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  Pharmaceutical Patents  

 

Title:  Use of ADNF III polypeptides for treating mental diseases and disorders, including schizophrenia
United States Patent: 
7,960,334
Issued: 
June 14, 2011

Inventors:
 Gozes; Illana (Ramat Hasharon, IL), Alcalay; Roy N. (Tel Aviv, IL), Divinski; Inna (Petach Tikva, IL), Giladi; Eliezer (Netanya, IL)
Assignee:
  Ramot at Tel-Aviv University Ltd. (Tel-Aviv, IL)
Appl. No.:
 10/547,986
Filed:
 March 11, 2004
PCT Filed:
 March 11, 2004
PCT No.:
 PCT/IL2004/000232
371(c)(1),(2),(4) Date:
 April 10, 2006
PCT Pub. No.:
 WO2004/080957
PCT Pub. Date: 
September 23, 2004


 

Covidien Pharmaceuticals Outsourcing


Abstract

This invention relates to the use of ADNF III polypeptides in the treatment of mental diseases or disorders, including schizophrenia. Embodiments of the invention provide methods for treating mental disorders, including schizophrenia, in a subject by administering a NAP, an 8-amino-acid peptide derived from Activity Dependent Neurotrophic Factor (ADNF III), in an amount sufficient to reduce or eliminate symptoms. The ADNF III polypeptides include polypeptides, analogs, subsequences, and D-amino acid versions (either wholly D-amino acid peptides or mixed D- and L-amino acid peptides), and combinations thereof which contain the active core sites and provide neuroprotective and anti-schizophrenic functions.

Description of the Invention

SUMMARY OF THE INVENTION

This invention discloses the surprising finding that NAP, and consequently, NAP related peptides, e.g., ADNF polypeptides, can provide novel therapeutic treatments for serious diseases and disorders, particularly anxiety disorders and mood disorders such as depression. This invention further discloses for the first time the molecular target for NAP, tubulin, a novel target platform for drug discovery, neuroprotection, anxiety and depression.

In one aspect, the present invention provides a method of treating or preventing anxiety or depression in a subject, the method comprising the step of administering a therapeutically effective amount of an ADNF polypeptide to a subject in need thereof.

In one embodiment, the ADNF polypeptide is a member selected from the group consisting of: (a) an ADNF I polypeptide comprising an active core site having the following amino acid sequence: Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:1); (b) an ADNF III polypeptide comprising an active core site having the following amino acid sequence (NAP): Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (SEQ ID NO:2); and (c) a mixture of the ADNF I polypeptide of part (a) and the ADNF III polypeptide of part (b).

In one embodiment, the ADNF polypeptide is a member selected from the group consisting of a full length ADNF I polypeptide, a full length ADNF III polypeptide (ADNP), and a mixture of a full length ADNF I polypeptide and a full length ADNF III polypeptide.

In one embodiment, the ADNF polypeptide is an ADNF I polypeptide. IN another embodiment, the active core site of the ADNF I polypeptide comprises at least one D-amino acid. In another embodiment, the active core site of the ADNF I polypeptide comprises all D-amino acids. In another embodiment, the ADNF I polypeptide is Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:1). In another embodiment, the ADNF I polypeptide comprises up to about 20 amino acids at at least one of the N-terminus and the C-terminus of the active core site. In another embodiment the ADNF I polypeptide is selected from the group consisting of -- see Original Patent.

In one embodiment, the ADNF polypeptide is an ADNP III polypeptide. In another embodiment, the ADNF polypeptide is a full length ADNF III polypeptide. In another embodiment, the ADNP III polypeptide is Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (SEQ ID NO:2). In another embodiment, the active core site of the ADNF III polypeptide comprises at least one D-amino acid. In another embodiment, the active core site of the ADNF III polypeptide comprises all D-amino acids. In another embodiment, the ADNF III polypeptide comprises up to about 20 amino acids at least one of the N-terminus and the C-terminus of the active core site. In another embodiment, the ADNF III polypeptide is a member selected from the group consisting of -- see Original Patent.

In one embodiment, at least one of the ADNF polypeptides is encoded by a nucleic acid that is administered to the subject.

In one embodiment, an ADNF I polypeptide and an ADNF III polypeptide are administered to the subject.

In one embodiment, the ADNF I or ADNF III polypeptide contains a covalently bound lipophilic moiety to enhance penetration or activity.

In one embodiment, the subject suffers from anxiety or depression. In another embodiment, the ADNF polypeptide is administered to prevent-anxiety or depression. In another embodiment, the disease is selected from the group consisting of: panic disorder, obsessive-compulsive disorder, post-traumatic stress disorder, social phobia, social anxiety disorder, specific phobias, generalized anxiety disorder, Major depression, dysthymia, and bipolar disorder.

In one embodiment, the ADNF polypeptide is administered intranasally. In another embodiment, the ADNF polypeptide is administered orally. In another embodiment, the ADNF polypeptide is administered intravenously or subcutaneously.

In one aspect, the present invention provides use of an ADNF polypeptide in the manufacture of a medicament for the treatment of depression or anxiety.

In one aspect, the present invention provides the use of the NAP-tubulin binding site(s) to identify anxiolytic drugs and drugs that alleviate depression and provide neuroprotection.

DETAILED DESCRIPTION OF THE INVENTION

This invention relates to the therapeutic use of NAP and ADNF polypeptides in the treatment of diseases and disorders including anxiety and depression, and disorders related thereto. The invention is based on the finding set out in Example 1 that treatment of mice with NAP peptide significantly reduces anxiety-like behavior in a widely used and accepted industry standard model of anxiety, the Elevated plus-maze (see Rodgers & Dalvi, Neurosci. Biobehav. Rev. 21(6) 801-801(1997)). The invention further discloses that while providing anxiolytic effects, NAP does not inhibit cognitive functions. In another embodiment, this invention further discloses NAP's mechanism of action provides drug assays for compounds that also can be used to treat anxiety and depression. In such assays, compounds that modulate the interaction between NAP and tubulin are identified.

ADNF Polypeptides

In one embodiment the ADNF polypeptides of the present invention comprise the following amino acid sequence: (R.sup.1).sub.x-Asn-Ala-Pro-Ser-Ile-Pro-Gln-(R.sup.2).sub.y(SEQ ID NO: 13) and conservatively modified variations thereof. In this designations, R.sup.1 denotes the orientation of the amino terminal (NH.sub.2 or N-terminal) end and R.sup.2 represents the orientation of the carboxyl terminal (COOH or C-terminal) end.

In the above formula, R.sup.1 is an amino acid sequence comprising from 1 to about 40 amino acids, wherein each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid analogs. The term "independently selected" is used herein to indicate that the amino acids making up the amino acid sequence R.sup.1 may be identical or different (e.g., all of the amino acids making up the amino sequence may be threonine, etc.). Moreover, as previously explained, the amino acids making up the amino acid sequence R.sup.1 may be either naturally occurring amino acids, or known analogues of natural amino acids that functions in a manner similar to the naturally occurring amino acids (i.e., amino acid mimetics and analogs). Suitable amino acids that can be used to form the amino acids sequence R.sup.1 include, but are not limited to, those listed in Table I (see Original Patent), infra. The indexes "x" and "y" are independently selected and can be equal to one or zero.

As with R.sup.1, R.sup.2, in the above formula, is an amino acid sequence comprising from 1 to 40 amino acids, wherein each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid analogs. Moreover, as with R.sup.1, the amino acids making up the amino acid sequence R.sup.2 may be identical or different, and may be either naturally occurring amino acids, or known analogues of natural amino acids that function in a manner similar to the naturally occurring amino acids (i.e., amino acid mimetics and analogs). Suitable amino acids that can be used to form R.sup.2 include, but are not limited to, those listed in Table I, infra.

As used herein, "NAP" or "NAP peptide" refers to the formula above where x and y both equal 0. "NAP related peptide" refers to any of the other variants of NAP which are described the formula.

R.sup.1 and R.sup.2 are independently selected. If R.sup.1R.sup.2 are the same, they are identical in terms of both chain length and amino acid composition. For example, both R.sup.1 and R.sup.2 may be Val-Leu-Gly-Gly-Gly (SEQ ID NO:14). If R.sup.1 and R.sup.2 are different, they can differ from one another in terms of chain length and/or amino acid composition and/or order of amino acids in the amino acids sequences. For example, R.sup.1 may be Val-Leu-Gly-Gly-Gly (SEQ ID NO:14), whereas R.sup.2 may be Val-Leu-Gly-Gly (SEQ ID NO:15). Alternatively, R.sup.1 may be Val-Leu-Gly-Gly-Gly (SEQ ID NO:14), whereas R.sup.2 may be Val-Leu-Gly-Gly-Val (SEQ ID NO:16). Alternatively, R.sup.1 may be Val-Leu-Gly-Gly-Gly (SEQ ID NO:14), whereas R.sup.2 may be Gly-Val-Leu-Gly-Gly (SEQ ID NO:17).

Within the scope of the above formula, certain NAP and NAP related polypeptides are preferred, namely those in which x and y are both zero (i.e. NAP). Equally preferred are NAP and NAP related polypeptides in which x is one; R.sup.1 Gly-Gly; and y is zero (SEQ ID NO:9). Also equally preferred are NAP and NAP related polypeptides in which x is one; R.sup.1 is Leu-Gly-Gly; y is one; and R.sup.2 is -Gln-Ser (SEQ ID NO:10). Also equally preferred are NAP and NAP related polypeptides in which x is one; R.sup.1 is Leu-Gly-Leu-Gly-Gly- (SEQ ID NO:18); y is one; and R.sup.2 is -Gln-Ser (SEQ ID NO:11). Also equally preferred are NAP and NAP related polypeptides in which x is one; R.sup.1 is Ser-Val-Arg-Leu-Gly-Leu-Gly-Gly- (SEQ ID NO:19); y is one; and R.sup.2 is -Gln-Ser (SEQ ID NO:12). Additional amino acids can be added to both the N-terminus and the C-terminus of the active peptide without loss of biological activity.

In another aspect, the present invention provides pharmaceutical compositions comprising one of the previously described NAP and NAP related polypeptides in an amount sufficient to exhibit anxiolytic (e.g. anxiety reducing) or anti-depressant activity, in a pharmaceutically acceptable diluent, carrier or excipient. In one embodiment, the NAP or NAP related peptide has an amino acid sequence selected from the group consisting of SEQ ID NO:2, and 9-12, and conservatively modified variations thereof.

In another embodiment, the ADNF polypeptide comprises the following amino acid sequence: (R.sup.1).sub.x-Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala-(R.sup.2).sub.y (SEQ ID NO:20) and conservatively modified variations thereof. In this designation, R.sup.1 denotes the orientation of the amino terminal (NH.sub.2 or N-terminal) end and R.sup.2 represents the orientation of the carboxyl terminal (COOH or C-terminal) end.

In the above formula, R.sup.1 is an amino acid sequence comprising from 1 to about 40 amino acids, wherein each amino acid is independently selected from the group consisting of naturally occurring amino acids and amino acid analogs. The term "independently selected" is used herein to indicate that the amino acids making up the amino acid sequence R.sup.1 may be identical or different (e.g., all of the amino acids in the amino acid sequence may be threonine, etc). Moreover, as previously explained, the amino acid making up the amino acid sequences R.sup.1 may be either naturally occurring amino acids, or known analogues of natural amino acids that functions in a manner similar to naturally occurring amino acids (i.e., amino acid mimetics and analogs). Suitable amino acids that can be used to form the amino acid sequence R.sup.1 include, but are not limited to, those listed in Table I, infra. The indexes "x" and "y" are independently selected and can be equal to one or zero.

As with R.sup.1, R.sup.2, in the above formula, is an amino acid sequence comprising from 1 to about 40 amino acids, wherein each amino acid is independently selected form the groups consisting of naturally occurring amino acids and amino acid analogs. Moreover, as with R.sup.1, the amino acids making up the amino acid sequence R.sup.2 may be identical or different, and may be either naturally occurring amino acids, or known analogues of natural amino acids that functions in a manner similar to the naturally occurring amino acids (i.e., amino acid mimetics and analogs). Suitable amino acids that can be used to form R.sup.2 include, but are not limited to those listed in Table I, infra.

As used herein, "SAL" or "SAL peptide" refers to the formula above where x and y both equal 0. "SAL related peptide" refers to any of the other variants of SAL which are described the formula.

R.sup.1 and R.sup.2 are independently selected. If R.sup.1R.sup.2 are the same, they are identical in terms of both chain length and amino acid composition. Additionally amino acids can be added to both the N-terminus and the C-terminus of the active peptide without loss of biological activity.

In an another aspect, the present invention provides pharmaceutical compostions comprising on of the previously described SAL and SAL-related polypeptides in an amount sufficient to exhibit anxiolytic (e.g., anxiety reducing) or anti-depressant activity, in a pharmaceutically acceptable diluent, carrier or excipient. In one embodiment, the SAL or SAL related peptide has an amino acid sequence selected form the group consisting of SEQ ID NO:1 and 3-8, and conservatively modified variations thereof.

Design and Synthesis of ADNF Polypeptides

Polypeptides and peptides comprising the core NAPVSIPQ (SEQ ID NO:2) or SALLRSIPA (SEQ ID NO:1) active site can be easily made, e.g., by systematically adding one amino acid at a time and screening the resulting peptide for biological activity, as described herein. In addition, the contributions made by the side chains of various amino acid residues in such peptides can be probed via a systematic scan with a specified amino acid, e.g., Ala.

One of skill will recognize many ways of generating alterations in a given nucleic acid sequence. Such well-known methods include site-directed mutagenesis, PCR amplification using degenerate oligonucleotides, exposure of cells containing the nucleic acid to mutagenic agents or radiation, chemical synthesis of a desired oligonucleotide (e.g., in conjunction with ligation and/or cloning to generate large nucleic acids) and other well-known techniques (see Giliman & Smith, Gene 8:81-97 (1979); Roberts et al., Nature 328:731-734 (1987)).

Most commonly, polypeptide sequences are altered by changing the corresponding nucleic acid sequence and expressing the polypeptide. However, polypeptide sequences are also optionally generated synthetically using commercially available peptide synthesizers to produce any desired polypeptide (see Merrifield, Am. Chem. Soc. 85:2149-2154 (1963); Stewart & Young, Solid Phase Peptide Synthesis (2nd ed. 1984)).

One of skill can select a desired nucleic acid or polypeptide of the invention based upon the sequences provided and upon knowledge in the art regarding proteins generally. Knowledge regarding the nature of proteins and nucleic acids allows one of skill to select appropriate sequences with activity similar or equivalent to the nucleic acids and polypeptides disclosed herein. The definitions section, supra, describes exemplar conservative amino acid substitutions.

Modifications to the NAP and ADNF polypeptides are evaluated by routine screening techniques in suitable assays for the desired characteristic. For instance, changes in the immunological character of a polypeptide can be detected by an appropriate immunological assay. Modifications of other properties such as nucleic acid hybridization to a target nucleic acid, redox or thermal stability of a protein, hydrophobicity, susceptibility to proteolysis, or the tendency to aggregate are all assayed according to standard techniques.

More particularly, it will be readily apparent to those of ordinary skill in the art that the small peptides of the present invention can readily be screened for anxiolytic and anti-depressant activity by employing suitable assays and animal models known to those skilled in the art. Among the animal models employed to evaluate the anxiolytic or anxiogenic effects of drugs, the elevated plus-maze is probably the most popular. (See Rodgers and Dalvi, supra). For factors controlling measures of anxiety and responses to novelty in the mouse, see File, Behav. Brain Res. 125:151-157 (2001). For a review of the validity and variability of the elevated plus-maze as an animal model of anxiety, see Hogg, Pharmacol. Biochem. Behav. 54:21-30 (1996); and Lister, Psychopharmacology (Berlin) 92: 180-185 (1987). The Elevated plus-maze model is described in some detail in Example 1. Still, those skilled in the art are aware of a wide range of alternative models which are also available to measure the anxiolytic effect of therapeutic agents. Such models nay require measurement of physiological or endocrine functions (e.g., hyperthermic or corticosterone responses to stress) while others analyze behavior. Broadly speaking, suitable behavioral models for testing anxiolytic effects of a test compound involve exposure of animals to stimuli (exteroceptive or interoceptive) that appear capable of causing anxiety in humans. The animals are then treated with the test compound to determine if it generates an anxiolytic effect. The models may also be grouped into two general categories involving either conditioned (e.g. Geller-Seifter conflict, potentiated startle) or unconditioned (social interaction and light/dark exploration tests) responses. Those in the art are aware that any of these standard behavioral models may be used to test NAP or ADNF polypeptides to identify or confirm anxiolytic activity of test peptides.

Using these assays and models, one of ordinary skill in the art can readily prepare a large number of NAP and ADNF polypeptides in accordance with the teachings of the present invention and, in turn, screen them using the foregoing animal models to find ADNF polypeptides, in addition to those set forth herein, which possess the desired activity. For instance, using the NAP peptide (i.e., Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (SEQ ID NO:2)) or SAL peptide Ser-Ala-Leu-Lou-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:1) as a starting point, one can systematically add, for example, Gly-, Gly-Gly-, Leu-Gly-Gly- to the N-terminus of the peptide and, in turn, screen each of these NAP or ADNP polypeptides in the foregoing assay to determine whether they possess anxiolytic or anti-depressant activity. In doing so, it will be found that additional amino acids can be added to both the N-terminus and the C-terminus of the active site, i.e., Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (SEQ ID NO:2) or Ser-Ala-Leu-Leu-Arg-Ser-Ile-Pro-Ala (SEQ ID NO:1), without loss of biological activity.

The peptides of the invention may be prepared via a wide variety of well-known techniques. Peptides of relatively short size are typically synthesized on a solid support or in solution in accordance with conventional techniques (see, e.g., Merrifield, Am. Chem. Soc. 85:2149-2154 (1963). Various automatic synthesizers and sequencers are commercially available and can be used in accordance with known protocols (see, e.g., Stewart & Young, Solid Phase Peptide Synthesis (2nd 1984)). Solid phase synthesis in which the C-terminal amino acid of die sequence is attached to an insoluble support followed by sequential addition of the remaining amino acids in the sequence is the preferred method for the chemical synthesis of the peptides of this invention. Techniques for solid phase synthesis are described by Barany & Merrifield, Solid-Phase Peptide Synthesis; pp. 3-284 in The Peptides: Analysis, Synthesis, Biology, Vol. 2: Special Methods in Peptide Synthesis, Part A.; Merrifield et al 1963; Stewart et al. 1984). NAP and related peptides are synthesized using standard Fmoc protocols (Wellings & Atherton, Methods Enzymol. 289:44-67 (1997)).

In addition to the foregoing techniques, the peptides for use in the invention may be prepared by recombinant DNA methodology. Generally, this involves creating a nucleic acid sequence that encodes the protein, placing the nucleic acid in an expression cassette under the control of a particular promoter, and expressing the protein in a host cell. Recombinantly engineered cells known to those of skill in the art include, but are not limited of bacteria, yeast, plant, filamentous fungi, insect (especially employing baculoviral vectors) of mammalian cells.

The recombinant nucleic acids are operably linked to appropriate control sequences for expression in the selected host. For E. coli, example control sequences include the T7, trp, or lambda promoters, a ribosome binding site and, preferably, a transcription termination signal. For eukaryotic cells, the control sequences typically include a promoter and, preferably, an enhancer derived from immunoglobulin genes, SV40, cytomegalovirus, etc., and a polyadenylation sequence, and may include splice donor and acceptor sequences.

The plasmids of the invention can be transferred into the chosen host cell by well-known methods. Such methods include, for example, the calcium chloride transformation method for E. coli and the calcium phosphate treatment or electroporation methods for mammalian cells. Cells transformed by the plasmids can be selected by resistance to antibiotics conferred by genes contained on the plasmids, such as the amp, gpt, neo, and hyg genes.

Once expressed, the recombinant peptides can be purified according to standard procedures of the art, including ammonium sulfate precipitation, affinity columns, column chromatography, gel electrophoresis and the like (see, e.g., Scopes, Polypeptide Purification (1982); Deutscher, Methods in Enzymology Vol. 182: Guide to Polypeptide Purification (1990)). Once purified, partially or to homogeneity as desired, the NAP and ADNF polypeptides may then be used, e.g., to prevent neuronal cell death or as immunogens for antibody production. Optional additional steps include isolating the expressed protein to a higher degree, and, if required, cleaving or otherwise modifying the peptide, including optionally generating the protein.

After chemical synthesis, biological expression or purification, the peptide(s) may possess a conformation substantially different than the native conformations of the constituent peptides. In is case, it is helpful to denature and reduce the peptide and then to cause the peptide to re-fold into the preferred conformation, Methods of reducing and denaturing peptides and inducing re-folding are well known to those of skill in the art (see Debinski et al., Biol. Chem. 268:14065-14070 (1993); Kreitman & Pastan, Bioconjug. Chem. 4:585 (1993); and Buchner et al., Anal Biochem. 205:263-270 (1992)). Debinski et al., for example, describe the denaturation and reduction of inclusion body peptides in guanidine-DTE. The peptide is then refolded in a redox buffer containing oxidized glutathione and L-arginine.

One of skill will recognize that modifications can be made to the peptides without diminishing their biological activity. Some modifications may be made to facilitate the cloning, expression, or incorporation of the targeting molecule into a fusion peptide. Such modifications are well known to those of skill in the art and include, for example, a methionine added at the amino terminus to provide an initiation site, or additional amino acids (e.g., poly His) placed on either terminus to create conveniently located restriction sites or termination codons or purification sequences.

Use of NAP and ADNF Polypeptides for Treating Anxiety and/or Depression, Including Other Mood Disorders and Anxiety Disorders

This invention discloses for the first time the surprising finding that NAP and ADNF polypeptides that were shown before to be neuroprotective and providing cognitive enhancements can be used in the treatment and/or prevention of a broad range of human clinical disorders such as anxiety and depression and a broad range of related disorders. As current medications used for treatment of anxiety disorders may adversely affect alertness, this surprising discovery offers an obvious advantage. Furthermore, anxiety is common in the elderly and can present as a primary anxiety disorder or as a symptom of another disorder. Generalized anxiety disorder (GAD), in particular, is a common syndrome in late life. Anxiety symptoms are also common features of late-life depression and dementia.

Treatment of anxiety in elderly persons has typically involved the use of benzodiazepines, which are often effective but problematic because they are associated with increased risk of cognitive impairment, falls, and fractures (Lenze et al., CNS Spectr. 12 Suppl 3:6-13 (2003)). Benzodiazepines interact with the gamma-aminobutyric acid (GABA) receptor. Previously, gephyrin, a tubulin-binding protein, was found as the core of inhibitory postsynaptic scaffolds stabilizing glycine receptors (GlyRs) and/or GABA(A) receptors (Hanus, et al., J. Neurosci. 24(5):1119-28 (2004)). Here, a mechanism for NAP is disclosed and the molecular target--tubulin, the subunit protein of microtubules is identified as the NAP binding protein. The direct interaction of NAP with tubulin may circumvent the adverse side effects associated with benzodiazepines treatments and further offers a target platform for novel drug discovery.

Anxiety is a cardinal symptom of many psychiatric disorders as well as a disease in itself. Symptoms of anxiety commonly are associated with depression and especially with dysthymic disorder (chronic depression of moderate severity), panic disorder, agoraphobia; and other specific phobias, obsessive-compulsive disorder, eating disorders and many personality disorders. Anxiety in human includes those further divisions set out in the Diagnostic and Statistical Manual of Mental Disorders (American Psychiatric Association, DSM-IV, 4th Ed. 1994).

Anxiety disorders are serious medical illnesses that affect approximately 19 million American adults. (Narrow et al., NIMH epidemiology note: prevalence of anxiety disorder. One-year prevalence best estimates calculated from ECA and NCS data. Population estimates based on U.S., Census estimated residential population age 18 to 54 on Jul. 1, 1998. Unpublished). These disorders fill people's lives with overwhelming anxiety and fear. Anxiety disorders are acute attacks or are chronic, relentless, and can grow progressively worse if not treated. Examples include: panic disorder, obsessive-compulsive disorder, attention deficit disorder and attention deficit hyperactivity disorder, post-traumatic stress disorder, social phobia (or social anxiety disorder), specific phobias, and generalized anxiety disorder.

Major depression is characterized by clinically significant depressions of mood and impairment of functioning as its primary clinical manifestations. Its clinical manifestations and current treatment overlap the anxiety disorders including panic-agorophobia syndrome, sever phobias, generalized anxiety disorder, social anxiety disorder, post-traumatic stress disorders and obsessive-compulsive disorder. Extremes of mood may be associated with psychosis, manifested as disordered or delusional thinking and perceptions, often congruent with the predominant mood.

In any given 1-year period, 9.5 percent of the population, or about 18.8 million American adults, suffer from a depressive illness (Robins & Regier (Eds). Psychiatric Disorders in America, The Epidemiologic Catchment Area Study, 1990; New York: The Free Press). Depression often accompanies anxiety disorders (Regier et al., British Journal of Psychiatry Supplement 34: 248 (199.degree.)) and, when it does, it needs to be treated as well. Symptoms of depression include feelings of sadness, hopelessness, changes in appetite or seep, low energy, and difficulty concentrating. Most people with depression can be effectively treated with antidepressant medications, certain types of psychotherapy, or a combination of both.

Depressive disorders is expressed in different forms:

Major depression is manifested by a combination of symptoms (see symptom list) that interfere with the ability to work, study, sleep, eat, and enjoy once pleasurable activities. Such a disabling episode of depression may occur only once but more commonly occurs several times in a lifetime.

A less severe type of depression, dysthymia, involves long-term, chronic symptoms that do not disable, but keep one from functioning well or from feeling good. Many people with dysthymia also experience major depressive episodes at some time in their lives.

Another type of depression is bipolar disorder, also called manic-depressive illness. Not nearly as prevalent as other forms of depressive disorders, bipolar disorder is characterized by cycling mood changes: severe highs (mania) and lows (depression). Sometimes the mood switches are dramatic and rapid, but most open they are gradual. When in the depressed cycle, an individual can have any or all of the symptoms of a depressive disorder. When in the manic cycle, the individual may be overactive, overtalkative; and have a great deal of energy. Mania often affects thinking, judgment, and social behavior in ways that cause serious problems and embarrassment. For example, the individual in a manic phase may feel elated, full of grand schemes that might range from unwise business decisions to romantic sprees. Mania, left untreated, may worsen to a psychotic state.

Gamma-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the mammalian Central Nervous System (CNS). GABA participates in the regulation of neuronal excitability through interaction with specific membrane proteins (the GABAA receptors). The binding of GABA to these postsynaptic receptors, results in an opening of a chloride channel integrated in the receptor which allows the entry of Cl-- and consequently leads to hyperpolarization of the recipient cell. The action of GABA is allosterically modulated by a wide variety of chemical entities which interact with distinct binding sites at the GABAA receptor complex.

One of the most thoroughly investigated modulatory site is the benzodiazepine binding site. The benzodiazepines constitute a well-known class of therapeutics displaying hypnotic, anxiolytic and anticonvulsant effects. Their usefulness, however, is limited by a broad range of side effects comprising sedation ataxia, amnesia, alcohol and barbiturate potentiation, tolerance development and abuse potential. Consequently, there has been an intensive search for modulatory agents with an improved profile, and a diversity of chemical entities distinct from the benzodiazepines, but with GABA modulatory effects have been identified. The existence of endogenous ligands for the GABAA receptor complex beside GABA has often been described, but their role in the regulation of GABA action is still a matter of controversy.

The progress of molecular biology during the last decade has contributed enormously to the understanding of benzodiazepine receptor pharmacology. A total of 14 GABAA receptor subunits have been cloned from mammalian brain and have been expressed/co-expressed in stable cell lines. These transfected cells constitute an important tool in the characterization of subtype selective ligands. In spite of the rapidly expanding knowledge of the molecular and pharmacological mechanisms involved in GABA/benzodiazepine related CNS disorders, the identification of clinically selective acting drugs is still to come (Teuber et al., Curr Pharm Des 5(5):317-43(1999)).

Control of neurotransmitter receptor expression and delivery to the postsynaptic membrane is of great importance for neural signal transduction at synapses. The GABA type A (GABA(A)) receptor-associated protein GABARAP was reported to have an important role for movement and sorting of GABA(A) receptor molecules to the postsynaptic membrane. GABARAP not only binds to GABA(A) receptor gamma2-subunit but also to tubulin, gephyrin, and ULK1, suggesting regulation through the interaction with the microtubular network (Stangler et al. J Biol. Chem. 19:277 (2002), 16:13363-6. Epub 2002 Mar. 1)

Anxiety is often defined as an organism's response to potential threat, as opposed to direct or immediate threat. Anxiety and depression also encompass disorders of mood such as affective disorders. The severity of these conditions covers an extraordinarily broad range from normal grief reactions and dysthymia to severe, incapacitating illnesses that may result in death.

Thus, according to the instant invention, NAP and ADNF polypeptides may be used to treat anxiety and/or depression and diseases or disorders related thereto, as defined herein.

Drug Discovery Using Nap-Tubulin Binding

The identification of tubulin as the NAP-biding site allows the use of tubulin and tubulin--derived peptides as targets for further drug discovery. e.g., for the treatment of diseases related to ADNF polypeptides such as anxiety, depression, disease related to neuronal cell death and oxidative stress, neurodegenerative diseases such as Alzheimer's disease, AIDS-related dementia, Huntington's disease, and Parkinson's disease, HIV-related dementia complex, stroke, head trauma, cerebral palsy, conditions associated with fetal alcohol syndrome. Such therapeutics can also be used in methods of enhancing learning and memory both pre- and post-natally. Experiments can be carried out with the intact tubulin structure and NAP as a displacing agent or by further identification of the precise tubulin-NAP interacting site (e.g., as described Katchalski-Katzir et al., Biophys Chem. 100(1-3):293-305 (2003); Chang et al., J Comput Chem, 24(16):1987-98 (2003)).

Preliminary screens can be conducted by screening for agents capable of binding polypeptide of the invention, as at least some of the agents so identified are likely modulators of polypeptide activity. The binding assays usually involve contacting a polypeptide of the invention with one or more test agents and allowing sufficient time for the protein and test agents to form a binding complex. Any binding complexes formed can be detected using any of a number of established analytical techniques. Protein binding assays include, but are not limited to, methods that measure co-precipitation, co-migration on non-denaturing SDS-polyacrylamide gels, and co-migration on Western blots (see, e.g., Bennet and Yamamura (1985) Neurotransmitter, Hormone or Drug Receptor Binding Methods, in Neurotransmitter Receptor Binding (Yamamura et al., eds.), pp. 61-89. The protein utilized in such assays can be naturally expressed, cloned or synthesized.

Agents that are initially identified by any of the foregoing screening methods can be further tested to validate the apparent activity. Preferably such studies are conducted with suitable animal models. The basic format of such methods involves administering a lead compound identified during an initial screen to an animal that serves as a model for humans and then determining if expression or activity of a polynucleotide or polypeptide of the invention is in fact upregulated. The animal models utilized in validation studies generally are mammals of any kind. Specific examples of suitable animals include, but are not limited to, primates, mice, and rats. In one embodiment, the Elevated plus maze and the Morris water maze tests are used, as described in Example 1.

The agents teased as modulators of the polypeptides of the invention can be any small chemical compound, or a biological entity, such as a protein, sugar, nucleic acid, RNA, or lipid. Typically, test compounds will be small chemical molecules and peptides. Essentially, any chemical compound can be used as a potential modulator or ligand in the assays of the invention, although most often compounds that can be dissolved in aqueous or organic (especially DMSO-based) solutions are used. The assays are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g., in microtiter formats on microtiter in robotic assays). It will be appreciated that there are many suppliers of chemical compounds, including Sigma (St Louis, Mo.), Aldrich (St. Louis, Mo.), Sigma-Aldrich (St. Louis, Mo.), Fluka Chemika-Biochemica Analytika (Buchs, Switzerland) and the like. Modulators also include agents designed to reduce the level of mRNA of the invention (e.g. antisense molecules, ribozymes, DNAzymes and the like) or the level of translation from an mRNA.

In one preferred embodiment, high throughput screening methods involve providing a combinatorial chemical or peptide library containing a large number of potential therapeutic compounds (potential modulator or ligand compounds). Such "combinatorial chemical libraries" or "ligand libraries" are then screened in one or more assays, as described herein, to identify those library members (articular chemical species or subclasses) that display desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics. Libraries available for screening for small active molecules include the Available Chemical Directory (ACD, 278,000 compounds), ACD screening library (>1,000,000 compounds), CRC Combined Chemical Dictionary (.about.350,000 compounds) Anisex (115,000 compounds) Maybridge (62,000 compounds) Derwent and NCI libraries.

Pharmaceutical Administration

The pharmaceutical compositions of the present invention are suitable for use in a variety of drug delivery systems. Peptides that have the ability to cross the blood brain barrier can be administered, e.g., systemically, nasally, etc., using methods known to those of skill in the art. Larger peptides that do not have the ability to cross the blood brain barrier can be a administered to the mammalian brain via intracerebroventricular (ICV) injection or via a cannula using techniques well known to those of skill in the art (see, e.g., Motta & Martini, Proc. Soc. Exp. Biol. Med. 168:62-64 (1981); Peterson et al., Biochem. Pharmacol. 31:2807-2810 (1982); Rzepczynski et al., Metab. Brain Dis. 3:211-216 (1939); Leibowitz et al., Brain Res. Bull. 21:905-912 (1988); Sramka et al., Stereotact. Funct. Neurosurg. 58:79-83 (1992); Peng et al., Brain Res. 632:57-67 (1993); Chem et al., Exp. Neurol. 125:72-81 (1994); Nikkhah et al., Neuroscience 63:57-72 (1994); Anderson et al., J Comp. Neurol. 357:196-317 (1995); and Brecknell & Fawcett, Exp. Neurol. 138:338-344 (1996)).

Suitable formulations for use in the present invention are found in Remington's Pharmaceutical Sciences (17th ed. 1985)), which is incorporated herein by reference. In addition, for a brief review of methods for drug delivery, see Langer, Science 249:1527-1533 (1990), which is incorporated herein by reference. Suitable dose ranges are described in the examples provided herein, as well as in WO 9611948, herein incorporated by reference in its entirety.

As such, the present invention provides for therapeutic compositions or medicaments comprising one or more of the NAP or ADNF polypeptides described hereinabove in combination with a pharmaceutically acceptable excipient, wherein the amount of the NAP or ADNF polypeptide is sufficient to provide a therapeutic effect.

In a therapeutic application, the NAP and ADNF polypeptides of the present invention are embodied in pharmaceutical compositions intended for administration by any effective means, including parenteral, topical, oral, pulmonary (e.g. by inhalation) or local administration. Preferably, the pharmaceutical compositions are administered parenterally, e.g., intravenously, subcutaneously, intradermally, or intramuscularly, or intranasally.

Thus, the invention provides compositions for parenteral administration that comprise a solution of NAP or ADNF polypeptide, as described above, dissolved or suspended in an acceptable carrier, preferably an aqueous carrier. A variety of aqueous carriers may be used including, for example, water, buffered water, 0.4% saline, 0.3% glycine, hyaluronic acid and the like. These compositions may be sterilized by conventional, well known sterilization techniques or, they may be sterile filtered. The resulting aqueous solutions may be packaged for use as is or lyophilized, the lyophilized preparation being combined with a sterile solution prior to administration. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions including pH adjusting and buffering agents, tonicity adjusting agents, wetting agents and the like, such as, for example, sodium acetate, sodium lactate, sodium chloride potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, etc.

For solid compositions, conventional nontoxic solid carriers may be used that include, for, example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like. For oral administration, a pharmaceutically acceptable nontoxic composition is formed by incorporating any of the normally employed excipients, such as those carriers previously listed, and generally 10-95% of active ingredient and more preferably at a concentration of 25%-75%.

For aerosol administration, the NAP or ADNF polypeptides are preferably supplied in finely divided from along with a surfactant and propellant. The surfactant must, of course, be nontoxic, and preferably soluble in the propellant. Representative of such agents are the esters or partial esters of fatty acids containing from 6 to 22 carbon atoms, such as caproic, octanoic lauric, palmitic, stearic, linoleic, linolenic, olesteric and oleic acids with an aliphatic polyhydric alcohol or its cyclic anhydride. Mixed esters, such as mixed or natural glycerides may be employed. A carrier can also be included, as desired, as with, e.g., lecithin for intranasal delivery. An example includes a solution in which each milliliter included 7.5 mg NaCl, 1.7 mg citric acid monohydrate, 3 mg disodium phosphate dihydrate and 0.2 mg benzalkonium chloride solution (50%) (Gozes et al., J Mol Neurosci. 19(1-2):167-70 (2002)).

In therapeutic applications, the NAP or ADNF polypeptides of the invention are administered to a patient in an amount sufficient to reduce or eliminate symptoms of anxiety ad/or depression. An amount adequate to accomplish this is defined as "therapeutically effective dose." Amounts effective for this use will depend on, for example, the particular NAP or ADNF polypeptide employed, the type of disease or disorder to be prevented, the manner of administration, the weight and general state of health of the patient, and the judgement of the prescribing physician.

For example, an amount of polypeptide falling within the range of a 100 ng to 10 mg dose given intranasally once a day (e.g., in the evening) would be a therapeutically effective amount. Alternatively, dosages may be outside of this range, or on a different schedule. For example, dosages may range from 0.0001 mg/kg to 10,000 mg/kg, and will preferably be about 0.001 mg/kg, 0.1 mg/kg, 1 mg/kg 5 mg/kg, 50 mg/kg or 500 mg/1 g per dose. Doses may be administered hourly, every 4, 6 or 12 hours, with meals, daily, every 2, 3, 4, 5, 6, for 7 days, weekly, every 2, 3, 4 weeks, monthly or every 2, 3 or 4 months, or any combination thereof. The duration of dosing may be single (acute) dosing, or over the course of days, weeks, months, or years, depending on the condition to be treated. Those skilled in the art can determine the suitable dosage, and may rely on preliminary data reported in Gozes et al., 2000, Gozes et al., 2002), Bassan et al. 1999, Zemlyak et al.; Regul. Pept. 96:39-43 (2000); Brenneman et al., Biochem, Soc. Trans. 28: 452-455 (2000); Erratum Biochem Soc. Trans. 28:983; Wilkemeyer et al. Proc Natl. Acad., Sci. USA 100:3543-8548 (2003)).
 

Claim 1 of 12 Claims

1. A method of treating schizophrenia in a subject in need thereof, the method comprising the step of administering a therapeutically effective amount of an ADNF III polypeptide comprising an active core site having the following amino acid sequence: Asn-Ala-Pro-Val-Ser-Ile-Pro-Gln (SEQ ID NO:2) to the subject.
 

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