WO2000044273A2

WO2000044273A2 - Diagnosis and prediction of light therapy response in seasonal affect disorder patients using g-protein subunit measurements

Info

Publication number: WO2000044273A2
Application number: PCT/IL2000/000066
Authority: WO
Inventors: Sofia Avissar
Original assignee: Ben-Gurion University Of The Negev
Priority date: 1999-01-31
Filing date: 2000-01-31
Publication date: 2000-08-03
Also published as: IL128304A0; WO2000044273A3; AU2317300A

Abstract

The present invention is concerned with the use of biochemical measurements, in particular the levels of certain G-protein subunits of interest, in the diagnosis of seasonal affective disorder (SAD) and the prediction of the response of SAD patients to light therapy.

Description

METHODS AND KITS FOR THE DIAGNOSIS OF SEASONAL

AFFECTIVE DISORDER AND FOR MONITORING THE

RESPONSE OF PATIENTS WITH THIS DISORDER TO

LIGHT THERAPY

FIELD OF THE INVENTION The present invention is concerned with the use of biochemical measurements, in particular the levels. of certain G-protein subunits of interest, in the diagnosis of seasonal affective disorder (SAD) and the prediction of the response of SAD patients to light therapy.

BACKGROUND OF THE INVENTION SAD is a mood disorder characterized by recurrent winter depressive episodes, with remission or hypomanic periods in the spring and summer seasons, when average daily light levels are higher. SAD patients differ from patients with other forms of mood change or depressive disorders with respect to their clinical profiles, the former being characterized by overeating, carbohydrate craving, weight gain, increased sleep, and fatigue, as well as in relation to biological characteristics (for example, normal dexamethasone suppression test responses; normal responses to TRH challenge tests and normal REM sleep latency). Both the clinical and biochemical profiles of patients with SAD more closely resemble those profiles found in individuals with atypical depression (Rosenthal, N.E. et al., Arch Gen. Psychiatry, 1984; 41 :72-80).

The most common form of treatment for SAD is phototherapy, which may be given either as full spectrum light or with light sources restricted to certain frequency ranges. The high efficacy of phototherapy for SAD is now generally acknowledged by practitioners in the field (Rosenthal, N.E., J. Am Med Assoc 1993;270: 2717-2720). Although there is no current consensus as to the pathophysiology of SAD, nor to ^"the mechanism of action of light therapy, the mechanisms that have been put forward to account for these phenomena include: altered primary messenger function, abnormal brain serotonergic transmission, reduced sympathetic system arousal and underactive HPA axis functioning (Rosenthal, N.E., Biologic Effects of Light (eds. Holick MF, Jung EG) Walter de Gruyter, Berlin, 1996 p. 311-324).

Currently used diagnostic and prognostic methods for psychiatric disorders tend to be phenomenological and empirical in type. This is in sharp contrast to many other branches of medicine, where advances in the understanding of molecular mechanisms of disease have led to more rational approaches to both its classification and control.

In recent years, however, research addressing biochemical mechanisms underlying the pathogenesis and treatment of neuropsychiatric disorders has focused on alterations in the post-receptor information transduction elements. The family of heterotrimeric G proteins is a crucial point of convergence in the transmission of signals from a variety of hormone and neurotransmitter membrane receptors to a series of downstream cellular events: intracellular second messenger effector enzymes and ionic channels. It has been shown that receptor binding of agonists leads to displacement of GDP from the α subunit of the G-protein by GTP. In the case of the stimulatory G proteins (Gs), this change in nucleotide binding causes activation of the G-protein, wherein there is dissocation of the β and γ subunits. Gsα moves towards adenylate cyclase, leading to its activation and the subsequent catalytic production of the 2nd messenger cAMP from ATP.

The increasing interest in the clinical perspective of altered G protein function has yielded important findings concerning the involvement of G proteins in the pathophysiology of mood disorders and in the biochemical mechanisms underlying the treatment of these disorders. The function of receptor-coupled G proteins was found- to be altered by lithium, and other antibipolar treatments (Avissar S., Schreiber G., Pharmacopsychiatry 1992;25:44-50). Increased activity of Gs and Gi proteins, and elevated levels of Gsα and Giα subunit proteins were detected in mononuclear leukocytes (MNL) of patients with mania (Avissar S. et al., J Affect Disord. 1997;43:85-93.). In contrast to the findings in manic patients, reduced functional measures of Gs and Gi proteins have been reported in MNL of patients with major depressive disorder (ibid.). Although conflicting results were obtained concerning G proteins immunoreactive levels in MNL of patients with major depression, a larger scale study has detected reduced levels of Gsα and Giα proteins in MNL of depressed patients which were in correlation with the severity of depression and with the reductions in the functional measures of these proteins (Avissar S, et al. Am J Psychiat

1997;154:21 1-217.). These novel and unexpected alterations in G-protein activity and concentration in psychiatric diseases form the basis of the differential diagnostic methods disclosed in a co-owned patent application, WO 97/20211.

Quantitative - and functional measures of G proteins, have also been undertaken in mononuclear leukocytes of patients undergoing treatment with antipsychotic agents, as a means for monitoring the extra-pyramidal side effects of these drugs. The results of these studies are disclosed in co-owned Israeli patent application no. 125346.

SUMMARY OF THE INVENTION It has now been found, that the changes in G-protein levels observed in patients with SAD may be used to assist in the diagnosis of SAD. Furthermore, the measurement of G-protein levels may also be used to determine whether a patient already diagnosed with SAD is responsiveness to light treatment. The invention is directed to a method for diagnosing SAD in an untreated subject - or determining the responsiveness to light treatment of a subject already diagnosed with SAD, comprising the steps of: a) determining the level of at least one G-protein in a sample taken from the subject; and b) diagnosing SAD in an untreated subject, or determining responsiveness to light treatment of a subject already diagnosed with SAD, based on said determination in a).

In one embodiment, the above-described method is used to diagnose SAD. In this embodiment, a diagnosis of SAD is made when the results of said G-protein level determination in an untreated subject indicate a decrease in the level of G_αs or G_α„ compared to a group of healthy subjects.

In another embodiment, the method of the invention is used to determine the responsiveness to light treatment of subjects already diagnosed as having SAD. The subject is determined as being, responsive to light treatment when the results of said G-protein level determination in a SAD patient following two weeks of light treatment, indicate levels of G_αs or G_α„ that have risen to fall within the normal ran-e of those obtained from a group of healthy individuals.

While the method of the invention may be carried out in any convenient cellular or tissue source from which G-proteins may be isolated, in a preferred embodiment, the determination of G-protein levels is made in mononuclear leukocytes isolated from peripheral blood.

In a preferred embodiment, the determination of G-protein levels is made by measurement of immunoreactive levels of at least one G-protein subunit. In one aspect, the invention is directed to measuring the immunoreactive levels of G_sα. In another aspect, the invention is directed to measuring the immunoreactive levels of Gj_α.

In a further aspect, the invention is directed to the provision of a kit for monitoring the therapeutic response to light therapy of SAD patient, by the use of G-protein concentration measurements, comprising: a) one or more monoclonal or polyclonal antibodies selected from the group comprising anti-G_sα and anti-G;_α; b) detectable probe which is capable of specifically binding to the antibody of a); and c) standard samples, for comparison with patients' samples.

In a preferred embodiment, this kit may further comprise separation media for the purification of MNL and/or membrane homogenization buffer.

Furthermore, these measurements may also be used as a determinative measure of the response to light therapy of a patient already diagnosed as suffering from SAD.

It is a purpose of this invention to provide a quantitative biochemical method for use in establishing a diagnosis of SAD.

It is another purpose of this invention to provide for the use of measurements of immunoreactive levels of the G-protein subunits in order to help establish a diagnosis of SAD.

It is a further purpose of the invention to provide the aforementioned method for use in determining the response of an individual suffering from SAD to light therapy. It is a further purpose of the invention to provide kits for use in the aforementioned method, for predicting whether a subject with SAD is likely to benefit from treatment with light therapy and for the diagnosis of SAD.

All the above and other characteristics and advantages of the invention will be further understood from the following illustrative examples of preferred embodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS The present invention will be more clearly understood from the detailed description of the preferred embodiments and from the attached drawings in which:

Fig. 1 is a graph summarizing the results of measurements of G-protein subunit measurements in SAD patients before, and two weeks after light treatment, and in normal subjects.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS For purposes of clarity and as an aid in the understanding of the invention, as disclosed and claimed herein, the following terms and abbreviations are defined below:

Seasonal Affective Disorder (SAD) - a mood disorder, clinically and biochemically distinguishable from typical depressive disorders, which is characterized by recurrent episodes of winter depression, with remission in the spring and summer.

Mononuclear Leukocytes (MNL) - herein used to refer to the isolated fraction of peripheral blood cells containing lymphocytes and monocytes. "G-Protein" means GTP-binding regulatory proteins involved in cell signaling processes. G-proteins are known to those skilled in the art. G-proteins include but are not limited to receptor coupled G protein. Examples of such receptor coupled G-protein include β-adrenergic receptor coupled G protein or muscarnic receptor coupled G-protein. In one embodiment the G protein is a α subunit G-proteins. As contemplated herein, a number of sub-types including stimulatory (Gs), inhibitory (Gi) and phosphoinositide-specific phospholipase C-related G proteins (Gp) are included. In the preferred embodiment the G-protein is Gα. Antibodies specific for binding to specific G proteins are known in the art. For example, commercially available anti Gα protein antibodies include but are not limited to: anti Gial by Chemico catalog number AB1617; anti Human Gαs clone K-20 Santa Cruz catalog number SC-823; anti Human Giα2 clone T-19 Santa Cruz catalog number SC-7276; anti Giαl clone 159-168 Calbochem catalog number 371720-s; anti Giαl/2 clone 345035 4/364-3 55 Calbochem catalog number 371720-s; or anti Giα2 clone L5.6 NeoMarkers catalog number MS-244-P.

Moreover, the antibody used for the determination according to the present invention may be directly label with the preferred fluorescence label, or may be indirectly labeled with the preferred fluorescence label. In the last case, the preferred fluorescence label is conjugated to a secondary antibody, which is directed against the first antibody, such as an anti species Ig antibody.

Labels are known to those skilled in the art. For example, Examples of labels encompassed by the present invention include, but are not limited to, radioisotopic labels (e.g., sup.3 H, .sup.125 I, .sup.131 I, .sup.35 S, .sup.14 C, etc.), non-radioactive isotopic labels (e.g., .sup.55 Mn, .sup.56 Fe, etc.), fluorescent labels (e.g., a fluorescein label, an isothiocyanate label, a rhodamine label, a phycoerythrin label, a phycocyanin label, an allophycocyanin label, art O-phthaldehyde label, a fluorescamine label, etc.) for example, as in peridinin chlorophyll protein (PerCP), chemiluminescent labels, enzyme labels (e.g., alkaline phosphatase, horse radish peroxidase, etc.), protein labels, labels useful in radioimaging and radioimmunoimaging. Further, as used herein, the term "label" refers to a molecule, which may be conjugated or otherwise attached (i.e., covalently or non-covalently) to a binding protein as defined herein. Particularly suitable labels include those, which permit analysis by flow cytometry, e.g., fluorochromes. Preferred fluorochromes include phycoerythrin (P.E., Coulter Corp., Hialeah, FL), phycoerythrin-cyanin dye 5 (PECy5, Coulter), and fluorescein isothiocyanate (FITC, International Biological Supplies, Melbourne, FL). Other suitable detectable labels include those useful in colorimetric enzyme systems, e. g., horseradish peroxidase (HRP) and alkaline phosphatase (AP). Other proximal enzyme systems are known to those of skill in the art, including hexokinase in conjunction with glucose-6-phosphate dehydrogenase. Chemiluminescent labels, such as green fluorescent proteins, blue fluorescent proteins, and variants thereof are known. Also bioluminescence or chemiluminescence can be detected using, respectively, NAD oxidoreductase with luciferase and substrates NADH and FNIN or peroxidase with luminol and substrate peroxide. Other suitable label systems useful in the present invention include radioactive compounds or elements, or immunoelectrodes.

Example 1 Measurement of G-protein subunit levels in SAD patients and controls

Subjects

Twenty-six patients (18 women, 8 men) fulfilling the following required inclusion criteria were selected for the study:

(a) Rosenthal et al. (Arch Gen. Psychiatry, 1984;41 :72-80) diagnostic criteria for SAD: a pattern of recurrent depressions, at least one of which met criteria for major depressive episode, and at least two of which occurred in consecutive years;

(b) a score of at least 15 on the Structured Interview Guide for the Hamilton Depression Rating Scale-Seasonal Affective Disorders Version (SIGH-SAD; Williams, J.B.W. et al.. Structured Interview Guide for the Hamilton Depression Scale - Seasonal Disorder Version. New York State Psychiatric Institute, NY, 1988); (c) absence of any additional current Axis I disorders;

(d) good physical health as determined by physical examination and routine blood work; and

(e) no use of light therapy or medications for the current winter depression. The mean age of onset of SAD in cases where it could definitely be determined was 28 years. Patients, therefore, had SAD for an average period of 13 years. Of the 26 patients, 17 (13 women) presented unipolar and 9 (5 women) bipolar II characteristics. Twenty-eight healthy controls (20 women) were matched with patients on the basis of age, gender, body mass index (BMI, kg/m²), menstrual phase and birth control status for women (4 women). Controls were required to have no personal or family history of Axis I psychiatric disorder and to be physically healthy. There were no significant differences (control vs. SAD) in age (mean age 46, range: 26-51 vs. 41, range-. 26-60) and BMI (25.9+11.4 vs. 26.4±11.2). Patients and healthy subjects were free of psychotropic medications for one month prior to the study. They were free of non-psychotropic medications for two weeks prior to the study. Sub3'ects were allowed no more than 2 caffeinated beverages per day for two weeks prior to the study.

Study Design

Subjects were all studied during the winter ("mean date"; 2/6/96 + 29 days vs. 2/6/96 + 32 days) in an untreated condition. Of the 26 SAD patients, 19 were studied again after two weeks of standard light therapy (10,000 lux from a light box, 2 feet by 2 feet, manufactured by the SunBox Company). The distance from center of patient's forehead to center of the light box was 12 inches. Light therapy was administered at home for 45 minutes, twice a day: once between 6:00-9:00 A.M. and once between 6:00-9:00 P.M. The summer group of the same patients included 22 patients, as four could not be contacted. Remission is defined as not meeting criteria for major depression plus having SIGH-SAD scores of less than 10. Non-responders are defined as meeting criteria for major depression plus having SIGH-SAD scores of more than 15. The same group of healthy volunteers was studied twice: during winter (28 subjects), as well as summer (25 subjects). Healthy volunteers did not receive light treatment in this study. Patients and healthy volunteers were studied during the summer period between May 28 and July 25. For each study, 30 ml of blood was drawn in the morning (7:00-9:00 A.M.). SIGH-SAD ratings were administered within a day of the bipod draw. Written informed consent was obtained after the procedures had been fully explained to the patients and the healthy subjects.

From 26 untreated SAD patients with winter depression, 21 were examined for G_αs and G_αj immunoreactivities, and 22 for Gβ immunoreactivity. Of the 19 patients after 2 weeks of light therapy 15 were evaluated for G_αs 16 for G_αi, and 19 for Gβ. Of the 22 summer-SAD patients, 17 were analyzed for G_αs, 20 for G_αi and for Gβ. Of the 28 winter group of healthy volunteer subjects 21 were assessed for G_αs> 17 for G_αi and 15 for Gβ immunoreactivities, while of the 25 summer group of the same sub'ects, 19 were measured for G_αs and 18 for G_αi and Gβ.

Protein concentrations of the samples were determined, following which the proteins in the samples were separated electrophoretically, subjected to i munoblotting and densitometry. All of these procedures were performed on a blind basis. Gel protocols were pre-prepared in advance to include matched controls in each gel.

MNL isolation

Mononuclear leukocytes (MNL) were isolated from 30 ml heparinized fresh blood, using a Ficoll-Paque gradient. Cells were homogenized in 25 mM Tris-HCI, pH 7.4, containing 1 mM dithiothreitol (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 10 min. Membranes were then suspended in homogenization buffer containing ImM EGTA and 30% sucrose w/v and frozen at -70° until assayed. Aliquots were taken for protein concentration determination using the Bradford assay.

Immunoblot analysis

On the day of assay, membranes were thawed, aliquots of 10 4g membranes taken for protein separation by SDS-(10%) polyacrylamide gel electrophoresis and the resulting proteins transferred to nitrocellulose paper by use of electroblotting apparatus. Blots were washed in Tris-buffered saline (TBS) containing 3% Tween-20, and blocked by incubation with 5% bovine serum albumin (BSA) for 1 hr in TBS containing 0.1% Tween-20 (TTBS). After two washes in TTBS, blots were incubated overnight with ing antisera (Santa Cruz Biotechnology, Inc.) directed specifically against G_αs, G_αii_,2, and Gβ (all diluted 1 : 100), followed by subsequent incubation with goat anti-rabbit IgG labeled with horseradish peroxidase (Jackson Immunoresearch Laboratories, Inc.).

Immunoreactivity was detected with the Enhanced Chemiluminescence Western Blot Detection System (Araersham) followed by exposure to Kodak X-Omat film.

Assay linearity was found for membrane protein concentrations of between 2.5 - 15 μg. Peak heights of immunoreactive bands were determined with an image analysis system, and semi-quantitative analysis was carried out. The optical density of the immunoreactive bands was normalized against 10 μg rat cortical membranes, run in each blot as a standard value. Although anti-G_αs detects both 52 and 45 kDa G_αs species, only the 45 kDa species is consistently detected in leukocytes, while rat cortex membranes show predominantly the 52 kDa species. The other subunits assayed in leukocytes were found to migrate at the expected molecular weights similarly to those labeled in the rat cortex membranes. Statistical analysis

The Wilcoxon Signed-Rank test was used for intra-blot comparisons, which are singular matched-comparisons within immunoblots, with alpha level of significance of 5%. For inter-blot average-comparisons, the Mann Whitney rank sum test was used, corrected for multiple comparisons by the Bonferroni inequality, with alpha level of significance of 1.6%. All the above statistical tests were performed in accordance with Glantz, S.A. (Biostatistics, 3^rd ed., N.Y., 1992).

Results

There were no significant differences between G protein levels of the control healthy subjects during winter to those measured during the summertime. Normalizing the winter samples as our comparison reference, the results show the following features: similar G_αs, G_α, and Gβ immunoreactivities for the control subjects during winter and summer (for Gas: (100.0±20.4)% versus (99.3±18.3)%, W=4, n=19, N.S.; for G_αι: (100.0±29.5)% versus (97.2±28.4)%, W=30, n=17, N.S.; for Gβ: (100.0±24.1)% versus (102±22.2)%. W=-12, n=15, N.S., Wilcoxon signed rank test).

The G_α, immunoreactive levels (figure 1A). and the G_α, immunoreactive levels (figure IB), in MNL of winter-depressed SAD patients (71.9±22.4)% and (79.5±23.2)%, respectively (open circles), were significantly reduced in comparison with the corresponding levels in healthy subjects (100.0±15.8)% and (100±7.2)% (open squares), using both intra-blot matched comparisons (for G_αs: W=195, n=21, p<0.01; for G_α,: W=221, n=21, p<0.01, Wilcoxon Signed-Rank test) and inter-blot average comparisons (for G_αs: Us=379, ts=3.99, df=40, p<0.01 ; for G_αι: Us=274, ts=2.8, df=36, p<0.01, Mann Whitney rank sum test). In contrast, MNL G_β levels (figure 1C) of depressed SAD patients (100.6±16.6)% (open circles), were similar to levels in healthy volunteers (open squares), (100.0±9.4)%, using both inter-blot average comparison (Us=170, ts=0.15, df=35, N.S., Mann Whitney rank sum test), and intra-blot matched comparisons (W=37, n=22, N.S., Wilcoxon Signed-Rank test).

Two weeks of light therapy resulted in clinical remission in SAD patients with decreases in typical, atypical, and total SIGH-SAD scores (Table I).

After two weeks of light therapy the reduced G_αs and G_α, levels in the depressed SAD patients (closed circles, figures 1A and IB respectively) were significantly elevated to normal-like levels (for G_αs: (95.7±24.2)%, W=85, n=15, p<0.02; for G_αι: (103.4±23.6)%, W=90, n=16, P<0.02, Wilcoxon signed-rank test), while G_β levels (100.3±14.3), remained similar to the control-like values obtained for depressed SAD patients (W=5, n=T8, N.S., Wilcoxon signed-rank. test) (figure 1C). Table I shows that G_αs and G_α, protein normalization paralleled clinical remission in all treated patients as well as in the subgroups of responders and non-responders.

During the summer the SIGH-SAD scores show the SAD patients to be in remission (Table I), with the levels of their MNL G_αs, (102.3±19.5)%, G_αι (102.9+21.7)%, and G_β, (101.5+18.7)%, all found to be normalized, similar to levels obtained for healthy subjects (for G_αs: Us=169, ts=0.24, df=34, N.S.; for G_αι: Us=218, ts=l.l 1, df=36, N.S.; for GP: Us=170, ts=0.25. df-36, N.S., Mann Whitney rank sum test).

Table I

Claims

1. A method for diagnosing seasonal affective disorder (SAD) in an untreated subject, or determining the responsiveness to light treatment of a subject already diagnosed with SAD, comprising the steps of: a) determining the level of at least one G-protein in a sample taken from the subject; and b) diagnosing SAD in an untreated subject, or determining responsiveness to light treatment of a subject already diagnosed with SAD, based on said determination in a).

2. A method according to claim 1, wherein when the results of said G-protein level determination in an untreated subject indicate a decrease in the level of G_αs or G_α„ compared to a group of healthy subjects, a diagnosis of SAD is made.

J . A method according to claim 1, wherein when the results of said G-protein level determination in a SAD patient following two weeks of light treatment, indicate levels of G_αs or G_α„ that have risen to fall within the normal range of those obtained from a group of healthy individuals, the subject is determined as being responsive to light treatment.

4. A method according to any one of claims 1 to 3, wherein the determination of G-protein levels is made in mononuclear leukocytes (MNL) isolated from the subject.

5. A method according to any one of claims 1 to 4, wherein the determination of G-protein levels is made by measurement of immunoreactive levels of at least one G-protein subunit.

6. A method according to claim 5, wherein G-protein levels are determined by measurement of the immunoreactive level of G_sα.

7. A method according to claim 5, wherein G-protein levels are determined by measurement of the immunoreactive level of G,_α,.

8. A kit for monitoring the therapeutic response to light therapy of SAD patient, by the use of G-protein concentration measurements according to any one of the claims 1 to 7, comprising: a) 1 or 2 monoclonal or polyclonal antibodies selected from the group comprising anti-G_sα, and anti-Gj_α,; b) detectable probe which is capable of specifically binding to the antibodv of a); and c) standard samples, for comparison with patients' samples.

9. A kit according to claim 8, further comprising separation media for the purification of MNL.

10. A kit according to claims 8 or 9, further comprising membrane homogenization buffer.