WO1998042872A1

WO1998042872A1 - Estrogen and the mental state: molecular variations within the estrogen receptor gene modulate clinical susceptibility to depression

Info

Publication number: WO1998042872A1
Application number: PCT/US1998/005789
Authority: WO
Inventors: Stephen J. Peroutka
Original assignee: Spectra Biomedical
Priority date: 1997-03-24
Filing date: 1998-03-23
Publication date: 1998-10-01
Also published as: AU6771598A; EP0983382A1

Abstract

The application provides evidence that genetic variations in the human estrogen receptor gene (ESR) correlate with depression. The invention provides methods of diagnosing depression or susceptibility thereto by detecting variations in the human estrogen receptor gene. The invention also provides methods of treating depression by administering an agonist of estrogen binding to the estrogen receptor.

Description

ESTROGEN AND THE MENTAL STATE: MOLECULAR VARIATIONS WITHIN THE ESTROGEN RECEPTOR GENE MODULATE CLINICAL SUSCEPTIBILITY TO DEPRESSION

CROSS-REFERENCE TO RELATED APPLICATIONS The present application derives priority from 60/041,449, filed March 24, 1997.

TECHNICAL FIELD

The present invention relates generally to the diagnosis and treatment of depression.

BACKGROUND OF THE INVENTION Depression is a clinical disorder that may begin at any age, although it usually begins in the mid-20s and 30s. These symptoms may develop over days to weeks. Some people have only a single episode, with a full return to premorbid function. However, more than 50 percent of those who initially suffer a single major depressive episode eventually deve1op another .

The point prevalence for major depressive disorder in the Western industrialized nations is 2.3 to 3.2 percent for men and 4.5 to 9.3 percent for women. The lifetime risk for major depressive disorder is 7 to 12 percent for men and

20 to 25 percent for women. Risk factors for major depressive disorder include female gender (especially during the postpartum period) , a history of depressive illness in first- degree relatives and prior episodes of major depression. Patients with major depressive disorder have substantial amounts of physical and psychological disability, as well as occupational difficulties. Untreated major depressive disorder has a substantial effect on health and functioning. Physical complaints are also common during a major depressive episode .

Although a genetic component has been suggested in depression, it has not been confirmed, much less characterized (U.S. Department of Health and Human Services; AHCPR Publication No. 93-0550, 1993). Further, it has not been reported whether the genetic bases and corresponding biochemical mechanisms underlying the different forms of depression are different in kind or only in degree. At present no specific genetic or biochemical tests are available for the positive diagnosis of depression. Diagnosis and treatment is presently based solely on patient self-reporting and symptom description. The clinical heterogeneity associated with depression has complicated patient reporting as well as the diagnosis and treatment of the disorder. As a result, no clear modality of treatment for all individuals with depression has emerged, and treatment as well as diagnosis varies greatly not only from patient to patient but from physician to physician. Thus, many sufferers of depression are not diagnosed or not effectively treated.

Identification of inheritance pattern (s) and genetic bases for depression would greatly facilitate the diagnosis and treatment of this disorder. SUMMARY OF THE CLAIMED INVENTION

In one aspect, the invention provides methods of diagnosing a patient for susceptibility to depression. Such methods entail detecting a variant allele of the human estrogen receptor gene (ESR) . For example, homozygous PvuII T/T alleles of the receptor gene indicate increased susceptibility to depression. Patients having a variant form of the human estrogen receptor gene can be treated with an antidepressive agent .

In another aspect, the invention provides methods of treating a patient suffering from depresion due to a variant allele of the human estrogen receptor gene. The methods entail administering to the patient a therapeutically effective amount of an antidepressive agent. In some methods the agent is administered prophylatically and in other methods therapeutically. In some methods, the agent binds to the estrogen receptor. Preferred agents agonize binding of estrogen to the estrogen receptor and thereby compensate for reduced sensitivity of the estrogen receptor to signal transduction due to a variation in the receptor gene.

In another aspect, the invention provides methods of screening potential drugs for antidepressant activity. ^• Such methods entail administering a potential drug to a population of patients having a variation in an estrogen receptor gene indicating increased susceptibility to depression, and determining the existence of depression in members of the population relative to a control population.

The invention further provides for the use of an agent effective to agonize estrogen binding to an estrogen receptor in the manufacture of a medicament for use in the treatment of depression. The invention further provides for the use of an agonist of estrogen binding to the estrogen receptor for treatment of depression. Typically, in such uses, the patient has a variant estrogen receptor gene associated with increased susceptibility to depression.

The invention further provides a diagnostic agent for detecting a variant estrogen receptor gene for use in therapy, prophylaxis or diagnosis of depression. The diagnostic agent can be an allele-specific oligonucleotide . or an allele-specific monoclonal antibody.

The invention further provides for the use of an agent for detecting a variation in the human estrogen receptor gene ESR in the manufacture of an agent for treatment, prophylaxis or diagnosis of depression.

DEFINITIONS Polymorphism refers to the occurrence of two or more genetically determined alternative sequences or alleles in a population. A polymorphic marker or site is the locus at which divergence occurs . Preferred markers have at least two alleles, each occurring at frequency of greater than 1%, and more preferably greater than 10% or 20% of a selected population. A polymorphic locus may be as small as one base pair. The allelic form occurring most frequently in a selected population is referred to as the wildtype form. Other forms are referred to as variant forms .

An allele-specific oligonucleotide hybridizes to a segment of target DNA from one individual but does not hybridize to the corresponding segment from another individual due to the presence of different polymorphic forms in the respective segments from the two individuals. Hybridization conditions should be sufficiently stringent that there is a significant difference in hybridization intensity between alleles, and preferably an essentially binary response, whereby a probe hybridizes to only one of the alleles. Likewise, an allele-specific monoclonal antibody shows specific binding to one allele without binding to another.

DETAILED DESCRIPTION The present invention provides methods of diagnosing depression and susceptibility thereto in women. These methods are premised, in part, on the insight that polymorphic variations in the human estrogen receptor gene (ESR) having a prototypical sequence described by Walter, Proc . Natl . Acad . Sci . USA 82, 7089-7093 (1985) cause increased susceptibility to depression.

The ESR gene encodes an intracelluar receptor for estrogen. The receptor functions as a transcriptional activator. The receptor has one domain that binds to estrogen and another than binds to palindromic sequences called estrogen responsive elements (ERE) to activate transcription (see Mukherjee et al . , Nucleic Acids Res . 21, 655-6 (1993)). Note that the ESR gene is to be distinguished from a second estrogen gene termed ERB, which has recently been discovered, and has a distinct in vivo role to ESR. Variations in the estrogen receptor cause depression by producing a form of receptor having altered sensitivity to estrogen signal transduction or by producing altered levels of receptor. Typically, variations cause the receptor to be less sensitive to estrogen signal transduction or to be expressed in reduced amounts relative to receptors in the general population. For example, detection of T/T PvuII alleles of the estrogen receptor gene indicates increased susceptibility to depression relative to individuals with C/c or C/T genotypes and to the general population.

Several additional variations in the human estrogen receptor gene are described by Wang et al . , Breast Cancer - Res . Treat . 2, 145-51 (1997); Sano et al . , Bioc em. Biophys . Res . Commun . 217, 378-83 (1995); Schmutzler et al . , Breast

Cancer Res . Treat . 19, 111-117 (1991); Lehrer et al . , Lancer 335, 622-624 (1990) . Other variant genes can be detected, for example, by sequencing, allele-specific amplification (Gibbs, Nucleic Acid Res . 17, 12427-12448 (1989)), restriction enzyme analysis, allele-specific probe hybridization assays (Saiki et al . , Nature 324, 163-166 (1986)) or single-stranded conformational analysis (Orita et al . , Proc . Nat . Acad . Sci . 86, 2766-2770 (1989)), or use of monoclonal antibodies with allele-specific binding specificity. Reagents used for detecting variant alleles, such as allele specific probes and primers can be packaged as diagnostic reagents or kits. The diagnostic reagents or kits can bear labels or instructions indicating their suitability for use in diagnosis of depression or symptoms thereof . Additional polymorphisms in the estrogen receptor gene correlated with susceptibility to depression can be identified as follows. The first step is to identify additional polymorphic sites within one of these genes. Such polymorphic sites can be identified either by comparative sequencing of these genes in a population of individuals or from the published literature and databases. A prototypical sequence of the coding region of the estrogen receptor gene is described by Walter, Proc . Natl . Acad . Sci . USA 82, 7089-7093 (1985) . Additional sequence data is described by Green, Science 231, 115-54 (1986) and Nature 320, 134-9 (1986) . Having identified the location of a polymorphism and the nature of its polymorphic forms, a correlation is performed between type of polymorphic form and presence or absence of depression in a population. Optionally, the correlation can be determined with respect to combinations of two or more polymorphisms within the same gene.

There are undoubtedly other genes besides the estrogen receptor described above having variant forms associated with depression. The existence of variant forms of such genes can be detected and correlated with probabilities of susceptibility to the syndrome in similar fashion to the analysis of estrogen receptor genes. Combined statistical analysis of the estrogen repeptor gene with other genes still increases the predictive value of the diagnosis.

Analysis of variations in the estrogen receptor gene is useful in identifying at least a subset of depression patients having a common genetic basis giving rise to the disease. Such patients are amenable to treatments with agents that anonize or antagonise estrogen binding to estrogen receptor. The selection of agonist or antagonist depends whether the variation reduces or increases sensitivity of the estrogen receptor to estrogen. Typically, agonists are used. An agonist can act alone to transduce a signal through the human estrogen receptor or can act in a cooperative manner with estrogen. Treatment with agents directed to the estrogen receptor may be ineffective in other classes of depression patients with a genetic basis unrelated to the human estrogen receptor.

II. Therapeutic Agents

Therapeutic agents useful in prophylaxis or therapeutic treatment of depression include estrogen and agonists or antagonists of estrogen binding to the estrogen receptor hRE . Some known agonists and antagonists of estrogen-estrogen receptor binding are described by Zysk et al . , Endocrinology 136, 1323-26 (1995) . Other agents having efficacy in treating at least some forms of depression include 5-HT1 agonists, monoamine oxidase inhibitors, tricyclic antidepressants (TCAs) , and serotonin-selective re-uptake inhibitors (SSRIs) , such as Prozac. Agonists that increase signal transduction of estrogen can be obtained by producing and screening large combinatorial libraries. Combinatorial libraries can be produced for many types of compound that can be synthesized in a step by step fashion. Such compounds include polypeptides, beta-turn mimetics, polysaccharides, phospholipids, hormones, prostaglandins , steroids, aromatic compounds, heterocyclic compounds, benzodiazepines, oligomeric N-substituted glycines and oligocarbamates . Large combinatorial libraries of the compounds can be constructed by the encoded synthetic libraries (ESL) method described in Affymax, WO 95/12608, Affymax, WO 93/06121, Columbia University, WO 94/08051, Pharmacopeia, WO 95/35503 and Scripps, WO 95/30642 (each of which is incorporated by reference for all purposes) . Peptide libraries can also be generated by phage display methods. See, e . g . , Devlin, W0 91/18980. The libraries of compounds can be initially screened for specific binding to the estrogen receptor, optionally in competition with estrogen. Preferred agents bind with a Kd < μM. The assay can be performed using cloned estrogen receptor immobilized to a support such as a microtiter well. Optionally, compounds can be screened for lack of specific binding to other cellular receptors, such as human estrogen receptors nonallelic with ESR. Assays for agonist activity can then be performed by contacting a compound under test with a reporter cell. The reporter cell has one construct expressing a reporter gene linked to an ERE (estrogen response element) , and a second construct expressing the human estrogen receptor. Transfection of a human estrogen receptor cDNA into mammalian cell lines is described by e.g., Levenson et al . , J. Steroid Bioche . Mol . Biol . 51, 229-39 (1994). The compound is taken up by the cell and activates the estrogen receptor. The activated estrogen receptor binds to the ERE element causing expression of the reporter gene.

III. Pharmaceutical Compositions Therapeutic agents can be formulated in pharmaceutical compositions to be used for prophylactic or therapeutic treatment of depression. A pharmaceutical composition comprises an active therapeutic agent, e . g. , estrogen or an agonist of estrogen binding to the estrogen receptor and a variety of other components. The preferred form depends on the intended mode of administration and therapeutic application. The compositions can also include, depending on the formulation desired, pharmaceutically- acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like. For parenteral administration, the therapeutic agents of the invention can be administered as injectable dosages of a solution or suspension of the substance in a physiologically acceptable diluent with a pharmaceutical carrier which can be a sterile liquid such as water and oils with or without the addition of a surfactant and other pharmaceutically preparations are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, and mineral oil. The agents of this invention can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained release of the active ingredient.

IV. Therapeutic Methods

Therapy can be initiated on diagnosis of depression and continued at regular intervals (e.g., weekly) until the symptoms are reduced, arrested or reversed. In some instances, therapy can be administered prophylactically to patients susceptible to depression before symptoms are apparent. The methods are also useful in pediatric patients (i.e., less than two years old) or disabled patients with genetic susceptibility to depression but who are unable to articulate their symptoms. In therapeutic applications, compositions are administered to a patient suspected of, or already suffering from depression in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease and its • complications. An amount adequate to accomplish this is defined as a therapeutically- or pharmaceutically-effective dose. In prophylactic applications, pharmaceutical compositions are administered to a patient susceptible to, or otherwise at risk of, disease in an amount sufficient to eliminate or reduce the risk or delay the outset of the disease. Such an amount is defined to be a prophylactically effective dose. Effective doses of the compositions vary depending upon many different factors, including means of administration, target site, physiological state of the patient, and other medicants administered. Agents can be administered intravenously, intramuscularly, subcutaneously, intranasally, cutaneously, via suppository, by inhalation or orally. The pharmaceutical carrier can be any compatible, non-toxic substance suitable to deliver the antagonist to the patient. Sterile water, alcohol, fats, waxes, and inert solids can be used as the carrier. Pharmaceutically-acceptable adjuvants, buffering agents, dispersing agents, and the like, can also be incorporated into the pharmaceutical compositions. The concentration of the active agent in the pharmaceutical composition can vary widely, i.e., from less than about 0.1% by weight, usually being at least about 1% by weight to as much as 20% by weight or more. Methods for preparing parenterally administrable compositions are described in more detail in, for example, Remington ' s Pharmaceutical Science (15th ed. , Mack Publishing Company, Easton, Pennsylvania, 1980) (incorporated by reference in its entirety for all purposes) . For oral administration, the active ingredient can be administered in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. Active component (s) can be encapsulated in gelatin capsules together with inactive ingredients and powdered carriers, such as glucose, lactose, sucrose, mannitol, starch, cellulose or cellulose derivatives, magnesium stearate, stearic acid, sodium saccharin, talcum, magnesium carbonate and the like. Examples of additional inactive ingredients that may be added to provide desirable color, taste, stability, buffering capacity, dispersion or other known desirable features are red iron oxide, silica gel, sodium lauryl sulfate, titanium dioxide, edible white ink and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric-coated for selective disintegration in the gastrointestinal tract. Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.

EXAMPLES Following informed consent, DNA samples were obtained from 144 unrelated adult females (average age = 50 ± 2 years) . A diagnosis of current or previous major depression was made in 95 of the 144 women (66%) based on a direct psychiatric interview using DSM-IV criteria (3) . Genotypes were determined independently for the PvuII T-C polymorphism located in intron 1 of the ESR gene as described previously (4) . As summarized in Table 1, the frequency of the ESR PvuII T/T genotype was significantly higher (Chi-square = 8.28; p < 0.002) in individuals with a history of depression (35%; 33/95) than in screened control individuals who had never suffered from depression (12%; 6/49) .

These data indicate that molecular variations within the ESR gene result in differential modulation of the physiological effects of estrogen. Biological effects of estrogen are thus mediated differentially in individuals with the ESR PvuII T/T genotype than in individuals with and ESR PvuII C allele. A different concentration of estrogen is necessary to achieve the same physiological effect in ESR

PvuII T/T individuals than in individuals with an ESR PvuII C allele. As such, an ESR PvuII T/T individual is more susceptible to the clinical effects of variations in estrogen levels . It is possible that the PvuII polymorphism in ESR, which occurs in an intronic region, is itself a cause of depression, perhaps due to an influence on splicing or expression. Alternatively, the PvuII polymorphism may be in equilibrium dislinkage with another polymorphism, which is the causative locus of depression. Other allelic variants in the ESR gene can be tested by association analysis. The allelic variant (s) displaying the highest level of statistical association is/are most likely causative mutations.

References

1. Fink, & Sumner, Nature 383, 306 (1996).

2. Sumner & Fink, J. Steroid Biochem . Mol . Biol . 54, 15-20 (1995) . 3. American Psychiatric Association in Diagnostic and Statistical Manual of Mental Disorders vol 4: 317-391 (American Psychiatric Association, Washington, D.C., 1994) .

4. Yaich et al . , Cancer Res . 52, 77-83 (1992).

The foregoing description of the preferred embodiments of the present invention has been presented for purposes of illustration and description. They are not intended to be exhaustive or to limit the invention to the precise form disclosed, and many modifications and variations are possible in light of the above teaching. All publications and patent applications cited herein are incorporated by reference in their entirety to the same extent as if each individual publication or patent application was specifically and individually so denoted.

TABLE 1 ESR PvuII GENOTYPE DISTRIBUTION IN SCREENED CONTROLS AND INDIVIDUALS WITH A HISTORY OF DEPRESSION (n = 144 females)

ESR PvuII Genotypes

Group C/C C/T T/T

Screened Controls

(n = 49) 8 (17%) 35 (71%) 6 (12%)

History of depression

(n = 95) 16 (14%) 46 (48%) 33 (35%)*

* Chi -square = 8.28 (p < 0.002) vs. screened control group

Claims

What is claimed is:

1. A method of diagnosing a patient for susceptibility to depression comprising detecting a variant allele of the human estrogen receptor gene ESR.

2. The method of claim 1, wherein the method detects homozygous PvuII T/T alleles to indicate increased susceptibility to depression.

3. The method of claim 1 or 2 further comprising treating a patient with a variant allele indicating increased susceptibility to depression with an antidepressive agent.

4. A method of treating a patient suffering from depression due to a variant allele of the human estrogen receptor gene ESR, the method comprising administering to the patient a therapeutically effective amount of an antidepressive agent.

5. The method of claim 4 or 5 , wherein the agent is administered prophylatically.

6. The method of claim 5, wherein the agent binds to the estrogen receptor.

7. The method of claim 4, wherein the agent agonizes estrogen binding to the human estrogen receptor.

8. The method of claim 4, wherein the agent is estrogen.

9. A method of screening potential drugs for antidepressant activity, comprising administering a potential drug to a population of patients having a variation in the human estrogen receptor gene ESR indicating increased susceptibility to depression, and determining the existence of depression in members of the population relative to a control population.

10. Use of an agent effective to agonize estrogen binding to the human estrogen receptor ESR in the manufacture of a medicament for use in the treatment of depression.

11. Use of an agonist of estrogen binding to the human estrogen receptor ESR for treatment of depression.

12. The use according to claim 10 or 11 for treating a patient having a variant estrogen receptor gene associated with increased susceptibility to depression.

13. A diagnostic agent for detecting a variation in the human estrogen receptor gene ESR for use in therapy, prophylaxis or diagnosis of depression.

14. The diagnostic agent of claim 13 that is an allele-specific oligonucleotide.

15. The diagnostic agent of claim 13 that is an allele-specific monoclonal antibody.

16. Use of an agent for detecting a variation in the human estrogen receptor gene ESR in the manufacture of an agent for treatment, prophylaxis or diagnosis of depression.