JP2000502179A - Methods and kits for monitoring the diagnosis and treatment of mental disorders - Google Patents

Abstract

  (57) [Summary] (a) measuring the function and / or level of at least one receptor-coupled G protein; (b) diagnosing a psychiatric disorder based on the measurement in (a) or measuring the effect of treatment on the patient A method for diagnosing a psychiatric disorder or measuring the effect of treatment on a psychiatric patient.

Description

DETAILED DESCRIPTION OF THE INVENTION         Methods and kits for monitoring the diagnosis and treatment of mental disorders Field of the invention   The present invention relates to biochemical differential diagnosis of mental disorders (psychiatric disorders), Especially in the accurate diagnosis of various mental disorders, and the course of treatment of such disorders Use specific protein function and level measurements to accurately track About things.Background of the Invention   Differential diagnostic methods currently used to classify mental disorders are primarily phenomenological. Things. Diagnosis is based on the observation of certain signs and the course of the disorder. Currently used The Manual for Diagnosis and Statistics of Mental Disorders (DSM IIIR) is based on experience. And mainly uses phenomenological operating standards.   There have been many attempts to decipher the biological basis of mental disorders. Mental disorders Pathogenesis of harm and various biotherapeutic mechanisms used to eradicate such disorders Hypotheses about the involvement of various neurotransmitters and their receptors in the mechanism Has been advocated. However, biological or psychiatric diagnoses can be made Lab-based cross-over diagnostic tests that can be routinely used in biochemistry are still available. Not. For laboratory tests of biochemical properties useful for differential diagnosis in psychiatry There is a great need for this and it is well recognized.   Need for scientifically established biochemical assays for differential diagnosis of mental disorders The necessity is widely recognized, and enabling it has great advantages. That Such assays can be performed on inpatients, outpatients, and patients treated by GPs. Helps with differential diagnosis of Of particular importance are the two major mental illnesses, schizophrenia and Differential diagnosis after the onset of the first psychotic symptoms during manic depression.   For example, using currently known phenomenological diagnostic methods, schizophrenia is clearly diagnosed. There are no characteristic signs of disease. Therefore, in order to reach the final judgment of schizophrenia Requires a follow-up diagnosis of at least 6 months after the initial diagnosis.   Differentiating major psychiatric illnesses has important prognostic and therapeutic implications, and more Has very desirable social consequences. About mental disorders like severe depression An important aspect of all biochemical assays is that the general population treating half of severely depressed patients Practitioners and mental health professionals will determine the adequacy of pharmacological antidepressant treatment. And that is possible.   It is usually treated first by a general practitioner and requires one to rule out physical disability. Another obstacle that usually requires a series of physical examinations and a costly group of laboratory tests Have a panic disorder. Once a biochemical assay is established for this disorder, Use available pharmacological treatments available to help treat these patients early.   Various specific therapies have been applied to emotional disorders. Lithium-like emotional stability Drugs and electroconvulsive therapy (ECT) are used to treat mania and depression and to predict both disease states. It is effective for prevention. Antidepressants are used to treat and prevent depression. No need for these treatments They do not immediately show the therapeutic effect, but require 3 weeks to 1 month. Approximately 30% of patients do not respond to one treatment but may respond to another There is. Therefore, to examine a patient's response to psychiatric treatment biochemically It is of special importance that biochemical tests exist that allow for   Also, biochemical parameters measured by diagnostic assays in psychiatric patients Data changes are indicative of the disease state of the disease and become normal after effective psychiatric treatment. If such assays are available, practitioners will be able to assess the efficacy of such treatments biochemically. Tracking and measuring.   Signal transduction in psychiatric disorders, especially the family of receptor-coupled G proteins Possible involvement of members of the art has been pointed out in the prior art. For example, white blood cells Decreased β-adrenergic receptor density in membrane preparations is observed in depressed patients Reduced and decreased β-adrenergic receptor responsiveness Measurement of stimulated cAMP production in leukocytes of patients with depression It recognized. The magnitude of these changes in β-adrenergic receptor density and responsiveness Degree was found to be correlated with the severity of depression (Mazzola-Pomi etto et al., 1994).   Beta-adrenergic receptor and other receptors for signal transduction The earliest events include coupling of activated receptors to G proteins. No. The family of G proteins now comprises 12-15 known individual proteins . G-proteins consist of three subunits, α, β and γ. α-sub uni The kit contains a guanine nucleotide binding site and has GTPase activity. Ma Α-subunit is a nicotinamide adenine nucleus catalyzed by bacterial toxins Contains a site for reotide (NAD) -dependent ADP-ribosylation. α-subunit G-proteins are classified into major classes (Gs, Gi, Gq) due to the heterogeneity of You.   The receptor for stimulating hormone, β-adrenergic receptor, is G-protein that activates Rase, interacts with Gs and inhibits ligand, eg For example, M_Two-Muscarinic receptor interacts with adenylate cyclase inhibitory regulator Gi I do. Gq is phosphatidylinositol-4,5-biphos by phospholipase C It may be involved in receptor activation leading to fate degradation.   The stimulatory effect of the agonist is caused by the binding of guanine nucleotide to G protein. And leads to its activation. Based on this feature, guanine nucleotide binding The assay has been used as an established test for G protein function.   The function of the receptor-coupled G-protein is lithium (Avissar et al., 1988), Antibipolar treatment and antidepressants (Avissar and Schreiber 1992a, b) has been found to weaken in various ways. Furthermore, β-adrenergic conjugate and Hyperactivity of muscarinic conjugated G-protein is detected in MNL of mania patients ( Schreiber et al., 1991), the α-subunit of Gs-protein (Gα_s) Rose Bells were found in the postmortem cerebral cortex of bipolar patients (Young et al., 1991).   All of these publications may use these findings to diagnose mental disorders. No mention is made of the efficacy of Certainly, there is no suggestion for the possibility of differential diagnosis.   In the present invention, measurement of the function and level of receptor-coupled G-protein Used for the differential diagnosis of patients with disabilities and the response of patients to psychiatric treatment Used for gender evaluation.Summary of the Invention   According to the present invention, (a) the function and / or function of at least one receptor-coupled G protein Or level, and diagnose the mental disorder based on the measurements in (b) (a), or Or diagnosing mental disorders, including measuring the effect of treatment on a patient Or a method for measuring the effect of a treatment on a psychiatric patient.   According to a preferred embodiment of the present invention, the function of the receptor-coupled G-protein is Agonist-induced increase in guanine nucleotide binding capacity in mononuclear leukocytes (MNL) Measured as   According to another preferred embodiment of the invention, the level of the receptor-coupled G protein is α_sOr Gα_iQuantification using antibodies against subunits.   The present invention also provides a kit for performing the method of the present invention.   Other aspects and aspects of the present invention are described below or in the following description of the invention. It is understood from.BRIEF DESCRIPTION OF THE FIGURES   FIG. 1: Against MNL obtained from healthy volunteers as described in Example 1.^ThreeH-Gpp (NH) Graph showing non-specific basal binding of p and agonist-induced specific binding.   Figure 2 (a-e): Healthy volunteers and four different spirits as described in Examples 1 and 2. In MNL membranes from impaired patients^ThreeBasal and agonist-induced binding of H-Gpp (NH) p 4 is a representative Scatchard plot showing the results.   FIG. 3 (a-d): Various comparisons with those of healthy volunteers described in Examples 1 and 2. In MNL membranes from patients with mental illness^ThreeAgonist-induced increase in H-Gpp (NH) p binding capacity Scatter plots showing the results showing the change in loading.   Figure 4: Healthy volunteers (A) described in Examples 1 and 2, healthy volunteers after exercise (B), mania (C), depression (D), panic disorder (E), and schizophrenia (F) Agonist-induced increase in Gpp (NH) p binding capacity in MNL membranes from Bar chart showing results showing different patterns.   FIG. 5: Gα described in Example 3._sAnd Gα_iSpirit obtained by antiserum against By MNL protein samples from diseased patients (A, C) and healthy volunteers (B) Representative immunoblot.   Fig. 6 (a, b): From healthy volunteers and various mental disorders described in Example 3. Gα in MNL_s(6a) and Gα_i(6b) Results showing relative immunoreactivity levels Scatter plot showing the results.   FIG. 7 (a, b): In the depressed patients and healthy volunteers described in Example 4,^ThreeH-Gp Isoproterenol (7a) and carbamylcholine (7b) -induced increases in p (NH) p binding capacity Is a graphical representation of the results showing the correlation between and Beck scores.   FIG. 8: β-address in depressed patients treated with antidepressants as described in Example 5 Narin receptor-coupled G-protein (β-G_P) A graph showing the results of functional recovery What you did.   FIG. 9 (a, b): Incarnation of a depressed patient treated with electroconvulsive therapy as described in Example 5 Graphical representation of the results showing the improvement over time of the biological and psychiatric parameters.Detailed description of the invention   The present invention relates to receptor coupling in MNL from patients with major psychiatric disorders as follows: Based on the discovery of different patterns of G-protein function and immunoreactivity levels Follow.   maniaShows that β-adrenergic and muscarinic receptors are higher than normal (control) individuals. Body-conjugated G-protein (β-G_PAnd MG_P) Function is enhanced and Gα_sAnd Gα_iExemption Increases epidemic reactivity levels.   depressionIn comparison with normal (control) individuals, β-G_PAnd MG_PFunction is reduced, Gα_s And Gα_iImmunoreactivity levels are reduced.   schizophreniaShows that the dopamine receptor-coupled G-tan Park (DG)_P) Function is enhanced, β-G_PAnd MG_PThe level of function is the same as the control Yes, Gα_sAnd Gα_iIs similar to the control.   Panic disorderIn comparison with normal (control) individuals, β-G_PFunction progressed, MG_PMachine Performance decreases and Gα_sAnd Gα_iIs similar to the control.   The present invention further provides for alteration of receptor-coupled G protein function and level in MNL. Is a biochemical indicator of the disease state of the disease, and treated asymptomatic patients have normal values Based on the discovery that   The present invention provides a method for measuring the G-protein based on functional and / or quantitative measurement. A biochemical cross-diagnostic assay is provided that can distinguish disorders. The present invention Biochemical assays to assess patient responsiveness to psychiatric treatment I will provide a.   The above “function of G-protein” refers to G protein induced by agonist. It implies activation of the material. According to a preferred embodiment of the present invention, the G protein function is Measured as agonist-induced increase in guanine nucleotide binding capacity It is.   Preferred agonists for performing the method of the invention are isoproterenol, Carbamylcholine and dopamine, which are β-adrenaline, Activates muscarinic and dopamine receptors. However, similar types of receptors Other stimulating β-adrenaline, M_Two-Muscarin and D_1/5-Dopamine Gonists are also suitable and their use is within the scope of the invention .   According to another preferred embodiment of the present invention, the above "quantitative measurement" of G protein Alternatively, the “immunoreactivity level” refers to the immunoreactivity level of the α-subunit. Measured.   According to another preferred embodiment of the present invention, a mental disorder diagnosed by the method of the present invention. The harms are mania, depression, panic disorder and schizophrenia.   According to a preferred method of the present invention, receptor-coupled G-protein function and level Measured in the MNL from the patient, but other Cell types may also be suitable. For example, other peripheral blood cells or adult Minutes (eg, platelets) or skin cells may be suitable for diagnosis. However, different cells It should be noted that different levels and types of condition can be indicated, and The survey is performed on a population of patients already diagnosed initially and on a population of healthy subjects, Determine the suitability of each cell type for these purposes and use these different cells You have to adjust the way you do.   In the present invention, measurement of the function of one, two or three kinds of receptor-coupled G proteins To diagnose psychiatric disorders or to measure the effects of psychiatric treatment It can be used appropriately and effectively. In fact, depression is β-G_POf the function Mania can be diagnosed by MG with high specificity and sensitivity._PFunction of Diagnosis can be based on hyperactivity. But in most cases, The function of more than one receptor-coupled G-protein to enable accurate diagnosis Should. These include β for mania, depression and panic disorder -G_PAnd MG_PDG for diagnosis of function, schizophrenia_PFunction and β-G_POr MG_P Function or both. Therefore, for the differential diagnosis of patients, three types All (β-G_P, MG_PAnd DG_PIt is preferable to measure the function of ()). specific The specificity of a test for diagnosing a disorder is to determine the minimum requirements and thresholds. You have more control.   However, to monitor the effects of psychiatric treatment, one receptor Normal function of the conjugated G protein, preferably in the acute state of the disease It is sufficient to measure the function of the one that deviates the most from the bell. Evaluation of the treatment Based on a comparison of the values obtained before and during treatment, a shift to normal It can be said that it is reactive.   According to a preferred embodiment of the present invention, the receptor-coupled G-protein function is Measured by a nucleotide binding assay. However, to carry out the method of the invention In addition, it allows the measurement of G protein agonist-induced activation. Any other assay can be used.   According to another preferred embodiment of the present invention, the guanine used in the assay Reotide is Gpp (NH) p. Other guanine nucleotides or analogs are suitable. And their use is within the scope of the present invention. That Such a substance is, for example, GTPγS (Wieland T. and Jakobs KH, 1994) or Is GTP-azidanilide (irreversibly binds to G proteins when exposed to UV light) Photolabile compounds, Laugitz KL et al., 1994).   According to yet another aspect of the invention, a guanine nucleotide for use in an assay is provided. Label the otide. Guanine nucleotide labels include radioactive labels, fluorescent labels, etc. Any type of label that can be detected by appropriate detection means, such as You. Alternatively, instead of labeling, the labeled guanine nucleotide may be labeled using a labeled probe. May be detected, in which case the labeled probe is used only after separating unbound material. Specific for complex G protein-guanine nucleotides by contact with the sample That can bind specifically to the guanine nucleotide itself. Things. To carry out the method of the present invention, a guanidine Utilize any method that produces a detectable change proportional to nucleotide binding be able to.   G-protein as measured by agonist-induced increase in binding capacity Function is calculated by general procedure and multi-point measurement as described in Example 1. Can be. However, using single point measurements at a single concentration of guanine nucleotides To simplify the assay. For Gpp (NH) p, at a concentration of 5 mM, Similar values were obtained based on multipoint and single point measurements.   The diagnosis of mental disorders or the tracking of the effects of psychiatric treatment according to the present invention can be performed by It may be based on measuring the level of the α-subunit. Gα_sAnd Gα_iImmune response The measurement of the sex level was performed by first measuring the MNL membrane protein isolated by SDS-PAGE. Performed by Munoblot analysis. However, in routine assays, sample Measurement of Gα-subunit immunoreactivity without separation . Quantitative immunoassay for different types of Gα-subunits in crude membrane preparations (See, for example, Lesch K.P., Manjii H.K., 1992). Their use is within the scope of the present invention.   Also, Gα for the method of the present invention_sAnd Gα_iOf the immunoreactivity level of It may be performed by an ISA test. Latching using a competitive ELISA-based method Various types of Gα-subunits in membranes from human brain have been quantified (Les ch K.P. and Manjii H.K., 1992). However, other forms of ELISA testing Alternatively, other immunoassays can be used, and their use Are within the range of   In order to carry out the method of the present invention, many types of G protein functions are measured. Kinds of things are possible. For example, at the end of the binding reaction, immobilized antibodies or magnetic particles Contacting the sample with the antibody bound to the guanine nucleotide -G protein complex can be captured. This allows the cleaning of unbound substances This avoids the need to filter the sample. G-tan by various methods Immunoseparation of the protein-guanine nucleotide complex has been disclosed (Friedman E. et al. , 1993). The bound guanine nucleotide can then be added directly (if it is labeled ) Or indirectly (specifically binds to the G protein-guanine nucleotide complex). (With a compatible labeled probe).   Another aspect of the present invention relates to a kit for performing the method of the present invention. kit Is the measurement of G protein function, or Gα_sAnd / or Gα_iImmunoreactive levels of This is based on the measurement of Kit can be used to diagnose or treat psychiatric disorders Can be used for tracking effects.   An exemplary kit based on G protein function measurement according to the method of the present invention comprises: Including:   (a) (i) β-adrenergic receptor       (ii) Muscarinic receptor       (iii) Dopamine receptor       An agonist of one, two or three receptors selected from the group comprising   (B) labeled guanine nucleotide   (c) Manufacturer's instructions for using the kit.   The criteria for the diagnosis of each major psychiatric disorder shall be included in the manufacturer's instructions. You may.   Gα by the method of the present invention_sAnd / or Gα_iAn exemplary kit based on measurements is Including:   (a) Anti-Gα_sAnd anti-Gα_iOne or two monochromes selected from the group containing Null or polyclonal antibodies,   (b) a detectable probe that can specifically bind to the antibody of (a),   (c) a standard sample,   (d) Manufacturer's instructions for using the kit.   The standard sample normalizes the results and compares them to the known mean of normal subjects Must be included to make it possible. Detailed theory for standardization The statement must be included in the manufacturer's instructions along with the diagnostic criteria.   The present invention is further described by the following illustrative, non-limiting examples.General procedure Patients: sample of mentally ill patients and control group of healthy volunteers   All patients should be referred to at least two experienced psychiatrists according to DSM-IIIR criteria. Was diagnosed. Physical examination, ECG, and kidney, liver, blood and thyroid function The criterion used was to show normal results in all laboratory tests. Depression Be Further use of ck survey (Beck Inventory of Depression) for patients and healthy volunteers When the evaluation of the person was performed, it was specified. The patient agreed to donate 60 ml of blood for the experiment It is. Patient has not received any psychiatric treatment during the month prior to being referred to the experiment. won. If you have a history of treatment with depot antipsychotics (of schizophrenic patients 3) had not received such medication for at least 2 months prior to the referral It was confirmed.   A group of schizophrenic patients with positive signs was hospitalized with 13 men and 10 women With an average age of 31.4 years (19-59) and acute or subchronic acute relapse Or delusional or disorganized forms with a course classified as chronic with acute relapse A patient diagnosed with schizophrenia. Patients in this group have positive and negative syndrome scales. (Positive and Negative Syndrome Scale, PANSS) Showed symptomatic features (total PANSS score = 92.8 + 5.5, positive scale = 26.7 ± 1.5) . Eight of the patients had previously been treated with dopamine antagonists. Unexpectedly, examined during the first episode of psychosis and at least one year of clinical follow-up She had been diagnosed with schizophrenia after contemplation.   The group of mania patients consisted of 13 male and 7 female inpatients, the average age She was 34.8 years old (20-57) and had acute mania.   Untreated patients with severe depression (monopolar and bipolar) consisted of 15 women and 13 men Consisting of subjects, the average age was 42.7 years (20-68), 18 were outpatients, Ten were inpatients.   The group of outpatients with panic disorder, including those with agoraphobia, included six men and The average age was 37.5 years (23-55).   The group of healthy volunteers consisted of 17 men and 13 women with an average age of 38.9 years ( 20-77).MNL isolation   MNL is based on Boyum (Boyum A., 1968) and uses Ficoll-Paque gradients to The donor was isolated from 60 ml of heparinized fresh blood. Cells were 25 mM Tris-HCl, Homogenized in pH 7.4 and 1 mM dithiothreitol (DTT). Homogene Pass through two layers of cheesecloth to remove debris and expose at 18,000 g for 10 minutes. The membrane was recovered by centrifugation. Then the membrane is fresh for functional binding measurements. Use as it is or 1mM ATP, 1mM EGTA, 2mMg²⁺And 30% sucrose w / v Suspend in homogenization buffer and assay by quantitative measurement Frozen at -70 ° C. Protein using standard Bradford assays An aliquot was taken for quality determination.Guanine nucleotide binding assay   Guanine nucleotide binding assays are described in Avissar S. et al. (Avissar S. et al., 1988). So we did. Guanosine β, γ, imidotriphosphate (Gpp (NH) p), In other words, non-hydrolyzable GTPs showing higher affinity for G-proteins Analogs were used for guanine nucleotide binding assays.   The binding reaction is^ThreeThe experiments were performed at various concentrations of H-Gpp (NH) p (0.05-5 μM). Membrane protein Of a 50 μg aliquot of the reaction buffer (25 mM Tris-HCl, pH 7.4, 1 mM ATP , 1mM Mg²⁺, 1 mM EGTA and 1 mM DTT)^ThreeH-Gpp (NH) p at various concentrations Total binding was measured as a final volume of 200 μl in a series of tubes. 10 tubes For 5 minutes at room temperature (18 ° C. to 25 ° C.) and 5 ml of ice-cold buffer (10 mM The reaction was stopped with squirrel-HCl (pH 7.4, 100 mM NaCl). Transfer the sample to GF / C The mixture was filtered with a filter. Filter twice, After washing with 3 ml of cooling buffer, the radioactivity was measured. each^ThreeH-Gpp (NH) p concentration Non-specific binding was measured in parallel in the presence of 100 μM unlabeled GTPγS . The binding reaction is performed in triplicate, and specificity is determined by subtracting nonspecific binding from total binding. Target binding was calculated.Agonist-induced binding   Three agonists, β-adrenergic agonist isoproterenol, Mus Carbamylcholine, a kalin agonist, and dopamine, a dopamine agonist It was used. Isoproterenol (25 μM), carbamylcholine (50 μM) or By adding dopamine (50 μM) to the reaction mixture^ThreeH-Gpp (NH) p bond The effects of the agonists on were investigated. These are the smallest that show the maximum effect of the agonist. Indicates the concentration.Effects of antagonists on agonist-induced binding ^ThreeThe effect of the antagonist on agonist-induced increase in H-Gpp (NH) p binding ability, Add the antagonist to the reaction mixture to the final concentration shown below. I checked. 1 μM propranolol (β-adrenergic antagonist), 10 μM  ADFX116 and 10 μM pirenzepine (M_TwoAnd M₁Muscarinic antagonists), 1 μM SCH23390 and 1 μM sulpiride (D_1/5And D_TwoDopamine receptor Antagonist).Effects of cholera and pertussis toxin on agonist-induced binding   The MNL membrane (3-4 mg) was treated with 25 mM Tris-HCl, pH 7.4, 10 mM NAD, 1 mM ATP, 10 mM Suspended in 1 ml buffer containing thymidine and 100 μM GTP. 20mM DTT 10 minutes with cholera toxin (20 μg / ml) or 20 mM DTT preactivated at 37 ° C for 10 minutes Addition of pertussis toxin (10 μg / ml) pre-activated at 30 ° C for The silation was performed for 15 minutes at 30 ° C. Add 25 ml of ice-cold 25 mM Tris-HCl, pH 7.4 The reaction was stopped by immediately centrifuging at 18,000 g for 10 minutes. Then on As noted^ThreeH- Gpp (NH) p binding was performed.Immunoblot analysis   Thaw membranes and separate proteins by SDS- (10%) polyacrylamide gel electrophoresis An aliquot of 10 μg of membrane was taken for Rita by electric blotting device The protein was transferred to nitrocellulose paper. Blot with Tris containing 3% Tween-20 Wash with buffered saline (TBS) and add 5% BSA and 1% in TBS containing 0.1% Tween-20 (TTBS). Blocked by incubating for hours. After two washes in TTBS, The blot was prepared using the following antiserum (NEN-DuPont), α_sSpecific to (dilution 1: 2,500), α_{I, 1,2}Each of which is specific (dilution 1: 5,000) Incubate overnight, then horseradish peroxidase-labeled goat anti-rabbit Ig Incubated with G. Enhanced Chemiluminescence Western Blo t Detection System (Amersham), then exposed to Kodak X-Omat film Was. The peak height of the immunoreactive band was measured with an image analysis system. Immunoreactivity The optical density of the band was determined as Standardized for membrane. 2.5 to 15 μg membrane protein from healthy volunteers And linearity of the assay with respect to protein concentration was observed.Statistical analysis   Using Dunn's test, receptor-coupled G-protein function and immunity in patient MNL Non-parametric reaction volume for a single control group with non-identical sample size Multiple comparisons were made and the test statistics were compared to a table of critical values for Q '(Zar JH, 1984).Example 1: Receptor coupled G-protein function in healthy volunteers   From healthy applicants to MNL films^ThreeThe binding of H-Gpp (NH) p^ThreeH-Gpp (NH) p concentration Without an agonist, or with isoproterenol, carbamylcholine or Was measured in the presence of dopamine. Specific binding (from total binding to non-binding (Calculated by reducing specific binding) reaches equilibrium within 5 minutes and at least 30 minutes It remained in a steady state.   In the absence of agonist (open circles), the presence of isoproterenol (open triangles) Healthy volunteers in the presence of bamilcholine (closed squares) and dopamine (closed circles) For MNL film from applicant^ThreeRepresentative saturation curve for specific binding of H-Gpp (NH) p, FIG. 1 shows non-specific binding curves (closed triangles) of the parallel experiments. All three kinds of agoni Strike-induced increase in Gpp (NH) p binding capacity substantially increases binding affinity Has no effect.   FIG. 2a shows basal and agonist-induced binding curves in MNL membranes from healthy volunteers. Shown is a representative Scatchard plot of a line. Specific binding Gpp (NH) p free Gpp ( The ratio to NH) p is plotted against specific binding Gpp (NH) p. Most Large binding capacity, Bmax, can be estimated from the horizontal axis intercept of the extrapolated linear regression line. Can be.   Mean values for all populations of healthy volunteer subjects were examined.Bmax- Baseline Bmax in the absence of agonist is 40.3 ± 1.0 pmole / mg protein Quality.β-Bmax- Bmax in the presence of the β-adrenergic agonist isoproterenol The value is 50.9 ± 1.9 pmole / mg protein (Q ′ = 3.2 p <0.01).M-Bmax -Bmax value in the presence of the muscarinic agonist carbamylcholine is 52 0.5 ± 1.6 pmole / mg protein (Q ′ = 3.57p <0.01).D-Bmax- Bmax value in the presence of L-dopamine was 54.9 ± 2.0 pmole / mg protein. Quality (Q ′ = 4.34, p <0.01).   The increase in Gpp (NH) p binding capacity induced by all three agonists was statistically Very significant.   Bmax values also depend on age (L. Barki-Harrington et al., 1996, in press) or There were no significant changes after physical exercise.   The agonist-induced increase in Gpp (NH) p binding capacity is       ([Bmax in the presence of agonist] / [Bmax-basal level] -1) x 100 Is calculated.   Isoproterenol (β), carbamylco for healthy volunteers (solid circles) Scattering of increased Gpp (NH) p binding capacity induced by phosphorus (M) and dopamine (D) The plot is shown in FIG. 3d.   The mean and standard error of the mean of the agonist-induced increase in Gpp (NH) p binding capacity were calculated. At the same time, isoproterenol 26.4% ± 1.3 (white bar), carbamylcholine About 28.3% ± 1.7 (hatched bar) and about 26.8% ± 1.9 for dopamine (black Bar) (FIG. 4).   Table I shows the effects of β-adrenergic, muscarinic and dopamine antagonists , And the effects of cholera and pertussis toxin on agonist-induced increase in Gpp (NH) p binding Represents a fruit. Cholera toxin activates the Gs protein by ribosylation of the α subunit. It is known to inhibit. As shown in Table 1, dopamine and isoprotein Renol-induced increase in Gpp (NH) p binding capacity was abolished by cholera toxin, Suggests the effect of dopamine and isoproterenol on Gs protein function ing. Human MNL activates adenylyl cyclase function via Gs protein. It is known to have surface β-adrenergic receptors that become sexual. Guanine Dopamine-induced increase in nucleotide binding is dependent on selective D₁And D_FiveA Specifically blocked by the antagonist SCH-23390,_TwoAnn It was not affected by the agonist sulpiride. D₁And D_FiveReceptor selection SKF-383931 μM induces a concentration-dependent increase in Gpp (NH) p binding ability This was similar to the dopamine-induced increase (not shown). D₁And D_FiveHas now confirmed that it activates adenylyl cyclase through Gs It is the only dopamine receptor that has been. But D_ThreeAnd D_FiveReceptor is human powder It has previously been shown to be expressed only in peripheral blood lymphocytes. Therefore, Dopamine is probably D_FiveGenerating Gs protein function in MNL via receptor I guess.   As shown in Table 1, the effect of carbamylcholine on Gpp (NH) p binding was Inhibited by toxin pretreatment but not affected by cholera toxin_TwoAntagoni Specifically blocked by ADFX116, while M₁Antagonist It was not affected by Renzepine. M_TwoThe receptor is G_i-Conjugate to proteins. G_i -The protein is blocked by pertussis toxin via ribosylation of its alpha subunit. It is known to be harmed. Therefore, these findings suggest that the carbamylcholine effect Which inhibits adenylyl cyclase_iM to combine with_TwoDemonstrated via receptors Suggest that.a^ThreeNo statistically significant increase in H-Gpp (NH) p binding capacity was detected.Example 2: Receptor-coupled G protein function in psychotic patients   Assay of receptor-induced G-protein function in various psychiatric patients did. FIG. 2 (a-e) shows mania (b), depression (c), Alterations in Gpp (NH) p binding in individual patients with schizophrenia (d) and panic disorder (e) Here is a tabular example. Binding was performed under basal conditions (open circles) or with Proterenol (open triangle), carbamylcholine (closed square) or dopamine ( Assays were performed in the presence of (closed circles) ((a) and (d) only). Basal among 5 individuals No significant change in binding ability was observed. However, each patient has It showed a specific and different pattern of Nist-induced increase.   Figures 3 (a-d) and 4 show the agonism of binding ability in groups of patients with various mental disorders. These are summaries of the results of the G-induced increase. Manic compared to healthy volunteers (black circles) Patients with disease (3a), depression (3b), schizophrenia (3c) and panic disorder (3d) The scattering plot (open circles) is shown in FIG.   Isoproterenol (white bar), carbamylcholine (hatched bar), and Mean and mean markers for the increase in Gpp (NH) p binding capacity induced by papamine (black bars) Calculated quasi-errors are calculated as mania patient (C), depression patient (D), panic disorder patient ( E), schizophrenic patients (F) and health at rest (A) or after exercising (B) FIG. 4 shows various applicants.   Each group of patients is characterized by a different pattern of receptor-coupled G protein function. Be charged. Compared to control subjects, the MNL of manic patients has a higher β-ad Renalin receptor coupled G protein (β-G_P) Function and high muscarinic receptor Role G protein (MG_P) Function is characterized by β- G_PAnd MG_PIt was detected that all functions were reduced. Patients with panic disorder MNL is high β-G_PFunction and degraded MG_PFeatures features. Listed above In contrast to the group of patients with schizophrenia, β-G_PAnd MG_PMachine No difference in performance was detected. In these patients, compared to normal subjects High dopamine receptor-coupled G protein (DG_P) Function detected.   Gpp (8) in patients with neuroleptic congenital schizophrenia (8 of 23 patients) The pattern of agonist-induced increases in NH) p binding capacity is similar to that of other schizophrenic patients. It was similar.   Normal subjects after intense physical exercise have different G protein functions than control subjects Not shown, changes in G-protein function in patients are simply motor activity It's not just excess or underactivity.   The results are summarized in Table II.  Specific binding at 5 mM (basal and agonist induced) was determined by the ability of Gpp (NH) p binding Equivalent results were obtained when used as a single point measurement to calculate nyst-induced increases. Was done.   Empirical findings may be used for the differential diagnosis of mentally ill patients.   Sensitivity and specificity of potential diagnostic assays based on G-protein function measurements Is determined by any given threshold and predetermined test criteria. Can be evaluated using efficacy tests.   For example, Gp is defined as a threshold that reflects hyperactivity and hypofunction of G protein, respectively. By measuring the values ≧ 45% and 15% ≦ of the agonist-induced increase in p (NH) p binding capacity The following results were obtained.   β-G_PAnd MG_PSensitivity for the diagnosis of mania requiring both hyperactivity Was 0.8 and the specificity was 0.93.   D-G_PHyperfunction and normal β-G_PAnd MG_PDiagnosis of schizophrenia requiring As for the sensitivity, it was found that the sensitivity was 0.87 and the specificity was 0.97.   β-G_PAnd G_PFor the diagnosis of depression requiring reduced function of the sensitivity is 0.89, The specificity was found to be 0.90.   β-G_PHyperfunction and normal or impaired MG_PNeed for panic disorder The diagnosis was found to have a sensitivity of 0.75 and a specificity of 0.93.   Analysis of the results shows that 1 (only in 2 cases), 2 or 3 agonists Can be based on the values obtained from the Depending on the fabric, different thresholds can be used for each of the agonists. Asse Other processing of the results of (a) is also possible and may result in a reliable diagnosis. For example , The evaluation is not based on each parameter separately, but β-G_P/ M-G_PBased on the ratio of This can increase the sensitivity and specificity of diagnosing panic disorder.Example 3: G.alpha in psychiatric patients _s and G.alpha _i Level   The levels of Gs and Gi proteins in MNL were_sAnd Gα_iPo for It was determined by immunoblot analysis using a clonal antibody. Manic patients (A) Membranes from depressed patients (C) and healthy volunteers age- and gender-matched (B) A typical immunoblot with proteins is shown in FIG. Anti-Ga_sRecognized by Gα_sOf the two types (45 kDa and 52 kDa)_sOnly to MNL Was detected.   The normalized data is shown by a scatter plot (FIG. 6). Gα_s45kDa species level (6a) and Gα_iLevel (6b) was lower than that of control subjects (open circles) (Solid squares) significantly increased and decreased in depressed patients (solid circles). schizophrenia Yes in MNL from patients (solid triangles) or panic disorder patients (not shown) No significant change was detected.   Compared to the mean of the control group, Gα_sAnd Gα_i≥15% increase in immunoreactivity level Alternatively, the specificity and selectivity of the test can be evaluated using the value of the decrease or the value as a threshold. Little And one kind of Gα-subunit (Gα_s, Or Gα_i) Immunoreactivity level For the diagnosis of mania requiring a ≥15% increase in sensitivity, the sensitivity is 0.73 and the selectivity is 0.81. At least one Gα-subunit (Gα_sOr Gα_i) Immunity For the diagnosis of depression requiring a ≧ 15% decrease in reactivity level, the sensitivity is At 0.73, the selectivity is 0.90.Example 4: Correlation between biochemical parameters and psychiatric assessment of depression   Normal subjects, outpatients with mild depression, patients with moderate and severe depression, Beck Survey and β-G_PAnd MG_PFunction and evaluate Gα_sAnd Gα_iLes The bell was analyzed.   As shown in FIG. 7, isoproterenol (7a) and carbamido Lecoline (7b) -induced increase in the Beck survey score in a statistically very significant form (Spearman correlation coefficient = -0.781p <0.001 and -0. 749p <0.001). Also, Gα_sAnd Gα_iBetween immunoreactivity level and Beck score Significant correlation was also obtained (-0.63p <0.001 and -0.693p <0.001 respectively) (Not shown).Example 5: Effect of psychiatric treatment on G-protein function and levels   Β-G in euphoric bipolar patients treated with lithium_PAnd MG_PFunction is It has been found to be similar to that of control subjects (Schreiber et al., 1991, Avi ssar and Schreiber 1992a).   FIG. 8 shows in depressed patients before (black circle) and after (open circle) treatment with antidepressants. β-G_PIndicates the function. Β-G was present in most of the depressed patients examined (7 out of 8)._PMachine Changes in performance became normal after treatment with antidepressants. The only exception is a psychiatrist It was also found to be non-responsive to treatments based on biological parameters.   Β-G in MNL membranes from depressed patients during ECT treatment_PFunction (open circle) and MG_P Functions (open squares) were measured at regular intervals.   In parallel, Beck study of depression (filled circles), or Hamilton & Brief psychiatry Use a rating scale (BPRS) score (not shown) to assess mental illness Valued.   FIG. 9a shows β-G in four depressed patients on electroconvulsive therapy [ECT]._PFunction (white circle ), MG_PRepresentative examples of dynamics of changes in functions (open squares) and Beck scores (closed circles) Is shown.   FIG. 9b shows Gα in four depressed patients on electroconvulsive therapy [ECT]._s(White circle), G α_i(Open squares) Representative of the kinetics of changes in immunoreactivity levels and Beck scores (closed circles) Here is a typical example.   From the above figures, normalization of all four biochemical parameters leads to clinical improvement It is clear that it will be seen in advance. Responded to treatment according to psychiatric criteria Similar results were obtained in another 10 patients. ETC according to psychiatric standards Non-responders did not show reduced function and reduced immunoreactivity (Not shown).   Thus, functional and quantitative measurements of G-proteins are available in various psychiatric It can be a predictor of the responsiveness of mentally ill patients to treatment.References Avissar S., Schreiber G., Danon A, Belmaker RH, (1988a): "Lithium is Inhibits adrenergic and cholinergic increases in GTP binding in the rat cortex. "(L ithium Inhibits Adrenergic and Cholinergic Increases in GTP Binding inRa t Cortex) Nature; 331: 440-442. Avissar S. and Schreiber G., (1992b): "Receptors for both anti-bipolar and antidepressant treatments. Interaction with Anti-G protein "(Interaction of Antibipolar and Antidepressa ntTreatments with Receptor-Coupled G Proteins) (Anna Monika Award Paper) Pharmacopsychiatry, 25: 44-50. Avissar S., Schreiber G., (1992b): "G et al. In the etiology and treatment of affective disorders. Involvement of G Proteins in the Pathogenesis and Treatment of Affective Disorders) (Ziskind-Somerfeld Award Paper) .BiolPs ychiatry 31: 435-459. Barki-Harrington L., Nechamkin Y., Schreiber G., Avissar S., (1996 (printed Middle)): `` Functional and quantitative measurement of receptor-coupled G-protein in human mononuclear leukocytes “No change due to age” (Functional and Quantitative Measures of Recept or-Coupled G-Proteins in Human Mononuclear Leukocytes: No Change with Age ) Exp.Gerontol. Boyum A., (1968): "Separation of leukocytes from blood and bone marrow" ytes from Blood and Bone Marrow) Scand.J.Clin.Lab.Invest.; 21 (Suppl 97): 7 . Friedman E., Butkerait P., Wang HY, (1993): "Receptor stimulation and basal guar. Sodium dodecyl sulfate-polyamide binding of nin nucleotides to membrane G proteins K Analysis of Lilamide Gel Electrophoresis (Analysis of Receptor-Stimulated and Bass al Guanine Nucleotide Binding to Membrane G Proteins by SodiumDodecyl S (ulfate-Polyacrylamide Gel Electrophoresis) Analytical Biochemishy 214: 17 1-178. Laugwitz KL, Spicher K., Schultz G., Offermanns S., (1994): "Receptor activity. Of G-protein-selective immunoprecipitation of photolabeled G-protein α subunit Down) (Identification of Receptor-Activated G Proteins-Selective Immunoprecipitation of Photolabeled G Protein α Subunits) Methods inEnz ymology237: 283-294. Lesch, K.P., Manji H.K., (1992): "G protein and antidepressant" (G Proteins a ndAntidepressants) Biol Psychiatry 32: 549-579. Mazzola-Pomietto P., Azorin J.M., Tramoni V., Jeanningros R., (1994): Relationship between lymphocyte beta-adrenergic response and severity of depression disorder "(Relation Bet weenLymphocyteβ-Adrenergic Responsivity and the Severity of DepressiveD isorders) Biol Psychiatry, 35: 920-925. Schreiber G., Avissar S., Danon A, Belmaker RH, (1991): Functional G-Proteins in Nuclear Leukocytes "(Hyperfunctional G-Proteins inMo nonuclear Leukocytes of Patients with Mania) Biol. Psychiatry, 29: 273-280. Wieland T., Jakobs KH, (1994): "Receptor stimulated G proteins. Measurement of Guanosine 5'-O- (γ-thio) triphosphate Bond "(Measurement o fReceptor-Stimulated Guanosine 5'-O- (γ-Thio) triphospbate Binding by G Proteins) Methods in Enzymology, 237: 3-13. Young LT, Li PP, Kish SJ, Siu KP, Warsh JJ, (1991): "After death Cerebral cortex G_sAlpha-subunit levels are elevated in bipolar affective disorder. '' (Post-mortem Cerebral Cortex G_sα-Subunit Levels are Elevated in Bipol arAffective Disorder) Brain Res .; 553: 323-326. Zar JH, (1984): Biostatistical Analysis, 2nd ed., Pren tice-Hall, Englewood Cliffs, NJ, p. 569,

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, LS, MW, SD, S Z, UG), UA (AM, AZ, BY, KG, KZ, MD , RU, TJ, TM), AL, AM, AT, AU, AZ , BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, FI, GB, G E, HU, IL, IS, JP, KE, KG, KP, KR , KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, MW, MX, NO, NZ, P L, PT, RO, RU, SD, SE, SG, SI, SK , TJ, TM, TR, TT, UA, UG, US, UZ, VN

Claims (1)

Claims: 1. (a) measuring the function and / or level of at least one receptor-coupled G protein; (b) diagnosing a mental disorder based on the measurement in (a); or A method for diagnosing a psychiatric disorder, including measuring the effect of treatment on a patient, or for measuring the effect of treatment on a psychiatric patient. 2. (a) measuring the function and / or level of at least one receptor-coupled G protein in mononuclear leukocytes (MNL) isolated from the patient; (b) based on the measurements in (a) A method for diagnosing a psychiatric disorder or measuring the effect of a treatment on a psychiatric patient, including diagnosing a psychiatric disorder or measuring the effect of a treatment on a patient. 3. One, two or three types of receptors selected from (a) (i) β-adrenergic receptor coupled G protein, (ii) muscarinic receptor coupled G protein, and (iii) dopamine receptor coupled G protein 3. Mental disorder according to claim 1 or 2, comprising measuring the function of the conjugated G protein and diagnosing the mental disorder based on the measurements in (b) (a) or measuring the effect of treatment on the patient. A method for diagnosing or measuring the effect of treatment on a psychiatric patient. 4. The method according to any one of claims 1 to 3, wherein the receptor-coupled G protein function is measured by a guanine nucleotide binding assay. 5. The method according to claim 4, wherein the guanine nucleotide for measuring the binding is Gpp (NH) p. 6. The method according to claim 4 or 5, wherein the guanine nucleotide is labeled. 7. The psychiatric disorder according to any one of claims 1 to 6, wherein the disorder is selected from the group comprising: (i) mania, (ii) depression, (iii) panic disorder, and (iiii) schizophrenia. How to diagnose. 8. The method of any one of claims 1 to 7, wherein the function of the at least one receptor-coupled G protein is measured as an agonist-induced increase in guanine nucleotide binding capacity. 9. A value of ≧ 45% increase in Gpp (NH) p binding ability induced by an agonist indicates hyperactivity, and a value of ≦ 15% increase in Gpp (NH) p binding ability induced by an agonist indicates a decrease in function. 9. The method of claim 8, wherein a value of greater than 15% increase and less than 45% increase in Gpp (NH) p binding ability induced by the agonist is indicative of normal function. Ten. The method according to any one of claims 7 to 9, wherein the function of at least two receptor-coupled G proteins is measured and used for diagnosis. 11． The method according to claim 10, wherein the receptor-coupled G protein comprises a β-adrenergic receptor-coupled G protein and a muscarinic receptor-coupled G protein. 12． 12. The method according to claim 11, wherein when the result of the measurement indicates hyperactivity of β-adrenergic receptor-coupled G-protein and hyperactivity of muscarinic receptor-coupled G protein, the mental disorder is diagnosed as mania. 13. 12. The method according to claim 11, wherein the mental disorder is diagnosed as depression when the result of the measurement indicates decreased function of β-adrenergic receptor-coupled G protein and decreased function of muscarinic receptor-coupled G protein. 14. 12. The method according to claim 11, wherein the mental disorder is diagnosed as a panic disorder if the result of the measurement indicates hyperactivity of β-adrenergic receptor-coupled G protein and hypofunction or normal function of muscarinic receptor-coupled G protein. . 15. The method according to any one of claims 7 to 9, wherein the functions of three receptor-coupled G-proteins are measured and used for diagnosis. 16． 16. The method according to claim 15, wherein the receptor-coupled G-protein comprises a β-adrenergic receptor-coupled G protein, a muscarinic receptor-coupled G protein, and a dopamine receptor-coupled G protein. 17． If the result of the measurement indicates hyperactivity of dopamine receptor-coupled G protein, normal function of β-adrenergic receptor-coupled G protein, and normal function of muscarinic receptor-coupled G protein, the disorder is diagnosed as schizophrenia 17. The method of claim 16, wherein the method comprises: 18． (a) measuring the function of at least one receptor-coupled G protein before and during treatment; and (b) predicting the function of the at least one receptor-coupled G protein in a healthy person. A method for determining the effect of a treatment on a psychiatric patient according to any of claims 1 to 6, comprising comparing 19. 19. The method according to claim 18, wherein the psychiatric disorder is mania and a decrease in β-adrenergic conjugated G protein function and / or muscarinic conjugated G protein function to a normal value indicates a positive effect of the treatment. 20. 19. The method according to claim 18, wherein the psychiatric disorder is depression and increasing β-adrenergic conjugated G protein function and / or muscarinic conjugated G protein function to a normal value indicates a positive effect of the treatment. twenty one. 20. The method of claim 18, wherein the psychiatric disorder is schizophrenia and the dopamine-coupled G protein function is reduced to normal, thereby indicating a positive effect of treatment. twenty two. Claims wherein said psychiatric disorder is a panic disorder, wherein a positive effect of treatment is indicated by a decrease in β-adrenergic G protein function to normal and / or an increase in muscarinic G protein function to normal. Item 19. The method according to Item 18. twenty three. Methods for the measurement to measure the effect of treatment for mental disorders diagnosing or psychotic patient, according to claim 1 or 2 which is a measurement of G.alpha _s and / or G.alpha _i level. twenty four. The G.alpha _s and / or G.alpha _i level-specific method of claim 23 monoclonal or is due to polyclonal antibodies measured against its subunits. twenty five. 26. The method for diagnosing a psychiatric disorder according to claim 23 or 25, wherein the disorder is selected from the group comprising (i) mania and (ii) depression. 26. Wherein the result of the measurement, relative to the average value of the control group, indicating an increase of over 15% of the level of increase and / or G.alpha _s exceeds 15% of the level of G.alpha _i, to diagnose psychiatric disorders and mania Item 26. The method according to Item 25. 27. 26. The method of claim 25, wherein the psychiatric disorder is diagnosed as depression if the result of the measurement indicates a> 15% decrease in the level of G [alpha] _i and / or the level of G [alpha] _s relative to the mean of the control group. . 28. Said mental disorder is mania, methods for measuring the effect of treatment for mental disorders a patient according to claim 23 or 24 reduction in G.alpha _s and / or G.alpha _i indicates the positive effect of the treatment. 29. 25. The method for measuring the effect of a treatment on a psychiatric patient according to claim 23 or 24, wherein the psychiatric disorder is depression and an increase in Gα _s and / or Gα _i indicates a positive effect of the treatment. 30. 23. A kit for diagnosing a psychiatric disorder by monitoring the function of at least one receptor-coupled G-protein in MNL isolated from a patient according to any one of claims 1 to 22. (A) (i) β-adrenergic receptor (ii) muscarinic receptor (iii) dopamine receptor agonists of one, two or three receptors selected from the group comprising: (b) labeled guanine nucleotide ( c) said kit comprising the manufacturer's instructions for using the kit. 31. 31. The kit according to claim 30, wherein the β-adrenergic receptor agonist is isoproterenol, the muscarinic receptor agonist is carbamylcholine, and the dopamine receptor agonist is dopamine. 32. A kit for diagnosing a psychiatric disorder by monitoring the amount of at least one Gα-subunit in MNL isolated from a patient according to any one of claims 1, 2 and 24-29. a is, (a) anti G.alpha _s and one or two monoclonal or polyclonal antibodies are selected from the group comprising _{anti-Gα i, (b) (a} ) the detectable probe capable of specific binding to an antibody of (C) a standard sample, (c) the manufacturer's instructions for using the kit.