WO1997032037A1

WO1997032037A1 - Assessment of the responsiveness of individuals to modulators of the 5-ht2 receptors, especially the 5-ht2a receptor

Info

Publication number: WO1997032037A1
Application number: PCT/EP1997/000993
Authority: WO
Inventors: Robert Kerwin; David Collier; Gareth Wyn Roberts
Original assignee: Smithkline Beecham Plc
Priority date: 1996-03-01
Filing date: 1997-02-26
Publication date: 1997-09-04
Also published as: AU1878997A; ZA971775B; GB9604465D0; JP2000506009A

Abstract

A method for use in assessing in a subject suffering from a condition which may be treated with a 5-HT2 modulator the likelihood whether said subject will be responsive or non-responsive to treatment with a 5-HT2 modulator, the method comprising detecting the presence or absence of DNA encoding the Tyr452 and/or His452 alleles of the 5-HT2A gene in a biological sample obtained from said subject.

Description

ASSESSMENT OF THE RESPONSIVENESS OF INDIVIDUALS TO MODULATORS OF THE 5-HTj RECEPTORS, ESPECIALLY THE 5-HT.A RECEPTOR

The present invention relates to methods of assessing the responsiveness of individuals to therapeutic agents which interact with 5-HT2 receptors, in particular the 5- HT2A receptor, and especially to neuroleptic agents such as clozapine.

Schizophrenia is a devastating psychiatric disease for which there is currently no cure, although advances are now being made in understanding its causes and controlling its symptoms. In general the age of onset is in late adolescence and it is a lifelong illness with a very poor prognosis. Subjects suffering from schizophrenia may exhibit positive symptoms, for example delusions and hallucinations, and /or negative symptoms such as withdrawal, isolation and demotivation leading ultimately to social decline and suicide. There are around 600,000 schizophrenics at any one time in the UK and their care has been reported to cost approximately 1.6% of the total healthcare budget. Most of these costs are non-drug, community and hospital costs. Therefore better targetting of effective drug treatments has the potential for considerable economic savings. (British Journal of Psychiatry, (1994) 165 (suppl.25), 18-21).

Since the 1950's antipsychotic drugs (neuroleptics) have been available and are used with varying degrees of success to treat the positive symptoms of schizophrenia. However, the majority of these agents ("typical" neuroleptics) have little effect on the negative symptoms of schizophrenia and furthermore have a number of side effects, the most distressing of which are movement disorders known as extrapyramidal side effects (eps). Examples of typical neuroleptics include haloperidol and sulpiride. Over 90% of patients in the UK are treated with such traditional antipsychotics but some 30% of patients fail to respond. The therapeutic effect of typical antipsychotic agents is believed to be exerted principally via blockade of dopamine D2 receptors; however this mechanism is also thought to be responsible for the extrapyramidal side effects.

More recently second generation antipsychotic agents, so-called "atypical" neuroleptics, having enhanced efficacy and fewer side effects have been developed. These compounds appear to have a lower affinity for D2 receptors than do the typical neuroleptics but they also interact with other receptors, eg the dopamine D4 receptor and serotonin receptors, notably the serotonin 5-HT2A and 5-HT2C receptors. Atypical neuroleptics provide advantages in that they improve both the positive and negative symptoms of schizophrenia and cause virtually no eps. An example of this type of drug is clozapine. However, its use has been severely limited by controversy over its propensity to produce neutropenia and its expense; hence it is reserved for the treatment of schizophrenia in subjects who do not respond to other neuroleptics. In addition there remains a proportion of patients who are resistant to treatment even with clozapine. A test, therefore, that helps to predict those patients most likely to benefit would be a valuable clinical decision making tool.

Further examples of "atypical" neuroleptics include risperidone, olanzapine, seroquel, sertindole and ziprasidone; all are antagonists at both dopamine D2 and 5- HT2A receptors.

Once schizophrenia has been diagnosed it is clearly desirable to select and administer the most appropriate therapy as quickly as possible. At present treatment with neuroleptics is largely a matter of trial and error, as there is no way of determining in advance whether a patient is likely to be responsive to drug treatment. Hence a patient may undergo several courses of treatment with various antipsychotic agents before non- responsiveness is established. In view of the side effects of these drugs it would be highly beneficial to avoid giving them to patients who may never respond to the treatment (non-responders); this is particularly important in the case of clozapine, in view of its known toxicity profile. Furthermore, it is generally found that the long-term outcome of the disease is improved if a patient is given the most effective drug therapy at the outset, rather than after one or more inappropriate drugs. Therefore a means of targetting those patients more likely to respond to drug therapy would be advantageous. Recently it has been found that the human serotonin receptor gene encoding for the 5-HT2A receptor exhibits a coding mutation His452Tyr, comprising two alleles, His452 and Tyr452, which have been found to occur in the general population with a frequency of 91% and 9% respectively, with an observed heterozygosity of 16.4%. These alleles give rise to three distinct genotypes, His452/His452; His452/Tyr452 and Tyr452/Tyr452. (Lancet, Vol 346, No. 8979, p908-909, 30 Sept 95). However, no association between these variants and response to clozapine amongst a group of schizophrenic patients has been reported.

It has now surprisingly been found that in a population of diagnosed schizophrenic patients treated with clozapine the distribution of alleles His452 and Tyr452 between patients who respond to treatment with clozapine (hereinafter "responders") and those who fail to respond to such treatment (hereinafter "non- responders") is significantly different from that in the general population. In particular the proportion of non-responders found to be homozygous for the Tyr452 allele was higher than would be expected from the frequency of this genotype in the general population. However, amongst the patients found to be homozygous for the His452 allele or those who were heterozygous, there was no significant difference between the proportions of responders and non-responders. Thus there is a correlation between homozygosity for the Tyr452 allele and failure to respond to treatment with clozapine. This allelic variation can therefore be utilised to predict which patients are least likely to repond to treatment with clozapine.

The present invention therefore provides an objective method for use in assessing in a subject the likelihood whether said subject will be non-responsive to treatment with clozapine, the method comprising detecting the presence of DNA encoding the Tyr452 allele and/or the His452 allele of the 5-HT2A gene in said subject.

The method of the present invention may also be used more generally in assessing whether a subject is likely to be responsive or non-responsive to a therapeutic agent which acts at 5-HT2 receptors, in particular the 5-HT2A receptor. Compounds acting at these receptors will hereinafter be referred to as 5-HT2 and 5-HT2A modulators. Such compounds include both typical and atypical neuroleptic agents

The present invention therefore provides a method for use in assessing whether a subject will be responsive or non-responsive to treatment with a 5-HT2 modulator, the method comprising testing for and detecting the presence or absence of DNA comprising the Tyr452 allele and/or the His452 allele of the 5-HT2A g^ene in said subject.

Preferably, the method is used in assessment of the likelihood of response or non-response to a 5-HT2A modulator. 5-HT2 and 5-HT2 modulators include agonists and antagonists at these receptors. Such compounds may exert a variety of therapeutic effects. Thus for example 5-HT2A antagonists may be of potential use in the treatment of CNS disorders such as schizophrenia, depression, manic-depressive illness, obsessive compulsive disorders, panic disorders, post-traumatic stress disorders, generalised anxiety disorders, feeding disorders such as anorexia and bulimia, anhedonia, sexual dysfunction, premenstrual syndrome, migraine, epilepsy, Alzheimers disease, sleep disorders, withdrawal from drug abuse such as cocaine, ethanol, nicotine and benzodiazepines, drug eg cocaine craving, and also disorders associated with spinal trauma and/or head injury such as hydrocephalus.

In a particularly preferred embodiment the invention provides a method for use in assessing in a subject diagnosed as suffering from schizophrenia the likelihood that said subject will be non-responsive or responsive to a neuroleptic agent, the method comprising testing for and detecting the presence or absence of DNA comprising the Tyr452 allele and/or the His452 allele of the 5-HT2 gene in said subject. The neuroleptic agent is preferably a modulator of a 5-HT2 receptor, especially a 5-HT2A receptor modulator. Advantageously the neuroleptic agent is an 'atypical' neuroleptic agent such as, clozapine, risperidone, olanzapine, seroquel, sertindole or ziprasidone. Most preferably the neuroleptic agent is clozapine. Preferably the method is used to assess likelihood of non-response. In the case of clozapine, the presence of 5-HT2A Tyr452 alone, ie where the subject is homozygous for the Tyr452 allele, indicates that the subject will be less likely to respond (ie will have a lower probability of response) to treatment with this agent. In this context 'lower probability' means that the likelihood of response is lower than the 'normal' response rate, ie the response rate observed for a random group of schizophrenic patients. It will of course be understood by practitioners skilled in the treatment of conditions such as those listed above, in particular schizophrenia, that the method of the present invention does not give a precise or absolute identification of responders and non- responders but rather indicates the degree or likelihood of responsiveness and so can be used to aid and guide the clinical judgement of the physician. Those skilled in the treatment of schizophrenia will also appreciate that the terms "responders" and "non-responders" as used herein are terms well known in the art. In the clinic the degree of responsiveness to a neuroleptic agent such as clozapine is generally assessed according to well-established rating scales, such as the Global Assessment Scale (GAS) or the Brief Psychiatric Rating Scale (BPRS). In a further embodiment the present invention provides a method for assessing whether a subject suffering from a condition which may be treated with a 5-HT2 modulator will be responsive or non-responsive to treatment with a 5-HT2 modulator, said method comprising the steps of:

(i) testing for the presence or absence of DNA encoding the Tyr452 and/or His452 allele of the 5-HT2A g^ene in sample containing DNA obtained from said subject, and

(ii) comparing the result with a pre-determined correlation between the distribution of Tyr452 and/or His452 alleles and the response to said 5-HT2 modulator obtained for a population of subjects suffering from said condition. The pre-determined correlation utilised in step (ii) may itself be obtained by the following series of steps: selecting a population or cohort of subjects diagnosed as suffering from a specified condition; treating said cohort with a specified 5-HT2 (eg 5-HT2A) modulator; monitoring the outcome of said treatment and identifying responders and non-responders to the said treatment, taking from said cohort biological samples containing DNA and testing this for the presence or absence of the Tyr452 and/or His452 alleles of the 5-HT2A gene; analysing the distribution of alleles as between responders and non-responders; making a comparison with the distribution of alleles in a control group of subjects, not suffering from said condition; performing a statistical analysis to determine if there is a statistically significant association between presence or absence of the Tyr452 and/or His452 alleles and response to treatment.

The present invention thus also provides a method for assessing whether a subject suffering from a condition which may be treated with a 5-HT2 (eg a 5-HT2A) modulator will be responsive or non-responsive to treatment with a specified 5-HT2 modulator, said method comprising the steps of:

(i) correlating the distribution of 5-HT2A Tyr452 and 5-HT2A His452 alleles in a population of subjects suffering from a specified condition requiring treament with a 5-HT2 modulator with observed clinical response to said modulator; (ii) testing for the presence or absence of DNA encoding the Tyr452 and/or His452 allele of the 5-HT2A g^ene in ^a sample containing DNA obtained from said subject, and (iii) comparing the result obtained in (ii) with the correlation between the distribution of Tyr452 and His452 alleles and the response to said 5-HT2 modulator obtained in (i). Step (i) will itself comprise taking from the subjects biological samples containing DNA and testing this for the presence or absence of the alleles. It will be appreciated that where the correlation in (i) is known , eg as in the case of clozapine, the process comprises only steps (ii) and (iii).

In a yet further aspect the invention provides a method for generating a model to assess whether a subject is likely to be responsive or non-responsive to treatment with a 5HT2 (eg a 5-HT2 ) modulator which method comprises: (i) treating a cohort of patients diagnosed as suffering from a specified condition (eg schizophrenia) which may be treated with a 5HT2 modulator (eg clozapine) with said 5HT2 modulator;

(ii) assessing the outcome of treatment so as to define responders and non-responders according to pre-determined criteria; (iii) obtaining DNA samples from the cohort of patients in (i);

(iv) obtaining DNA samples from a control group of subjects diagnosed as not suffering from said condition;

(v) analysing the samples obtained in (iii) and (iv) to identify whether they encode the Tyr452 allele or the His452 allele of the 5-HT2A gene or both alleles; (vi) calculating the distribution of Tyr452 and His452 alleles in the samples from

(iii) for responders and non-responders; (vii) calculating the distribution of Tyr452 and His452 alleles in the samples from (iv);

(viii) comparing the distributions obtained in (vi) and (vii); (ix) comparing the distribution obtained in (vi) as between responders and non- responders

(x) performing statistical analysis on the results from (viii) and (ix) to generate a model for asssessing the probability of response to treatment with said 5HT2 modulator, based on whether a subject possesses the Tyr452 and/or His452 allele. The invention also provides a method for assessing whether a subject suffering from a condition which may be treated with a 5-HT2 (eg a 5HT2 ) modulator will be responsive or non-responsive to treatment with a specified 5-HT2 modulator, which comprises comparing said subject's genotype with the model described above.

It will be appreciated that in any of the above methods, one or more of the steps may be effected by a computer-controlled system. Thus, for example, once the biological samples have been obtained, genotyping may be carried out by a computer- controlled robotic system. A computer-controlled system may also be configured to make the comparison between the subject to be assessed and a pre-determined correlation or model. A computer controlled system may also be configured to give either a positive or negative readout depending on the outcome of the comparison. The present invention therefore extends to such computer-controlled or computer-implemented methods. The step of testing for and detecting the presence of DNA encoding Tyr452 and/or His452 alleles may be carried out either directly or indirectly by any suitable means, such as by techniques well known in the art, and is preferably carried out ex vivo All generally involve the step of collecting a sample of biological material containing DNA from the subject, and then detecting which alleles the subject possesses from that sample. For example, the detecting step may be carried out by collecting a biological sample containing DNA from the subject, and then determining the presence of DNA comprising a Tyr452 and/or His452 allele in the biological sample. Any biological sample which contains the DNA of that subject may be employed, including tissue samples and blood samples, with blood cells being a particularly convenient source. Determining the presence of DNA comprising a Tyr452 and or His452 allele may be carried out with an oligonucleotide probe labelled with a suitable detectable group; by means of an amplification reaction such as a polymerase chain reaction or ligase chain reaction (the product of which amplification reaction may then be detected with a labelled oligonucleotide probe) or by means of restriction nuclease digestion and electrophoretic separation to detect restriction fragment length polymorphism (RLFP). Further, the detecting step may include the step of detecting whether the subject is heterozygous or homozygous for the gene encoding a Tyr452 or His452 allele. Numerous different oligonucleotide probe assay formats are known which may be employed to carry out the present invention.

It will readily be appreciated that the detecting steps may be carried out directly or indirectly. Thus, for example, any of the techniques described above may be used to detect either the Tyr452 allele or the His452 allele. If only the Tyr452 allele is detected in the subject, then it is determined that the subject is homozygous for allele Tyr452; but if allele His452 is detected in the subject, either alone or in addition to allele Tyr452 then it is determined that the subject is either homozygous for His452 or heterozygous. The present invention has utility in enabling improvements in the clinical management of patients suffering from schizophrenia. By identifying in advance of treatment those who are not likely to respond to neuroleptics such as clozapine, it will be possible to avoid unecessary and non-beneficial adminstration of such drugs together with the associated side effects and costs and instead to select a more appropriate form of therapy. Thus the invention provides direct benefits to the patient in terms of indicating the most appropriate therapy as early as possible in the treatment process and is of wider benefit in terms of health economics.

In a further embodiment therefore the present invention provides a method of treating a condition which requires treatment with a 5-HT2 modulator, said method comprising:

(i) testing for DNA comprising allele Tyr452 and/or His452 in a sample of biological material containing DNA obtained from a subject suffering from said condition, and

(ii) if DNA comprising the His452 allele, either alone or in the presence of the Tyr452 allele, is present in the sample, treating said subject with a 5-HT2 modulator. Preferably the condition is schizophrenia. The 5-HT2 modulator is preferably a neuroleptic agent, such as clozapine.

In addition the invention has utility in enabling effective and efficient design of clinical trials with 5-HT2 modulators such as neuroleptic agents. Thus in comparative trials with two or more neuroleptics, patients who are not likely to respond to either or any of the agents can be excluded.

It is believed that the presence of the Tyr452 allele is also associated with a greater probability or risk of developing schizophrenia. Thus the presence of this allele can be used prognostically in assessing the likelihood that a given subject will develop schizophrenia. The present invention therefore also provides a method for use in assessing whether a subject is likely to develop schizophrenia, the method comprising testing for and detecting the presence or absence of DNA comprising the Tyr452 allele and/or the His452 allele of the 5-HT2 ne in said subject.

In a further embodiment the invention also provides an assay suitable for use in the methods of the present invention said assay comprising means for determining the presence of DNA encoding allele Tyr452 and/or allele His452 in a biological sample.

The present invention also provides a kit suitable for use in the methods of the present inventions, said kit comprising

(a) means for testing for the presence or absence of DNA encoding allele Tyr452 and/or His452 in a sample of human DNA; (b) reagents for use in the detection process.

The means for testing preferably comprises a labelled probe or a restriction enzyme. The reagents may comprise for example diluents, wash solutions and control solutions.

In a further embodiment the present invention provides a cell line which expresses DNA encoding the human 5-HT2A Tyr452 and/or His452 alleles. Preferably the cell line expresses DNA encoding the human 5-HT2A Tyr452 allele. Such cell lines can be obtained using known recombinant techniques and methodology.

In a yet further embodiment the present invention also provides a transgenic animal, in particular a transgenic mammal such as a mouse, which expresses the human 5-HT2A gene containing the Tyr452 allele. Such a transgenic animal may be used to screen for and identify novel antipsychotic agents that are likely to be effective in patients who do not respond to currently available neuroleptics such as clozapine. Transgenic animals homozygous for the Tyr452 allelic variant of the h5HT2A gene may be obtained using procedures which are standard in the field of genetic engineering.

SUBSTITUTE SHEET (RULE 2^β) EXAMPLE

Method Genotyping for His452Tyr polymorphism was carried out using blood samples obtained from individuals diagnosed as suffering from schizophrenia (DSM III) and undergoing treatment with clozapine. The individuals were also separately assessed for responsiveness to clozapine treatment: non-response is defined as a drop of <20 points on GAS or <20% on BPRS after six weeks treatment. The results were correlated as shown in the table below.

Detection of His452Tyr polymorphism

Methods described by Erdmann et al (1996, Human Genetics, ref: (1996) 22(5) p614- 619) with modification of PCR conditions as follows: Standard PCR carried out in 25μl volume containing lOOng genomic DNA, lOpmol each primer, 50mM KC1, lOnM Tris-HCl, 2.0mM MgC_2, 0.01% gelatin, 200μM dNTPs and 1 U Taq DNA polymerase. Primers:

5'-CAAAGCAAGATGCCAAGACA-3' 5-GGCATACAGATATGATCGTTGG-3'

PCR programme:

95°C for 1', followed by 35 cycles of 95°C for 45", 58°C for 45", 72°C for 45", final elongation step of 72°C for 10'. Allele detection: PCR product digested with Bbvl (15μl of PCR product, 2μl 1OX RE buffer, 2μl of

H2O, lμl (10 units) of Bbvl) at 37°C for a minimum of 2 hours digested products run in a 3% Agarose gel for 3 hours at 70v: PCR product: 248bp

Allele His452: digested in two fragments of 180bp and 68bp Allele Tyr452: uncut (248 bp).

Results

5-HT2_a Receptor Gene and Clozapine Analysis of His452Tyr Polymorphism

Genotypic frequencies: χ = 2.84 p = 0.24 Allelic frequencies: χ² = 1.92 p = 0.16

Claims

Claims:

1. A method for use in assessing in a subject suffering from a condition which may be treated with a 5-HT2 modulator the likelihood whether said subject will be responsive or non-responsive to treatment with a 5-HT2 modulator the method comprising detecting the presence or absence of DNA encoding the Tyr452 and or His452 alleles of the 5-HT2A gene in a biological sample obtained from said subject.

2. A method according to Claim 1 wherein the 5-HT2 modulator acts at the 5-HT2A receptor.

3. A method according to Claim 1 or Claim 2 wherein the 5-HT2 modulator is an antagonist at said 5-HT2 receptor.

4. A method according to any of claims 1 to 3 wherein the condition is schizophrenia.

5. A method according to any of claims 1 to 4 wherein the5-HT2 modulator is a neuroleptic agent.

6. A method as claimed in claim 5 wherein the neuroleptic agent is atypical.

7. A method as claimed in claim 6 wherein the neuroleptic agent is clozapine.

8. A method as claimed in claim 6 wherein the neuroleptic agent is risperdone.

9. A method as claimed in claim 6 wherein the neuroleptic agent is selected from olanzapine, seroquel, sertindole and ziprasidone.

10. A method for assessing whether a subject suffering from a condition which may be treated with a 5-HT2 modulator will be responsive or non-responsive to treatment with a 5-HT2 modulator, said method comprising the steps of: (i) testing for the presence or absence of DNA encoding the Tyr452 and/or His452 alleles of the 5-HT2A g^enc in a sample containing DNA obtained from said subject, and

(ii) comparing the result with the correlation between the distribution of Tyr452 and His452 alleles and the response to said 5-HT2 modulator obtained for a population of subjects suffering from said condition.

11. A method according to claim 10, said method comprising the steps of: (i) correlating the distribution of Tyr452 and His452 alleles in a population of subjects suffering from a specified condition requiring treament with a 5-HT2 modulator with observed clinical response to said modulator; (ii) testing for the presence or absence of DNA encoding the Tyr452 and/or His452 allele of the 5-HT2A gene in a sample containing DNA obtained from said subject, and (iii) comparing the result obtained in (ii) with the correlation between the distribution of Tyr452 and His452 alleles and the response to said 5-HT2 modulator obtained in (i) for a population of subjects suffering from said condition.

12. A method for generating a model to assess whether a subject is likely to be responsive or non-responsive to treatment with a 5HT2 modulator which method comprises:

(i) treating a cohort of patients diagnosed as suffering from a specified condition which may be treated with a 5HT2 modulator with said 5HT2 modulator; (ii) assessing the outcome of treatment so as to define responders and non-responders according to pre-determined criteria;

(iii) obtaining DNA samples from the cohort of patients in (i);

(iv) obtaining DNA samples from a control group of subjects diagnosed as not suffering from said condition; (v) analysing the samples obtained in (iii) and (iv) to identify whether they encode the Tyr452 or His452 allele of the 5-HT2A gene or both alleles;

(vi) calculating the distribution of Tyr452 and His452 alleles in the samples from

(iii) for responders and non-responders;

(vii) calculating the distribution of Tyr452 and His452 alleles in the samples from (iv);

(viii) comparing the distributions obtained in (vi) and (vii); (ix) comparing the distribution obtained in (vi) as between responders and non- responders;

(x) performing statistical analysis on the results from (viii) and (ix) to generate a model for asssessing the probability of response to treatment with said 5HT2 modulator, based on whether a subject possesses the Tyr452 and/or His452 allele of the 5-HT2A gene.

13. A method for assessing whether a subject suffering from a condition which may be treated with a 5-HT2 modulator will be responsive to treatment with a specified 5-HT2 modulator, which comprises comparing said subject's genotype with a model as claimed in claim 12.

14. A method according to any of claims 10 to 13 wherein at least one step is effected by a computer controlled system.

15. A method according to any of claims 10, 11, 13 or 14 which is configured to detect the Tyr452 allele.

16. A method according to any of claims 1 to 15 wherein detection of the Tyr452 allele is indicative of poor likelihood response to treatment with said 5-HT2 modulator.

17. A method according to any of claims 10 to 16 wherein the condition is schizophrenia.

18. A method according to any of claims 10 to 17 wherein the 5-HT2 modulator is a neuroleptic agent.

19. A method for use in assessing whether a subject is likely to develop schizophrenia, the method comprising testing for and detecting the presence or absence of DNA comprising the Tyr452 allele and/or the His452 allele of the 5-HT2A gene in said subject.

20. A cell line which expresses DNA encoding the human -HT2A Tyr452 and/or His452 alleles of the 5-HT2A gene.

21. An assay comprising means for determining the presence or absence of DNA comprising Tyr452 and/or His452 alleles in a biological sample.

22. A kit suitable for use in assessing the responsiveness of a subject to a 5HT2 modulator, said kit comprising:

(a) means for testing for the presence or absence of DNA encoding Tyr452 and/or His452 alleles in a sample of human DNA;

(b) reagents for use in the detection process.

23. A transgenic mammal which expresses the human 5-HT2A gene comprising the Tyr452 allele.

24. A transgenic mammal which expresses the human 5-HT2A gene comprising the His452 allele.

25. Use of a transgenic mammal according to claim 23 or claim 24 in screening compounds for antipsychotic activity.