WO 2007/024946

PCT/US2006/032913

METHODS FOR THE TREATMENT OF ANXIETY
AND FOR IDENTIFICATION OF ANXIOLYTIC AGENTS
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This Application claims benefit of U.S. Provisional Application No. 60/710,385 filed
August 23, 2005, which is hereby incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0002] The invention relates generally to the field of neuropharmacology. The invention
features methods for the treatment of neuropsychiatric disorders such as anxiety. Also featured
are methods to identify compounds that reduce anxiety in a subject.
BACKGROUND OF THE INVENTION
[0003] Various publications, including patents, published applications, technical articles and
scholarly articles are cited throughout the specification. Each of these cited publications is
incorporated by reference herein in its entirety.
[0004] The angiotensin IV receptor (AT4R), also known as insulin-regulated membrane
aminopeptidase (IRAP), was first described in 1992 as a high-affinity binding site for the
hexapeptide angiotensin IV (AT4). (Swanson, GN et al. Regul Pept. (1992) 40:409-19). The
AT4R is a member of the Ml family of zinc metallopeptidases and is a type II membrane-
spanning protein, i.e., its active site is extracellular. (Keller, SR et al. J. Biol. Chem. (1995)
270:23612-18). Localization studies have demonstrated that AT4Rs are found in the kidney,
heart, and adrenal tissue. (Baker, KM et al. Am. J. Physiol. (1990) 259:H324-32; Slinker, BK et
al. Cardiovasc. Res. (1999) 42:660-9; Hamilton, TA et al. Peptides (2001) 22:935-44; and,
Abrahamsen, CT et al. J. Pharmacol. Exp. Ther. (2002) 301:21-8). Within the central nervous
system, western blot and in situ hybridization experiments showed that AT4R axe found at high
levels in the hippocampus and the entorhinal, prefrontal, and insular cortices. Levels in the
substantia nigra, hypothalamus, and limbic areas, such as the amygdala, are also moderately
high. (Thomas, WG et al. Int. J. Biochem. Cell Biol. (2003) 35:774-9). The differential

WO 2007/024946 PCT/US2006/032913
distribution of AT4.R in the brain has prompted considerable investigation into identifying a
biological role for the receptors in central nervous system function.
[0005] Several literature reports indicate that AT4Rs influence various facets of cognitive
function. For example, central infusions of one AT4R ligand, AT4, facilitate memory retention
and retrieval in rodent passive avoidance paradigms. (Wright, JW et al. Brain Res. Bull. (1993)
32:497-502; and, Braszko, JJ et al. Pharmacol. Res. (1998) 38:461-8). Similarly, chronic AT4
infusions were found to improve performance in the Morris Water Maze. (Pederson, ES et al.
Regal, Pept. (1998) 74:97-103). Moreover, synthetic analogs of AT4 were found to reverse
memory deficits induced by either scopolamine or bilateral perforant pathway lesion.
(Perderson, ES (1998); and, Wright, JW et al. J. Neurosci. (1999) 19:3952-61). Consistent with
the cognitive-enhancing role of AT4R, it has been reported that AT4Rs enhance both long term
potentiation and potassium-evoked acetylcholine release in hippocampal slices. (Wayner, MJ et
al. Peptides (2001) 22:1403-14).
[0006] It is believed that the mechanism by which AT4 affects cognitive processes is by
turning off the constitutively active peptidase activity of AT4R. Inhibition of AT4R activity
results in elevated synaptic levels of several neuropeptides involved in cognitive processes
Kovacs, GL and De Wied, D Pharmacol. Rev. (1994) 46:269-291). These neuropeptides include
oxytocin, somatostatin, cholecystokinin 8, vasopressin, and substance P. (Herbst, J.J. et al. Am. J
Physiol. (1997) 272, E600-E606; and, Matsumoto et al. Eur. J. Biochem. (2000) 267:46-52).
While the exact recognition sequence for the peptidase activity has yet to be elucidated, blocking
AT4R peptidase activity does not appear to affect other neuropeptides such as GnRH,
neuropeptide Y, TRH, melanocortin, alpha-MSH, galanin, or calcitonin. Moreover, AT4 does
not seem to inhibit AT4R by binding to the active site of the enzyme. Rather, AT4 binds to a
juxtamembrane region to induce a conformational change in AT4R. The consequence of AT4
binding to AT4R is the inhibition of the peptidase activity of the AT4R. (Albiston, AL et al.
Trends in Endo. Metabol (2003) 43: 72-77).
[0007] Some reports suggest that oxytocin, one of the neuropeptides elevated as a result of
AT4R repression, may exert an anxiolytic effect. Oxytocin knock-out mice showed higher levels
of anxiety-related behavior when tested in the elevated plus maze test (EPM) for anxiety relative
to wild-type mice. (Amico, JA et al. J. Neuroendocrinal. (2004) 16:319 24). In addition, central
administration of synthetic oxytocin to oxytocin knock-out mice reduced anxiety levels as
measured by EPM, and administration of an oxytocin receptor antagonist in addition to oxytocin
in the knock-out mouse model abrogated the anxiolytic effects of the oxytocin. (Mantella, RC
(2003)). Similarly, central administration of oxytocin to rats was found to attenuate the stress-
-2-

WO 2007/024946 PCT/US2006/032913
induced neuroendocrine and molecular response in the brain. (Windle, RJ et al. J. Neurosci,
(2004) 24:2974-82).
[0008] In contrast, vasopressin, which is also elevated when AT4R is inhibited, is an
anxiogenic neuropeptide. (Bhattacharya, SK et al. Biogenic Amines (1998) 14:367-86). Given
the fact that the AT4R cleaves vasopressin more efficiently than it cleaves oxytocin (Lew, RA et
al. J. Neurochem. (2003) 86:344-50), it seems that inhibition of AT4R in the central nervous
system would be more likely to exert an anxiogenic, rather than an anxiolytic effect. However,
studies reported heretofore have not addressed any relationship between AT4R activity and
neuropsychiatric conditions such as anxiety.
[0009] Although most individuals experience feelings of anxiety within their lives, especially
around new or important events, anxiety disorders are characterized by chronic and unremitting
episodes of fear and nervousness that generally interfere with the individual's everyday life
activities and experiences. Anxiety disorders are among the most common mental illness in the
United States, affecting more than 19 million, or roughly 13% of adults between the ages of 18
and 54. (Source: U.S. National Institute of Mental Health). Anxiety disorders fall into several
classes: Generalized Anxiety Disorder, characterized by constant, exaggerated worrisome
thoughts about everyday routine life activities, and physical symptoms such as trembling,
fatigue, insomnia, headaches, and nausea; Panic Disorders, characterized by repeated episodes of
intense terror, and physical symptoms such as pounding heart, chest pains, lightheadedness,
trembling, sweating, and hot flashes or chills; Phobias, characterized by disabling and irrational
fears of specific objects or situations, which can lead to an individual avoiding such objects or
situations unnecessarily; Obsessive Compulsive Disorder, characterized by repeated unwanted
thoughts or compulsive behaviors that seem impossible to stop or control; and Post-Traumatic
Stress Disorder, which generally occurs after witnessing or taking part in a terrifying event such
as a rape, abuse, war, disaster, or serious accident, and physical symptoms such as insomnia,
nightmares, flashbacks, depression, and irritability.
[0010] Anxiety disorders are typically treated with cognitive behavioral therapy and various
medications. However, given the side effects of many drugs currently used to treat anxiety
disorders, newer drugs and methods of treatment with fewer or less severe side effects are
desirable. Moreover it is also desirable to obtain drags that can work synergistically with
existing therapies to enhance their efficacy, or that can target the underlying molecular,
biochemical, or physiological basis for the anxiety disorder in question.
-3-

WO 2007/024946 PCT/US2006/032913
SUMMARY OF THE INVENTION
[0011] The present invention describes methods for the treatment of neuropsychiatric disorders
such as anxiety and methods to identify compounds for the treatment of neuropsychiatric
disorders such as anxiety.
[0012] Some aspects of the invention feature methods for treating neuropsychiatric disorders in
a subject in need of such treatment by administering'to the subject a composition comprising a
pharmaceutically acceptable carrier and an angiotensin IV receptor antagonist in an amount
effective to diminish the biological activity of the AT4R. In a detailed embodiment, the
neuropsychiatric disorder is anxiety. In a further detailed embodiment, the antagonist is
angiotensin IV, divalinal-angiotensin IV, LVV-hemorphin 7, Nle-angiotensin IV, norleucinal-
angiotensin IV, or any derivatives thereof.
[0013] The invention also features methods for treating neuropsychiatric disorders in a subject
in need of such treatment by modulating the expression of the AT4R in the subject. In a detailed
embodiment, the neuropsychiatric disorder is anxiety. In a further detailed embodiment,
expression of the AT4R is reduced. In some embodiments, the expression of the AT4R on cell
membranes is diminished. In some aspects, expression of the AT4R is modulated by an
oligonucleotide that is antisense to a nucleic acid encoding the AT4R. In some embodiments
expression of the AT4R is diminished by preventing localization of the AT4R to the cell surface
by removing or altering the membrane translocation signal peptide, or by targeting the expressed
AT4R for proteasome degradation.
[0014] The invention also provides methods for treating neuropsychiatric disorders in a subject
in need of such treatment by blocking the active site of the AT4R with antibodies to the AT4R
such that other molecules such as AT4R substrates cannot access the active site of the AT4R. In
some embodiments, the neuropsychiatric disorder is anxiety.
[0015] Another aspect of the invention features methods for identifying antagonists of the
AT4R. In some embodiments, the methods involve contacting a test compound with the AT4R
and determining a decrease in the biological activity of the AT4R in the presence of the test
compound relative to the biological activity of the AT4R in the absence of the test compound. In
some embodiments, the method will utilize purified AT4R. In other embodiments, the method
will he performed on a cell membrane comprising AT4R. In other embodiments, the method
will be performed on whole cells expressing the AT4R. Compounds identified by this inventive
method are also contemplated to be within the scope of the invention, as well as pharmaceutical
compositions that comprise compounds identified by the inventive methods admixed with a
pharmaceutically acceptable carrier.
-4-

WO 2007/024946 PCT/US2006/032913
[0016] Also provided are methods for identifying compounds that reduce anxiety in a subject
by administering a test compound to the subject and determining a decrease in the level of
anxiety in the subject relative to the level of anxiety in the subject in the absence of the test
compound. Anxiety in a subject can be determined using such models as the four-plate model,
elevated zero maze, elevated plus maze, light-dark transition test, Geller-type anticonflict test,
Vogel-type anticonflict test, hole-board test, Morris water maze test, schedule-induced
polydipsia model, stress-induced hyperthermia model, fear-potentiated startle model, maternal
separation test, swim-despair test, or microdialysis. Compounds identified by this inventive
method are also contemplated to be within the scope of the invention, as well as pharmaceutical
compositions that comprise compounds identified by the inventive methods admixed with a
pharmaceutically acceptable carrier.
[0017] The invention features methods for identifying compounds that reduce anxiety in a
subject by contacting a test compound with the AT4R and determining a decrease in the
biological activity of the AT4R in the presence of the test compound relative to the biological
activity of the AT4R in the absence of the test compound, and then administering the test
compound to a subject and determining a decrease in the level of anxiety in the subject relative
to the level of anxiety in the subject in the absence of the test compound. Compounds identified
by this inventive method are also contemplated to be within the scope of the invention, as well as
pharmaceutical compositions that comprise compounds identified by the inventive methods
admixed with a pharmaceutically acceptable carrier.
BRIEF DESCRIPTION OF THE DRAWINGS
[0018] Figure 1. Bar graph showing anxiolytic-like effect of AT4R blockade by AT4 in
the mouse 4-plate model of anxiety. Acute AT4 administration produces anxiolytic-like effects
in mice in a dose-dependent manner. Mice were administered systemic subcutaneous injections
of vehicle or AT4 at 1, 3, and 10 mg/kg body weight (X axis), and evaluated in the mouse 4-plate
model for anxiety, measuring number of punished crossings (Y axis). (* P<0.05 compared to
vehicle.)
[0019] Figure 2. Bar graph showing reversal of anxiolytic-like effects of AT4
administration by an oxytocin receptor antagonist. Mice were administered either vehicle,
3mg/kg of AT4, lOmg/kg of WAY-162720, an oxytocin receptor antagonist, or 3mg/kg AT4 and
lOmg/kg of WAY-162720 (X axis), and evaluated in the mouse 4-plate model for anxiety,
measuring number of punished crossings (Y axis). (*P<0.05 compared to vehicle.)
-5-

WO 2007/024946 PCT/US2006/032913
[0020] Figure 3. Bar graph showing reversal of anxiolytic-like effects of AT4
administration by an AT4 receptor antagonist. Mice were administered either vehicle,
3mg/kg of AT4, 5 nmol (icv) of divalinal, a AT4 receptor antagonist, or 3mg/kg AT4 and 5 nmol
(icv) of divalinal (X axis), and evaluated in the mouse 4-plate model for anxiety, measuring
number of punished crossings (Y axis). (*P<0.05 compared to vehicle).
DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS
[0021] As described herein, the inventors have demonstrated that inhibition of the AT4R
produces anxiolytic effects in a widely used rodent model of anxiety that is predictive of effects
in primates and humans. The anxiolytic effects observed by blocking AT4R activity parallel the
effects observed by administering the anti-anxiety drug diazepam. Moreover, the anxiolytic
effect has been shown by the inventors to be mediated through the neuropeptide oxytocin,
inasmuch as those effects are reversed if the animal is co-administered an oxytocin antagonist.
[0022] Previous in vitro studies demonstrated that inhibition of the AT4R inhibited cleavage of
both oxytocin and vasopressin. (Herbst (1997) and Matsumoto (2000)). Oxytocin is believed to
be anxiolytic, but vasopressin is anxiogenic. (Bhattacharya, SK et al. Biogenic Amines (1998)
14:367-86). Because the AT4R cleaves vasopressin more efficiently than it cleaves oxytocin
(Lew, RA et al. J. Neurochem. (2003) 86:344-50), it would have been expected prior to the
present invention that inhibition of the AT4R protease activity would result in elevated synaptic
levels of vasopressin, thereby inducing an anxiogenic effect, or at a minimum, offsetting any
potential anxiolytic effects that may result from an increase in the level of oxytocin. The effects
observed by the present inventors are contrary to these expectations.
[0023] The inventors' discovery that inhibition of the AT4R exerts an anxiolytic effect enables
the practice of several methods in accordance with the present invention. These include methods
of treating an individual for anxiety, as well as methods of identifying anxiolytic compounds that
act through the AT4R pathway, as described in greater detail below.
Definitions
[0024] Various terms relating to the methods and other aspects of the present invention are
used throughout the specification and claims. Such terms are to be given their ordinary meaning
in the art unless otherwise indicated. Other specifically defined terms are to be construed in a
manner consistent with the definition provided herein.
[0025] The term "treating" or "treatment" refers to any indicia of success in the attenuation or
amelioration of a pathology or condition, including any objective or subjective parameter such as
-6-

WO 2007/024946 PCT/US2006/032913
abatement, remission, or reduction of symptoms; increased tolerance by the subject to the
pathology or condition; and improved physical or mental well-being of a subject. The indicia of
success in the attenuation amelioration of a pathology or condition can be based on any objective
or subjective parameters; including the results of a physical examination, neurological
examination, and/or psychological or psychiatric evaluations.
[0026] The term "reduce anxiety" or "reducing anxiety" or "reduction of anxiety" refers to any
measurable decrease, attenuation, or amelioration, including the elimination, of the symptoms of
or the underlying psychological, molecular, biochemical, cellular, or physiological bases for
anxiety.
[0027] "Effective amount" refers to an amount of a compound, material, or composition, as
described herein effective to achieve a particular biological result. Such results may include, but
are not limited to, treating neuropsychiatric disorders such as anxiety in a subject.
[0028] "In vivo" means within a living organism.
[0029] "In vitro" means within an artificial environment.
[0030] "Anxiety" refers to an emotional state comprising psychological, molecular,
biochemical, cellular, and physiological responses to apprehension or fear of unreal or imagined
danger. Anxiety includes, but is not limited to a generalized anxiety disorder, panic anxiety,
obsessive compulsive disorder, social phobia, performance anxiety, post-traumatic stress
disorder, acute stress reaction, adjustment disorders, hypochondriacal disorders, separation
anxiety disorder, agoraphobia and specific phobias. Specific anxiety-related phobias which may
be treated with the methods of the present invention are those commonly experienced in clinical
practice including, but not limited to, fear of animals, insects, storms, driving, flying, heights or
crossing bridges, closed or narrow spaces, water, blood or injury, as well as extreme fear of
inoculations or other invasive medical or dental procedures.
[0031] "Anxiolytic" means any tendency to reduce anxiety.
[0032] "Anxiogenic" means any tendency to induce anxiety.
[0033] "Neuropeptide" means any molecule found in tissue from the peripheral or central
nervous system comprised of at least two amino acids.
[0034] "Synapse" refers to the site of functional apposition between neurons, at which an
impulse is transmitted from one neuron to another.
[0035] "Pharmaceutically acceptable" refers to those properties and/or substances which are
acceptable to the patient from a pharmacological/toxicological point of view and to the
manufacturing pharmaceutical chemist from a physical/chemical point of view regarding
composition, formulation, stability, patient acceptance and bioavailability. "Pharmaceutically
-7-

WO 2007/024946 PCT/US2006/032913
acceptable carrier" refers to a medium that does not interfere with the effectiveness of the
biological activity of the active ingredient(s) and is not toxic to the host to which it is
administered.
[0036] The term "AT4R antagonist" is used in the broadest sense, and includes any molecule
that partially or fully blocks, inhibits, reduces, or neutralizes a biological activity of the
angiotensin IV receptor.
[0037] "Biological activity" refers to any function or action of a molecule or ability to produce
an effect in vitro or in vivo. With respect to the AT4R, such activity includes the
protease/peptidase activity and all downstream effects thereof, including without limitation,
anxiolytic or anxiogenic effects, signaling, glucose transport, enhancement of memory, reversal
of amnesia, and the like.
[0038] As used herein, "test compound" refers to any purified molecule, substantially purified
molecule, molecules that are one or more components of a mixture of compounds, or a mixture
of a compound with any other material that can be analyzed using the methods of the present
invention. Test compounds can be organic or inorganic chemicals, or biomolecules, and all
fragments, analogs, homologs, conjugates, and derivatives thereof. Biomolecules include
proteins, polypeptides, nucleic acids, lipids, polysaccharides, and all fragments, analogs,
homologs, conjugates, and derivatives thereof. Test compounds can be of natural or synthetic
origin, and can be isolated or purified from their naturally occurring sources, or can be
synthesized de novo. Test compounds can be defined in terms of structure or composition, or
can be undefined. The compound can be an isolated product of unknown structure, a mixture of
several known products, or an undefined composition comprising one or more compounds.
Examples of undefined compositions include cell and tissue extracts, growth medium in which
prokaryotic, eukaryotic, and archaebacterial cells have been cultured, fermentation broths,
protein expression libraries, and the like.
[0039] As used herein, "measure" or "determine" refers to any qualitative or quantitative
determinations.
[0040] "Stable cell" or "stable cell line" refers to any cell in which any subunit of the AT4R or
combinations thereof, including the whole AT4R, can be expressed so that antagonists of the
AT4R can, be identified and tested, and the roles of the AT4R in neuropsychiatric disorders such
as anxiety can be examined.
[0041] "Antibodies" as used herein includes polyclonal and monoclonal antibodies, chimeric,
single chain, and humanized antibodies, as well as antibody fragments {e.g., Fab, Fab', F(ab')2
and Fv), including the products of a Fab or other immunoglobulin expression library. With
-8-

WO 2007/024946 PCT/IJS2006/032913
respect to antibodies, the term, "immunologically specific" or "specific" refers to antibodies that
bind to one or more epitopes of a protein of interest, but which do not substantially recognize and
bind other molecules in a sample containing a mixed population of antigenic biological
molecules. Screening assays to determine binding specificity of an antibody are well known and
routinely practiced in the art. For a comprehensive discussion of such assays, see Harlow et al.
(Eds.), ANTIBODIES A LABORATORY MANUAL; "Cold Spring Harbor Laboratory; Cold Spring
Harbor, NY (1988), Chapter 6.
Methods of Treatment
[0042] One aspect of the invention features methods for the treatment of neuropsychiatric
disorders in a subject in need of such treatment. In some embodiments the method involves
administering to the subject a composition comprising a pharmaceutically acceptable carrier and
an angiotensin IV receptor antagonist in an amount effective to diminish the biological activity
of the angiotensin IV receptor. In one preferred embodiment, the neuropsychiatric disorder is
anxiety.
[0043] The AT4R antagonist can modulate the activity of the AT4R by inhibiting the active
site of die AT4R, or by inducing a conformational change in the AT4R. The antagonist can be
any organic or inorganic chemical, or biomolecule, or any fragment, analog, homolog, conjugate,
or derivative thereof. Preferred examples of AT4R antagonists include, but are not limited to,
angiotensin IV (Val-Tyr-Ile-His-Pro-Phe) (SEQ ID NO:l), Divalinal-Angiotensin IV, Nle-
Angiotensin IV, Norleucinal Angiotensin IV, LVV-hemorphin-7 (Leu-Val-Val-Tyr-Pro-Trp-Thr-
Gln-Arg-Phe) (SEQ ID NO:2), peptide analogs of LW-hemorphin-7, including Leu-Val-Val-
Tyr-Pro-Trp-Thr-Gln-Arg (SEQ ID NO:3), Val-Val-Tyr-Pro-Trp-Thr-Gln (SEQ ID NO:4), Val-
Val-Tyr-Pro-Trp-Thr (SEQ ID NO:5), Val-Val-Tyr-Pro-Trp (SEQ ED NO:6), Val-Val-Tyr-Pro,
Val-Val-Tyr (SEQ ID NO:7), Val-Val-Tyr-Pro-Trp-Thr-Gln-Arg-Phe (SEQ ID NO:8), Val-Tyr-
Pro-Trp-Thr-Gln-Arg-Phe (SEQ ID NO:9), Tyr-Pro-Trp-Thr-Gln-Arg-Phe (SEQ ID NO:10),
Val-Tyr-Pro-Trp-Thr-Gln-Arg (SEQ ID NO:l 1), Val-Tyr-Pro-Trp-Thr-Gln (SEQ ID NO:12),
Val-Tyr-Pro-Trp-Thr (SEQ ID NO: 13), Val-Tyr-Pro-Trp (SEQ ID NO: 14), Val-Tyr-Pro, Leu-
Val-Val-Ala-Pro-Trp-Thr-Gln-Arg-Phe (SEQ ID NO: 15), Leu-Val-Val-Tyr-Ala-Trp-Thr-Gln-
Arg-Pbe (SEQ ID NOrl 6), Leu-Val-Val-Tyr-Pro-Ala-Thr-Gln-Arg-Phe (SEQ ID NO: 17), Leu
Val-Val-T>T-Pro-Trp-Ala-Gln-Arg-Phe (SEQ ID NO: 18), Leu-Val-Val-Tyr-Pro-Trp-Thr-Gln
(SEQ ID NO: 19), Leu-Val-Val-Tyr-Pro-Trp-Thr (SEQ ID NO:20), Leu-Val-Val-Tyr-Pro-Trp
(SEQ ID NO:21), Leu-Val-Val-Tyr-Pro (SEQ ID NO:22), or Leu-Val-Val-Tyr (SEQ ID NO:23).
(Lee, J et al. J. Pharmacol. Exp. Therapeutics (2003) 305:205-11; and, Lew, RA.. (2003)).
-9-

WO 2007/024946 PCT/IJS2006/032913
Antibodies to the AT4R can also be used as antagonists. Such antibodies may be monoclonal or
polyclonal, or may be in the form of an antisera.
[0044] The subject can be any animal, and preferably is a mammal such as a mouse, rat,
hamster, guinea pig, rabbit, cat, dog, monkey, cow, horse, pig, and the like. Most preferably, the
mammal is a human.
[0045] Preferred antagonists are those that provide a reduction in the peptidase activity of the
AT4R of at least about 5%, and more preferably at least about 10%, at least about 15%, at least
about 20%, at least about 25% at least about 30%, at least about 35%, at least about 40%, at least
about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at
least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%,
at least about 95%, or greater than 95% reduction in the peptidase activity of the AT4R, at a
specified concentration of the antagonist. In one preferred embodiment, the reduction of
peptidase activity of the AT4R modulates the concentration of anxiolytic or anxiogenic
neuropeptides in the synapse. In a detailed embodiment, the synaptic concentration of anxiolytic
neuropeptides are increased in the subject. In another detailed embodiment, the synaptic
concentration of anxiogenic neuropeptides are decreased in the subject. In a more preferred
embodiment, the synaptic concentration of anxiolytic neuropeptides are increased and the
synaptic concentration of anxiogenic neuropeptides are decreased in the subject. Non-limiting
examples of anxiolytic neuropeptides include oxytocin, galanin, and neuropeptide Y. Non-
limiting examples of anxiogenic neuropeptides include vasopressin, somatostatin, corticotrophin
releasing factor (CRF), and substance P.
[0046] The concentration of antagonist required to reduce the peptidase activity of the AT4R
may vary with the species, breed, size, height, weight, age, overall health of the subject, the type
of antagonist used, or the severity of the neuropsychiatric disorder. Determination of the proper
concentration of antagonist required for a particular situation is within the skill of the art. In the
inventive methods, the compositions comprise a concentration of antagonist in a range of about
0.001% to about 90%> of the dry weight of the composition, or from about 1 pM to about 1 M.
Dosage ranges may vary, e.g., from about 1 pg/kgbody weight to about 1 g/kg body weight of
the subject. A daily dose range of about 1 |J.g/kg to about 100 mg/kg of the weight of the subject
is used-in some embodiments, while a daily dosage range of at least about 0.01 mg/kg is used in
other embodiments. Treatment can be initiated with smaller dosages that are less than the
optimum dose of the antagonist, followed by an increase in dosage over the course of the
treatment until the optimum effect under the circumstances is reached. If needed, the total daily
dosage may be divided and administered in portions throughout the day.
-10-

WO 2007/024946 PCT/US2006/032913
[0047] The compositions can be prepared in a wide variety of dosage forms according to any
means suitable in the art for preparing a given dosage form. Pharmaceutically acceptable
carriers can be either solid or liquid. Non-limiting examples of solid form preparations include
powders, tablets, pills, capsules, lozenges, cachets, suppositories, dispersible granules, and the
like. A solid carrier can include one or more substances which may also act as diluents,
flavoring agents, buffering agents, binders, preservatives, tablet disintegrating agents, or an
encapsulating material. Suitable carriers are magnesium carbonate, magnesium stearate, talc,
sugar, lactose, pectin, dextrin, starch, gelatin, tragacanth, methylcellulose, sodium
carboxymethyl-cellulose, a low melting wax, cocoa butter, and the like. Non-limiting examples
of liquid form preparations include solutions, suspensions, and emulsions, for example, water,
alcohol, water propylene glycol solutions, and the like.
[0048] Administration of the compositions can be by infusion, injection (intravenously,
intramuscularly, intracutaneously, subcutaneously, intraduodenally, intraperitoneally, and the
like), intranasally, rectally, orally, or transdermally. Preferably, the compositions are
administered orally.
[0049] For effective treatment of anxiety, one skilled in the art may recommend a dosage
schedule and dosage amount adequate for the subject being treated. It may be preferred that
dosing occur one to four times daily for as long as needed. The dosing may occur less frequently
if the compositions are formulated in sustained delivery vehicles. The dosage schedule may also
vary depending on the active drug concentration, which may depend on the needs of the subject.
[0050] Another aspect of the invention features methods for the treatment of neuropsychiatric
disorders in a subject in need of such treatment by modulating the expression of the AT4R in the
subject. In one preferred embodiment, the neuropsychiatric disorder is anxiety. In some
embodiments, expression of the AT4R is modulated at the molecular level, for example, by
diminishing the expression of the AT4R protein.
[0051] Expression of the AT4R may be specifically suppressed at the molecular level by
utilizing antisense nucleic acids or RNA interference (RNAi). A review of RNAi is found in
Marx, J. (2000) Science, 288:1370-1372. In brief, traditional methods of gene suppression,
employing anti-sense RNA or DNA, operate by binding to the reverse sequence of a gene of
.- interest such that binding, interferes with subsequent cellular processes and blocks synthesis of
the corresponding protein. Exemplary methods for controlling or modifying gene expression are
provided in WO 99/49029, WO 99/53050 and WO 01/75164, the disclosures of which are hereby
incorporated by reference in their entirety for all purposes. In these methods, post-transcriptional
gene silencing is brought about by a sequence-specific RNA degradation process which results in
-11-

WO 2007/024946 PCT/US2006/032913
the rapid degradation of transcripts of sequence-related genes. Studies have shown that double-
stranded RNA may act as a mediator of sequence-specific gene silencing (see, for example,
Montgomery and Fire, Trends in Genetics, 14:255-258,1998). Gene constructs that produce
transcripts with self-complementary regions are particularly efficient at gene silencing.
[0052] It has been demonstrated that one or more ribonucleases specifically bind to and cleave
double-stranded RNA into short fragments. The ribonuclease(s) remains associated with these
fragments, which in turn specifically bind to complementary mRNA, i.e., specifically bind to the
transcribed mRNA strand for the gene of interest. The mRNA for the gene is also degraded by
the ribonuclease(s) into short fragments, thereby obviating translation and expression of the
gene. Additionally, an RNA polymerase may act to facilitate the synthesis of numerous copies
of the short fragments, which exponentially increases the efficiency of the system. Gene-
silencing may extend beyond the cell in which it is initiated such that the inhibition can result in
biochemical, molecular, physiological, or phenotypic changes in other cells and systems
throughout the organism.
[0053] Thus, available genetic information such as the nucleotide sequence, etc. of the AT4R
can be used to generate gene silencing constructs and/or gene-specific self-complementary,
double-stranded RNA sequences that can be delivered by conventional art-known methods. A
gene construct may be employed to express the self-complementary RNA sequences.
Alternatively, cells are contacted with gene-specific double-stranded RNA molecules, such that
the RNA molecules are internalized into the cell cytoplasm to exert a gene silencing effect. The
double-stranded RNA must have sufficient homology to the targeted gene to mediate RNAi
without affecting expression of non-target genes. The double-stranded DNA is at least 20
nucleotides in length, and is preferably 21-23 nucleotides in length. Preferably, the double-
stranded RNA corresponds specifically to a polynucleotide of the present invention. The use of
small interfering RNA (siRNA) molecules of 21-23 nucleotides in length to suppress gene
expression in mammalian cells is described in WO 01/75164. Tools for designing optimal
inhibitory siRNAs include that available from DNAengine Inc. (Seattle, Wash.). See WO
01/68836. See also: Bernstein et al, RNA (2001) 7: 1509-1521; Bernstein et al, Nature (2001)
409:363-366; Billy et al, Proc. Nat'lAcad. Sci USA (2001) 98:14428-33; Caplan et al, Proc.
Nat'lJeart Sci USA (2001) 98;9742-7; Carthew etal, Curr. Opin. Gell Bid {2W\) 13:244-«;
Elbashir et al., Nature (2001) 411: 494-498; Hammond et al., Science (2001) 293:1146-50;
Hammond et al, Nat. Ref. Genet. (2001) 2:110-119; Hammond et al, Nature (2000) 404:293-
296; McCaffrrey et al, Nature (2002): 418-38-39; and McCaffrey et al, Mol Ther. (2002)
5:676-684; Paddison et al, Genes Dev. (2002) 16:948-958; Paddison et al, Proc. Nat'lAcad. Sci
-12-

WO 2007/024946 PCT/US2006/032913
USA (2002) 99:1443-48; Sui et al, Proc. Nat'lAcad. Sci USA (2002) 99:5515-20. U.S. Patents
of interest include U.S. Pat. Nos. 5,985,847 and 5,922,687. Also of interest is WO/11092.
Additional references of interest include: Acsadi et al, New Biol. (January 1991) 3:71-81; Chang
et al, J. Virol. (2001) 75:3469-3473; Hickman eta!., Hum. Gen. Ther. (1994) 5:1477-1483; Liu
et al, Gene Ther. (1999) 6:1258-1266; Wolffs al, Science (1990) 247: 1465-1468; and Zhang
et at, Hum. Gene Ther. (1999) 10:1735-1737: and Zhang et al, Gene Ther. (1999)"7:1344-1349.
These disclosures are herein incorporated by reference in their entirety for all purposes.
[0054] In gene therapy applications, genes are introduced into cells in order to achieve in vivo
synthesis of a therapeutically effective genetic product, for example for replacement of a
defective gene. "Gene therapy" includes both conventional gene therapy where a lasting effect is
achieved by a single treatment, and the administration of gene therapeutic agents, which involves
the one time or repeated administration of a therapeutically effective DNA or mRNA. Antisense
RNAs and DNAs can be used as therapeutic agents for blocking the expression of certain genes
in vivo. It has already been shown that short antisense oligonucleotides can be imported into
cells where they act as inhibitors, despite their low intracellular concentrations caused by their
restricted uptake by the cell membrane. (Zamecnik et al, Proc. Natl. Acad. Sci. USA, 83:4143-
4146 (1986)). The oligonucleotides can be modified to enhance their uptake, e.g., by
substituting their negatively charged phosphodiester groups by uncharged groups.
[0055] There are a variety of techniques available for introducing nucleic acids into viable
cells. The techniques vary depending upon whether the nucleic acid is transferred into cultured
cells in vitro, ex vivo, or in vivo in the cells of the intended host. Techniques suitable for the
transfer of nucleic acid into cells in vitro include the use of liposomes, electroporation,
microinjection, cell fusion, DEAE-dextran, the calcium phosphate precipitation method, etc. The
currently preferred in vivo gene transfer techniques include transfection with viral vectors and
viral coat protein-liposome mediated transfection (Dzau et al, 1993, Trends in Biotechnology,
11:205-210). Viral vector mediated techniques may employ a variety of viruses in the
construction of the construct for delivering the gene of interest. The type of viral vector used is
dependent on a number of factors including immunogenicity and tissue tropism. Some non-
limiting examples of viral vectors useful in gene therapy include retroviral vectors (see e.g., U.S.
Patents 6,3.1 ?.,682,6,235,522,.5,672,510 and 5,952,225), adenoviral.(Ad)vectors (see e.g., U.S,•---
Patents 6,482,616, 5,846,945 ) and adeno-associated virus (AAV) vectors (see, e.g., U.S. Patents
6,566,119, 6,392,858, 6,468,524 and WO 99/61601). In some situations it is desirable to
provide the nucleic acid source with an agent that targets the target cells, such as an antibody
specific for a cell surface membrane protein or the target cell, a ligand for a receptor on the target
-13-

WO 2007/024946 PCT/US2006/032913
cell, and the like. Where liposomes are employed, proteins which bind to a cell surface
membrane protein associated with endocytosis can be used for targeting and/or to facilitate
uptake, e.g., capsid proteins or fragments thereof tropic for a particular cell type, antibodies for
proteins which undergo internalization in cycling, and proteins that target intracellular
localization and enhance intracellular half-life. The technique of receptor-mediated endocytosis
is described, for example, by Wu et al., J. Biol. Chem., 262:4429-4432 (1987); and Wagner et
al, Proc. Natl. Acad. Sci. USA, 87:3410-3414 (1990). For review of the currently known gene
marking and gene therapy protocols see Anderson et al., Science, 256:808-813 (1992).
[0056] Another aspect of the invention features methods for the treatment of neuropsychiatric
disorders in a subject in need of such treatment by modulating the localization of the AT4R to
the cell surface. In one preferred embodiment, the neuropsychiatric disorder is anxiety. In some
embodiments, localization of the AT4R to the cell surface is modulated by targeting expressed
AT4R for protease degradation. For example, ubiquitination of the AT4R can be utilized to
target expressed AT4R to proteasomes. In some embodiments, localization of the AT4R to the
cell surface is modulated by removing cell surface translocation signal peptides. Such signal
peptides can be removed pre-transcriptionally or post-translationally.
[0057] Another aspect of the invention features methods for the treatment of neuropsychiatric
disorders in a subject in need of such treatment by blocking the active site of the AT4R. By
"blocking the active site" of the AT4R, it is meant that a chemical or biomolecule is utilized to
obstruct the active site of the AT4R such that substrates of the AT4R cannot access the active
site of the AT4R and thus are not cleaved by the AT4R. In one preferred embodiment, the
neuropsychiatric disorder is anxiety. In some embodiments, the active site of the AT4R is
blocked by antibodies to AT4R.
Methods for Screening Compounds
[0058] Another aspect of the invention features methods for identifying antagonists of the
AT4R comprising contacting a test compound with the AT4R and determining a decrease in the
biological activity of the AT4R in the presence of the test compound relative to the biological
activity of the AT4R in the absence of the test compound.
[0059] For the screening assays, AT4R can be obtained from anysource suitable in the art.
The AT4R can be purified or bound to a cell membrane or membrane fragment. Purified AT4R,
or subunits thereof, can be synthesized de novo, or obtained from any mammalian cell that
naturally expresses the AT4R such as kidney, heart, adrenal, or brain tissue. Methods for
purifying membrane-bound proteins are well established in the art, and commercial kits are also
-14-

WO 2007/024946 PCT/US2006/032913
available such as the ProteoPrep Extraction Kit (Sigma, St. Louis, MO) and the Qprotome Cell
Compartment Kit (Qiagen, Valencia, CA). Purified AT4R can also be obtained from the
membranes of stable cells or cell lines that express the AT4R, such as transfected HEK 293T
cells. (Lew, RA (2003)). Purified AT4R can also be obtained from recombinant expression
systems, such as bacterial, yeast, insect cell systems, and the like. Screening assays can also be
carried out on AT4R still bound to the cell membrane. Techniques of recombinant cloning and
protein expression and purification are well established in the art.
[0060] . Membrane-bound AT4R, or subunits thereof, can be obtained from any cell expressing
the AT4R or subunits thereof. The cells can naturally express AT4R, such as mammalian kidney
cells, cardiac cells, adrenal gland cells, or brain cells. The cells can be stable cells or stable cell
lines induced to express AT4R such as transfected HEK 293T cells. (Lew, RA (2003)). Stable
cells can be produced by any means suitable in the art for cloning and recombinant gene
expression. Isolation of cell membranes or membrane fragments containing the AT4R can be
carried out according to any means suitable in the art, including the membrane extraction method
described by Mustafa et al. (Mustafa, T et al. J. Neurochem. (2001) 76:1679-87. In the
alternative, whole cells whose membranes contain the AT4R can be used.
[0061] In one embodiment, interaction of a test compound with the AT4R is determined by any
qualitative or quantitative technique known in the art. Determination of whether the test
compound interacts with the AT4R can be carried out using binding assays wherein the test
compound is labeled. The label can be any label suitable in the art such as radioisotopes,
including 3H, 1251,35S, 33P, 32P,,77Lu, 90Y, and the like; fluorophores, including FITC,
phycoerythrin, rhodamine, Cyl, Cy2, Cy3, Cy4, Cy5, allophycocyanin, AlexaFluor® dyes
(Invitrogen, Carlsbad, CA), fluorescent proteins, and the like; or enzyme labels, including
phosphatase, luciferase, urease, peroxidase, oxidase, P-galactosidase, and the like. The binding
assay can determine the equilibrium constant, dissociation constant, binding constant. Binding
determinations can be made by any means suitable in the art, including without limitation,
microscopy, equilibrium dialysis, ultrafiltration, spectroscopic analysis, chromatography, and
calorimetry such as isothermal titration calorimetry. Competition assays may also be employed
to determine the interaction with the test compound and the AT4R, such as those described by
Mustafa et al (Mustafa, T. (2001)), Lee et al (Lee, J (2003)), and Lew et al (Lew, RA (2003)).
[0062] The effect of the test compound on the biological activity of the AT4R can be
determined by any means suitable in the art. The test compound can be assessed at multiple
concentrations. A decrease in the biological activity of the AT4R can be measured in terms of a
decrease in fluorescence resulting from cleavage of Leu-p-NA, a substrate of the AT4R, relative
-15-

WO 2007/024946 PCT/US2006/032913
to the level of fluorescence observed in the absence of a test compound, or upon contacting the
AT4R with a negative control compound. (Lew, RA (2003)). Alternatively, a decrease in the
biological activity of the AT4R can be measured in terms of a decrease in cleavage of any other
substrate of the AT4R. Such measurements can be carried out by any means suitable in the art,
such as chromatography/HPLC, polyacrylamide gel electrophoresis, or mass spectroscopy.
(Zhu,L,etaI.J.Bioi: Chem. (2003)278:22418-23). Modulation of the biological activity of the
AT4R can also be determined by measuring modulation of the concentration of neuropeptides
that are known AT4R substrates. The modulation concentration of such neuropeptides can be
measured in the synapse.
[0063] Another aspect of the invention features methods for identifying compounds that reduce
anxiety in a subject by administering a test compound to the subject and determining a decrease
in the level of anxiety in the subject relative to the level of anxiety in the subject in the absence
of the test compound.
[0064] Baseline levels of anxiety and any reduction in anxiety resulting from the
administration of the test compound to the subject can be measured using any means acceptable
in the art. Such means may be with or without punishment to the subject. Non-limiting
examples of assays used in the art for measuring anxiety include the Four-Plate Model, Elevated
Zero Maze, Elevated Plus Maze, Light-Dark Transition Test, Geller-Type Anticonfiict Test;
Vogel-Type Anticonfiict Test, Hole-Board Test, Morris Water Maze Test, Schedule-Induced
Polydipsia Model, Stress-Induced Hyperthermia Model, Fear-Potentiated Startle Model,
Maternal Separation Test, Swim-Despair Test, Microdialysis, and the like.
[0065] An additional aspect of the invention features methods for identifying compounds that
reduce anxiety in a subject by a combination of an in vitro and in vivo screening assay. In one
embodiment, a test compound is first screened in vitro to determine its physiologic, cellular,
biochemical, or molecular effect, and then screened further in vivo to determine if the compound
can reduce anxiety. In another embodiment, a test compound is first screened in vivo to
determine if the compound can reduce anxiety, and then screened further in vifro to determine its
physiologic, cellular, biochemical, or molecular effect.
[0066] In a detailed embodiment, the in vitro screening assay comprises identifying antagonists
of the AT4R comprising contacting a test compound with the AT4R and determining a decrease ------
in the biological activity of the AT4R in the presence of the test compound relative to the
biological activity of the AT4R in the absence of the test compound. This embodiment can be
practiced according to the details described herein. In a further detailed embodiment, the in vivo
screening assay comprises identifying compounds that reduce anxiety in a subject comprising
-16-

WO 2007/024946 PCT/US2006/032913
administering a test compound to the subject and determining a decrease in the level of anxiety
in the subject relative to the level of anxiety in the subject in the absence of the test compound.
This embodiment can be practiced according to the details described herein.
[0067] Compounds identified by any of the foregoing inventive screening methods are
contemplated to be within the scope of this invention. Such compounds are preferably
anxiolytic. Such compounds may be formulated as a pharmaceutical composition by admixing
such compound in an amount effective to reduce anxiety in the subject to which it is
administered and a pharmaceutically acceptable carrier, as described herein. Such
pharmaceutical compositions can be administered to a subject according to the methods of the
invention in order to treat anxiety in the subject.
[0068] The following examples are provided to illustrate the invention in greater detail. The
examples are intended to illustrate, not to limit, the invention.
EXAMPLE 1
Effect of AT4 Receptor Blockade on Anxiety Behavior in Mouse 4-PIate Model
[0069] The effects of AT4 receptor blockade by AT4 were investigated in the mouse 4-plate
model of anxiety.
[0070] Male Swiss Webster mice weighing 18-24 g were used inihe 4 plate studies. Animals
were housed in groups of 15 in an AAALAC-accredited facility (Wyeth Research, Princeton, NJ)
with food and water available ad libitum. Animals were maintained on a 12-hour light/dark
cycle (lights on at 0600) with all studies performed during the light phase. On the day of
experiments, mice were injected with AT4 (0, 1, 3 and 10 mg/kg) 30 minutes before the start of
the study. Initially, mice were individually placed in a plexiglass cage (18 x 25 x 16 cm) with a
floor consisting of four rectangular metal plates (8x11 cm), which are wired to a shock
generator (Med Associates). In each experiment, mice were placed into the chamber and given
an 18-sec habituation period, which was followed by a 1-min test session. After the habituation
period, an electric shock (0.8 mA) was delivered for 3.0 sec when mice crossed from one plate to
another. The crossing from one plate to the next is referred to as a "punished crossing." The
number of punished crossings during a 1-min test period was recorded by a computer. The mean
number ofpimished crossings for each group was expressed as a percentage of the value -
observed in the control animals. Data were subjected to an overall one-way analysis of variance
(ANOVA) and post-hoc comparisons were made by a contrast using least squares. Significant
differences in treatment occurred when p<0.05 compared to vehicle.
-17-

WO 2007/024946 PCT/US2006/032913
[0071] Results are shown in Figure 1. As can be seen, acute treatment with 3 and 10 mg/kg of
AT4 significantly (p<0.05) increased the number of punished crossing compared to animals
treated with vehicle alone. The results are similar to those observed with known anti-anxiety
drugs such as Valium (diazepam) in this model.
EXAMPLE 2
Reversal of Anxiolvtic-Like Effects of AT4 by an Antagonist of Oxytocin
[0072] To determine whether the anxiolytic-like effects of AT4 Receptor Blockade were
mediated, at least in part, by oxytocin, the procedures set forth in Example 1 were repeated in the
presence of a known oxytocin receptor antagonist, WAY-162720.
[0073] For these studies, the same 4-plate procedures were used as described in Example 1,
above. The only difference was that animals were injected with 10 mg/kg of the oxytocin
receptor antagonist, WAY-162720. This injection was given at the same time as AT4 (3 mg/kg)
which was administered 30 minutes before mice were placed in the 4-plate cage. After the 18
sec habituation period, an electric shock (0.8 mA) was delivered for 3.0 sec when mice crossed
from one plate to another. A 3-sec time followed the delivery of each shock and a computer
recorded the number of punished crossings during a 1-min test period. The mean number of
punished crossings for each group was expressed as a percentage of the value observed in the '
control animals. Data were subjected to an overall one-way analysis of variance (ANOVA) and
post-hoc comparisons were made by a contrast using least squares. Significant differences in
treatment occurred when p<0.05 compared to vehicle.
[0074] Results are shown in Figure 2. As can be seen, acute treatment with WAY-162720
produced no effect on behavior when tested alone, and acute treatment with AT4 increased the
number of punished crossings compared to animals administered the vehicle control. Acute
treatment with WAY-162720 and AT4 showed that this oxytocin receptor antagonist completely
blocked the anxiolytic effects of AT4 in the 4-plate model.
EXAMPLE 3
Effect of AT4 Receptor Blockade on Oxytocin Levels in Rat Amygdala
[0075] hi vitr.o,.hTA inhibits, the peptidase activity of the AT4 receptor, leading to increases in
levels of several peptides including oxytocin. To confirm this observation in vivo, microdialysis
coupled to immunoassay techniques were used to monitor basal and AT4-induced changes in
extracellular levels of oxytocin in the rat amygdala.
-18-

WO 2007/024946 PCT/US2006/032913
[0076] For microdialysis protocols, male Sprague-Dawley rats, weighing between 280 and 350
g, were group housed in an AAALC-accredited facility and maintained on a 12 hr light/dark
cycle. All procedures were conducted during the light period (lights on at 0600 h). Using 2-3%
halothane (Fluothane; Zeneca, Cheshire, UK) anesthesia, animals were secured in a stereotaxic
frame with ear and incisor bars (David Kopf, Tujunga, CA). A microdialysis guide cannula
(CMA/12; CMA Microdialysis, Stockholm, Sweden) was directed toward the rat amygdala using
the following coordinates: A/P - 2.7mm M/L - 4.6mm and D/V - 7.2mm (Paxinos, G and
Watson, C. The Rat Brain in Stereotaxic coordinates, 1986, Academic Press). Guide cannula
was fixed to the skull with two stainless-steel screws (Small Parts, Roanoke, VA) and dental
acrylic (Plastics One, Roanoke, VA). Following surgery, animals were individually housed in
Plexiglass cages (45 cm sq.) for approximately 24 hours and had access to food and water ad
libitum. Following a 24 hr post-operative recovery, a pre-washed microdialysis probe (CMA/12;
OD 0.5 mm, membranes length 2mm, 20 kD cut-off) was perfused with artificial CSF (aCSF;
125mM NaCl, 3mM KC1, 0.75mM MgS04 and 1.2mM CaCl2, pH 7.4) at flow rate of 0.2ml/min
for at least 18 hours prior to experimentation. On the day of procedures, microdialysis probes
were inserted, via the guide cannula, into the amygdala and perfused with aCSF at a flow rate of
l(il/min. A 3-hour stabilization period was allowed following probe insertion before any
neurochemical were measured. Thirty-minute samples were collected for 2 hours to establish a
steady baseline. These samples were immediately placed on dry ice. Next, AT4 was infused
directly thru the probe into the amydala for 60 minutes. Once the injection was complete,
samples were collected for 3 hours post-infusion to evaluate a timecourse of AT4 effects.
Following collection, all samples were stored on dry ice. Oxytocin levels from dialysis samples
were quantified by an oxytocin immunoassay (cat no. DE1900; R&D Systems, Inc) according to
conditions specified by the manufacturer.
[0077] Intra-amygdala infusion (60 min) of 1 and 10 uM Nle-AT4 resulted in a concentration-
dependent increase in amygdala levels of oxytocin (83% and 128% above baseline, respectively).
Additionally, a systemic injection ofNle-AT4 (0.5 mg/kg, s.c.) produced marked elevations in
amygdala levels of oxytocin (5-fold) compared to vehicle-treated animals, suggesting that this
peptide readily enters the central nervous system.
-19-

WO 2007/024946 PCT/US2006/032913
EXAMPLE 4
AT4 receptor antagonist, divalinal, blocks the anxiolytic properties of angiotensin IV
[0078] To determine whether the AT4 receptor mediates the anxiolytic-like properties of AT4, the
procedures set forth in Example 2 were repeated in the presence of the known AT4 receptor
antagonist, divalinal.
[0079] For these studies, the same 4-plate procedures were used as described in Example 1,
above. The only difference was that animals were injected intracerebroventricularly (icv) with 5
nmol of the AT4 receptor antagonist, divalinal. AT4 (3 mg/kg) and divalinal were administered
30 and 20 min, respectively, prior to being placed in the 4-plate cage to habituate. After the 18
sec habituation period, an electric shock (0.8 mA) was delivered for 3.0 sec when mice crossed
from one plate to another. A computer recorded the number of punished crossings during a 1-
min test period. The mean number of punished crossings for each group was expressed as a
percentage of the value observed in the control animals. Data were subjected to an overall one-
way analysis of variance (ANOVA) mdpost-hoc comparisons were made by a contrast using
least squares. Significant differences in treatment occurred when p<0.05 compared to vehicle.
[0080] The results are shown in Figure 3. Acute treatment with 5 nmol (icv) of the AT4
receptor antagonist, divalinal, had no effect on behavior when tested alone. However, divalinal
completely blocked the anxiolytic-like effects of angiotensin IV in the 4-plate model. These data
show that the AT4 receptor, in part, mediates the anxiolytic-like properties of Ang IV.
[0081] The present invention is not limited to the embodiments described and exemplified
above, but is capable of variation and modification within the scope of the appended claims.
-20-

WO 2007/024946
What is Claimed:

PCT/US2006/032913

1. A method for treating a neuropsychiatric disorder in a subj ect in need of such treatment
comprising administering to the subject a composition comprising a pharmaceutically acceptable
carrier and an angiotensin IV receptor (AT4R) antagonist in an amount effective to diminish the
biological activity of the AT4R.
2. The method of claim 1, wherein the AT4R antagonist induces a conformational change in
the AT4R.
3. The method of claim 1, wherein the AT4R antagonist inhibits the active site of the AT4R.
4. The method of any one of claims 1 to 3, wherein the AT4R antagonist is angiotensin IV,
Divalinal-Angiotensin IV, LW-hemorphin 7, Nle-Ang IV, or Norleucinal Ang IV.
5. A method for treating a neuropsychiatric disorder in a subject in need of such treatment
comprising modulating the expression of an AT4R in the subject.
6. The method of claim 5 wherein expression of the AT4R is diminished by utilizing an
oligonucleotide molecule that is antisense to a nucleic acid encoding the AT4R.
7. The method of claim 6, wherein the molecule that is antisense to a nucleic acid encoding
the AT4R is a siRNA.
8. The method of any one of claims 1 to 7, wherein the neuropsychiatric disorder is anxiety.
9. A method for treating anxiety in a subject in need of such treatment comprising
modulating the localization of the AT4R to the cell membrane.
10. A method for treating anxiety in a subj ect in need of such treatment comprising
modulating the concentration of anxiolytic or anxiogenic neuropeptides in the subject.
11. The method of claim 10, wherein the concentration of an anxiolytic neuropeptide is
increased.
-21-

WO 2007/024946 PCT/US2006/032913
12. The method of claim 11, wherein the anxiolytic neuropeptide is oxytocin.
13. The method of claim 10, wherein the concentration of an anxiogenic neurop eptide is
decreased.
14. The method of claim 13, wherein the anxiogenic neuropeptide is vasopressin.
15. The method of any one of claims 10 to 14, wherein the concentration of anxiolytic or
anxiogenic neuropeptides is in synapses.
16. A method for identifying compounds that reduce anxiety in a subject comprising
administering a test compound to the subject and determining a decrease in the level of anxiety
in the subject relative to the level of anxiety in the subject in the absence of the test compound.
17. A method for identifying compounds that reduce anxiety in a subject comprising
contacting a test compound with the AT4R and determining a decrease in the biological activity
of the AT4R in the presence of the test compound relative to the biological activity of the AT4R
in the absence of the test compound, and, administering the test compound to the subject and
determining a decrease in the level of anxiety in the subject relative to the level of anxiety in the
subject in the absence of the test compound.
18. The method of any one of claims 1 to 17, wherein the subject is a mammal.
19. The method of claim 18, wherein the mammal is a human.
20. A method for identifying antagonists of the AT4R comprising contacting a test
compound with the AT4R and determining a decrease in the biological activity of the AT4R in
the presence of the test compound relative to the biological activity of the AT4R in the absence
of the test compound.
21. The method of claims 17 or 20, wherein the ATR4 is bound to a cell membrane or cell
membrane fragment.
22. The method of claim 21, wherein the cell membrane or cell membrane fragment is from a
mammalian cell.
-22-

WO 2007/024946 PCT/US2006/032913
23. The method of claim 22, wherein the mammalian cell is a kidney cell, cardiac cell,
adrenal gland cell, or brain cell.
24. The method of claim 22, wherein the mammalian cell is a stable cell or stable cell line
expressing the AT4R.
25. A compound identified by the method of any one of claims 16,17 or 20 to 24.
26. A pharmaceutical composition comprising the compound of claim 25 and a
pharmaceutically acceptable carrier.
-23-

Methods for the treatment
of neuropsychiatric disorders such
as anxiety are disclosed. The methods
involve modulating the expression of the
angiotensin IV receptor or modulating
the biological activity of the angiotensin
IV receptor by utilizing antagonists to the
receptor. Also disclosed are methods for
identifying antagonists of the angiotensin
IV receptor that are effective to reduce
anxiety in a subject.