WO2011091418A1 - Screening method for adverse side effects of a therapeutic pharmaceutical drug - Google Patents

Abstract

Disclosed are methods for identifying whether a test pharmaceutical drug causes depression, psychosis, or anxiety in a conscious, non-human, mammalian subject.

Description

SCREENING METHOD FOR ADVERSE SIDE EFFECTS

OF A THERAPEUTIC PHARMACEUTICAL DRUG

[0001] This Application claims the benefit of priority to U.S. Provisional Application No. 61/297,875, filed January 25, 2010 and to U.S. Provisional Application No. 61/384,948, filed September 21, 2010, the specifications of which are incorporated by reference in their entirety.

FIELD OF THE INVENTION

[0002] The present invention is in the field of medicine. More specifically, the disclosure is in the field of drug development and screening to identify therapeutic pharmaceutical drugs that do not cause mental illness-related side effects or which are useful for treating psychosis, depression and/or anxiety.

BACKGROUND OF THE INVENTION

[0003] In 2008, the pharmaceutical industry spent over $65 billion U.S. dollars ("USD") on research to discover and develop new drugs to allow patients to live longer, healthier, and more productive lives. On average, it takes around 10-15 years and more than $800 million USD to bring a new drug to market. Most drugs, however, never make it to market. A survey of the 10 largest pharmaceutical companies demonstrated that only 11% of the drugs that entered clinic trials in the United States were approved (Kola et al. (2004) Nature Rev. : Drug Develop. 3(8):71 1-15).

[0004] As a potential new drug moves further along in clinical trials, the cost to bring the drug to market increases exponentially. The earlier a drug fails in clinical trials, the less expensive the failure becomes. For example, the cost associated with a new drug that fails in Phase I or II pales in comparison to a drug that fails in Phase III. New drugs typically fail in clinical trials because they do not meet efficacy benchmarks or because they have adverse side effects.

[0005] For example, rimonobant, a ligand antagonist directed to the cannabinoid CB 1/CB2 receptors, was developed as an anti-obesity drug. However, rimonobant (Acomplia™) was pulled from clinical trials in the United States because it increased the risk of depression and suicide.

[0006] Nevertheless, the Food and Drug Administration ("FDA") may approve drugs with adverse side effects if the benefit to the patient significantly outweighs the consequences of these adverse side effects. Ideally, pharmaceutical companies would like to identify and develop highly efficacious drugs with little to no side effects. To that end, pharmaceutical companies would also prefer to identify drug candidates that are likely to fail in clinical trials due to adverse side effects earlier in the process, thus saving both time and money spent on research and development.

[0007] One of the primary areas pharmaceutical companies are focusing their research and development dollars is on mental health. Since Americans are spending their health care dollars on mental health/illness at a faster pace than any other health care category (Olfson et al. (2009) Arch. Gen. Psychiatry 66(8):848-56), pharmaceutical companies are actively researching and developing drugs to treat mental health diseases and illnesses such as, but not limited to, schizophrenia, attention deficit hyperactive disorder, psychosis, anxiety, bipolar disorders, anorexia, bulimia, addiction, and depression.

[0008] What is needed are better ways to identify and test therapeutically effective

pharmaceutical compounds that have few, if any, adverse side effects and to do this screening early in the drug development process. In addition, a method to identify and test pharmaceutical drugs useful for the treatment of mental illness/disorders is desirable. Furthermore, there is a need to identify the neural networks involved in mental disorders to gain a better understanding of these conditions as well as to develop drugs which target these brain areas.

SUMMARY OF THE INVENTION

[0009] A drug that causes a certain type of behavior does so by altering the activity of different areas of the brain. The drug could be said to have a finger print. Drugs that share a common action would be predicted to have similar (not necessarily identical) finger prints. Thus, this disclosure is based, at least in part, on the ability of certain therapeutically effective pharmaceutical drugs to induce detectable changes in neuronal/glial activity that can be compared to against known pharmaceutical drugs with known adverse side effects. This method has been exploited to develop the present disclosure, which is directed in part, to methods of drug screening.

[0010] In one aspect, the method comprises treating a non-human, control mammal with a control drug, the control drug being a known psychotic, a known anxiogenic, and/or a known depressant; measuring the neuronal/glial activity in the brain of the treated control by functional imaging; treating the subject with a therapeutically effective amount of the test pharmaceutical drug; measuring the neuronal/glial activity in the brain of the treated subject by functional imaging; and comparing the neuronal/glial activity in the brain of the treated subject with the neuronal/glial activity in the brain of the control animal. A comparable pattern of neuronal/glial activity in the brain of the treated subject relative to the pattern of neuronal/glial activity in the brain of the control after administration of the control drug indicates that the test pharmaceutical drug with therapeutic activity causes depression and/or psychosis and/or anxiety.

[0011] In certain embodiments, the subject is a non-human mammal selected from the group consisting of a mouse, rodent, and primate.

[0012] In some embodiments, the control psychotic is selected from the group consisting of apomorphine, cocaine, amphetamine, methamphetamine, methylenedioxymethamphetamine ("MDMA"), and phenylcyclohexylpiperidine ("PCP").

[0013] In other embodiments, the control anxiogenic is selected from the group consisting of DMCM, FG-7142, ZK-93426, mCPP, and yohimbine.

[0014] In yet other embodiments, the control anxiogenic is selected from the group consisting of alcohol, barbiturates, benzodiazepines, non-benzodiazepines, and opioids.

[0015] In another aspect, the disclosure features a method for determining whether a test pharmaceutical drug with therapeutic activity is also a psychosis-inducing drug. In this method, a conscious, non-human, mammalian subject is treated with a therapeutically effective amount of the test pharmaceutical drug; measuring the neuronal/glial activity in the integrated neural network of the subject by functional imaging of the subject's brain; and comparing the neuronal/glial activity in the subject after administration of the test pharmaceutical drug with the neuronal/glial activity in the integrated neural network of a control animal after administration of a control drug known to induce psychosis. A comparable pattern of neuronal/glial activity in the subject relative to the pattern of neuronal/glial activity in the control animal after administration of the control drug indicates that the test pharmaceutical drug is a psychosis-inducing drug.

[0016] In certain embodiments, the nonhuman, mammalian subject is selected from the group consisting of a mouse, rodent, and primate.

[0017] In some embodiments, the known psychosis-inducing drug is selected from the group consisting of apomorphine, cocaine, amphetamine, methamphetamine,

methylenedioxymethamphetamine ("MDMA"), and phenylcyclohexylpiperidine ("PCP").

[0018] In a further aspect, a method of determining whether a test pharmaceutical drug with therapeutic activity is also an anxiogenic is disclosed. This method comprises treating a conscious, non-human, mammalian subject with a therapeutically effective amount of the test pharmaceutical drug; measuring the neuronal/glial activity in the integrated neural network of the subject by functional imaging of the subject's brain; and comparing the neuronal/glial activity in the subject after administration of the test pharmaceutical drug with the neuronal/glial activity in the integrated neural network of a control animal after administration of a control angiogenic. A comparable pattern of neuronal/glial activity in the subject relative to the pattern of neuronal/glial activity in the control animal after administration of the control anxiogenic indicates that the test pharmaceutical drug is an anxiogenic.

[0019] In some embodiments, the mammalian subject is selected from the group consisting of a mouse, rodent, and primate.

[0020] In certain embodiments, the known anxiogenic is selected from a group consisting of DMCM, FG-7142, ZK-93426, mCPP, and yohimbine.

[0021] In another aspect, the disclosure provides a method for identifying whether a test pharmaceutical drug with therapeutic activity is also an anti-depressant. The method disclosed comprises treating a conscious, non-human, mammalian subject with a therapeutically

effective amount of the test pharmaceutical drug; measuring the neuronal/glial activity in the integrated neural network of the subject by functional imaging of the subject's brain; and comparing the neuronal/glial activity in the subject after administration of the test

pharmaceutical drug with the neuronal/glial activity in the integrated neural network of a control animal after administration of a known depressant. A comparable pattern of neuronal/glial activity in the subject relative to the pattern of neuronal/glial activity in the control animal after

administration of the known depressant indicates that the test pharmaceutical drug is a depressant.

[0022] In certain embodiments, the mammalian subject is selected from the group consisting of a mouse, rodent, and primate.

[0023] In some embodiments, the known depressant is selected from a group consisting of alcohol, barbiturates, benzodiazepines, non-benzodiazepines, and opioids.

[0024] In yet another aspect of the disclosure, a method of identifying areas of the integrated neural network of the brain of a conscious, non-human, mammalian control that elicit psychosis is disclosed. The method comprises treating the control with an amount of a control psychotic known to cause psychosis; identifying a region of the brain of the treated control subject which shows neuronal/glial activity by functional imaging; treating a conscious, non-human, mammalian subject of the same species as the control with a therapeutically-effective amount of an anti-psychotic drug; allowing the anti-psychotic drug to achieve pharmacokinetic equilibrium in the subject; treating the subject with an amount of the control psychotic known to cause psychosis; identifying a region in the brain of the treated subject having neuronal/glial activity by functional imaging; and comparing the regions of neuronal/glial activity in the brain of the treated control with the regions of neuronal/glial activity in the brain of the treated subject. Detectable differences in the regions of the brain having neuronal/glial activity in the treated control relative to the treated subject indicate that these brain regions are involved in psychosis.

[0025] In certain embodiments, the mammalian control and subject are selected from the group consisting of a mouse, rodent, and primate.

[0026] In some embodiments, administration of either the psychotic or anti-psychotic is chronically administered over 1 to 12 weeks, individually or simultaneously.

[0027] The disclosure also discloses a method of identifying areas of the integrated neural network of the brain of a conscious, non-human, mammalian subject that elicit anxiety. The method comprises treating a control animal with an amount of a control anxiogenic known to cause anxiogenic; identifying a region of the brain of the treated subject which shows neuronal/glial activity by functional imaging; treating a conscious, non-human, mammalian subject of the same species as the control with a therapeutically effective amount of an anxiolytic drug; allowing the anxiolytic drug to achieve pharmacokinetic equilibrium in the subject; treating the subject with a an amount of the control anxiogenic which is known to cause anxiety; identifying a region in the brain of the treated subject having neuronal/glial activity by functional imaging; and comparing the regions of neuronal/glial activity in the treated control with the regions of neuronal/glial activity in the brain of the treated subject. A detectable difference in the regions of the brain having neuronal/glial activity in the treated control relative to the treated subject indicates that these brain regions are involved in anxiety.

[0028] In certain embodiments, the mammalian control and subject are selected from the group consisting of a mouse, rodent, and primate.

[0029] In some embodiments, administration of either the anxiogenic or anxiolytic is chronically administered over 1 to 12 weeks, individually or simultaneously.

[0030] A method of identifying areas of the integrated neural network of the brain of a conscious, non-human, mammalian control that elicit depression is also disclosed herein. The method comprises treating the control with an amount of a control depressant; identifying a region of the brain of the treated subject which shows neuronal/glial activity by functional imaging; treating a conscious, non-human, mammalian subject of the same species with a therapeutically effective amount of an anti-depressant drug; allowing the anti-depressant drug to achieve pharmacokinetic equilibrium in the subject; treating the subject with a an amount of the control depressant; identifying a region in the brain of the treated subject having neuronal/glial activity by functional imaging; and comparing the regions of neuronal/glial activity in the treated control with the regions of neuronal/glial activity in the brain of the treated subject. A detectable difference in the regions of the brain having neuronal/glial activity in the treated control relative to the treated subject indicates that these brain regions are involved in depression.

[0031] In certain embodiments, the mammalian control and subject are selected from the group consisting of a mouse, rodent, and primate.

[0032] In some embodiments, administration of either the depressant or anti-depressant is chronically administered over 1 to 12 weeks, individually or simultaneously.

[0033] In other aspects, methods of screening a test drug for increased risk of one or more of depression, psychosis, or anxiety in a conscious, non-human, mammalian subject are disclosed. The methods comprise measuring a neural/glial activity in a brain of a first conscious, non-human, mammalian subject treated with a drug used to treat one or more of a psychotic, an anxiogenic, or a depression disorder and identifying a pattern of neural/glial activity of the brain of the first subject treated with the drug. The methods also entail treating a second conscious, non-human, mammalian subject with a test drug and measuring the neural/glial activity in the brain of the second subject treated with the test drug and identifying a pattern of the neural/glial activity of the brain of the second subject. The pattern of the neural/glial activity of the brain of the first subject is compared to the pattern of the neural/glial activity of the brain of the second subject. A

determination of a pattern of neural/glial activity associated with one or more of depression, psychosis, or anxiety is made.

[0034] In certain embodiments, the neuronal/glial activity is measured by functional MRI. In other embodiments, the test drug is used to treat mental illness. In particular embodiments, the mammalian subject is selected from the group consisting of a mouse, rodent, and primate.

[0035] In further embodiments, the control psychotic is selected from the group consisting of apomorphine, cocaine, amphetamine, methamphetamine, methylenedioxymethamphetamine ("MDMA"), and phenylcyclohexylpiperidine ("PCP"). In additional embodiments, the control anxiogenic is selected from the group consisting of DMCM, FG-7142, ZK-93426, mCPP, and yohimbine. In still further embodiments, the control depressant is selected from the group consisting of alcohol, barbiturates, benzodiazepines, non-benzodiazepines, and opioids.

[0036] In particular embodiments, the methods further comprise measuring a neural/glial activity in a brain of a first conscious, non-human, mammalian subject treated with a drug used to treat one or more of a psychotic, an anxiogenic, or a depression disorder in the brains of a plurality of conscious, non-human, mammalian subjects with a plurality of drugs used to treat one or more of a psychotic, an anxiogenic, or a depression disorder. In more particular embodiments, the methods further comprise compiling the patterns identified into a database. In other embodiments, determining a pattern of neural/glial activity comprises identifying differences between the pattern of neural/glial activity of the first subject and the pattern of the neural/glial activity of the second subject.

[0037] In certain embodiments, patterns are identified for a plurality of test drugs. In additional embodiments, the methods further comprise generating a composite pattern of neural/glial activity from the patterns identified for the plurality of test drugs, the composite pattern representing a consensus of neural/glial activity associated with one or more of depression, psychosis, or anxiety as determined from the patterns identified for the plurality of test drugs.

[0038] In additional embodiments, the methods further comprise refining the composite pattern based on additional patterns compiled in the database. In still more embodiments, the methods further comprise measuring a neural/glial activity in a brain of a third conscious, non-human, mammalian subject treated with a drug having no known effect on one or more of a psychotic, an anxiogenic, or a depression disorder and identifying a pattern of neural/glial activity of the brain of the first subject treated with the drug having no known effect. In more embodiments, the methods further comprise determining whether the drug having no known effect has a pattern of neural/glial activity similar to any of the composite patterns in the database.

[0039] In another aspect, methods of identifying a drug for increased risk of suicide in a subject are disclosed. The methods comprise administering the drug to a subject and detecting the level of activity of cells in one or more brain regions of the subject, the region selected from prelimbic cortex, gustatory cortex, insular cortex, secondary somatosensory cortex, parietal cortex, medial dorsal striatum, anterior thalamus, ventral anterior lateral thalamus, reuniens thalamus, ventral medial thalamus, lateral posterior thalamus, lateral dorsal thalamus, central thalamus, CA1 hippocampus, posterior hypothalamus, and central raphe. An increased level of activity relative to a control is indicative of the drug having an increased risk of suicide by the subject.

BRIEF DESCRIPTION OF THE FIGURES

[0040] The foregoing and other objects of the present invention, the various features thereof, as well as the invention itself may be more fully understood from the following description, when read together with the accompanying drawings in which:

[0041] Figure 1 is a representation showing a methodological approach to screen drugs for the potential to increase the risk for a disorder such as suicidal ideation. Figure 1 specifically shows the generation of a map detailing a pattern of brain activity attributed to increasing the risk of the disorder by determining a common brain activity pattern following test drug administration of five hypothetical drugs to brain activity patterns following administration of drugs known to treat the specific disorder.

[0042] Figure 2A is a graphical representation showing the median activated voxels in the insular cortex following administration of vehicle, 0.25 mg/kg body mass of corticosterone, or 1.0 mg/kg body mass of corticosterone in male, Long Evans rats.

[0043] Figure 2B is a graphical representation showing the median activated voxels in the motor cortex following administration of vehicle, 0.25 mg/kg body mass of corticosterone, or 1.0 mg/kg body mass of corticosterone in male, Long Evans rats.

[0044] Figure 3 is a graphical representation showing the change in BOLD signal from a stimulus (vehicle, 0.25 mg/kg or 1.0 mg/kg body mass of corticosterone) over seven minutes of monitoring by fJVIRI.

[0045] Figure 4A is a graphical representation showing the change in respiratory rate (respirations/minute) of male, Long Evans rats over eight days of acclimation to the imaging protocol.

[0046] Figure 4B is a graphical representation showing the change in heart rate (beats/minute) of male, Long Evans rats over eight days of acclimation to the imaging protocol.

[0047] Figure 4C is a graphical representation showing the change in corticosterone levels (ng of corticosterone/ml of serum) of male, Long Evans rats over eight days of acclimation to the imaging protocol.

[0048] Figure 5 is a photographic representation showing the total BOLD signal in 154 brain areas (>16,000 voxels) after administration of test drugs, Venlafaxine, Gabapentin, and

Rimonabant, as well as the pattern of expression after administration of the control drugs, Clozapine and Buspirone. The representation also shows the 67 areas of common brain activity after administration of the test drugs and the 17 brain areas unique to the test drugs excluding the BOLD signal in brains exposed to the control drugs.

[0049] Figure 6 is a photographic representation showing a 3D activation map of the brain areas unique to the test drugs, with rostral at the top of the representation and caudal at the bottom of the representation.

[0050] Figure 7 is a photographic representation showing 2D activation maps used to generate the 3D activation map. DETAILED DESCRIPTION OF THE INVENTION

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

Definitions

[0052] As used herein, an "activation map" is an image showing one or more regions of a brain, the image showing the areas of the brain having increased or decreased activity. Activation maps can be either two-dimensional ("2D") or three-dimensional ("3D"). An activation map can be generated using any imaging device disclosed herein or known to those of ordinary skill in the art.

[0053] As used herein, an "adverse side effect" is a harmful, undesirable, and unintended secondary consequence resulting from medication administered to address the primary symptom(s). For example, in the context of psychosis, administration of an antipsychotic to quell hallucinations (primary symptom) can lead to tardive dyskinesia (the unintended and undesirable consequence of the antipsychotic).

[0054] "Depression" is defined as a mental state of altered mood characterized by feelings of sadness, despair, loneliness, discouragement, low self-esteem, and self-reproach, often

accompanied by psycho-motor retardation, agitation, withdrawal from social contact, loss of appetite, and/or insomnia. Depression may be induced by environmental factors (e.g., seasonal affective disorder, situational depression).

[0055] An "anti-depressant" is a medication used to alleviate mood disorders, such as major depression and dysthymia, associated with depression. Conversely, "depressants" are compositions that lower one's mood and induce feelings of sadness, lethargy or ambivalence.

[0056] "Anxiety" is the experience of fear or apprehension in response to anticipated internal or external danger accompanied by cognitive, somatic, emotional, and of behavioral components.

Anxiety is a generalized mood condition that occurs without an identifiable triggering stimulus.

[0057] As used herein, an anxiolytic is any drug that is prescribed to treat the symptoms of anxiety. Conversely, an "anxiogenic" is any drug that can induce anxiety. [0058] "Psychosis" is a symptom or feature of mental illness typically characterized by radical changes in personality, impaired functioning, and a distorted or nonexistent sense of objective reality. Psychosis may appear as a symptom of a number of mental disorders, including mood and personality disorders. It is also the defining feature of schizophrenia, schizophreniform disorder, schizoaffective disorder, delusional disorder, and the psychotic disorders.

[0059] "Anti-psychotics" are used to treat symptoms of psychosis (e.g., hallucinations, paranoia) and are divided into two classes: typical and atypical anti-psychotics. "Psychotics," on the other hand, are drugs that can induce psychosis.

[0060] As used herein, "chronic administration" is defined as dosing sustained for over one week.

[0061] A "test pharmaceutical drug" refers to a compound whose therapeutic abilities are unknown, e.g., whose ability to decrease the symptoms of a mental illness is unknown.

[0062] A "test pharmaceutical drug with therapeutic efficacy" refers to a compound whose therapeutic benefits are known.

[0063] A "control drug" refers to a drug with known efficacy and/or known adverse side effects. In the context of this disclosure, control drugs may be known psychotics, anxiogenics, and depressants.

[0064] A "control animal," as used herein, describes an animal that is administered a control drug to be used as a reference in comparison to a test animal of the same species and age that is treated with a test pharmaceutical drug.

[0065] As used herein, the phrase "neuronal/glial activity" refers to a measurable change in the cerebral blood flow in a particular brain region. "Increased neuronal/glial activity" in a particular brain region corresponds to increased blood flow to that brain region to meet the metabolic demands of the neuronal/glial activity. Likewise, a "decrease in neuronal/glial activity" within a particular brain region correlates to diminished cerebral blood flow due to the decrease in neuronal/glial activity. Detectable changes in neuronal/glial activity act as a surrogate marker for cerebral blood flow and, in turn, can provide a "molecular fingerprint" of neuronal/glial activity in response to various stimuli.

[0066] A "comparable pattern" of neuronal/glial activity in the brain is defined as when at least 70% of the area of defined integrated neural network are activated as measured by functional imaging when compared from one subject to another.

[0067] A "detectable difference" of neuronal/glial activity is defined as a measurable

(detectable) change in the neuronal/glial activity. [0068] An "integrated neural network" is a basic unit of the nervous system controlling behavior, comprised of neural pathways conveying sensory and motor information to and from brain areas involved in integrating this information with past memories, feelings, mood, and temperament. For example, the integrated neural network of aggression can comprise sensory information coming from any of the sensory modalities (smell, vision, sound, etc.) about a potential competitor, directed to an area of the brain that organizes an aggressive respond based upon, past history of the competitor, history of fighting and winning, context of the agonist encounter (e.g., protecting young, fighting for food and sex, defense of home territory), mood of the protagonist, endocrinology of the protagonist etc. This information is part of the emotional experience of an aggressive encounter. When the decision is made to fight or run there are parts of the brain involved in the organizing the motor component of attack, the output of the behavior.

[0069] A "therapeutically effective amount" of a test pharmaceutical drug or control drug is a dosage quantity of a drug that has beneficial effects that proves to be measurably and significantly different from effects in subjects/patients treated with a placebo. This dosage amount can be determined by titration of a drug to identify the most efficacious dose.

[0070] The test pharmaceutical drug with therapeutic activity can be administered by any protocol and in a variety of unit dosage forms depending upon the method of administration.

Dosages for test pharmaceutical compositions are well known to those of skill in the art. The amount of the test pharmaceutical drug adequate to generate the desired response is defined as a "therapeutically effective dose." The dosage schedule and amounts effective for desired uses, i.e., the "dosing regimen," will depend upon a variety of factors, including the pharmaceutical formulation and concentration of excipient, and the like. The dosage regimen also takes into consideration pharmacokinetics, i.e., the test pharmaceutical drug's rate of absorption,

bioavailability, metabolism, clearance, and the like.

[0071] As used herein, "pharmacokinetic equilibrium" is a plateau in the brain concentration of a drug administered at which time the drug achieves its highest levels based upon the dosing regimen.

[0072] The term "about" or "approximately" means within 20%, within 10%, or within 5% of a given value or range. Alternatively, particularly in the measurement of biological processing, the term "about" or "approximately" means within an order of magnitude, preferably within a factor of 2, of a given value, e.g., a concentration of a compound that causes a half-maximal biological effect. Thus, the term "about" or "approximately" means that a value can fall within a scientifically acceptable error range for that type of value, which will depend on how quantitative a measurement can be given the available tools.

[0073] "Functional imaging" as described herein is the study of brain function and activity based on the analysis of data acquired using brain imaging modalities.

Selection Methods

[0074] One approach to discovering new drugs for the treatment of depression, psychosis, and/or anxiety is screening the drugs in different animal models. This has been the traditional route practiced by pharmaceutical companies. The present disclosure is directed, in part, to a method for identifying a test pharmaceutical drug that causes depression, psychosis, and/or anxiety in a conscious, non-human mammalian subject. This screening method uses brain activity, not behavior, as a surrogate marker for efficacy of the test pharmaceutical drug.

[0075] The test pharmaceutical drug identified in this method may be a compound whose therapeutic benefits are known, but whose potential side affects are unknown. For example, test pharmaceutical drugs with side effects associated with depression, psychosis, and anxiety are screened herein.

[0076] In certain embodiments, the disclosed methods identify a particular area of the brain that renders a subject more likely to suffer from a particular disorder, such as those disorders described herein. In specific embodiments, fMRI is used to identify a pattern of brain activity (i.e., activation map) following the administration of one or more test drugs 100 to subjects (Figure 1). In addition, control drugs known to treat the particular disorder are provided to a control group of subjects. The patterns of activity (i.e., activation map) induced by the control drugs are determined 110 and compared to the common brain activity identified in the activation maps of brains exposed to the test drugs 120. The comparison shows the areas in brains (i.e., also an activation map) exposed to the test drugs that have a higher activity than the areas of brains exposed to the control drugs 130.

[0077] The activation maps can be stored in a database. By "database," it is meant a collection of information or data stored in a system. Databases are typically organized based on a database model. For instance, common database models include row-based, column-based, hierarchical, relational, object-oriented and network models. The database is organized to store activation maps such that particular activation maps are associated with particular disorders. In addition, activation maps (i.e., brain activity patterns or "fingerprints") can be stored such that the activation maps are associated with a particular drug. Such database capabilities are well-known in the computer arts. For instance, database search software products include, but are not limited to, Exorbyte Master Data Server, IMP Database Search Engine, Google Search Appliance, and Dieselpoint. Therefore, a user can search the database for activation maps relating to a particular disorder or a particular drug.

[0078] In certain embodiments, the database stores activation maps for particular disorders. The activation maps can be refined as more information is obtained for drugs increasing or decreasing the likelihood of a particular disorder. For instance, an activation map is generated when a test drug associated with a particular disorder exhibits a unique activation pattern as compared to a control drug that treats a particular disorder. The activation map is further modified and refined as information for additional drugs associated with a particular disorder are added to the database. This information is used to modify the activation map associated with a particular disorder.

[0079] Accordingly, there are at least two sets of activation maps in particular embodiments. The first set of activation maps are associated with particular drugs. The second set of maps are composite activation maps associated with each particular disorder. The composite activation maps are continually refined as more drugs are tested and their activation maps are added to the database. Such composite activation maps can be generated using the algorithms disclosed herein and in Example 1.

Depression

[0080] Depression is a common but serious illness. There are several forms of depressive disorders including major depressive disorder, dysthymic disorder, and psychotic depression.

Major depressive disorder is characterized by a combination of sy mptoms that interfere with a person's ability to work, sleep, study, eat, and enjoy once pleasurable activities. Dysthymic disorder, also called dysthymia, is characterized by long-term (i.e., longer than two years), but less severe, symptoms that may not disable a person but can prevent one from functioning normally. Psychotic depression, which occurs when a severe depressive illness is accompanied by some form of psychosis, such as a break with reality, hallucinations, and delusions. Symptoms of depression include: persistent sad. anxious or "empty" feelings; feelings of hopelessness and/or pessimism; feelings of guilt, worihlessness and/or helplessness; irritability, restlessness; loss of interest in activities or hobbies once pleasurable, including sex; fatigue and decreased energy; difficulty concentrating, remembering details and making decisions; insomnia, early-morning wakefulness, or excessive sleeping; overeating, or appetite loss; thoughts of suicide, suicide attempts; and persistent aches or pains, headaches, cramps or digestive problems that, do not ease even with treatment.

[0081] In particularly severe depression and aggression, suicidal ideation and increased risk of self-harm are possible. The disclosed methods allow for identification of regions of the brain that increase the risk of suicidal ideation. In particular embodiments, pharmacological MRI is used to compare BOLD signal changes across brain areas. The methods are used to reveal differences in neural activity between drugs that potentially increase the risk for suicide and those used to treat suicidal ideation and aggression. In certain embodiments, the methods identify specific brain areas comprising a putative neural circuit characteristic of drugs that increase the risk. Exemplary areas of the brain include prelimbic cortex, gustatory cortex, insular cortex, secondary somatosensory cortex, parietal cortex, medial dorsal striatum, anterior thalamus, ventral anterior lateral thalamus, reuniens thalamus, ventral medial thalamus, lateral posterior thalamus, lateral dorsal thalamus, central thalamus, CA1 hippocampus, posterior hypothalamus, and central raphe.

Psychosis

[0082] Psychosis is a loss of contact with reality, usually including false ideas about what is taking place or who one is (delusions) and seeing or hearing things that aren't there (hallucinations). Symptoms include: abnormal displays of emotion; confusion; depression and sometimes suicidal thoughts; disorganized thought and speech; extreme excitement (mania); false beliefs (delusions); loss of touch with reality; mistaken perceptions (illusions); seeing, hearing, feeling, or perceiving things that are not there (hallucinations); and unfounded fear/suspicion.

Anxiety

[0083] Anxiety is a feeling of nervousness, apprehension, fear, or worry. While some fears and worries are justified, problem anxiety interferes with the ability to sleep or otherwise function. Anxiety may occur without a cause, or it may occur based on a real situation but may be out of proportion to what would normally be expected. For panic anxiety, symptoms usually include at least four of the following: palpitations, sweating, trembling, shortness of breath, sense of choking, chest pain, nausea or other stomach upset, dizziness, derealization, being unable to think, feeling as if the mind has gone blank, a fear of dying, numbness or tingling, and chills or hot flashes. For generalized anxiety disorder, which is defined as excessive, unrealistic, and difficult to control worry over a period of at least six months, symptoms usually are associated with three of the following: restlessness, being easily tired, trouble concentrating, irritability, muscle tension and issues with sleeping. [0084] In the present method, the test pharmaceutical drug may also be a drug with unknown therapeutic benefits. The therapeutic benefit of the test pharmaceutical drug can be ascertained by one of ordinary skill in the art. For example, a therapeutic benefit may be to treat, alleviate or reduce the symptoms associated with any disease or disorder.

[0085] In the present method, the side effects of the test pharmaceutical drug are tested in non- human mammalian subjects. Examples of non-human mammalian subjects include rodents, such as rats and mice, rabbits, and primates, and are commercially available from a number of sources.

[0086] In the present method, a control animal is first treated with a control drug that is a known psychotic, a known anxiogenic or a known depressant. The control animal should be of the same species and age as the non-human, mammalian subject. In some instances, the control animal and the test subject may be the same animal.

[0087] According to the method, the control drug used to treat the control animal is a known psychotic, a known anxiogenic, or a known depressant. These drugs may be obtained from commercial sources such as Sigma-Aldrich or Fisher Scientific.

[0088] Known psychotic drugs include, but are not limited to, AK-Pentolate, amphetamines, ancobon, anxiolytics, Ativan, cannabis, centrax, cyclopentolate hydrochloride, cycloserine, depacon, depakene, depakote, depakote ER, depakote sprinkle, diastat, diazemuls, diazepam, diazepam intensol, flucytosine, hallucinogens, hypnotics, I-Pentolate, lodosyn, lorazepam intensol, marijuana, nimodipine, nimotop, opioids, paxipam, pentolair, prograf, sedative withdrawal, sedatives, serax, seromycin pulvules, sinemet, sinemet CR, tacrolimus, valcyte, valganciclovir, valium, valproic acid, and valrelease.

[0089] Known anxiogenics include, but are not limited to, asthma medicines (such as albuterol, salmeterol, and theophylline), blood pressure medicines (such as methyldopa), hormones (such as oral contraceptives), amphetamines, medicines containing amphetamines (such as Benzedrine, Dexedrine, and Ritalin), steroids (such as cortisone, dexamethasone, and prednisone), thyroid medicines, phenytoin, levodopa, quinidine, caffeine, decongestants (such as phenylephrine), and illegal drugs such as cocaine and crack.

[0090] Known depressants include, but are not limited to, Zovirax, alcohol, anticonvulsants such as Celontin and Zarontin, barbiturates, benzodiazepines such as Ativan, Dalman, Halcion, Klonopin, Librium, Valium and Xanax, beta-adrenergic blockers such as Lopressor, Tenormin and Coreg, bromocriptine (Parlodel), calcium-channel blockers such as Calan, Cardizem, Tiazac and Procardia, antabuse, estrogens including Premarin and Prempro, fluoroquinolone antibiotics, statins, interferon alpha, accutane, codeine, morphine, Demerol, Darvocet, Percodan, Oxycodone, and Norplant.

[0091] The control drug is administered at a dosage which induces psychosis, anxiety, or depression. The amount of the control drug to be administered to the control animal is known to one of ordinary skill in the art (see, e.g., Remington: The Science and Practice of Pharmacy, 21^st edition, Lippincott Williams & Wilkins, Gennaro, ed. (2006) ("Remington's")) based on published reports studying psychosis, anxiety and depression in laboratory animals. In general, the dosage will be proportional to the body weight of the control animal. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any drug used in the method disclosed herein, the therapeutically effective dose can be estimated initially from the literature and/or titration studies. Information for preparing and testing such compositions are known in the art (see, e.g., Remington's supra), which is incorporated herein by reference in its entirety.

[0092] After administration of the control drug to the control animal, the neuronal/glial activity in the brain of the control animal is measured by functional imaging. Functional imaging techniques include, but are not limited to, functional magnetic resonance imaging (fMRI), positron emission tomography (PET), single photon emission computed tomography (SPECT), optical imaging, thermal imaging, electroencephalogram (EEG), magnetoencephalogram (MEG) and two- photon laser-scanning microscopy. Due to its non-invasive nature, quick scan times, and image resolution, blood oxygen-level dependent (BOLD) fMRI is often used for neuroimaging experiments.

Functional Magnetic Resonance Imaging

[0093] BOLD fMRI measures the blood flow to the local vasculature that accompanies brain activity. Blood oxygen is released to active neurons and glia at a greater rate than to inactive neurons and glia. The difference in magnetic susceptibility between oxyhemoglobin and deoxyhemoglobin, and thus oxygenated or deoxygenated blood, leads to magnetic signal variation which can be detected using an MRI scanner. MRI scanners are available at Oxford Instrument (Oxford, U.K.).

[0094] However, there are times where an increase in BOLD signal may be caused without any neuronal/glial activity, e.g., a CO₂ challenge. Upon inhalation of CO₂, the arteries dilate and this in turn causes an increase of blood flow to the area. The increase of blood flow to the area is not caused by an increase of neuronal/glial activity but is a consequence of CO₂ challenge. However, this "false-positive" result can be circumvented by measuring the cerebral blood flow (CBF) of the proton molecules in the water molecules of blood as the tracer. Functional MRI can thus measure direct changes in CBF, irrespective of neuronal/glial activity.

[0095] Another fMRI method employs paramagnetic contrast agents that alter local magnetic susceptibility and enhance the sensitivities of fMRI signals. Using this method, regional and global changes in cerebral blood volume (CBV) can be detected. Examples of paramagnetic contrast agents include, but are not limited to, metalloporphyrins such as gadolinium-based contrast agents (including, but not limited to, Omniscan™ or gadodiamide (GE Healthcare, UK), Magnevist™ or gadopentetate dimeglumine (Berlex Laboratories, Inc., Trenton, NJ), Optimark™ or

gadoversetamide (Mallinckrodt Inc., St. Louis, MO)) and monocrystalline iron oxide nanocolloid (MION) (The Center for Molecular Imaging Research, Massachusetts General Hospital,

Charlestown, MA). Other paramagnetic contrast agents include, but are not limited to,

gadopentetic acid, gadoteric acid, gadoteridol, mangafodipir, ferric ammonium citrate, gadobenic acid, gadobutrol, gadoxetic acid, Photofrin™ (porfimer sodium), gold-coated and dextran-coated MIONs.

[0096] Subject motion is an issue in fMRI data analysis; even the slightest movement during the scan can displace voxel location corresponding to a distinct physical area. Unlike human fMRI, this issue is more prevalent in small animals like rats, because voxel size is much larger than physical (anatomical) area in the brain. The change in signal intensity due to motion can be greater than the BOLD signal, especially at the edge of the brain and tissue boundaries which essentially leads to artifact in the activation map. To avoid this, "motion correction" has become common preprocessing step in fMRI data analysis. Commonly used motion correction tools include, but are not limited to, automated image registration (AIR) (Woods et al, 1992, J. Comput. Assist. Tomogr. 16:620-33; Woods et al, 1998, J. Comput. Assist. Tomogr. 22: 139-52; Woods et al., 1998, J.

Comput. Assist. Tomogr. 22: 153-65), analysis of functional neuroimages (AFNI) (Cox, 1996, Comput. Biomed. Res. 29(3): 162-73), and statistical parametric mapping (SPM) realign tools (Friston et al., 1996, Magn. Reson. Med. 35:346-55).

[0097] Motion correction may induce spurious activation in motion-free fMRI data (Freire and Mangin, 2001, Neurolmage 14:709-22). This artifact stems from the fact that activated areas behave like biasing outliers for the difference of square-based measures usually driving such registration methods. This problem is amplified in case of small mammals where the BOLD signal change can be 10% or greater over baseline. If motion parameters are included in the general linear model for event-related data, it makes little difference if motion correction is actually applied to the data (Johnstone et al, 2000, Hum. Brain Map 27:779-88).

[0098] Image resolution using fMRI depends on the strength of the magnet. Magnets employed for fMRI studies range from 1.5 Tesla (T) to 11.7 T. The more powerful the magnet, the greater the resolution of the image. For brain imaging studies, the typical magnet strength is about 4.7 T and 7.0 T (GE Healthcare, U.K.; Bruker BioSpin, U.S.). Using a magnet field strength greater than 7.0 T may be problematic as there are limitations with high magnetic field strengths. For example, stronger magnetic field strengths shorten the T2 relaxation time, thereby making it difficult to delineate boundaries in fMRI studies that favor T2-weighted sequences.

Positron Emission Tomography

[0099] Positron emission tomography (PET) also measures CBF using radiolabeled compounds. This invasive imaging modality takes advantage of the unstable positron-emitting isotopes (for example, ¹⁵0 and ⁿC ) incorporated in radiolabeled water or glucose. When injected into the bloodstream, the radiolabeled water or glucose is delivered to the active neurons and glia. As the unstable isotope decays, a positron is emitted and eventually collides with an electron, thereby emitting two gamma rays, which are then measured using gamma ray detectors. By reconstructing the sites of the positron-electron collisions, the location of active regions can be imaged. Cyclotrons, which are used to produce the positron-emitting isotopes, and PET imaging scanners may be purchased from GE Healthcare (U.K.).

Single Photon Emission Computed Tomography

[0100] Single photon emission computed tomography (SPECT) imaging also measures CBF using radiolabels that need to be injected into the subject. Red blood cells pick up and distribute the injected radiolabel (for example, ¹²³I-labeled iodoamphetamine) throughout the body, specifically to areas of high metabolic activity. As the radiolabel decays, photons are emitted and detected to recreate a three-dimensional image of neuronal/glial activity. Compared to fMRI or PET, image resolution from SPECT is low and is thus better suited to image large regions of the brain as opposed to finer features within. Because radiolabeled tracers, rather than positron- emitting isotopes, are used, a cyclotron is not needed. Gamma ray detectors, similar to the ones used in PET imaging, are then used to detect and image the neuronal/glial activity. Electroencephalogram

[0101] Electroencephalograms (EEGs) measure the electrical activity of the brain as a measure of time varying spontaneous potentials through a number of electrodes attached to the scalp. The information from the electrical activity obtained through EEG analysis is recorded as sets of traces of the amplitude of spontaneous potentials over time. While EEGs can capture oscillations created by brain electric potentials from the 10 millisecond to 100 millisecond range, its spatial resolution is quite poor. When the subjects are animals, surgery is typically required to mount the electrodes directly onto the animal's skull. Pinnacle Technology, Inc. (Lawrence, KS) manufactures a rat and mouse EEG system suitable for use with the method disclosed.

Magnetoencephalogram

[0102] Whereas EEG measures electrical activity of the brain, magnetoencephalograms (MEGs) measure the magnetic field changes associated with neuronal firing. Superconducting magnetic detectors detect rapidly changes in magnetic fields and translate them into detectable alterations in electric current. Like EEG, MEG also has superior temporal resolution and poor spatial resolution. Pinnacle Technology, Inc. (Lawrence, KS) sells MEG systems for rodents.

[0103] After the neuronal/glial activity of the control animal treated with the control drug has been measured, a non-human, mammalian subject of the same age and species as the control animal is treated with a therapeutically-effective amount of a test pharmaceutical drug. Test

pharmaceutical drugs include, but are not limited to, those found in drug libraries, combinatorial drug libraries, synthetic drug libraries and other known synthetic or natural drugs or compounds with known properties, including known properties for off-label uses. As described below, the therapeutically-effective amount of a test pharmaceutical drug may be determined by one with ordinary skill in the art (see, e.g., Remington's supra), by administering the drug in varying amounts to identify the most efficacious dose compared to a placebo.

Pharmaceutical Compositions and Administration

[0104] The test pharmaceutical drug described herein can be incorporated into pharmaceutical compositions to be used in the methods described herein. Such compositions typically include the test pharmaceutical drug and a pharmaceutically acceptable carrier.

[0105] As used herein, a "pharmaceutically acceptable carrier" means a carrier that can be administered to a control or subject together with a test pharmaceutical drug, which does not destroy the pharmacological activity thereof. Pharmaceutically acceptable carriers include, e.g., solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Supplementary active compounds can also be incorporated into the compositions.

[0106] Non-limiting examples of pharmaceutically acceptable carriers that can be used include poly(ethylene-co-vinyl acetate), PVA, partially hydrolyzed poly(ethylene-co-vinyl acetate), poly(ethylene-co-vinyl acetate-co-vinyl alcohol), a cross-linked poly(ethylene-co-vinyl acetate), a cross-linked partially hydrolyzed poly(ethylene-co-vinyl acetate), a cross-linked poly(ethylene-co- vinyl acetate-co-vinyl alcohol), poly-D,L-lactic acid, poly-L-lactic acid, polyglycolic acid, PGA, copolymers of lactic acid and glycolic acid (PLGA), polycaprolactone, polyvalerolactone, poly (anhydrides), copolymers of polycaprolactone with polyethylene glycol, copolymers ofpolylactic acid with polyethylene glycol, polyethylene glycol; and combinations and blends thereof.

[0107] Other carriers include, e.g., an aqueous gelatin, an aqueous protein, a polymeric carrier, a cross-linking agent, or a combination thereof. In another instances, the carrier is a matrix. In yet another instances, the carrier includes water, a pharmaceutically acceptable buffer salt, a pharmaceutically acceptable buffer solution, a pharmaceutically acceptable antioxidant, ascorbic acid, one or more low molecular weight pharmaceutically acceptable polypeptides, a peptide comprising about 2 to about 10 amino acid residues, one or more pharmaceutically acceptable proteins, one or more pharmaceutically acceptable amino acids, an essential-to-human amino acid, one or more pharmaceutically acceptable carbohydrates, one or more pharmaceutically acceptable carbohydrate-derived materials, a non-reducing sugar, glucose, sucrose, sorbitol, trehalose, mannitol, maltodextrin, dextrins, cyclodextrin, a pharmaceutically acceptable chelating agent, EDTA, DTPA, a chelating agent for a divalent metal ion, a chelating agent for a trivalent metal ion, glutathione, pharmaceutically acceptable nonspecific serum albumin, and/or combinations thereof.

[0108] A pharmaceutical composition containing a test pharmaceutical drug can be formulated to be compatible with its intended route of administration as known by those of ordinary skill in the art. Nonlimiting examples of routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, intracerebral ventricular injection, oral (e.g., inhalation), intranasal, intraocular, transdermal (topical), transmucosal, vaginal and rectal administration, or any mode that allows a test pharmaceutical drug to cross the blood-brain barrier. Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. The pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0109] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EL™ (BASF, Parsippany, NJ) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of

microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. It may be desirable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable compositions can be accomplished by including in the composition an agent that delays absorption, for example, aluminum monostearate and gelatin (see, e.g., Remington's supra).

[0110] Sterile injectable solutions can be prepared by incorporating test pharmaceutical drug in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the methods of preparation include, without limitation, vacuum drying and freeze-drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0111] Oral compositions generally include an inert diluent or an edible carrier. For the purpose of oral therapeutic administration, the test pharmaceutical drug can be incorporated with excipients and used in the form of tablets, pills, troches, or capsules, e.g., gelatin capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part of the composition. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0112] For administration by inhalation, the test pharmaceutical drug can be delivered in the form of an aerosol spray from pressured container or dispenser that contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0113] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, but are not limited to, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into, e.g., ointments, salves, gels, or creams as generally known in the art.

[0114] The pharmaceutical compositions containing a test pharmaceutical drug can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.

[0115] It may be advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

[0116] Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD5₀ (the dose lethal to 50% of the population) and the ED₅₀ (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD5₀/ED5₀. While compounds that exhibit toxic side effects can be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.

[0117] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in the test and control mammals disclosed herein. The dosage of such compounds lies generally within a range of circulating concentrations that include the ED5₀ with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC₅₀ (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in mammals. Levels in plasma can be measured, for example, by high performance liquid chromatography. Information for preparing and testing such compositions are known in the art (see, e.g., Remington's supra).

[0118] In some instances, a therapeutically effective amount or dosage of a test pharmaceutical drug can range from about 0.001 mg/kg body weight to about 500 mg/kg body weight, e.g., from about 0.01 mg/kg body weight to about 50 mg/kg body weight, from about 0.025 mg/kg body weight to about 25 mg/kg body weight, from about 0.1 mg/kg body weight to about 20 mg/kg body weight, from about 0.25 mg/kg body weight to about 20 mg/kg body weight, from about 0.5 mg/kg body weight to about 20 mg/kg body weight, from about 0.5 mg/kg body weight to about 10 mg/kg body weight, from about 1 mg/kg body weight to about 10 mg/kg body weight, or about 5 mg/kg body weight.

[0119] In other instances, a therapeutically effective amount or dosage of a test pharmaceutical drug can range from about 0.001 mg to about 500 mg total, e.g., from about 0.01 mg to about 40 mg total, from about 0.025 mg to about 30 mg total, from about 0.05 mg to about 20 mg total, from about 0.1 mg to about 10 mg total, or from about 1 mg to about 10 mg total.

[0120] A drug developer will appreciate that certain factors may influence the dosage required to effectively treat a subject, including but not limited to previous treatments, the general health and/or age of the subject, and various other factors. Moreover, treatment of a subject with a therapeutically effective amount of a test pharmaceutical drug can include a single treatment or a series of treatments. In one example, a subject is treated with a test pharmaceutical drug in the range of between about 0.06 mg to 120 mg, one time per week for between about 1 to 12 weeks, alternatively between 2 to 8 weeks, between about 3 to 7 weeks, or for about 4, 5, or 6 weeks. It will also be appreciated that the effective dosage of a test pharmaceutical drug used for treatment may increase or decrease over the course of a particular treatment.

[0121] Treatment of a subject with a therapeutically effective amount of a test pharmaceutical composition described herein can be a single treatment, continuous treatment, or a series of treatments divided into multiple doses. The treatment can include a single administration, continuous administration, or periodic administration over one or more years. Chronic, long-term administration can be indicated in many cases. In some instances, a subject is treated for up to one year. In other instances, a subject is treated for up to 6 months. In yet another situation, a subject is treated for up to 100 days. In one example, a subject is treated with a test pharmaceutical drug in a time frame of one time per week for between about 1 to 12 weeks, alternatively between 2 to 8 weeks, between about 3 to 7 weeks, or for about 4, 5, or 6 weeks. In other instances, a subject can be treated substantially continuously. In other situations, a subject can be treated once per day, twice per day, once per week, or once per month.

[0122] Generally, each formulation is administered in an amount sufficient to reduce the symptoms associated with psychosis, depression and/or anxiety.

[0123] After the neuronal/glial activity of the subject is measured the same way as described above for the control animal, the neuronal/glial activities of the subject and control animals are compared to one another. Images from the functional imaging can be compared by the human eye or by computer assisted imaging programs to identify and measure any increase or decrease increase or decrease in neuronal/glial activity of specific brain areas. As defined above, a comparable pattern of neuronal/glial activity is observed when at least 70% of the identified integrated neural network are activated in both the control animal and test subject. If a comparable pattern is observed between the test pharmaceutical drug and control drug, then the test pharmaceutical drug is a poor clinical candidate since it could induce depression, anxiety and/or psychosis.

[0124] The disclosure also provides a method for determining whether a test pharmaceutical drug with therapeutic activity also a psychotic. This method entails treating a conscious, non- human mammalian subject with a therapeutically effective amount of the test pharmaceutical drug. As noted previously, one of ordinary skill in the art could determine the therapeutically effective dosage of a test pharmaceutical drug based on the teachings of the prior art (see, e.g., Remington's supra). After administration of the test pharmaceutical drug, the neuronal/glial activity of the subject is measured by functional imaging. This image is then compared to a functional image of a control subject that has been administered a control drug known to induce psychosis. If the images are similar and reveal a comparable pattern of neuronal/glial activity in the same brain regions, the test pharmaceutical drug is a psychosis-inducing drug. This same method may be adapted to identify whether a test pharmaceutical drug with therapeutic activity is an anxiogenic and/or a depressant.

[0125] The present disclosure also provides a method for identifying areas of the mammalian brain involved in eliciting psychosis. In this method, a non-human, mammalian control animal is treated with an amount of a known psychotic known to cause psychosis. As described above, the dose of the known psychotic administered to induce psychosis is known in the art or can be determined using known methodology. Having treated the control animal with the known psychotic, the neuronal/glial activity is measured by functional imaging to identify the regions of the brains activated by the psychotic. The areas that are activated are part of the integrated neural network that is involved with psychosis.

[0126] Once these brain regions are identified, a test subject of the same species as the control animal is treated with a therapeutically-effective amount of an anti-psychotic. Anti-psychotics are commercially available by prescription form (e.g., Abilify and Clorazil) and over the counter (e.g., alcohol and codeine containing OTC medication). The therapeutically-effective amount of an antipsychotic will be apparent to one of ordinary skill in the art based on the scientific literature as well as by recommended dosages on the packaging label.

[0127] Known anti-psychotics include, but are not limited to haloperidol (Haldol, Serenace), droperidol (Droleptan), chlorpromazine (Thorazine, Largactil), fluphenazine (Prolixin), perphenazine (Trilafon), prochlorperazine (Compazine), thioridazine (Mellaril, Melleril), trifluoperazine (Stelazine), mesoridazine, periciazine, promazine, triflupromazine (Vesprin), levomepromazine ( ozinan), promethazine (Phenergan), pimozide (Orap), chlorprothixene , flupenthixol (Depixol, Fluanxol), thiothixene ( avane), zuclopenthixol (Clopixol, Acuphase), clozapine (Clozaril), olanzapine (Zyprexa), risperidone (Risperdal), quetiapine (Seroquel), ziprasidone (Geodon), amisulpride (Solian), asenapine, paliperidone (Invega), ilioperidone (Fanapt), zotepine (Nipolept, Losizopilon, Lodopin, Setous), sertindole (Serdolec), aripiprazole (Abilify), bifeprunox; norclozapine (ACP-104), tetrabenazine, and cannabidiol. Both typical and atypical anti-psychotics may have adverse side effects associated with them, including tardive dyskinesia, diabetes, weight gain, etc.

[0128] The anti-psychotics may be administered over a prolonged time period of time, that is, chronic administration, e.g., over 1 to 12 weeks, to allow for changes in brain plasticity to be taken into account in identifying the integrated neural networks that are involved in eliciting pyschosis. Drugs can be administered 1-3 times daily, at a dosage of 0.001 mg to 500 mg daily. Dose may depend on the level needed to achieve pharmokinetic equilibrium.

[0129] After the anti-psychotic has reached pharmacokinetic equilibrium, the subject is then administered a control psychosis-inducing drug and the neuronal/glial activity of the subject is measured by functional imaging as described above. The functional images from the control and subject animals are then compared to ascertain differences in neuronal/glial activity. Any discernible/detectable differences in neuronal/glial activity observed in the subject, but not observed in the control animal, identifies the brain regions that are involved in psychosis.

[0130] The method described above for identifying areas of the mammalian brain involved in eliciting psychosis can also be modified to identify areas of the mammalian brain involved in eliciting anxiety and depression. For example, to identify areas of the mammalian brain involved in either anxiety (or depression), one would administer an known anxiogenic (or known depressant) to the control animal, measure the neuronal/glial activity by functional imaging to identify the regions of the brains activated by the anxiogenic (or depressant), treat a test subject with a therapeutically- effective amount of an anxiolytic (or anti-depressant) (administration of the anxiolytic/anti- depressant may be chronic administration), administer a control anxiogenic (or depressant) and measure the neuronal/glial activity by functional imaging. As described above for the psychosis pathway, the functional images from the control and subject animals are compared and any discernible/detectable differences in neuronal/glial activity observed in the subject, but not observed in the control animal, identifies the brain regions that are involved in anxiety (or depression).

[0131] Known anxiolytics include, but are not limited to, alprazolam (Xanax), chlordiazepoxide (Librium), clonazepam (Klonopin), clorazepate (Tranxene), diazepam (Valium), lorazepam (Ativan), buspirone (BuSpar), barbiturates, meprobamate (Miltown), hydroxyzine (Atarax), cannabidiol, pineapple sage, chlorpheniramine, diphenhydramine and herbal treatments such as St. John's wort, valerian, kava, chamomile, kratom, blue lotus extracts, sceletium tortuosum (kanna), common skullcap and bacopa monniera.

[0132] Known anti-depressants include, but are not limited to, Celexa™ (citalopram),

Lexapro™ (escitalopram oxalate), Luvox™ (fluvoxamine), Paxil™ (paroxetine), Prozac™

(fluoxetine), Zoloft™ (sertraline), Cymbalta™ (duloxetine), Effexor™ (venlafaxine), Pristiq™ (desvenlafaxine), Nardil™ (phenelzine), Parnate™ (tranylcypromine), Adapin™ (doxepin), Anafranil™ (clomipramine), Elavil™ (amitriptyline), Endep™ (amitriptyline), Ludiomil™

(maprotiline), Norpramin™ (desipramine), Pamelor™ (nortryptyline), Pertofrane™ (desipramine), Sinequan™ (doxepin), Surmontil™ (trimipramine), Tofranil™ (imipramine), Vivactil™

(protriptyline), Buspar™ (buspirone), Desyrel™ (trazodone), Edronax™, Vestra™ (reboxetine), Remeron™ (mirtazapine), Serzone™ (nefazodone), and Wellbutrin™ (bupropion).

[0133] The invention is further illustrated by the following examples. The examples are provided for illustrative purposes only. They are not to be construed as limiting the scope or content of the invention in any way.

EXAMPLE 1

Live Animal Functional Imaging

[0134] To image the brain activity of live rats (Charles River Laboratories, Wilmington, MA), the rats were anesthetized with 2% - 3% isoflurane (Abbott Laboratories, North Chicago, IL). Nine (9) rats were used for each experimental condition. A topical anesthetic of 10% lidocaine gel was applied to the skin and soft tissue around the ear canals and over the bridge of the nose. A plastic semi-circular headpiece with blunted ear supports that fit into the ear canals was positioned over the ears. The head was placed into a cylindrical head holder with the rat's canines secured over a bite bar and ears positioned inside the head holder with adjustable screws fitted into lateral sleeves. An adjustable, receive-only surface coil built into the head holder was pressed firmly on the head and locked into place. The body of the rat was placed into a body restrainer. The body restrainer "floats" down the center of the chassis connecting at the front and rear end-plates and buffered by rubber gaskets. The head piece locks into a mounting post on the front of the chassis. This design isolates all of the body movements from the head restrainer and minimizes motion artifact. Once the rat was positioned in the body holder, a transmit-only volume coil was slid over the head restrainer and locked into position.

Acclimation to Imaging Protocol

[0135] When the rats were fully conscious, the restraining unit was placed into a black opaque tube mock scanner with a tape-recording of an MRI pulse sequence. This acclimation protocol lasted for 60 min in order to simulate the bore of the magnet and the imaging protocol. This procedure was repeated every other day for four days. With this procedure, rats show a significant decline in respiration, heart rate, motor movements, and plasma corticosteroid (CORT) when compared the first to the last acclimation periods (King et al, 2005, J. Neurosci. Methods

148(2): 154-60). Imaging Protocol

[0136] Experiments were conducted in a Bruker Biospec 4.7-T/40-cm horizontal magnet (Oxford Instrument, Oxford, U.K.) equipped with a Biospec Bruker console (Bruker, Billerica, MA, U.S.A.) and a 20 G/cm magnetic field gradient insert (internal diameter = 12 cm) capable of a 120 μ$ rise time (Bruker). Radiofrequency (RF) signals were sent and received with the dual coil electronics built into the animal restrainer (Ludwig et al, 2004, J. Neurosci. Methods 132(2): 125- 35). The volume coil for transmitting RF signal features an 8-element microstrip line configuration in conjunction with an outer copper shield. The arch-shaped geometry of the receiving surface coil provides excellent coverage and high signal-to-noise. To prevent mutual coil interference, the volume and surface coils were actively tuned and detuned.

[0137] Functional images were acquired using a multi-slice fast spin echo sequence. A single data acquisition including 12 1.2 mm slices collected in 6 seconds (field of view (FOV) 3.0 cm; data matrix 64 x 64; repetition time (TR) 1.43 sec, effective echo time (Eff TE) 53.3 msec, echo time (TE) 7 msec; rapid acquisition with relaxation enhancement (RARE) factor 16, number of excitations (NEX) 1). This sequence was repeated 100 times in a 10 minute imaging session, consisting of 5 min of baseline data followed by 5 min of stimulation data. At the beginning of each imaging session, a high resolution anatomical data set was collected using a RARE pulse sequence (12 slice; 1.2 mm; FOV 3.0 cm; 256 x 256; TR 2.1 sec; TE 12.4 msec; NEX 6; 7 min acquisition time).

Motion Artifact

[0138] The experiments conducted in this work are a single epoch event-related design. To assess false activation due to subject motion, fJVIRI data were collected from awake rats (n = 8) over a 10 min scanning session in the absence of any stimulation. From these empirical data, a series of virtual fMRI data were numerically generated using a tri-linear interpolation algorithm with Gaussian noise and a preset amount of rigid body motion in random direction. The amount of motion introduced was in increment of 1/10 of a voxel (ca. 50 μιη) up to one voxel (486 μιη). The data was analyzed with statistical t-tests on each subject within their original coordinate system.

On an average, approximately 3,500 voxels were tested for each subject. The control window was the first 50 time periods (5 min), whereas the stimulation window was the remaining 50 time periods (5 min) as described for the fMRI studies above. The t-test statistics used a 95% confidence level, two-tailed distributions, and heteroscedastic variance assumptions. In this case, a multiple comparison control (false detection rate) was not used to avoid suppression of any spurious activation. There is no significant change in BOLD signal or the number of activated voxels up to ca 300 μιη (or 6/10 of voxel) motion. Both, number of voxels and percent BOLD signal, increases dramatically as it approaches one voxel of motion.

[0139] For each subject, rigid body motion in x-, y- and z-direction was computed with Stimulate software (Strupp, 1996, Neurolmage 3:S607) using center of intensity method. Standard deviation of this data gives measure of how widely spread the motion is for each subject. A conservative criteria of 120 μιη standard deviation of motion in any direction was set as acceptance criteria. In these experiments, motion in the z- and x-direction was small as compared to y- direction. Animals showing an average displacement exceeding 25% of the total in-plane (x-y) voxel resolution (>120 μιη out of 468 μιη) or more than 25% displacement in the slice (z) direction (>300 μιη out of 1,200 μιη slice thickness) were excluded. Most of the motion was in y-direction (64 μιη ±42 μιη) and can be attributed to limitations in the design of the rat head holder.

Data Analysis

[0140] Anatomy images for each subject were obtained at a resolution of 256²x l2 slices and a FOV of 30 mm with a slice thickness of 1.2 mm. Subsequent functional imaging was performed at a resolution of 64²* 12 slices with the same FOV and slice thickness. Each subject was registered to a segmented rat brain atlas. The alignment process was facilitated by an interactive graphic user interface. The affine registration involved translation, rotation, and scaling in all three dimensions, independently. The matrices that transformed the subject's anatomy to the atlas space were used to embed each slice within the atlas. All transformed pixel locations of the anatomy images were tagged with the segmented atlas major and minor regions creating a fully segmented representation of each subject. The inverse transformation matrix [Ti]^"1 for each subject (i) was also calculated.

[0141] Twelve brain slices were collected extending from the tip of the forebrain to the end of the cerebrum stopping at the midbrain just rostral to the cerebellum. Within these rostral/caudal boundaries, 83 minor volumes were delineated. In addition, brain areas were grouped into "major volumes" (e.g., amygdala, hippocampus, hypothalamus, cerebrum, etc.). The volume of activation (number of significant voxels) can be visualized in these 3D major and minor anatomical groupings.

[0142] Each scanning session consisted of 100 data acquisitions with a period of 6 seconds each for a total lapse time of 600 seconds or 10 min. The control window was the first 50 scan repetitions, while the stimulation window was scans 51-100 after the stimulation period. Statistical t-tests were performed on each subject within their original coordinate system. The baseline threshold was set at 2%. The t-test statistics used a 95% confidence level, two-tailed distributions, and heteroscedastic variance assumptions. As a result of the multiple t-test analyses performed, a false-positive detection controlling mechanism was introduced (Genovese et al, 2002, Neurolmage 15:870-8). This subsequent filter guaranteed that, on average, the false-positive detection rate was below the cutoff of 0.05. The formulation of the filter satisfied the following expression:

p < ^_

w V c(V) where Ρφ is the p value based on the t-test analysis. Each pixel (i) within the region of interest (ROI) containing (V) pixels was ranked based on its probability value. The false-positive filter value q was set to be 0.05 for the analyses, and the predetermined constant c(V) was set to unity, which is appropriate for data containing Gaussian noise such as fMRI data (Genovese et al., 2002, Neurolmage 15:870-8). These analysis settings provided conservative estimates for significance. Those pixels deemed statistically significant retained their percentage change values (stimulation mean minus control mean) relative to control mean. All other pixel values were set to zero.

[0143] A statistical composite was created for each group of subjects. The individual analyses were summed within groups. The composite statistics were built using the inverse transformation matrices. Each composite pixel location (i.e., row, column, and slice), premultiplied by [Ti]^"1, mapped it within a voxel of subject (i). A tri-linear interpolation of the subject's voxel values (percentage change) determined the statistical contribution of subject (i) to the composite (row, column, and slice) location. The use of [Ti]^"1 ensured that the full volume set of the composite was populated with subject contributions. The average value from all subjects within the group determined the composite value. The BOLD response maps of the composite were somewhat broader in their spatial coverage than in an individual subject. Thus, only average number of activated pixels that has the highest composite percent change values in particular ROI was displayed in composite map. Activated composite pixels are calculated as follows:

N

Activated Pixels Subject(i) ROI(j)

Activated Composite Pixels ROI( j) =— —

The composite percent change for the time history graphs for each region was based

weighted average of each subject, as follows: N

Activated Pixel Subject(i) x Percent Change(i)

Composite Percent Change =—

Activated Composite Pixels where N is number of subjects.

EXAMPLE 2

Identifying the Neural Networks Associated with Anxiety

[0144] Animal models of anxiety usually involve testing inbred strains of rats that show strong behavioral phenotypes (e.g., high anxiety behavior and low anxiety behavior Wistar rat) or transgenic knock outs. Anxiety is associated with fear and avoidance behavior and is usually assayed in the elevated plus maze or light-dark shuttle box.

[0145] Treatment with conventional anxiolytics like the benzodiazepines (GABA agonists), buspirone (5HT1 agonist), and hydroxyzine (antihistamine) increases the time in the open arm of the plus maze or time spent in the light of the shuttle box. Anxiety can be chemically induced. The most common anxiogenics are GABA antagonists such as DMCM, FG-7142 and ZK-93426, serotonergic agents such as mCPP, adrenergic agents such as yohimbine. Conceptually, treatment with these different agents activates the integrated neural network involved in enhancing anxiety, fear and avoidance. Their anxiogenic activity has been shown in animals and humans showing the activation of a neural network common across species.

Method

[0146] Adult male rats are prepared for imaging as described in Example 1 above (Ferris et al. (2008) BMC Neurosci. 9: 1 1 1). While fully conscious, animals are given a tail vein injection of different doses of yohimbine in saline (1 μg - 100 mg) and imaged for changes in brain activity (see previous Methods) using fMRI. Similar studies are run for the different classes of anxiogenics. For example, other anxiogenics include, but are not limited to, asthma medicines (such as albuterol, salmeterol, and theophylline), blood pressure medicines (such as methyldopa), hormones (such as oral contraceptives), amphetamines, medicines containing amphetamines (such as Benzedrine, Dexedrine, and Ritalin), steroids (such as cortisone, dexamethasone, and prednisone), thyroid medicines, phenytoin, levodopa, quinidine, caffeine, decongestants (such as phenylephrine), and illegal drugs such as cocaine and crack.

[0147] Using a segmented 3D atlas of the rat brain, over 100 different areas are analyzed for changes in volume of activation and BOLD signal over time. Significant differences between these areas are determined generating an activity profile of an integrated neural network unique to the different classes of anxiogenics.

[0148] Because most neuropsychotropic agents require several weeks to work, it is likely that a change in brain plasticity is useful for efficacy. As such, this study described above is repeated after daily chronic treatment with the different classes of anxiogenics (1-12 weeks). Having established the neural network(s) for acute and chronic exposure to anxiogenics, it is now possible to "finger print" the conventional anxiolytics.

[0149] Animals (mice, rats, or primates) are pretreated (SC, IP, oral, IV) with conventional anxiolytics noted above (ranging in doses from 1 μg - 100 mg). After the drugs come to equilibrium, animals are imaged for changes in brain activity in response to SC, IP, oral, or IV administration of different anxiogenics. By examining the differences in brain activity between the anxiogenic sensitive neural network(s) and that with anxiolytics, areas are identified in the putative network of anxiety, fear and avoidance that are affected by anxiolytics. This method allows for delineating between different classes of anxiolytics and enables the screening of any new drug for anti-anxiety like activity. The cortical loop of Papez circuit, together with the amygdala, are affected by drug treatments.

EXAMPLE 3

Screening for Anxiogenics

[0150] Adult male rats are prepared for imaging as previously described (Ferris et al, 2008, BMC Neuroscience 9: 11 1). While fully conscious, animals are given a tail vein injection of different doses (1 μg - 100 mg) of a therapeutically effective amount of a test pharmaceutical drug, and imaged for changes in brain activity. This image or "finger print" is obtained by measuring a baseline metabolic activity (baseline signal intensity) of all voxels (3D pixels) in the brain are assessed as deduced from cerebral blood flow, cerebral blood volume or BOLD measurements for a period of 5-15 min (or longer) followed by the administration (tail vein, intraperitoneal, subcutaneous, oral, intranasal) of different doses (1 μg - 100 mg) of yohimbine. The change in signal intensity is determined for each voxel comparing the average signal intensity in its baseline period to the average signal intensity following drug administration. Voxels are assigned to one of hundreds of different regions of interests based upon their registration into a 3D segmented atlas. Each subject presents with a certain number of significantly activated voxels in a given region of interest or what we call volume of activation, i.e. number of 3D pixels in a 3D volume. Random effect analysis with different test statistics is used to compare the average (+/- SD) or median and range of all subjects in an region of interest across all doses. These data provide a dose-dependent profile of signal change or finger print identifying brain areas that are significantly activated (increase in signal) or deactivated (decrease in signal). From these profiles a map of an integrated neural circuit characterizing the action of yohimbine on the brain is determined. This represents the putative neural circuit of fear/anxiety.

[0151] This finger print is compared to that of an unknown drug to determine if the unknown has potential anxiogenic activity.

[0152] This fingerprint is also used to evaluate drug action by pretreating animals with a known anxiolytic (e.g., benzodiazepines), drugs that reduces or blocks fear and anxiety. After a period of time when the anxiolytic has achieved its effect, these animals are placed in the MR scanner and imaged in response administration of the know anxiogenic. If the finger print of the anxiogenic is altered appreciably (e.g., more than 30% - 40%) of its components (brain areas comprising the neural circuit), then the anxiolytic is effective in reducing or blocking the anxiogenic effect. Drugs thought to be anxiolytics are screened by pretreating animals with the test drug followed later by imaging brain activity in response to anxiogenic challenge. If the the know fingerprint of the anxiogenic is altered appreciably then the test drug has anxiolytic activity.

EXAMPLE 4

Identifying the Neural Networks Associated with Depression

[0153] Adult male rats are prepared for imaging as described in Ferris et al, 2008, BMC Neuroscience 9: 11 1. While fully conscious, animals are given a tail vein injection of different doses of rimonobant (1 μg - 100 mg) and imaged for changes in brain activity (as described supra in Example 3).

[0154] Using a segmented 3D atlas of the rat brain, over 100 different areas are analyzed for changes in volume of activation and BOLD signal over time. Significant differences between these areas are determined generating an activity profile of an integrated neural network unique to rimonobant.

[0155] Since rimonobant affects the brain to increase the risk of depression and suicide, the identified neural network contributes to these behavioral problems. Most neuropsychotropic agents require several weeks to work suggesting a change in brain plasticity is need for efficacy. As such, the same study described above would be repeated but only after daily chronic treatment with rimonobant (1-12 weeks). Having established the rimonobant sensitive neural network for acute and chronic drug exposure, a "finger print" of the conventional anti-depressants is obtained. [0156] Animals are pretreated (SC, IP, oral, IV) with conventional anti-depressants (ranging in doses from 1 μg - 100 mg). After the drugs come to equilibrium, animals are imaged for changes in brain activity in response to SC, IP, oral, or IV administration of different levels of rimonobant. By examining the differences in brain activity between the rimonobant sensitive neural network and that with anti-depressants, areas in the putative network of depression and suicide that are affected by anti-depressants are identified. This method allows for delineating between different classes of antidepressants and enables one to screen any new drug for anti-depressant like activity. The Papez circuit involved in emotional experience and memory together with areas of the mediobasal hypothalamus involved in emotional expression are affected by drug treatments.

EXAMPLE 5

Screening for Depressants

[0157] Adult male rats are prepared for imaging as described in Ferris et al, 2008, BMC Neuroscience 9: 11 1. While fully conscious, animals are given a tail vein injection of different doses (1 μg - 100 mg) of a therapeutically effective amount a test pharmaceutical drug and imaged for changes in brain activity (see previous Examples). These imaging results are compared to the images or finger print of the same animal which had been previously treated with a known depressant, such as benzodiazepine. If the two images exhibit a comparable pattern of

neuronal/glial activation, the test pharmaceutical drug is likely a depressant. To confirm the ability of the drug to act as a depressant, a behavioral test such as the tail suspension test and forced swim test is performed. Animals that have been administered a depressant become immobile, i.e., give up more quickly, than animals administered an anti-depressant.

EXAMPLE 61

Screening for Psvchotics

[0158] Adult male rats are prepared for imaging as previously described in Ferris et al, 2008, BMC Neuroscience 2008 9: 11 1. While fully conscious, animals are given a tail vein injection of different doses of a therapeutically effective amount a test pharmaceutical drug and imaged for changes in brain activity (see previous Examples). These imaging results are compared to the images of the same animal which had been previously treated with a known psychosis inducing drug, such as apomorphine. If the two images exhibit a comparable pattern of neuronal/glial activation, the test pharmaceutical drug is likely a psychosis inducing drug. To confirm that the test pharmaceutical drug is a drug that induces psychosis, a behavioral test such as, but not limited to, the tail-pinch test, pre-pulse inhibition of startle response test, passive avoidance test or learned helplessness test is performed.

EXAMPLE 7

Identifying an Increased Risk of Suicide

[0159] Experiments were performed using male, Long Evans rats (-350-450 g). Rats were acclimated using procedures described in Example 1. As shown in Figure 4, acclimated animals had decreasing respiratory rates, heart rates, and corticosterone levels over the course of eight days.

Method

[0160] All experimental procedures, including data analysis, statistical analysis, motion artifact assessment, and imaging, were performed as described in Example 1 except as otherwise provided below. All imaging was performed within one week of acclimation. Briefly, adult male rats were prepared for imaging as described in Example 1 above with the following modifications (Ferris et al. (2008) BMC Neurosci. 9: 1 11). Animals were positioned in the animal restrainer under 2% isoflurane and allowed to awaken for a six minute anatomical scan.

[0161] Prior to performing the tail injections, a five minute baseline control scan was performed to determine the baseline BOLD signal.

[0162] After the baseline was determined, fully conscious animals were given a tail vein injection of Venlafaxine (n=8), Rimonabant (n=9), and Gabapentin (n=10) at clinically relevant doses. phMRI was used to image changes in brain activity (see previous Methods) using phMRI. Imaging was performed using a Bruker Biospec 7.0-T/20-cm horizontal magnet (Oxford

Instrument, Oxford, U.K.). In addition, two groups of fully conscious animals were given tail vein injections of one of two control drugs known to decrease aggression and suicide risk. One group was given injections of Clozapine (n=10), which is a known anti-suicide drug prescribed for schizophrenics, and the other group was given injections of Buspirone (n=9), which is a known anti-aggression drug prescribed to victims of traumatic head injury.

[0163] The images shown in Figure 5 were obtained by first measuring a baseline metabolic activity (baseline signal intensity) of all voxels (3D pixels) in the brains as deduced from BOLD measurements for a period of 5 minutes followed by the administration of the drug. The change in signal intensity was determined for each voxel comparing the average signal intensity in its baseline period to the average signal intensity following drug administration. Voxels were assigned to one of hundreds of different regions of interest based upon their registration into a 3D segmented atlas. Each rat presented with a certain number of significantly activated voxels in a given region of interest or "volume of activation," i.e. number of 3D pixels in a 3D volume. Random effect analysis with different test statistics was used to compare the average (+/- SD) or median and range of all rats in a region of interest across all doses. These data provided a finger print (Figure 5).

[0164] Using a segmented 3D atlas of the rat brain, 154 different areas of the brain were analyzed for changes in volume of activation and BOLD signal over time (Figure 5). After comparing the activity of brains exposed to Venlafaxine 200, Rimonabant 210, and Gabapentin 220 to brains exposed to Clozapine 230 and Buspirone 240, 67 areas of common activity were identified 250 (Figure 5). Of these 67 areas, 17 areas were determined to be active exclusive of the control drugs 260 (Figure 5). These areas appear to be associated with an increased risk of suicide.

[0165] Figure 6 shows the results shown in a 3D activation map. The map provides a detailed view of the brain and the areas showing increased BOLD signal. The map was built from a compilation of 2D activation maps developed from the phMRI images generated during the experiments (Figure 7).

Translation of Rat Brain Regions To Human Brain Regions

[0166] The experiments identified particular areas of the rat brain activated by the test drugs. In particular, the particular regions of the rat brain were the Prelimbic cortex (corresponding to the human dorsal lateral and prefrontal cortex), medial dorsal striatum (corresponding to the human basal ganglia), the anterior, reuniens, and lateral posterior thalamus (corresponding to the human medial dorsal thalamus), dorsal and ventral CA1 (corresponding the human hippocampus), and insular, gustatory, and parietal cortices (corresponding to the human limbic and parietal cortices). The corresponding human region has been associated with schizophrenia, which has the highest incidence of suicide among mental diseases (Eisenberg & Berman, Neuropsychopharmacology (Reviews) 35:258-277 (2010)). Therefore, it appears that these regions are potentially associated with suicidal ideation.

EQUIVALENTS

[0167] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

Claims:

1. A method for identifying whether a test pharmaceutical drug also causes depression and/or psychosis and/or anxiety in a conscious, non-human, mammalian subject, comprising:

(a) treating a control animal with a control drug, the control drug being a known psychotic, a known anxiogenic, and/or a known depressant;

(b) measuring the neuronal/glial activity in the brain of the treated control by functional imaging;

(c) treating the subject with a therapeutically-effective amount of the test pharmaceutical drug;

(d) measuring the neuronal/glial activity in the brain of the treated subject by functional imaging; and

(e) comparing the neuronal/glial activity in the brain of the treated subject with the neuronal/glial activity in the brain of the control animal, a comparable pattern of neuronal/glial activity in the brain of the treated subject measured in (d) relative to the pattern of neuronal/glial activity in the brain of the control after administration of the control drug in (b) indicating that the test pharmaceutical drug also causes depression and/or psychosis and/or anxiety.

2. The method of claim 1, wherein the test pharmaceutical drug is used to treat mental illness.

3. The method of claim 1, wherein the mammalian subject is selected from the group consisting of a mouse, rodent, and primate.

4. The method of claim 1 , wherein the control psychotic is selected from the group consisting of apomorphine, cocaine, amphetamine, methamphetamine, methylenedioxymethamphetamine ("MDMA"), and phenylcyclohexylpiperidine ("PCP").

5. The method of claim 1, wherein the control anxiogenic is selected from the group consisting of DMCM, FG-7142, ZK-93426, mCPP, and yohimbine.

6. The method of claim 1, wherein the control depressant is selected from the group consisting of alcohol, barbiturates, benzodiazepines, non-benzodiazepines, and opioids.

7. A method of determining whether a test pharmaceutical drug is also a psychosis-inducing drug, comprising:

(a) treating a conscious, non-human, mammalian subject with a therapeutically

effective amount of the test pharmaceutical drug;

(b) measuring the neuronal/glial activity in the integrated neural network of the subject by functional imaging of the subject's brain; and

(c) comparing the neuronal/glial activity in the subject after administration of the test pharmaceutical drug with the neuronal/glial activity in the integrated neural network of a control animal after administration of a control drug known to induce psychosis, a comparable pattern of neuronal/glial activity in the subject measured in (b) relative to the pattern of neuronal/glial activity in the control animal after administration of the control drug in (c) indicating that the test pharmaceutical drug is a psychosis-inducing drug.

8. A method of determining whether a test pharmaceutical drug is also an anti-psychotic drug, comprising:

(a) treating a conscious, non-human, mammalian subject with a therapeutically effective amount of the test pharmaceutical drug;

(b) measuring the neuronal/glial activity in the integrated neural network of the subject by functional imaging of the subject's brain; and

(c) comparing the neuronal/glial activity in the subject after administration of the test pharmaceutical drug with the neuronal/glial activity in the integrated neural network of a control animal after administration of a control drug known to induce psychosis, a detectable difference in the regions of the brain having neuronal/glial activity in the subject measured in (b) relative to the pattern of neuronal/glial activity in the control animal after administration of the control drug in (c) indicating that the test pharmaceutical drug is an antipsychotic drug.

9. The method of claim 8, wherein the nonhuman mammalian subject is selected from the group consisting of a mouse, rodent, and primate.

10. The method of claim 8, wherein the known psychosis-inducing drug is selected from the group consisting of apomorphine, cocaine, amphetamine, methamphetamine,

methylenedioxymethamphetamine ("MDMA"), and phenylcyclohexylpiperidine ("PCP") .

11. A method of determining whether a test pharmaceutical drug is also an anxiogenic, comprising:

(a) treating a conscious, non-human, mammalian subject with a therapeutically effective amount of the test pharmaceutical drug;

(b) measuring the neuronal/glial activity in the integrated neural network of the subject by functional imaging of the subject's brain; and

(c) comparing the neuronal/glial activity in the subject after administration of the test pharmaceutical drug with the neuronal/glial activity in the integrated neural network of a control animal after administration of a control anxiogenic, a comparable pattern of neuronal/glial activity in the subject measured in (b) relative to the pattern of neuronal/glial activity in the control animal after administration of the control anxiogenic in (c) indicating that the test pharmaceutical drug is an anxiogenic.

12. The method of claim 1 1 wherein the mammalian subject is selected from the group consisting of a mouse, rodent, and primate.

13. The method of claim 1 1, wherein the known anxiogenic is selected from a group consisting of DMCM, FG-7142, ZK-93426, mCPP, and yohimbine.

14. A method of identifying whether a test pharmaceutical drug is also a depressant, comprising:

(a) treating a conscious, non-human, mammalian subject with a therapeutically

effective amount of the test pharmaceutical drug;

(b) measuring the neuronal/glial activity in the integrated neural network of the subject by functional imaging of the subject's brain; and

(c) comparing the neuronal/glial activity in the subject after administration of the test pharmaceutical drug with the neuronal/glial activity in the integrated neural network of a control animal after administration of a known depressant, a comparable pattern of neuronal/glial activity in the subject measured in (b) relative to the pattern of neuronal/glial activity in the control animal after administration of the known depressant in (c) indicating that the test pharmaceutical drug is a depressant.

15. The method of claim 14, wherein the mammalian subject is selected from the group consisting of a mouse, rodent, and primate.

16. The method of claim 14, wherein the known depressant is selected from a group consisting of alcohol, barbiturates, benzodiazepines, non-benzodiazepines, and opioids.

17. A method of identifying areas of the integrated neural network of the brain of a

conscious, non-human, mammalian subject that elicit psychosis, comprising:

(a) treating a non-human, mammalian control with an amount of a control psychotic known to cause psychosis;

(b) identifying a region of the brain of the treated control which shows neuronal/glial activity by functional imaging;

(c) treating the conscious, non-human, mammalian subject of the same species as the control with a therapeutically-effective amount of an anti -psychotic drug;

(d) allowing the anti-psychotic drug to achieve pharmacokinetic equilibrium in the subject;

(e) treating the subject with an amount of the control psychotic known to cause psychosis;

(f) identifying a region in the brain of the treated subject in (e) having neuronal/glial activity by functional imaging; and

(g) comparing the regions of neuronal/glial activity in the brain of the treated control of (b) with the regions of neuronal/glial activity in the brain of the treated subject in (f), detectable differences in the regions of the brain having neuronal/glial activity in the treated control relative to the treated subject indicating that these brain regions are involved in psychosis.

18. The method of claim 17, wherein the mammalian control and subject are selected from the group consisting of a mouse, rodent, and primate.

19. The method of claim 17, wherein (a) further comprises chronic administration of the psychotic over 1 to 12 weeks.

20. The method of claim 17, wherein (c) further comprises the chronic administration of the anti-psychotic drug over 1 to 12 weeks.

21. The method of claim 17, wherein (a) and (c) further comprise the chronic administration of both the psychotic and anti-psychotic drug over 1 to 12 weeks.

22. The method of claim 17, wherein the psychotic is selected from a group consisting of apomorphine, cocaine, amphetamine, methamphetamine, methylenedioxymethamphetamine ("MDMA"), and phenylcyclohexylpiperidine ("PCP").

23. The method of claim 17, wherein the anti-psychotic drug is selected from a group consisting of haloperidol, clozapine, chlorpromazine, olanzapine, risperidone, or aripiprazole.

24. A method of identifying areas of the integrated neural network of the brain of a

conscious, non-human, mammalian subject that elicit anxiety, comprising:

(a) treating a non-human, mammalian control with an amount of a control anxiogenic known to cause anxiogenic;

(b) identifying a region of the brain of the treated subject which shows neuronal/glial activity by functional imaging;

(c) treating the conscious, non-human, mammalian subject of the same species as the control with a therapeutically effective amount of an anxiolytic drug;

(d) allowing the anxiolytic drug to achieve pharmacokinetic equilibrium in the subject;

(e) treating the subject with a an amount of the control anxiogenic which is known to cause anxiety;

(f) identifying a region in the brain of the treated subject in (e) having neuronal/glial activity in (e) by functional imaging; and

(g) comparing the regions of neuronal/glial activity in the treated control of (b) with the regions of neuronal/glial activity in the brain of the treated subject in (f), detectable differences in the regions of the brain having neuronal/glial activity in the treated control relative to the treated subject indicating that these brain regions are involved in anxiety.

25. The method of claim 24, wherein the mammalian control and subject are selected from the group consisting of a mouse, rodent, and primate.

26. The method of claim 24, wherein (a) further comprises the chronic administration of the anxiogenic over 1 to 12 weeks.

27. The method of claim 24, wherein (c) further comprises the chronic administration of the anxiolytic over 1 to 12 weeks.

28. The method of claim 24, wherein (a) and (c) further comprise the chronic administration of both the anxiogenic and anxiolytic over 1 to 12 weeks.

29. The method of claim 24, wherein the anxiolytic drug is selected from a group consisting of benzodiazepines, buspirone, and hydroxyzine.

30 The method of claim 24, wherein the anxiogenic is selected from a group consisting of DMCM, FG-7142 ZK-93426, mCPP, and yohimbine.

31. A method of identifying areas of the integrated neural network of the brain of a conscious, non-human, mammalian subject that elicit depression, comprising:

(a) treating a non-human, mammalian control with an amount of a control depressant;

(b) identifying a region of the brain of the treated subject which shows neuronal/glial activity by functional imaging;

(c) treating the conscious, non-human, mammalian subject of the same species with a therapeutically effective amount of an anti-depressant drug;

(d) allowing the anti-depressant drug to achieve pharmacokinetic equilibrium in the subject;

(e) treating the subject with a therapeutically effective amount of the control depressant;

(f) identifying a region in the brain of the treated subject in (e) having neuronal/glial activity by functional imaging; and

(g) comparing the regions of neuronal/glial activity in the treated control of (b) with the regions of neuronal/glial activity in the brain of the treated subject in (f), detectable differences in the regions of the brain having neuronal/glial activity in the treated control relative to the treated subject indicating that these brain regions are involved in depression.

32. The method of claim 31, wherein the mammalian control and subject are selected from the group consisting of a mouse, rodent, and primate.

33. The method of claim 31, wherein (a) further comprises chronic administration of the depressant over 1 to 12 weeks.

34. The method of claim 31, wherein (c) further comprises the chronic administration of the anti-depressant over 1 to 12 weeks.

35. The method of claim 31, wherein (a) and (c) further comprise the chronic administration of both the depressant and anti-depressant over 1 to 12 weeks.

36. The method of claim 31, wherein the anti-depressant is selected from a group consisting of selective serotonin reuptake inhibitors, tricyclics, and monoamine oxidase inhibitors.

37. The method of claim 31, wherein the depressant is selected from a group consisting of alcohol, barbiturates, benzodiazepines, non-benzodiazepines, opioids, and rimonobant.

38. A method of screening a test drug for increased risk of one or more of depression, psychosis, or anxiety in a conscious, non-human, mammalian subject, comprising:

(a) measuring a neural/glial activity in a brain of a first conscious, non-human, mammalian subject treated with a drug used to treat one or more of a psychotic, an anxiogenic, or a depression disorder;

(b) identifying a pattern of neural/glial activity of the brain of the first subject treated with the drug;

(c) treating a second conscious, non-human, mammalian subject with a test drug;

(d) measuring the neural/glial activity in the brain of the second subject treated with the test drug and identifying a pattern of the neural/glial activity of the brain of the second subject;

(e) comparing the pattern of the neural/glial activity of the brain of the first subject to the pattern of the neural/glial activity of the brain of the second subject; and (f) determining a pattern of neural/glial activity associated with one or more of depression, psychosis, or anxiety.

39. The method of claim 38, wherein neuronal/glial activity is measured by functional MRI.

40. The method of claim 38, wherein the test drug is used to treat mental illness.

41. The method of claim 38, wherein the mammalian subject is selected from the group consisting of a mouse, rodent, and primate.

42. The method of claim 38, wherein the control psychotic is selected from the group consisting of apomorphine, cocaine, amphetamine, methamphetamine, methylenedioxymethamphetamine ("MDMA"), and phenylcyclohexylpiperidine ("PCP").

43. The method of claim 38, wherein the control anxiogenic is selected from the group consisting of DMCM, FG-7142, ZK-93426, mCPP, and yohimbine.

44. The method of claim 38, wherein the control depressant is selected from the group consisting of alcohol, barbiturates, benzodiazepines, non-benzodiazepines, and opioids.

45. The method of claim 38 further comprising performing (a) in the brains of a plurality of conscious, non-human, mammalian subjects with a plurality of drugs used to treat one or more of a psychotic, an anxiogenic, or a depression disorder.

46. The method of claim 45 further comprising compiling the patterns identified into a database.

47. The method of claim 38, wherein determining a pattern of neural/glial activity comprises identifying differences between the pattern of neural/glial activity of the first subject and the pattern of the neural/glial activity of the second subject.

48. The method of claim 46, wherein patterns are identified for a plurality of test drugs.

49. The method of claim 48 further comprising generating a composite pattern of neural/glial activity from the patterns identified for the plurality of test drugs, the composite pattern representing a consensus of neural/glial activity associated with one or more of depression, psychosis, or anxiety as determined from the patterns identified for the plurality of test drugs.

50. The method of claim 49 further comprising refining the composite pattern based on additional patterns compiled in the database.

51. The method of claim 50 further comprising measuring a neural/glial activity in a brain of a third conscious, non-human, mammalian subject treated with a drug having no known effect on one or more of a psychotic, an anxiogenic, or a depression disorder and identifying a pattern of neural/glial activity of the brain of the first subject treated with the drug having no known effect.

52. The method of claim 51 further comprising determining whether the drug having no known effect has a pattern of neural/glial activity similar to any of the composite patterns in the database.

53. A method of identifying a drug for increased risk of suicide in a subject, comprising: administering the drug to a subject; and detecting the level of activity of cells in one or more brain regions of the subject, the region selected from prelimbic cortex, gustatory cortex, insular cortex, secondary somatosensory cortex, parietal cortex, medial dorsal striatum, anterior thalamus, ventral anterior lateral thalamus, reuniens thalamus, ventral medial thalamus, lateral posterior thalamus, lateral dorsal thalamus, central thalamus, CA1 hippocampus, posterior hypothalamus, and central raphe; wherein an increased level of activity relative to a control is indicative of the drug having an increased risk of suicide by the subject.