptor activity in said schizophrenic patient, or an individual suffering from suicidal behaviour or mild cognitive impairment, whether the activity of the 5-HT2C receptor falls within the range of 5-HT2C receptor activity which is more prevalent in the patient population than the normal population.

Any appropriate 5HT2C receptor antagonist may be used in this aspect of the invention. Typical such antagonists are those listed in PCT/GB2004/001225 and include, for example, AHR- 16303B (AH Robins Co. Inc) , AP-792 and AT-1015 (Ajinomoto Co. Inc.), BMS-181102 (Bristol Myers Squibb), CV-5197 (Takeda Chemical Industries Ltd) , dotarizine (Ferrer Internacional SA) , E-2101 (Eisai Co Ltd) , eltoprazine (Solvay SA) , emopamil (Knoll AG) , HT-90B (Chugai Pharmaceutical Co Ltd) , ICI-169369 and ICI-170809 (Zeneca Group pic), LU-26042 and LU-29066 (H Lundbeck A/S), NPC-18166 (Scios Inc), Org-38457 (NV Organon) , pelanserin (Cinvestav) , perbufylline (Siegfried Group) , SB- 206553 and SB-242084 (SmithKline Beecham) , SR-46615A (Sanofi Recherche SA) , SUN-9221 (Suntory Ltd) tropoxin (Russian Academy Medical Science) and YM-992 (Yamanouchi Pharmaceutical Co Ltd) .

Most preferred such 5HT2C receptor antagonists for use in this aspect of the invention include Ro-60-0759, RS-102221, SDZ-SER-082, Amersergide, ICI-169369, Sergolexole, Deramciclane, N-desmethyl-deramciclane, CGS-18102A and LU- 26042. These compounds, together with methods for their preparation are described in WO 98/30546, US 5,739,336, EP 473,550, US 4,931,447, US 4,435,405, US 4,714,704, US 4,342,762, US 6,093,747, EP 161,218 and WO 93/14758 respectively.

EXAMPLES

Example 1: Indirect Tests to select patients with increased 5-HT2C receptor activity

These technologies include RT-PCR related methods such as by microarray, or by the ABI- Taqman™ technology (see WO- 00/05409) . Tissues which may be used include lymphocytes (see Gladkevich et al., 2004, Prog Neuropsychopharmacol Biol Psychiatry. 28 (3) : 559-76) .

Expression of messenger RNA

Expression of messenger RNA may be performed using the methods described in, for example, WO 00/05409.

RNA Extraction

Total RNA will be extracted from the tissues using Trizol according to the manufacturer's protocol. The RNA will only be used for cDNA synthesis if the optical absorbance ratio (A260/A280) >1.8 and it has intact 18 and 28S ribosomal RNA.

Primer/probe design

Primers and TaqMan probes are designed to amplify specific

GenBank sequences. These primers and probes are then homology searched against GenBank to confirm that they are specific for the targets from which they were designed. PCR reactions for the target gene are duplexed, with glyceraldehyde-3- phosphate dehydrogenase (GAPDH) , which is used as a marker of intact RNA.

The target probe is labelled with the fluor 6-FAM whilst the probe for GAPDH is labelled with the fluor VIC. Primers and probes will be designed across exon /exon boundaries or where this is not possible the samples will be DNase I treated. This is to avoid any amplification from genomic DNA, which has been co isolated with the total RNA.

cDNA synthesis

This will be synthesised from 50 ng of total RNA from each of the tissues being studied. The cDNA will synthesised using random primers, using a high capacity cDNA archive kit (Applied Biosystems 4322171) .

The cDNA derived from the 50ng total RNA for each sample will be subjected to PCR amplification in a single reaction to identify both target and GAPDH transcripts. Primers and probes for the target and GAPDH genes will be added to the reaction mix along with the appropiate buffer, nucleotides and Taq polymerase. The PCR conditions will be: 95°C for 10 minutes, followed by 45 cycles of 95°C for 15 seconds and 6O⁰C for 45 seconds. PCR amplification curves will be analysed to yield Ct values and these values will be used to determine the starting rriRNA copy number of both target and GAPDH genes by extrapolation from standard curves generated from known amounts of PCR product for both the target and GAPDH.

Example 2 : Genetic tests to select patients with genotype and resulting in increased 5-HT2C receptor activity

Genotyping Experiments

These may be performed as described in, for example, Brenga et al. 2002, Clin Chem. 48 (12) :2131-40) .

DNA samples

Genomic DNA was extracted and purified from blood samples and stored at -20 ⁰C before analysis. Selection of SNPs

Primers for PCR amplification of different SNPs in the genes of interest were designed.

PCR primers and DNA amplification

The allele-specific PCR primers and the COM (reverse) primers were designed from published gene sequences using Oligo™ v6.4 primer analysis software (Molecular Biology Insights) . PCR primer sequences were synthesized by Midland Certified Reagents .

PCR primers contained two allele-specific primers, wild type (WT) and mutant (MUT) , and a COM opposite primer per SNP, to amplify each of the SNP loci. The allele-specific primers contain 21-nucleotide (nt) regions (identical to the recognition site of each Universal Amplifluor primer; "tailed") that are different for one of two labeled primers (green or red) . A different sequence tail is then added to the 5' end of each allele-specific primer. The 21-nt tails on the allele-specific primers are identical with the 21-nt 3' region of the corresponding Universal Amplifluor (green or red) . Final concentrations of PCR reagents were 200 μM of each deoxynucleoside triphosphate, 1.0 U/reaction of either Taq DNA polymerase (Roche Biochemical) or Platinum® Taq DNA polymerase (Life Technologies) , 250 nM of both Universal Amplifluor primers and COM (reverse) primer, and 25 nM of both tailed allele-specific primers in 20 μL. The (Ix) reaction buffer was 1.8 TnMMgCl₂, 50 mM KCl, and 10 mM Tris, pH 8.30. The Amplifluor reagent system (Serologicals Corp.) includes two Universal Amplifluor primers [labeled with fluorescein (FAM) or sulforhodamine (SR)], 10x PCR buffer, and deoxynucleoside triphosphates] . PCR reactions were set up and performed in optically clear PCR microplates (VWR Scientific Products) and sealed with PCR plate-sealer adhesive tape (Robbins Scientific Corp.) .

Amplifications were performed in an PTC-200 gradient thermal cycler (MJ Research) with the following conditions: a pseudo- hot start of 5-10 s at 94 ⁰C, denaturation of 4 min at 95 ⁰C, then 35 cycles (10 s at 94 ⁰C, 20 s at 55 ⁰C, and 40 s at 72 ⁰C), followed by 3 min of final extension at 72 ⁰C. PCR reactions were held at 20 ⁰C until fluorescence measurements could be performed.

SNP PCR reactions were optimized by performing PCRs with several 10x PCR buffers [Buffer K: 600 mM Tris-HCl (pH 9.5), 150 mM (NH₄) ₂SO₄, and 25 mM MgCl₂; Buffer N: 600 mM Tris-HCl

(pH 10.0), 150 mM (NH₄)₂SO₄, and 20 mM MgCl₂; and Buffer I: 100 mM Tris-HCl (pH 8.3), 500 mM KCl, and 18 mM MgCl₂] and then analyzing the PCR products by gel electrophoresis for yield and specificity. One buffer that gave maximum amplicon yield and specificity was subsequently selected for all SNP PCRs. We also performed temperature-gradient PCRs to investigate the potential influence of Amplifluors on amplicon yield and specificity of PCR as a function of annealing temperature (range, 50-70 ⁰C) . The optimum combination of target amount and cycle number that provided the best yield of PCR amplicon was also determined.

Fluorescent measurements and data analysis Total fluorescence (as relative fluorescence units) of labeled Universal Amplifluor primer-containing amplicons was quantified through the top of each well of open PCR microplates using a Victor™ 1420 fluorescence microplate reader (Perkin-Elmer Wallac, Inc.) . The microplate reader was equipped with the narrow bandpass filters to quantify FAM (excitation, 485 nm; emission, 535 nm) and SR (excitation, 585 nm; emission, 620 nm) .

Fluorescence results were transferred to separate Excel worksheets for analysis, and scatterplots for each SNP locus were built as follows . Signals from WT (usually FAM-labeled primer) alleles were plotted along the x axes, whereas signals from MUT (usually SR-labeled primer) alleles were plotted along the y axes. In the typical labeling scheme, fluorescence of samples that have a homozygous WT genotype accumulate along the x axis, whereas signals from samples with the homozygous MUT genotype accumulate along the y axis. Signals from the heterozygous genotypes tend to cluster along a diagonal line between the x and y axes. Signals of no-template (blank) PCRs appear near the x,y origin.

Genotype frequencies were compared with Hardy-Weinberg expectations, and allele frequencies were compared between normotensive and hypertensive groups by the method of Roussett and Raymond (1995, Genetics 140 (4) : 1413-9) .

Sequence confirmation of snp amplicons

Confirmation of the WT and MUT amplicon sequences at five of six SNP loci was performed by use of a sequence-appropriate restriction endonuclease to digest the PCR products . After fluorescence quantification, PCR amplicons were typically- purified by precipitation using 2x volumes of absolute ethanol and then resuspended with deionized IH₂O and restricted with one to two units of an appropriate restriction endonuclease. After incubation, reaction mixtures (volume, 20 μL) were separated by gel electrophoresis on 4% agarose gels in Ix Tris-acetate-EDTA buffer, followed by staining with ethidium bromide. Sizes of digested amplicons were determined by comparison with a 10-bp size ladder (New England Biolabs) .

Claims

1. A method for determining whether a patient diagnosed with schizophrenia, or suffering from suicidal behaviour or from mild cognitive impairment, is suitable for treatment with a 5-HT2C receptor antagonist, which comprises:

2. A method according to claim 1 which further comprises treating the patient or individual with a 5HT2C receptor antagonist.

3. Use of a 5HT2C receptor antagonist in the manufacture of a medicament for the treatment of a patient diagnosed with schizophrenia, or an individual suffering from suicidal behaviour or from mild cognitive impairment, wherein said patient has been determined to be suitable for treatment with a 5HT2C receptor antagonist by a method comprising determining from the level of 5-HT2C receptor activity in said schizophrenic patient, or an individual suffering from suicidal behaviour or mild cognitive impairment, whether the activity of the 5-HT2C receptor falls within the range of 5-HT2C receptor activity which is more prevalent in the patient population than the normal population.

4. A method according to claim 1 or 2 or a use according to claim 3 wherein the 5HT2C receptor antagonist is selected from Ro-60-0759, RS-102221, SDZ-SER-082, Amersergide, ICI- 169369, Sergolexole, Deramciclane, N-desmethyl-deramciclane, CGS-18102A and LU-26042.

5. A method or use according to any one of the preceding claims wherein the level of 5HT2C receptor activity is determined from a blood, saliva, cerebrospinal fluid, liver, kidney, pancreatic, heart or urine sample taken from the patient or individual.

6. A method or use according to any one of the preceding claims wherein 5HT2C receptor activity is determined by measuring phospholipase C activity, calcium mobilisation, GTPgammaS incorporation or phospholipase A2-mediated arachidonic acid release, in a sample taken from the patient or individual.

7. A method or use according to any one of claims 1 to 5 wherein 5HT2C receptor activity is determined by measuring levels of 5HT2C receptor mRNA, 5HT2C receptor protein, or 5HT2C RNA editing products in a sample taken from the patient or individual .

8. A method or use according to any one of claims 1 to 5 wherein 5HT2C receptor activity is determined by means of a genetic test.

9. A method or use according to claim 8 wherein the genetic test comprises: - detection of one or more variable number tandem repeat (VNTR) sequence motifs listed in Table 1 or single nucleotide polymorphisms (SNPs) listed in Table 2;

- detection of the G/C-68 polymorphism (the 5HT2C-Ser allele) in the 5HT2C receptor gene; and/or

- assessment of the genotype of the mRNA editing enzyme ADAR, the G-protein G13, MAP kinase or G-protein kinase.

10. Use of a VNTR sequence motif listed in Table 1 or an SNP listed in Table 2 for the assessment of a genotype predictive of 5HT2C receptor activity in a patient diagnosed with schizophrenia, or suffering from suicidal behaviour or from mild cognitive impairment.

11. Use of a VNTR sequence motif listed in Table 1 or an SNP listed in Table 2 for the generation of a nucleic acid probe or primer for the assessment of a genotype predictive of 5HT2C receptor activity in a patient diagnosed with schizophrenia, or suffering from suicidal behaviour or from mild cognitive impairment.

12. Use according to claim 11 wherein the flanking sequence of the VNTR motif is used to generate the probe, and wherein the flanking sequence is the sequence from 1 to 250 bases upstream or downstream of the motif.

13. A nucleic acid probe or primer which is suitable for detecting a VNTR sequence motif listed in Table 1 or an SNP listed in Table 2.

14. A nucleic acid probe or primer according to claim 13 which comprises:

(a) a fragment of the flanking sequence of the VNTR motif; or (b) a sequence complementary to (a) ; wherein the flanking sequence is the sequence from 1 to 250 bases upstream or downstream of the motif.

15. A nucleic acid probe or primer according to claim 13 which comprises an SNP listed in Table 2.

16. A nucleic acid probe or primer generated according to claim 11 or 12 or as defined in any one of claims 13 to 15 for use in medicine.

17. Use of a probe or primer generated according to claim 11 or 12 or as defined in any one of claims 13 to 15 for the in vitro assessment of a genotype predictive of 5HT2C receptor activity in a sample from a patient diagnosed with schizophrenia, or suffering from suicidal behaviour or from mild cognitive impairment.

18. A kit for determining suitability for treatment with a 5-HT2C receptor antagonist of a patient diagnosed with schizophrenia, or suffering from suicidal behaviour or from mild cognitive impairment, which kit comprises a probe or primer generated according to claim 11 or 12 or as defined in any one of claims 13 to 15.