AU729117B2 - Methods and kits for diagnosis and monitoring treatments of psychiatric disorders - Google Patents

Description

WO 97/20211 PCT/IL96/00166 1 METHODS AND KITS FOR DIAGNOSIS AND MONITORING TREATMENTS OF PSYCHIATRIC DISORDERS FIELD OF THE INVENTION The present invention relates to differential biochemical diagnosis of psychiatric disorders and, more particularly, to the use of the determination of the function and level of certain proteins in the accurate diagnosis of a variety of psychiatric disorders and for accurately following the progress of treatment of such disorders.

BACKGROUND OF THE INVENTION Presently used differential diagnosis techniques for the classification of psychiatric disorders are largely phenomenological. Diagnosis is based on the observation of certain symptoms and the course of the disorder. The presently used Diagnostic and Statistical Manual of Mental Disorders (DSM IIIR) is empirically-oriented and makes use of operational criteria which are predominantly phenomenological, Considerable research effort has been directed at attempting to decipher the biological underpinnings of psychiatric disorders. Various hypotheses have been put forward as to the involvement of a variety of neurotransmitters and their receptors in the pathogenesis of psychiatric disorders and the mechanisms of various biological treatments used to combat these disorders. Yet, no biological or biochemical routine laboratory cross diagnostic tests which are capable of supporting psychiatric diagnosis are available. The need for laboratory tests of a biochemical nature, which will help in differential diagnosis in psychiatry is enormous and well recognized.

WO 97/20211 PCT/IL96/00166 2 There is a widely recognized need for, and it would be highly advantageous to have, a scientifically-established biochemical assay for the differential diagnosis of psychiatric disorders. Such an assay would help in the differential diagnosis of hospitalized patients, outpatients, and patients treated by general practitioners.

Of special importance is the differential diagnosis following the first psychotic episode between the two major psychoses, schizophrenia and manic-depressive illness.

For example, using presently known phenomenological diagnosis techniques there are no pathognomonic signs which unambiguously diagnose for schizophrenia. Hence, a follow-up diagnosis taking place at least six months following the initial diagnosis is required in order to reach a conclusive determination of schizophrenia.

Differentiating between the major psychoses has important prognostic and treatment implications and also has a number of highly desirable social outcomes. An important aspect of a biochemical assay for mental disorders such as major depression, is that it makes it possible for the general practitioner, who treats about half of major depression patients, and for mental health professionals, to decide on the desirability of pharmacological antidepressant treatments.

Another disorder which is normally first treated by general practitioners, and which usually requires a series of physical examinations and a battery of expensive laboratory studies to exclude a physical disorder, is panic disorder. An established biochemical assay for this disorder will aid in the early treatment of these patients using presently available, and effective, pharmacological treatments.

WO 97/20211 PCT/IL96/00166 3 Various specific treatments are applied against mood disorders. Mood stabilizers like lithium and electroconvulsive treatment (ECT) are effective for the treatment of mania and depression, and for the prevention of both affective states. Antidepressant drugs are used in the treatment and prevention of depression. All these treatments do not exert their therapeutic effects immediately, but within three weeks or one month. About 30% of the patients do not respond to a certain treatment, but may respond to another kind of treatment. It is therefore of prime importance also to have a biochemical test that enables to biochemically assess the responsiveness of a patient to a psychiatric treatment.

If the altered biochemical parameters determined in psychiatric patients by the diagnostic assay are indicators of the affective state of the illness, and are normalized following an efficient psychiatric treatment, then such an assay may also aid the practitioner to biochemically follow and gauge the effectiveness of these treatments.

There are indications in the prior art for possible involvement of signal transduction, and particularly for the involvement of members of the family of receptor-coupled G-proteins, in psychiatric disorders. For example, a reduction in the density of 1-adrenergic receptors in leukocyte membrane preparations has been shown in patients with depression, and a decreased B-adrenergic receptor responsiveness has been shown in leukocytes of patients suffering from depression, through measurements of B-adrenergic receptor-stimulated cAMP production. The degree of these changes in B-adrenergic receptor density and responsiveness were found to be correlated with the severity of depression (for discussion see Mazzola-Pomietto et al., 1994) The earliest recognized event in signal transduction beyond B-adrenergic as well as other receptors, involves coupling of the activated receptor with a G-protein.

WO 97/20211 PCT/IL96/00166 4 The family of G-proteins currently includes 12-15 already known individual proteins. G-proteins are composed of three subunits: a, P, and y. The a- subunit contains the binding site for guanine nucleotides, and possesses GTPase activity.

The a-subunit also contains the site for nicotinamide adenine nucleotide (NAD) dependent ADP-ribosylation catalyzed by bacterial toxins. The heterogeneity of the a-subunit serves to divide G-proteins into the major classes Gi, Gq).

Receptors for stimulatory hormones, P-adrenergic receptors, interact with Gs, the G-protein which activates adenylate cyclase, while those of inhibitory ligands, M2-muscarinic receptors, interact with adenylate cyclase inhibitory regulator Gi. Gq may be involved in coupling receptor activation to the breakdown of phosphatidylino-sitol -4,5-biphosphate by phospholipase

C.

Stimulation by an agonist increases the binding of guanine nucleotide to Gproteins and leads to their activation. Based on this characteristic, guanine nucleotide binding assay, is used as an established test for G-protein function.

The function of receptor-coupled G-proteins was found to be differentially attenuated by lithium (Avissar et al., 1988), other antibipolar treatments, and antidepressant drugs (Avissar and Schreiber 1992a,b). Moreover, hyperactivity of B-adrenergic coupled and muscarinic-coupled G-proteins was detected in MNL of patients with mania (Schreiber et al., 1991), and an elevated level of a subunit of Gs-protein (Gas) was found in postmortem cerebral cortices of bipolar patients (Young et al., 1991).

None of these publications address the possibility of using these findings for diagnosis of a psychiatric disorder, and certainly, even if taken together, there is no indication for the possibility of differential diagnosis of the major psychiatric disorders.

In the present invention measurements of receptor-coupled G-proteins function and level are used for differential diagnosis of a patient with a psychiatric disorder, and also for evaluation of the responsiveness of patients to psychiatric treatments.

SUMMARY OF THE INVENTION In a first aspect, the present invention provides a method for differential diagnosis of mania, depression, schizophrenia and panic, comprising the steps of: measuring the function of P-adrenergic receptor-coupled Gprotein muscarinic receptor-coupled G-protein and dopamine receptor-coupled G-protein (D-Gp) in a blood sample from a patient exhibiting psychiatric disorder symptoms, wherein the function is agonistinduced activation of G-protein; and comparing the function values of each G-protein from step a) with a set of G-protein function values obtained from normal individuals; whereby i) mania is diagnosed by an increase in the function of both p- Gp and M-Gp as compared to normal individuals; ii) depression is diagnosed by decrease in the function of both P-Gp and M-Gp as compared to normal individuals; iii) schizophrenia is diagnosed by an increase in the function of D-Gp and a normal like function in P-Gp and M-Gp as compared to normal individuals; and iv) panic is diagnosed by an increase in the function of P-Gp and a decrease in the function of M-Gp, as compared to normal individuals.

In a second aspect, the present invention provides a method for differential diagnosis of mania, depression and panic, comprising the steps of: measuring the function of p-adrenergic receptor-coupled Gprotein (P-Gp) and muscarinic receptor-coupled G-protein (M-Gp) to the level of either p-Gp or M-Gp in a blood sample from a patient exhibiting psychiatric disorder symptoms, wherein the function is agonist-induced activation of G-protein; and comparing the function or level values of each G-protein from step a) with a set of G-protein function or level values obtained from normal individuals; whereby i) mania is diagnosed by an increase in the function of both P- Gp and M-Gp or by an increase in the level of either p-Gp or M-Gp, as compared to normal individuals; ii) depression is diagnosed by a decrease in the function of both P-Gp and M-Gp or by a decrease in the level of either P- Gp or M-Gp, as compared to normal individuals; and iii) panic is diagnosed by an increase in the function of P-Gp and a decrease in the function of M-Gp or by a normal like level of either P-Gp or M-Gp, as compared to normal individuals.

In a third aspect, the present invention provides a method for differential diagnosis of mania, schizophrenia and panic comprising the steps of: measuring the function of muscarinic receptor-coupled G-protein (M-Gp) and dopamine receptor-coupled G-protein (D-Gp) in a blood sample from a patient exhibiting psychiatric disorder symptoms, wherein the function is agonist-induced activation of G-protein; and comparing the function values of each G-protein from step a) with a set of G-protein function values obtained from normal individuals; whereby i) mania is diagnosed by an increase in the function of M-Gp as compared to normal individual; ii) schizophrenia is diagnosed by an increase in the function of D-Gp and a normal like function of M-Gp as compared to normal individuals; and iii) panic is diagnosed by a decrease in the function of M-Gp, as compared to normal individuals.

In a fourth aspect, the present invention provides a method for differential diagnosis of depression, schizophrenia and panic, comprising the steps of: measuring the function of p-adrenergic receptor-coupled Gprotein (p-Gp) and dopamine receptor-coupled G-protein (D-Gp) in a blood sample from a patient exhibiting psychiatric disorder symptoms, wherein the function is agonist-induced activation of G-protein; and comparing the function values of each G-protein from step a) with a set of G-protein function values obtained from normal individuals; whereby i) depression is diagnosed by a decrease in the function of P- Gp as compared to normal individuals; ii) schizophrenia is diagnosed by an increase in the function of D-Gp and a normal like function of P-Gp as compared to normal individuals; and iii) panic is diagnosed by an increase in the function of P-Gp as compared to normal individuals.

In a fifth aspect, the present invention provides a method for differential diagnosis of mania and schizophrenia, comprising the steps of: measuring the function of dopamine receptor-coupled G-protein (D-Gp) and the function or level of either p-adrenergic receptor-coupled Gprotein (P-Gp) or muscarinic receptor-coupled G-protein (M-Gp) in a blood sample from a patient exhibiting psychiatric disorder symptoms, wherein the function is agonist-induced activation of G-protein; and comparing the function or level of each G-protein from step a) with a set of G-protein function or level values obtained form normal individuals; whereby i) mania is diagnosed by an increase in the function or level of either P-Gp or M-Gp as compared to normal individuals; and ii) schizophrenia is diagnosed by an increase in the function of D-Gp and a normal-like function or level of either P-Gp or M-Gp as compared to normal individuals.

In a sixth aspect, the present invention provides a method for differential diagnosis of mania and panic, comprising the steps of: measuring the function of muscarinic receptor-coupled G-protein (M- Gp) or the level of either p-adrenergic receptor-coupled G-protein (P-Gp) or (M-Gp) in a blood sample from a patient exhibiting psychiatric disorder symptoms, wherein the function is agonist-induced activation of G-protein; and comparing the function or level values of each G-protein from step a) with a set of G-protein function or level values obtained from normal individuals; whereby i) mania is diagnosed by an increase in the function of M-Gp or by an increase in the level of either P-Gp or M-Gp, as compared to normal individuals; and ii) panic is diagnosed by a decrease in the function of M-Gp or by a normal like level of either P-Gp or M-Gp, as compared to normal individuals.

In a seventh aspect, the present invention provides a method for differential diagnosis of depression and schizophrenia, comprising the steps of: measuring the function of dopamine receptor-coupled G-protein (D-Gp) and the function or level of either p-adrenergic receptor-coupled Gprotein (P-Gp) or muscarinic receptor-coupled G-protein (M-Gp) in a blood sample from a patient exhibiting psychiatric disorder symptoms, wherein the function is agonist-induced activation of G-protein; and comparing the function or level of each G-protein from step a) with a set of G-protein function or level values obtained from normal individuals; whereby i) depression is diagnosed by a decrease in the function or level of either P-Gp or M-Gp as compared to normal individuals; and ii) schizophrenia is diagnosed by an increase in the function of D-Gp and a normal-like function or level of either P-Gp or M-Gp as compared to normal individuals.

In an eighth aspect, the present invention provides a method for diagnosing mania, comprising the steps of: measuring the function of P-adrenergic receptor-coupled Gprotein (P-Gp) and muscarinic receptor-coupled G-protein (M-Gp) or measuring the level of either P-Gp or M-Gp in a blood sample from a patient exhibiting psychiatric disorder symptoms, wherein the function is agonistinduced activation of G-protein; and comparing the function or level of each G-protein from step a) with a set of G-protein function or level values obtained from normal individuals; whereby mania is diagnosed by an increase in the function of p-Gp and M-Gp or by an increase in the level of either P-Gp or M-Gp, as compared to normal individuals.

In a ninth aspect, the present invention provides a method for diagnosing depression, comprising the steps of: measuring the function of p-adrenergic receptor-coupled Gprotein (B-Gp) and muscarinic receptor-coupled G-protein (M-Gp) or measuring the level of either p-Gp or M-Gp in a blood sample from a patient exhibiting psychiatric disorder symptoms, wherein the function is agonistinduced activation of G-protein; and comparing the function or level of each G-protein from step (a) with a set of G-protein function or level values obtained from normal individuals; whereby depression is diagnosed by a decrease in the function of P-Gp and M-Gp or by a decrease in the level of either P-Gp or M-Gp, as compared to normal individuals.

In a tenth aspect, the present invention provides a method for diagnosing schizophrenia, comprising the steps of: measuring the function of dopamine receptor-coupled G-protein (D- Gp) and the function or level of P-adrenergic receptor-coupled G-protein (p- Gp) or muscarinic receptor-coupled G-protein (M-Gp) in a blood sample from a patient exhibiting psychiatric disorder symptoms, wherein the function is agonist-induced activation of G-protein; and comparing the function or level of each G-protein from step a) with a set of G-protein function or level values obtained from normal individuals; whereby schizophrenia is diagnosed by an increase in the function of D-Gp and a normal like function or level of P-Gp or M-Gp as compared to normal individuals.

In an eleventh aspect, the present invention provides a method for diagnosing panic, comprising the steps of: measuring the function of P-adrenergic receptor-coupled Gprotein (P-Gp) and muscarinic receptor-coupled G protein (M-Gp) in a blood sample from a patient exhibiting psychiatric disorder symptoms, wherein the function is agonist-induced activation of G-protein; and comparing the function of each G-protein from step a) with a set of G-protein function values obtained from normal individuals; whereby panic is diagnosed by an increase in the function of P-Gp and a decrease in the function of M-Gp, as compared to normal individuals.

In a twelfth aspect, the present invention provides a method for gauging the effect of a treatment upon a patient diagnosed with a psychiatric disorder selected from the group consisting of mania, depression and panic, the method comprising: measuring the function of at least one receptor-coupled G protein in a blood sample from the patient prior to the treatment, said receptor-coupled G-protein is selected from the group consisting of p-adrenergic receptorcoupled G-protein (P-Gp) and muscarinic receptor-coupled G-protein (p-Gp) and muscarinic receptor-coupled G-protein wherein the function is agonist-induced activation of G-protein; subjecting the patient to the treatment for a sufficient period of time; measuring the function of the receptor-coupled G-protein in a blood sample from the patient subjected to the treatment of step b); whereby a shift of the function toward the normal values obtained from normal individuals is indicative of an effective treatment.

In a thirteenth aspect, the present invention provides a method for gauging the effect of a treatment upon a patient diagnosed with schizophrenia, the method comprising: a) measuring the function of dopamine receptor-coupled G-protein (D-Gp) in a blood sample from the patient prior to the treatment, wherein the function is agonist-induced activation of G-protein; b) subjecting the patient to the treatment for a sufficient period of time; c) measuring the function of dopamine receptor-coupled G-protein (D-Gp) in a blood sample from the patient subjected to the treatment of step b); whereby a shift of the function toward the normal values obtained from normal individuals is indicative of an effective treatment.

In a fourteenth aspect, the present invention provides a method for gauging the effect of a treatment upon a patient diagnosed with a psychiatric disorder selected from the group consisting of mania and depression, the method comprising: a) measuring the level of at least one receptor-coupled G-protein in a blood sample from the patient prior to the treatment, said receptor-coupled Gprotein is selected from the group consisting of P-adrenergic receptor-coupled G-protein (P-Gp) and muscarinic receptor-coupled G-protein (M-Gp); b) subjecting the patient to the treatment for a sufficient period of time; c) measuring the level of the receptor-coupled G-protein in a blood sample from the patient subjected to the treatment of step b); whereby a shift of the level toward the normal values obtained from normal individuals is indicative of an effective treatment.

In a fifteenth aspect, the present invention provides a method for evaluating a patient's disease progression over a period of time, said patient having been diagnosed with a psychiatric disorder selected from the group consisting of mania, depression and panic, the method comprising: a) measuring the function of at least one receptor-coupled G-protein in a blood sample from the patient, said receptor-coupled G-protein is selected from the group consisting of p-adrenergic receptor-coupled G-protein (P-Gp) and muscarinic receptor-coupled G-protein wherein the function is agonist-induced activation of G-protein; b) measuring the function of the receptor-coupled G-protein in a blood sample from the patient at least one subsequent time; and c) comparing the function of the receptor-coupled G protein obtained in step b) with the function of the receptor-coupled G-protein obtained in step a); whereby a shift of the function over said period of time is indicative of said patient's disease progression.

In a sixteenth aspect, the present invention provides a method for evaluating a patient's disease progression over a period of time, said patient having been diagnosed with schizophrenia, the method comprising: a) measuring the function of dopamine receptor-coupled G-protein (D-Gp) in a blood sample from the patient, wherein the function is agonistinduced activation of G-protein; b) measuring the function of dopamine receptor-coupled G-protein (D-Gp) in a blood sample from the patient at least one subsequent time; and c) comparing the function of dopamine receptor-coupled G-Protein (D-Gp) obtained in step b) with the function of dopamine receptor-coupled Gprotein (D-Gp) obtained in step a): whereby a shift of the function of dopamine receptor-coupled G-protein (D-Gp) over said period of time is indicative of said patient's disease progression.

In a seventeenth aspect, the present invention provides a method for evaluating a patient's disease progression over a period of time, said patient having been diagnosed with a psychiatric disorder selected from the group consisting of mania and depression, the method comprising: a) measuring the level of at least one receptor-coupled G-protein in a blood sample from the patient, said receptor-coupled G-protein is selected from the group consisting of p-adrenergic receptor-coupled G-protein (P-Gp) and muscarinic receptor-coupled G-protein (M-Gp); b) measuring the level of the receptor-coupled G-protein in a blood sample from the patient at least one subsequent time; and c) comparing the level of the receptor-coupled G-protein obtained in step b) with the level of the receptor-coupled G-protein obtained in step a); whereby a shift of the level over said period of time is indicative of said patient's disease progression.

Preferably, the blood sample is a preparation of peripheral blood.

Preferably, the preparation is derived from mononuclear leukocyte (MNL) cells.

In a preferred embodiment, the G-protein function is determined as an agonist-induced increase in guanine nucleotide binding capacity. Preferably, the guanine nucleotide binding capacity is determined using a guanine nucleotide selected from the group consisting of Gpp(NH)p, GTPyS, and GTPazidoanilide. Preferably, the agonist is p-Gp agonist isoproterenol, M-Gp agonist carbamylcholine, or D-Gp agonist dopamine and analogs thereof.

Further, it is preferred that the G-protein level is measured by an immunoassay using an antibody specific to the a subunit of the receptorcoupled G-protein. Preferably, the a subunit is Gas or Gai.

The present invention also provides a kit when used for any method according to the present invention, said kit comprising: agonists of one, two or three receptors, wherein said receptors are selected from the group comprising: p-adrenergic receptor (ii) muscarinic receptor (iii) dopamine receptor, and labeled guanine nucleotide.

Preferably, the p-adrenergic receptor agonist is isoproterenol; muscarinic receptor agonist is carbamylcholine; and dopaminergic receptor agonist is dopamine.

In addition, the present invention provides a kit when used for a certain embodiments of the present invention, said kit comprising: 1 or 2 monoclonal or polyclonal antibodies selected from the group comprising anti-Gas and anti-Gai; detectable probe which is capable of specifically binding to the antibody of and standard samples.

Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.

Other embodiments and aspects of the present invention are set forth in, or arise from the following description of the invention.

WO 97/20211 PCT/IL96/00166 6 BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 A graphic representation depicting non-specific, basal and agonistinduced specific binding of 3 H-Gpp(NH)p to MNL obtained from a healthy volunteer, as described in Example 1.

Figs. 2(a-e) Representative Scatchard plots depicting the results showing basal and agonist-induced 3 H-Gpp(NH)p binding in MNL membranes from a healthy volunteer, and from 4 patients with different psychiatric disorders, as described in Examples 1 and 2.

Figs. 3(a-d) Scatter plots depicting the results showing altered agonist- induced increase in 3 H-Gpp(NH)p binding capacity in MNL membranes from patients with various psychiatric disorders compared to those of healthy volunteers, as described in Examples 1 and 2.

Fig. 4 Bar graphs depicting the results showing the differential pattern of agonist- induced increase in Gpp(NH)p binding capacity in MNL membranes from healthy volunteers healthy volunteers after physical exercise manic patients depressed patients patients with panic disorder and schizophrenic patients as described in Examples 1 and 2.

Fig. 5 A representative immunoblot with MNL protein samples from psychiatric patients and a healthy volunteer obtained with anti-sera against Gas and Gai, as described in Example 3.

Figs. 6(a,b) Scatter plots depicting the results showing the Gas (6a) and Gai (6b) relative immunoreactivity level in MNL from healthy volunteers and WO 97/20211 PCT/IL96/00166 from patients with various psychiatric Example 3.

disorders, as described in Figs. 7(a,b) Graphic representations of the results illustrating the correlation between isoproterenol (7a) and carbamylcholine (7b)-induced increase in 3 H-Gpp(NH)p binding capacity and Beck score, in patients with depression and in healthy volunteers, as described in Example 4.

Fig. 8 A graphic representation of the results illustrating the normalization of p-adrenergic receptor coupled G-protein (P-Gp) function in depressed patients treated with antidepressants, as described in Example Figs. 9(a,b) Graphic representations of the results illustrating time-course improvement of biochemical and psychiatric parameters in depressed patients treated by electroconvulsive treatment, as described in Example DETAILED DESCRIPTION OF THE INVENTION The invention is based on the discovery of a differential pattern of receptorcoupled G-proteins function and immunoreactive level in MNL from patients with the major mental disorders: Mania increase in p-adrenergic and muscarinic receptor-coupled Gproteins (p-Gp and M-Gp) function, and increase in immunoreactive level of Gas and Gai, as compared to normal (control) individuals.

WO 97/20211 PCT/IL96/00166 8 Depression decrease in P-Gp and M-Gp function, and decrease in immunoreactive levels of Gas and Gai, as compared to normal (control) individuals Schizophrenia Increase in dopamine receptor-coupled G-protein (D-Gp) function, as compared to normal (control) individuals; and control-like levels of p-Gp and M-Gp function, and control-like immunoreactive level of Gas and Gai.

Panic Disorder Increase in P-Gp function and decrease in M-Gp function as compared to normal (control) individuals; and control-like immunoreactive level of Gas and Gai.

The invention is further based on the discovery that altered receptor-coupled Gproteins function and level in MNL are biochemical indicators of the effective state of the illness, and that treated asymptomatic patients show normal values.

The present invention provides a cross diagnostic biochemical assay capable of differentiating between mental disorders, based on G-protein functional and/or quantitative measurements. The present invention also provides for biochemical assay for evaluating a patient's responsiveness to a psychiatric treatment.

By the above "function of G-protein" it is meant the agonist-induced activation of G-protein. According to a preferred embodiment of the present invention Gprotein function is determined as agonist-induced increase in guanine nucleotide binding capacity.

Preferred agonists for carrying out the method of the present invention are isoproterenol, carbamylcholine and dopamine, which activate p-adrenergic, muscarinic and dopaminergic receptors respectively. However, other padrenergic, M2-muscarinic and Dl/5-dopaminergic agonists stimulating receptors WO 97/20211 PCT/IL96/00166 9 of similar nature are suitable, and their use is within the scope of the present invention.

According to another preferred embodiment of the present invention the above "quantitative measures" or "immunoreactive level" of G-protein is determined as the immunoreactive level of its a-subunit.

According to a further preferred embodiment of the invention the psychiatric disorders that are diagnosed by the method of the invention are: mania, depression, panic disorder and schizophrenia.

According to a preferred method of the invention, receptor-coupled G-protein function and level are determined in MNL from a patient, however other types of cells may also be suitable for carrying out the method of the invention. For example other peripheral blood cells or elements (such as platelets), or skin cells may be suitable for diagnosis. However it should be noted that different cells may present different levels and types of receptors, and therefore survey must first be performed in an already diagnosed population of patients as well as in a population of healthy subjects, to assess the compatibility of each type of cells for these purposes, and to calibrate the method for when these different cells are used.

In the present invention the determination of the function of one, two or three receptor-coupled G-proteins may be suitably and effectively used for diagnosis of psychiatric disorders, or for gauging the effect of psychiatric treatments. Indeed, depression can be diagnosed based on hypofunction of P-Gp, and mania can be diagnosed based on hyperfunction of M-Gp, with high values of specificity and sensitivity. However, in most cases the function of more than one receptorcoupled G-protein should be determined to enable accurate diagnosis. These include P-Gp and M-Gp function, for mania, depression and panic disorder, and D-Gp function with either [-Gp or M-Gp function, or both, for diagnosing WO 97/20211 PCT/IL96/00166 schizophrenia. Therefore for differential diagnosis of a patient, it is preferred that the function of all three (P-Gp, M-Gp and D-Gp) be determined. The specificity of the test for diagnosing a certain disorder can be controlled by the determination of the minimal requirements and the threshold values.

However, for monitoring the effect of a psychiatric treatment, the determination of the function of one receptor-coupled G-protein is usually sufficient, preferably the one whose function in the acute state of the illness deviates most from the normal level. The evaluation is based on comparison of values obtained before and during the treatment, and a shift toward a normal value indicates responsiveness to the treatment.

According to a preferred embodiment of the present invention, receptor-coupled G-protein function is determined by guanine nucleotide binding essay. However, any other assay which enables the determination of agonist-induced activation of G-protein may also be used to carry out the method of the invention.

According to further preferred embodiment of the invention, the guanine nucleotide used in the assay is Gpp(NH)p. Other guanine nucleotides or analogs may be suitable, and their use is within the scope of the present invention.

These may include for example GTPyS (Wieland T. and Jakobs 1994), or GTP-azidoanilide (a photolable compound that binds irreversibly to the Gprotein following exposure to U V light, Laugwitz K. L. et al.,1994).

According to still another embodiment of the present invention the guanine nucleotide used in the assay is labeled. The label of the guanine nucleotide may be any kind of label which can be detected by appropriate detection means, such as radiolable, fluorescent label, etc. However, instead of being labeled, the bound guanine nucleotide may be detected using a labeled probe, which is capable of specifically binding to the complex G-protein-guanine nucleotide, or which is WO 97/20211 PCT/IL96/00166 11 capable of specifically binding to the guanine nucleotide itself, with the limitation that the probe be contacted with the sample only after separation of unbound material. Any procedure which generates a detectable change, which is proportionaly to the binding of the guanine nucleotide to the G-protein, can be exploited to carry out the method of the invention.

The function of G-proteins, determined as agonist-induced increase in binding capacity, may be calculated from multi-point measures of binding as described in the General Procedures and in Example 1. However, the assay may be simplified using single-point measures, at single concen-tration of guanine nucleotide. For Gpp(NH)p, similar values were obtained based on multiple-point measures and single-point measures, at concentration of 5 mM.

Diagnosis of psychiatric disorders or following the effect of psychiatric treatment according to the present invention may also be based on the determination of the level of Ga-subunits. Determination of the immunoreactive level of Gas and Gai has been performed by immunoblot analysis of MNL membrane-proteins which were first separated on SDS-PAGE. However, it is preferred for routine assays to determine the immunoreactivity of Ga-subunits without prior separation of the sample. Quantitative immuno-assay of different types of Ga-subunits can be performed in crude preparation of membranes (see for example Lesch K.P., Manjii 1992), and their use is in the scope of the present invention.

Determination of the immunoreactive level of Gas and Gai for the method of the invention may also be performed by an ELISA test. A method based on competitive ELISA has been used to quantify various types of Ga-subunits in membranes from brain of rats (Lesch and Manjii 1992). However, other variations of ELISA tests or other immuno-assays can be employed and their use is within the scope of the present invention.

WO 97/20211 PCT/IL96/00166 12 Many other variations in the determination of G-protein function are also possible, for carrying out the method of the invention. For example: guanine nucleotide-G-protein complex may be trapped by contacting the sample at the end of the binding reaction with an immobilized antibody, or with an antibody which is attached to magnetic particles. This enables the washing of the unbound material, and avoids the need for filtration of the sample. Immunoseparation of G-protein-guanine-nucleotide complex has been disclosed in a different method (Friedman et al., 1993). The bound guanine nucleotide may then be detected directly (if it is labeled) or indirectly (by a labeled probe which is capable of specifically binding G-protein-guanine-nucleotide complex).

In another aspect the invention is directed to kits for carrying out the method of the invention. Kits are based on the determination of G-protein function or on the determination of the immunoreactive level of Gas and/or Gai. The kits may be used for diagnosis of psychiatric disorders, or for following the effect of psychiatric treatments.

An illustrative kit based on G-protein function determination according to the method of the invention comprises: agonists of one, two or three receptors, wherein said receptors are selected from the group comprising: B-adrenergic receptor (ii) muscarinic receptor (iii) dopamine receptor labeled guanine nucleotide the manufacturer's instructions for using the kit WO 97/20211 PCT/IL96/00166 13 Criteria for diagnosis of each of the major psychiatric disorders may also be included in the manufacturer's instructions.

An illustrative kit based on Gas and/or Gai determination according to the method of the invention comprises: 1 or 2 monoclonal or polyclonal antibodies selected from the group comprising anti-Gas and anti-Gai; detectable probe which is capable of specifically binding to the antibody of standard samples; the manufacturer's instructions for using the kit.

Standard samples should be included to enable normalization of the results, and comparison to known mean-value of normal subjects. Detailed instructions for normalization should be included in the manufacturer's instructions along with the criteria for diagnosis.

The invention will be further illustrated by the following illustrative and nonlimiting examples.

GENERAL

PROCEDURES

Patients: Samples of Patients with Psychiatric Disorders and Control Group of Healthy Volunteers: All patients were diagnosed according to DSM-IIIR criteria by at least two senior psychiatrists. Inclusion criteria were normal results of physical examination, electrocardiogram, and laboratory tests for renal, hepatic, hematologic, and thyroid function. When indicated, patients and healthy volunteers were also evaluated through the use of Beck Inventory of Depression.

Patients consented to a 60 ml blood donation for the experiment. No psychiatric treatment was given to the patients during one month prior to referral. In case WO 97/20211 PCT/IL96/00166 14 of a history of treatment with depot antipsychotics (three of the patients with schizophrenia), it was verified that no such medication was given for at least two months prior to referral.

The group of schizophrenic patients with positive symptoms consisted of 13 male and 10 female hospitalized subjects, average age 31.4 (19-59) years, diagnosed as suffering from schizophrenia of paranoid or disorganized types with a course classified as acute, subchronic with acute exacerbation, or chronic with acute exacerbation. This group of patients presented predominantly positive symptomatology, assessed by the Positive and Negative Syndrome Scale, PANSS (Total PANSS score=92.8+5.5; Positive Scale=26.7+1.5). Eight of the patients, never before treated by dopamine antagonists, were examined during their first psychotic episode and the diagnosis of schizophrenia was reached after at least one year of clinical follow-up.

The group of manic patients consisted of 13 male and 7 female hospitalized subjects, average age 34.8 (20-57) years, suffering from acute mania.

The group of untreated patients with major depression (unipolar and bipolar) consisted of 15 female and 13 male subjects, average age 42.7 (20-68) years: 18 were outpatients and 10 were hospitalized.

The group of outpatients with panic disorder, with or without agoraphobia, consisted of 6 male and 7 female subjects, average age 37.5 (23-55) years.

The healthy volunteer group consisted of 17 men and 13 women, average age 38.9 (20-77) years.

MNL Isolation: MNL were isolated from 60 ml heparinized fresh blood of adult donors, using Ficoll-Paque gradient according to Boyum (Boyum 1968). Cells WO 97/20211 PCT/IL96/00166 were homogenized in 25 mM Tris-HCI, pH 7.4, and 1 mM dithiotreitol (DTT).

The homogenate was passed through two layers of cheesecloth to remove debris, and membranes were collected by further centrifugation at 18,000 g for minutes. Membranes were then either freshly used for the functional binding measures or suspended in homogenization buffer containing ImM ATP, 1mM EGTA, 2mM Mg2+ and 30% sucrose w/v, and frozen at -700C until assayed by the quantitative measures. Aliquots were taken for protein concentration determination using Bradford's standard assay.

Guanine Nucleotide Binding Assay: Guanine nucleotide binding assay was performed according to Avissar S. et al., (Avissar S. et al., 1988). Guanosine P,y, imido triphosphate [Gpp(NH)p], a nonhydrolyzable analog of GTP which has higher affinity for G-proteins, was used in guanine nucleotide binding assay.

Binding reaction was carried out at various concentrations of 3 H-Gpp(NH)p (0.05-5pM). Total binding was measured by adding aliquots of 50pg of membrane proteins to a series of tubes containing reaction buffer (25 mM Tris- HCI, pH 7.4, 1mM ATP, ImM Mg 2 1mM EGTA, and ImM DTT), with varying concentration of 3 H-Gpp(NH)p, to a final volume of 200pl. The tubes were incubated for 10 minutes at room temperature (180C 250C) and the reaction was terminated with 5 ml of ice-cold buffer (10mM Tris-HC1, pH 7.4; 100mM NaC1). The samples were filtered through GF/C Whatman filters. The filters were washed twice with 3 ml of cold buffer, and their radioactivity were determined. Non-specific binding, at each 3 H-Gpp(NH)p concentration, was measured in parallel, in the presence of 100pM unlabeled GTPyS. The binding reactions were carried out in triplicates, and specific binding was calculated by subtracting the nonspecific binding from the total binding.

Agonist-induced Binding: Three agonists were used, the P-adrenergic agonist isoproterenol, the muscarinic agonist carbamylcholine, and the dopamenergic WO 97/20211 PCT/IL96/00166 16 agonist-dopamine. The effects of the agonists on 3 H-Gpp(NH)p binding were assessed by adding isoproterenol (25 pM), carbamylcholine (50 pM) or dopamine PjM) to the reaction mixture. These represent minimal concentration resulting in maximal effect of the agonist.

Antagonists Effect on Agonist-induced Binding: Antagonists effects on agonistinduced increase in 3 H-Gpp(NH)p binding capacity were assessed by adding them to the reaction mixture, to the final concentrations indicated for each one.

1 pM propranolol, p-adrenergic antagonist; 10 pM ADFX116 and 10 pM pirenzepine, M 2 and M 1 muscarinic antagonists; 1 pM SCH23390, and 1 pM sulpiride, D 1 /5 and D 2 dopaminergic receptor antagonists, respectively.

The Effect of Cholera and Pertussis Toxins on Agonist-induced Binding: MNL membranes (3-4mg) were suspended in 1 ml buffer containing 25 mM Tris-HC1, pH 7.4, 10mM NAD, 1mM ATP, 10 mM thymidine, and 100 pM GTP. ADP ribosylation was carried out for 15 minutes at 300C by adding cholera toxin pg/ml) preactivated for 10 minutes at 370C with 20 mM DTT or pertussis toxin pg/ml) preactivated for 10 minutes at 300C with 20 mM DTT. The reaction was stopped by adding 25 ml ice-cold 25mM Tris-HC1, pH 7.4, immediately followed by centrifugation at 18,000 g for 10 minutes. 3 H-Gpp(NH)p binding was then carried out as described above.

Immunoblot Analysis: Membranes were thawed and aliquots of 10 (pg membranes were taken for protein separation on SDS- polyacrylamide gel electrophoresis. The proteins were transferred to nitrocellulose paper by electroblotting apparatus. Blots were washed in Tris-buffered saline (TBS) containing 3% Tween-20, and blocked by incubation for 1 hr with 5% BSA in TBS containing 0.1% Tween-20 (TTBS). After two washes in TTBS, blots were incubated overnight with each of the following antisera (NEN-DuPont) directed specifically against as (dilution 1:2,500), aC,1,2, (dilution 1:5,000), followed by WO 97/20211 PCT/IL96/00166 17 subsequent incubation with goat anti-rabbit IgG labeled with horseradish peroxidase. Immunoreactivity was detected with the Enhanced Chemiluminescence Western Blot Detection System (Amersham) followed by exposure to Kodak X-Omat film. Peak heights of immunoreactive bands were determined with an image analysis system. The optical density of the immunoreactive bands was normalized against 10 gg rat cortical membranes, run in each blot as a standard value. Linearity of the assay with respect to protein concentration, was found in the range of 2.5- 15 pg membrane protein from a healthy volunteer.

Statistical Analysis: Dunn's test was used for non-parametric multiple comparisons of receptor-coupled G-protein functions and immunoreactive quantities in patients MNL against a single control group with unequal sample sizes, and the value of the test statistic was compared with the table of critical values of Q' (Zar 1984).

Example 1: Receptors-coupled G-protein function in healthy volunteers.

Binding of 3 H-Gpp(NH)p to MNL membranes from healthy volunteers was determined at various 3 H-Gpp(NH)p concentrations in the absence of an agonist or in the presence of isoproterenol, carbamylcholine or dopamine. Specific binding (calculated by subtracting the non-specific binding from the total binding) reached equilibrium within 5 min. and remained constant for at least min.

Representative saturation curves of specific 3 H-Gpp(NH)p binding to MNL membranes from a healthy volunteer [in the absence of an agonist (open circles), and in the presence of isoproterenol (open triangles), carbamylcholine (closed squares), and dopamine (closed circles)and the parallel of non-specific binding curve (closed triangles) are presented in WO 97/20211 PCT/IL96/00166 18 Fig. 1. All three agonist-induced increase in Gpp(NH)p binding capacity without substantially effecting the affinity of binding.

Fig. 2a depicts representative Scatchard plots of basal and agonist-induced binding curves in MNL membranes from a healthy volunteer. The ratio of specifically bound Gpp(NH)p to free Gpp(NH)p is plotted against specifically bound Gpp(NH)p. Bmax, the maximal binding capacity can be estimated from the intercept of the extrapolated linear regression line with the horizontal axis.

Average over all the population of healthy volunteer subjects examined: Bmax A-Bmax M-Bmax D-Bmax Bmax basal value in the absence of an agonist is 40.3±1.0 pmole/mg protein Bmax value in the presence of P-adrenergic agonist isoproterenol is 50.9±1.9 pmole/mg protein p<0.01) Bmax value in the presence of the muscarinic agonist carbamylcholine is 52.5±1.6 pmole/mg protein (Q'=3.57 p<0.01) Bmax value in the presence of the dopamine is 54.9±2.0 pmole/mg protein p<0.01).

The increase in Gpp(NH)p binding capacity induced by all three agonists is statistically, highly significant.

The Bmax values did not show significant change with age, Barki-Harrington et al., 1996, in press) or after physical exercise.

Agonist-induced increase in Gpp(NH)p binding capacity is calculated as (Bmax in the presence of an agonist x 100 Bmax-basal level WO 97/20211 PCT/IL96/00166 19 Scatter plot of isoproterenol carbamylcholine and dopamine induced increase in Gpp(NH)p binding capacity for a group of healthy volunteers (closed circles) is shown in Fig. 3d.

The calculated means, and standard error of the means of agonists induced increase in Gpp(NH)p binding capacity (Fig. 4) were 26.4% 1.3 for isoproterenol(open column), 28.3%±1.7 for carbamylcholine (dashed column), and 26.8%±1.9 for dopamine (closed column).

Table I discloses the effect of P-adrenergic, muscarinic and dopaminergic antagonists, and the effect of cholera and pertussis toxins on agonist-induced increase in Gpp(NH)p binding. Cholera toxin is known to inhibit Gs protein through ribosylation of its a subunit. As depicted in Table 1 the increase in Gpp(NH)p binding capacity induced by dopamine and isoproterenol was abolished by cholera toxin, suggesting their effects on Gs protein function.

Human MNL are known to possess surface p-adrenergic receptors, which activate adenylyl cyclase function via Gs protein. Dopamine-induced increase in guanine nucleotide binding was specifically blocked by SCH-23390, a selective

D

1 and D 5 antagonist, and was not effected by sulpiride, a selective D 2 antagonist. The D 1 and D 5 receptor-selective agonist SKF-38393 1 M induced concentration-dependent increases in Gpp(NH)p binding capacity, which was similar to the increases induced by dopamine (not shown). D 1 and D 5 are the only dopamine receptors currently identified to activate adenylyl cyclase via Gs.

However, by now, there are indications to the expression of the D 3 and the D receptors only in human peripheral blood lymphocytes. Thus, dopamine probably effects Gs protein function in MNL through a D 5 receptor.

As depicted in Table 1 the effect of carbamylcholine on Gpp(NH)p binding was inhibited by pertussis toxin pretreatment, unaffected by cholera toxin, and specifically blocked by the M 2 antagonist ADFX116, while unaffected by the M 1 WO 97/20211 PCT/IL96/00166 antagonist pirenzepine. M 2 receptors are coupled to Gi-proteins. Gi-proteins are known to be inhibited by pertussis toxin, through ribosylation of their a subunit.

Thus, these findings suggest that carbamylcholine effects are exerted through

M

2 receptors, coupled to a Gi, which inhibits adenylyl cyclase.

TABLE I: Agonist Antagonist increase in 3 H-Gpp(NH)p binding capacity Cholera Pertussis __Toxin toxin Isoproterenol 25.1 3.1 <5 a 29.9 3.7 Isoproterenol Propranolol <5 a Carbamylcholine 29.8 2.1 25 2.6 Carbamylcholine ADFX116 Carbamylcholine Pirenzepine 31.9 Dopamine 26.4 2.5 <5a 19.5 3.9 Dopamine SCH23390 Dopamine Sulpiride 27.8 2.9 Dopamine Propranolol 22.6 2.3 a No statistically significant increases in 3 H-Gpp(NH)p binding capacity were detected.

Example 2: Receptor-coupled G-protein function in psychiatric patients.

Receptor induced G-protein functions were also assayed in patients with various psychiatric disorders. Fig. 2 depicts representative examples of Gpp(NH)p binding assay in individual patients with mania depression schizophrenia and panic disorder as compared with a healthy volunteer Binding was assayed under basal conditions (open circles) or in the presence of agonists: isoproterenol (open triangles), carbamylcholine (closed squares or dopamine (closed circles)-[in and only]. No significant change in basal binding capacity was found between the five individuals. However, each

C

WO 97/20211 PCT/IL96/00166 21 individual patient presented a specific, differential pattern of agonist induced increase in binding capacity.

Fig. 3(a-d) and Fig. 4 summarize the results of agonist-induced increase in binding capacity in groups of patients with the various psychiatric disorders.

Scatter plots for patients (open circles) with mania depression (3b); schizophrenia and panic disorder (3d) as compared to healthy volunteers (closed circles) are presented in Fig. 3.

The calculated means and standard error of the means for isoproterenol (open columns), carbamylcholine (dashed columns), and dopamine (closed columns) induced increase in Gpp(NH)p binding capacity, are presented in Fig. 4 for manic patients depressed patients patients with panic disorder schizophrenic patients and for healthy volunteers at rest or after physical exercise Each group of patients is characterized by a distinct pattern of receptor-coupled G-protein function. In comparison to control subjects, MNL of patients with mania were characterized by elevated P-adrenergic receptor-coupled G protein (P-Gp) function and elevated muscarinic receptor-coupled G protein (M-Gp) function; reduced function of both P-Gp and M-Gp were detected in MNL of depressed patients. MNL of patients with panic disorder were characterized by elevated p-Gp function and reduced M-Gp function. In contrast to the above mentioned groups of patients, no differences were detected in the function of P- Gp and M-Gp between patients with schizophrenia and control subjects. In these patients elevated dopaminergic receptor-coupled G protein (D-Gp) function was detected in comparison to normal subjects.

WO 97/20211 PCT/IL96/00166 22 The pattern of agonist-induced increase in Gpp(NH)p binding capacity in neuroleptic naive schizophrenic patients (8 of 23 patients) was similar to that of the other schizophrenic patients.

The changes in G-protein functions in the patients are not merely due to motor hyper- or hypoactivity, as normal subjects after intensive physical exercise showed G-proteins functions no different from the control subjects.

The results are summarized in Table II.

TABLE II: Agonist Agonist-induced Significance of increase in the difference Gpp(NH)p (in comparison binding capacity to control).

Control Subject Isoproterenol 26.4 1.3 Carbamylcholine 28.3 1.7 Dopamine 26.8 1.9 Mania Isoproterenol 72.7 6.5 Q' 5.00 p<0.01 Carbamylcholine 63.8 7.9 Q' 3.78 p<0.01 Depression Isoproterenol 2.0 1.2 4.44 p> 0 0 1 Carbamylcholine 10.5 2.9 Q' 3.79 p<0.01 Panic Isoproterenol 57.8 5.9 Q' 3.26 p<0.01 Carbamylcholine 14.3 3.2 Q' 2.65 p<0.01 Schizophrenia Isoproterenol 29.5 1.9% no significant differ.

Carbamylcholine 30.8 3.0% no significant differ.

Dopamine 76.5 8.2% Q' 5.56 p<0.01 Comparable results were obtained when specific binding (basal and agonistinduced) at 5mM was used as single point measure for calculating agonistinduced increase in Gpp(NH)p binding capacity.

WO 97/20211 PCT/IL96/00166 23 The empirical findings may potentially be used for differential diagnosis of psychiatric patients.

The sensitivity and specificity of a potential diagnostic assay which is based on a G-protein function measurements can be estimated using validity test, for any predetermined threshold values, and for predetermined test criteria.

For example, by determining the values of 45% and 15% agonist-induced increase in Gpp(NH)p binding capacity as threshold values reflecting G-protein hyperfunction and hypofunction respectively, the following results were obtained: For the diagnosis of mania requiring hyperfunction of both p-Gp and M-Gp the sensitivity was found to be 0.8 and the specificity 0.93.

For the diagnosis of schizophrenia, requiring hyperfunction of D-Gp and normal P-Gp and M-Gp, the sensitivity was found to be 0.87 and the specificity 0.97.

For the diagnosis of depression requiring hypofunctional of P-Gp and Gp, the sensitivity was found to be 0.89 and the specificity 0.90.

For the diagnosis of panic disorder requiring hyperfunction of P-Gp with normal or hypofunction of M-Gp, the sensitivity was found to be 0.75 and the specificity 0.93.

Analysis of the results may be based on values obtained with one (only in two cases), two or three agonists and different threshold may be used for each of them, according to the distribution of values found in patients and control subjects. Other manipulation of the assay results are also possible and may aid WO 97/20211 PCT/IL96/00166 24 in reliable diagnosis. For example, the sensitivity and specificity of the diagnosis of panic disorder may be elevated if evaluation is based on the ratio P-Gp/M-Gp, rather than on each parameter separately.

Example 3: Gas and Gai levels in psychiatric patients.

The level of Gs and Gi proteins in MNL was determined by immunoblot analysis using polyclonal antibodies against Gas and Gai. A representative immunoblot with MNL-membrane proteins from a patient with mania a patient with depression and an age and sex matched healthy volunteer is shown in Fig. 5. Of the two types of Gas recognized by anti- Gas (45kDa and 52 kDa), only the 45 kDa species Gas was detected in MNL.

Normalized data are presented by scatter plots (Fig. The level of the 45 kDa species of Gas (6a) and the level of Gai (6b) were significantly elevated in patients with mania (closed squares), and reduced in depressed patients (closed circles), in comparison with control subjects (open circles). No significant change was detected in MNL from patients with schizophrenia (closed triangles), or panic disorder (not shown).

Determining the value of 15% elevation or reduction in Gas and Gai immunoreactive level compared to the mean values of control group as a threshold, the specificity and selectivity of a test can be evaluated. For diagnosing of mania requiring elevation of 15% in the immunoreactive level of at least one type of Ga-subunit (Gas or Gai the sensitivity is 0.73 and the selectivity 0.81. For diagnosing of depression requiring reduction of 15% in the immunoreactive level of at least one type of Ga-subunit (Gas or Gai the sensitivity is 0.73 and the selectivity 0.90.

WO 97/20211 PCT/IL96/00166 Example 4: Correlation between the biochemical parameters and psychiatric evaluation of depression.

Normal subjects, outpatients with mild depression and patients with moderate and severe depression, were evaluated through the use of Beck Inventory of Depression and their p-Gp function and M-Gp function, as well as Gas and Gai level were simultaneously analyzed.

As depicted in Fig. 7, isoproterenol (7a) and carbamylcholine (7b)-induced increase in Gpp(NH)p binding capacity were found to be correlated, in a statistically highly significant manner with the scoring of Beck Inventory (Spearman correlation efficient -0.781 p<0.001 and -0.749 p<0.001 respectively). Significant correlations were also obtained between Gas and Gai immunoreactivity level and Beck Score (-0.63 p<0.001 and -0.693 p<0.001 respectively), (not shown).

Example 5: The effect of psychiatric treatments on the function and the level of G-proteins.

P-Gp and M-Gp function(s) in lithium treated euthymic bipolar patients were found to be similar to these of control subjects (Schreiber at al 1991, Avissar and Schreiber 1992a).

Fig. 8 depicts P-Gp function in depressive patients before (closed circles) and following treatments with antidepressants (closed circles). The alterations in P- Gp function in most (7of 8) of the depressive patients examined were normalized following treatments with antidepressants. The only exception being a patient who was also found to be non-responsive to the treatment based on psychiatric parameters.

P-Gp function (open circles) and M-Gp function (open squares), were measured in MNL membranes from depressed patients at intervals during ECT treatment.

WO 97/20211 PCT/IL96/00166 26 The state of the psychiatric illness was evaluated in parallel, using Beck Inventory for Depression (closed circles) or Hamilton and Brief Psychiatric Rating Scale (BPRS) Scores (not shown).

Fig. 9a depicts representative examples of the dynamics of the change in P-Gp function (open circles), M-Gp function (open squares), and Beck score (closed circles) in 4 depressed patients, on electroconvulsive treatment [ECT].

Fig. 9b depicts representative examples of the dynamics of the change in Gas (open circles), Gai (open squares) immunoreactive level, and Beck score (closed circles) in 4 depressed patients, on electroconvulsive treatment [ECT].

From the above mentioned Figures it is apparent that the normalization of all 4 biochemical parameters precedes the clinical improvement. Similar results were obtained in another 10 patients who, according to psychiatric criteria, were responsive to the treatment. Patients who, by psychiatric criteria, did not respond to ETC treatment, did not show normalization of the hypofunction and the reduced immunoreactivity (not shown).

Thus G-protein functional and quantitative measurements may serve as predictors for the responsiveness of psychiatric patients to various psychiatric treatments..

WO 97/20211 PCT/IL96/00166 27 References Avissar Schreiber Danon Belmaker (1988a): "Lithium Inhibits Adrenergic and Cholinergic Increases in GTP Binding in Rat Cortex.

Nature; 331:440-442.

Avissar and Schreiber (1992b): "Interaction of Antibipolar and Antidepressant Treatments with Receptor-Coupled G Proteins". (Anna Monika Award Paper) Pharmacopsychiatry, 25:44-50.

Avissar Schreiber (1992b): "The Involvement of G Proteins in the Pathogenesis and Treatment of Affective Disorders". (Ziskind-Somerfeld Award Paper). Biol Psychiatry 31:435-459.

Barki-Harrington Nechamkin Schreiber Avissar (1996 (in press)): "Functional and Quantitative Measures of Receptor-Coupled G-Proteins in Human Mononuclear Leukocytes: No Change with Age". Exp. Gerontol.

Boyum (1968): "Separation of Leukocytes from Blood and Bone Marrow".

Scand. J. Clin. Lab. Invest.;21 (Suppl 97):7.

Friedman Butkerait Wang H.Y. (1993): "Analysis of Receptor- Stimulated and Basal Guanine Nucleotide Binding to Membrane G Proteins by Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis". Analytical Biochemistry 2 1 4: 171-178.

Laugwitz Spicher Schultz Offermanns (1994): "Identification of Receptor-Activated G Proteins Selective Immunoprecipitation of Photolabeled G Protein a Subunits". Methods in Enzymology 2 3 7: 283-294.

WO 97/20211 PCT/IL96/00166 28 Lesch, Manji (1992): "G Proteins and Antidepressants". Biol Psychiatry 32: 549-579.

Mazzola-Pomietto Azorin Tramoni Jeanningros (1994): "Relation Between Lymphocyte P-Adrenergic Responsivity and the Severity of Depressive Disorders". Biol Psychiatry; 35:920-925.

Schreiber Avissar Danon Belmaker (1991): "Hyperfunctional G-Proteins in Mononuclear Leukocytes of Patients with Mania". Biol. Psychiatry; 29: 273-280.

Wieland Jakobs (1994): "Measurement of Receptor-Stimulated Guanosine (y-Thio)triphosphate Binding by G Proteins". Methods in Enzymology; 2 3 7:3-13.

Young Li Kish Siu Warsh (1991): "Post-mortem Cerebral Cortex Gsa-Subunit Levels are Elevated in Bipolar Affective Disorder".

Brain Res.; 553:323-326.

Zar (1984): Biostatistical Analysis, 2nd ed., Prentice-Hall, Englewood Cliffs, p. 569,

Claims (27)

1. A method for differential diagnosis of mania, depression, schizophrenia and panic, comprising the steps of: measuring the function of P-adrenergic receptor-coupled G- protein muscarinic receptor-coupled G-protein and dopamine receptor-coupled G-protein (D-Gp) in a blood sample from a patient exhibiting psychiatric disorder symptoms, wherein the function is agonist- induced activation of G-protein; and comparing the function values of each G-protein from step a) with a set of G-protein function values obtained from normal individuals; whereby i) mania is diagnosed by an increase in the function of both 3- Gp and M-Gp as compared to normal individuals; ii) depression is diagnosed by decrease in the function of both P-Gp and M-Gp as compared to normal individuals; iii) schizophrenia is diagnosed by an increase in the function of D-Gp and a normal like function in 3-Gp and M-Gp as compared to normal individuals; and iv) panic is diagnosed by an increase in the function of P-Gp and a decrease in the function of M-Gp, as compared to normal individuals.

2. A method for differential diagnosis of mania, depression and panic, comprising the steps of: measuring the function of P-adrenergic receptor-coupled G- protein (P-Gp) and muscarinic receptor-coupled G-protein (M-Gp) to the level of either P-Gp or M-Gp in a blood sample from a patient exhibiting psychiatric disorder symptoms, wherein the function is agonist-induced activation of G-protein; and comparing the function or level values of each G-protein from step a) with a set of G-protein function or level values obtained from normal individuals; whereby i) mania is diagnosed by an increase in the function of both P- Gp and M-Gp or by an increase in the level of either P-Gp or M-Gp, as compared to normal individuals; ii) depression is diagnosed by a decrease in the function of both P-Gp and M-Gp or by a decrease in the level of either P- Gp or M-Gp, as compared to normal individuals; and iii) panic is diagnosed by an increase in the function of P-Gp and a decrease in the function of M-Gp or by a normal like level of either P-Gp or M-Gp, as compared to normal individuals.

3. A method for differential diagnosis of mania, schizophrenia and panic comprising the steps of: measuring the function of muscarinic receptor-coupled G-protein (M-Gp) and dopamine receptor-coupled G-protein (D-Gp) in a blood sample from a patient exhibiting psychiatric disorder symptoms, wherein the function is agonist-induced activation of G-protein; and comparing the function values of each G-protein from step a) with a set of G-protein function values obtained from normal individuals; whereby i) mania is diagnosed by an increase in the function of M-Gp as compared to normal individual; ii) schizophrenia is diagnosed by an increase in the function of D-Gp and a normal like function of M-Gp as compared to normal individuals; and iii) panic is diagnosed by a decrease in the function of M-Gp, as compared to normal individuals.

4. A method for differential diagnosis of depression, schizophrenia and panic, comprising the steps of: measuring the function of p-adrenergic receptor-coupled G- protein (P-Gp) and dopamine receptor-coupled G-protein (D-Gp) in a blood sample from a patient exhibiting psychiatric disorder symptoms, wherein the function is agonist-induced activation of G-protein; and comparing the function values of each G-protein from step a) with a set of G-protein function values obtained from normal individuals; whereby i) depression is diagnosed by a decrease in the function of P- Gp as compared to normal individuals; ii) schizophrenia is diagnosed by an increase in the function of D-Gp and a normal like function of P-Gp as compared to normal individuals; and iii) panic is diagnosed by an increase in the function of P-Gp as compared to normal individuals.

5. A method for differential diagnosis of mania and schizophrenia, comprising the steps of: measuring the function of dopamine receptor-coupled G-protein (D-Gp) and the function or level of either P-adrenergic receptor-coupled G- protein (P-Gp) or muscarinic receptor-coupled G-protein (M-Gp) in a blood sample from a patient exhibiting psychiatric disorder symptoms, wherein the function is agonist-induced activation of G-protein; and comparing the function or level of each G-protein from step a) with a set of G-protein function or level values obtained form normal individuals; whereby i) mania is diagnosed by an increase in the function or level of either P-Gp or M-Gp as compared to normal individuals; and ii) schizophrenia is diagnosed by an increase in the function of D-Gp and a normal-like function or level of either P-Gp or M-Gp as compared to normal individuals.

6. A method for differential diagnosis of mania and panic, comprising the steps of: measuring the function of muscarinic receptor-coupled G-protein (M- Gp) or the level of either p-adrenergic receptor-coupled G-protein (P-Gp) or (M-Gp) in a blood sample from a patient exhibiting psychiatric disorder symptoms, wherein the function is agonist-induced activation of G-protein; and comparing the function or level values of each G-protein from step a) with a set of G-protein function or level values obtained from normal individuals; whereby i) mania is diagnosed by an increase in the function of M-Gp or by an increase in the level of either P-Gp or M-Gp, as compared to normal individuals; and ii) panic is diagnosed by a decrease in the function of M-Gp or by a normal like level of either P-Gp or M-Gp, as compared to normal individuals.

7. A method for differential diagnosis of depression and schizophrenia, comprising the steps of: measuring the function of dopamine receptor-coupled G-protein (D-Gp) and the function or level of either P-adrenergic receptor-coupled G- protein (P-Gp) or muscarinic receptor-coupled G-protein (M-Gp) in a blood sample from a patient exhibiting psychiatric disorder symptoms, wherein the function is agonist-induced activation of G-protein; and comparing the function or level of each G-protein from step a) with a set of G-protein function or level values obtained from normal individuals; whereby i) depression is diagnosed by a decrease in the function or level of either P-Gp or M-Gp as compared to normal individuals; and ii) schizophrenia is diagnosed by an increase in the function of D-Gp and a normal-like function or level of either p-Gp or M-Gp as compared to normal individuals.

8. A method for diagnosing mania, comprising the steps of: measuring the function of p-adrenergic receptor-coupled G- protein (P-Gp) and muscarinic receptor-coupled G-protein (M-Gp) or measuring the level of either p-Gp or M-Gp in a blood sample from a patient exhibiting psychiatric disorder symptoms, wherein the function is agonist- induced activation of G-protein; and comparing the function or level of each G-protein from step a) with a set of G-protein function or level values obtained from normal individuals; whereby mania is diagnosed by an increase in the function of P-Gp and M-Gp or by an increase in the level of either P-Gp or M-Gp, as compared to normal individuals.

9. A method for diagnosing depression, comprising the steps of: measuring the function of p-adrenergic receptor-coupled G- protein (B-Gp) and muscarinic receptor-coupled G-protein (M-Gp) or measuring the level of either P-Gp or M-Gp in a blood sample from a patient exhibiting psychiatric disorder symptoms, wherein the function is agonist- induced activation of G-protein; and comparing the function or level of each G-protein from step (a) with a set of G-protein function or level values obtained from normal individuals; whereby depression is diagnosed by a decrease in the function of P-Gp and M-Gp or by a decrease in the level of either P-Gp or M-Gp, as compared to normal individuals.

A method for diagnosing schizophrenia, comprising the steps of: measuring the function of dopamine receptor-coupled G-protein (D- Gp) and the function or level of p-adrenergic receptor-coupled G-protein (P- -A Gp) or muscarinic receptor-coupled G-protein (M-Gp) in a blood sample from a patient exhibiting psychiatric disorder symptoms, wherein the function is agonist-induced activation of G-protein; and comparing the function or level of each G-protein from step a) with a set of G-protein function or level values obtained from normal individuals; whereby schizophrenia is diagnosed by an increase in the function of D-Gp and a normal like function or level of P-Gp or M-Gp as compared to normal individuals.

11. A method for diagnosing panic, comprising the steps of: measuring the function of p-adrenergic receptor-coupled G- protein (P-Gp) and muscarinic receptor-coupled G protein (M-Gp) in a blood sample from a patient exhibiting psychiatric disorder symptoms, wherein the function is agonist-induced activation of G-protein; and comparing the function of each G-protein from step a) with a set of G-protein function values obtained from normal individuals; whereby panic is diagnosed by an increase in the function of P-Gp and a decrease in the function of M-Gp, as compared to normal individuals.

12. A method for gauging the effect of a treatment upon a patient diagnosed with a psychiatric disorder selected from the group consisting of mania, depression and panic, themethod comprising: measuring the function of at least one receptor-coupled G protein in a blood sample from the patient prior to the treatment, said receptor-coupled G-protein is selected from the group consisting of p-adrenergic receptor- coupled G-protein (p-Gp) and muscarinic receptor-coupled G-protein (P-Gp) and muscarinic receptor-coupled G-protein wherein the function is agonist-induced activation of G-protein; subjecting the patient to the treatment for a sufficient period of time; measuring the function of the receptor-coupled G-protein in a blood sample from the patient subjected to the treatment of step b); whereby a shift of the function toward the normal values obtained from normal individuals is indicative of an effective treatment.

13. A method for gauging the effect of a treatment upon a patient diagnosed with schizophrenia, the method comprising: a) measuring the function of dopamine receptor-coupled G-protein (D-Gp) in a blood sample from the patient prior to the treatment, wherein the function is agonist-induced activation of G-protein; b) subjecting the patient to the treatment for a sufficient period of time; c) measuring the function of dopamine receptor-coupled G-protein (D-Gp) in a blood sample from the patient subjected to the treatment of step b); whereby a shift of the function toward the normal values obtained from normal individuals is indicative of an effective treatment.

14. A method for gauging the effect of a treatment upon a patient diagnosed with a psychiatric disorder selected from the group consisting of mania and depression, the method comprising: a) measuring the level of at least one receptor-coupled G-protein in a blood sample from the patient prior to the treatment, said receptor-coupled G- protein is selected from the group consisting of p-adrenergic receptor-coupled G-protein (P-Gp) and muscarinic receptor-coupled G-protein (M-Gp); b) subjecting the patient to the treatment for a sufficient period of time; c) measuring the level of the receptor-coupled G-protein in a blood sample from the patient subjected to the treatment of step b); whereby a shift of the level toward the normal values obtained from normal individuals is indicative of an effective treatment.

A method for evaluating a patient's disease progression over a period of time, said patient having been diagnosed with a psychiatric disorder selected from the group consisting of mania, depression and panic, the method comprising: a) measuring the function of at least one receptor-coupled G-protein in a blood sample from the patient, said receptor-coupled G-protein is selected from the group consisting of p-adrenergic receptor-coupled G-protein if -Gp) and muscarinic receptor-coupled G-protein wherein the fnction is agonist-induced activation of G-protein; b) measuring the function of the receptor-coupled G-protein in a blood sample from the patient at least one subsequent time; and c) comparing the function of the receptor-coupled G protein obtained in step b) with the function of the receptor-coupled G-protein obtained in step a); whereby a shift of the function over said period of time is indicative of said patient's disease progression.

16. A method for evaluating a patient's disease progression over a period of time, said patient having been diagnosed with schizophrenia, the method comprising: a) measuring the function of dopamine receptor-coupled G-protein (D-Gp) in a blood sample from the patient, wherein the function is agonist- induced activation of G-protein; b) measuring the function of dopamine receptor-coupled G-protein (D-Gp) in a blood sample from the patient at least one subsequent time; and c) comparing the function of dopamine receptor-coupled G-Protein (D-Gp) obtained in step b) with the function of dopamine receptor-coupled G- protein (D-Gp) obtained in step a): whereby a shift of the function of dopamine receptor-coupled G-protein (D-Gp) over said period of time is indicative of said patient's disease progression.

17. A method for evaluating a patient's disease progression over a period of time, said patient having been diagnosed with a psychiatric disorder selected from the group consisting of mania and depression, the method comprising: a) measuring the level of at least one receptor-coupled G-protein in a blood sample from the patient, said receptor-coupled G-protein is selected from the group consisting of p-adrenergic receptor-coupled G-protein (P-Gp) and muscarinic receptor-coupled G-protein (M-Gp); b) measuring the level of the receptor-coupled G-protein in a blood sample from the patient at least one subsequent time; and c) comparing the level of the receptor-coupled G-protein obtained in step b) with the level of the receptor-coupled G-protein obtained in step a); 3 whereby a shift of the level over said period of time is indicative of said ipatient's disease progression.

18. The method of any one of claims 1-17, wherein the blood sample is a preparation of peripheral blood.

19. The method of claim 18, wherein the preparation is derived from mononuclear leukocyte (MNL) cells.

The method of any one of claims 1-19, wherein the G-protein function is determined as an agonist-induced increase in guanine nucleotide binding capacity.

21. The method of claim 20, wherein the guanine nucleotide binding capacity is determined using a guanine nucleotide selected from the group consisting of Gpp(NH)p, GTPyS, and GTP-azidoanilide.

22. The method of claim 21, wherein the agonist is p-Gp agonist isoproterenol, M-Gp agonist carbamylcholine, or D-Gp agonist dopamine and analogs thereof.

23. The method of claim 22, wherein the G-protein level is measured by an immunoassay using an antibody specific to the a subunit of the receptor- coupled G-protein.

24. The method of claim 23, wherein the a subunit is Gas or Gai.

A kit when used for a method according to any one of claims 1 to 24, said kit comprising: agonists of one, two or three receptors, wherein said receptors are selected from the group comprising: p-adrenergic receptor (ii) muscarinic receptor (iii) dopamine receptor, and labeled guanine nucleotide.

26. A kit according to claim 25 wherein the 3-adrenergic receptor agonist is isoproterenol; muscarinic receptor agonist is carbamylcholine; and dopaminergic receptor agonist is dopamine.

27. A kit when used for a method according to claim 23 or 24 said kit comprising: 1 or 2 monoclonal or polyclonal antibodies selected from the group comprising anti-Gas and anti-Gai; detectable probe which is capable of specifically binding to the antibody of and standard samples. Dated this twenty seventh day of November 2000 Ben-Gurion University of the Negev Patent Attorneys for the Applicant: F B RICE CO