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  Pharmaceutical Patents  

 

Title:  Screening and therapeutic methods relating to neurogenesis
United States Patent: 
7,323,334
Issued: 
January 29, 2008

Inventors: 
Zhou; Qun-Yong (Irvine, CA), Cheng; Michelle Y. (Irvine, CA)
Assignee: 
The Regents of the University of California (Oakland, CA)
Appl. No.: 
10/680,554
Filed: 
October 3, 2003


 

Woodbury College's Master of Science in Law


Abstract

The invention provides methods of identifying compounds that modulate neurogenesis. The methods involve providing a compound that modulates prokineticin receptor signaling; contacting a neural stem or progenitor cell with the compound; and determining the ability of the compound to modulate neurogenesis. The invention also provides methods for modulating neurogenesis. The methods involve contacting a neural stem or progenitor cell with an effective amount of a compound that modulates prokineticin receptor signaling. Such methods are useful for both ex vivo or in vivo therapeutic applications where neural regeneration is desirable.

Description of the Invention

SUMMARY OF THE INVENTION

The invention provides methods of identifying compounds that modulate neurogenesis. The methods involve providing a compound that modulates prokineticin receptor signaling; contacting a neural stem or progenitor cell with the compound; and determining the ability of the compound to modulate neurogenesis.

The invention also provides methods for modulating neurogenesis. The methods involve contacting a neural stem or progenitor cell with an effective amount of a compound that modulates prokineticin receptor signaling. Such methods are useful in both ex vivo and in vivo therapeutic applications.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the determination that prokineticins effectively promote neurogenesis in rats. Based on this determination of this important pharmacological role of prokineticins, the invention provides screening methods for identifying compounds that modulate neurogenesis and therapeutic methods for modulating neurogenesis in mammals.

As disclosed in Example V, administration of recombinant human prokineticin 2 (PK2) to adult rats via intracerebroventricular infusion resulted in an increased number of newly developed neurons in the forebrain. Specifically, incorporation of the cell proliferation marker BrdU was at least two-fold higher in the subventricular zone in rats treated with PK2 in comparison to control animals. Also disclosed herein are the observations that mRNAs for two related GPCRs, designated prokineticin receptor 1 (PKR1) and prokineticin receptor 2 (PKR2), are expressed at high levels in the subventricular zone (SVZ) of the lateral ventricle, the olfactory bulb/olfactory ventricle, and the dentate gyrus of the hippocampus of adult mouse brain (see Example I). Further disclosed in Example II is that mRNA for PKR2 is also expressed in the inner nuclear layer of the adult mouse retina. mRNA for prokineticin 2 (PK2), which is an agonist of both PKR1 and PKR2, is expressed in both the olfactory bulb and the dentate gyrus of the hippocampus of the adult mouse brain. Non-neurogenic surrounding tissues express PKR1, PKR2 or PK2 at much lower levels. These findings indicate a role for signaling through prokineticin receptors in the regulation of neurogenesis.

Methods for modulating neurogenesis have a variety of important applications, including for treating individuals having, or who are likely to develop, disorders relating to neural degeneration, neural damage and neural demyelination, as described in more detail below. Therapeutic methods of modulating neurogenesis include both drug-based therapies, in which compounds that modulate neurogenesis are administered to an individual, and cell-based therapies, in which neural cells generated, propagated or genetically modified ex vivo using compounds that modulate neurogenesis are transplanted into the nervous system of an individual. Developing methods for modulating neurogenesis ex vivo are also useful for advancing understanding of the complex processes of neural proliferation and differentiation, which can lead to development of additional compounds and approaches for diagnosing and treating disorders of the nervous system.

Accordingly, the invention provides methods of screening for compounds that modulate neurogenesis. The invention screening methods involve providing a compound that modulates PKR signaling, and determining the ability of the compound to modulate neurogenesis.

The invention also provides methods of modulating neurogenesis by contacting a neural stem or progenitor cell with an effective amount of a compound that modulates PKR signaling.

A compound that modulates PKR signaling will generally promote signaling in a suitable signaling assay, as described below, by at least about 10%, such as at least 25%, 50%, 100%, 500% or more, or alternatively reduce signaling in a suitable signaling assay by at least about 10%, such as at least 25%, 50%, 90% or more, in comparison to a control compound.

It will be appreciated that PKR signaling can be modulated either directly or indirectly, and by altering either activity or abundance of either a PKR receptor or of a PKR ligand. For example, PKR signaling can be modulated directly, such as by contacting the PKR with an agonist or antagonist. PKR signaling can also be modulated indirectly, such as by altering PKR expression, localization, stability, or ability to bind effector molecules, or by altering prokineticin (PK) expression, secretion, stability or ability to bind or activate a PKR. Various methods of modulating PKR signaling are described below, following a description of prokinetin receptors and prokineticins. Other general approaches to qualitatively or quantitatively modulate signaling through G-protein coupled receptors are known in the art, and can be applied in accordance with the invention to prokineticin receptors given the guidance herein.

As used herein, the term "prokineticin receptor" or "PKR" refers to a heptahelical membrane-spanning polypeptide that binds to a prokineticin and signals through a G-protein coupled signal transduction pathway in response to prokineticin binding. Prokineticin receptors are believed to couple exclusively to the G.alpha. subtype known as G.alpha.q, and thereby mediate intracellular calcium mobilization in response to agonists.

A prokineticin receptor can have the naturally-occurring amino acid sequence of a PKR from any species, or can contain minor modifications with respect to the naturally-occurring sequence. For example, a PKR can be a mammalian PKR, such as human PKR1 (SEQ ID NO:1; GenBank Accession No. AAM48127; also called. GPR73, fb41a, hZAQ, hGPRv21 and EG-VEGF receptor-1; Lin et al., J. Biol. Chem. 277:19276-19280 (2002), Masuda et al., Biochem. Biophys. Res. Commun. 293:396-402 (2002), WO 00/34334, WO 01/48188 and WO 01/16309); human PKR2 (SEQ ID NO:2; GenBank Accession No. AAM48128; also known as I5E, hRUP8 and hZAQ2; Lin et al., supra (2002), Masuda et al., supra (2002), WO 98/46620, WO 01/36471 and WO 02/06483); mouse PKR1 (SEQ ID NO:3; GenBank Accession No. AAM49570; Cheng et al., Nature 417:405-410 (2002) and WO 02/06483); mouse PKR2 (SEQ ID NO:4; GenBank Accession No. AAM49571; Cheng et al., supra (2002) and WO 02/06483); rat PKR1 (WO 02/06483); rat PKR2 (WO 02/06483); monkey PKR2 (also known as AXOR8; WO 01/53308); bovine PKR1 (Masuda et al., supra (2002), or a PKR of another mammalian species, such as other primate, dog, cat, pig, sheep or goat; or a PKR of another vertebrate species, such as an amphibian, reptile, fish or bird.

Based on the high degree of homology between the nucleotide sequences encoding PKR1 and PKR2, and between PKR types across species, the skilled person can readily identify and clone a PKR from any other species. For example, other PKRs can be identified from nucleic acid libraries using standard molecular biology approaches such as hybridization and polymerase chain reaction (PCR) techniques. Additionally, PKRs from other species can be identified from nucleic acid sequence databases specific for the species of interest. Methods of identifying, isolating and propagating homologous nucleic acid molecules are well known in the art and described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Plainview, N.Y. (2001) and Ausubel et al. (Current Protocols in Molecular Biology, John Wiley & Sons, New York ((current supplement)).

Likewise, based on the high degree of homology among PKR polypeptides, the skilled person can readily identify a cell from another species or from an expression library that expresses a PKR, using PKR-specific antibodies. Methods of preparing and using antibodies to identify polypeptides are well known in the art and described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988)

A prokineticin receptor can also contain minor modifications with respect to a naturally-occurring PKR, so long as the PKR retains its ability to bind to, and signal in response to, a prokineticin, such as a human PK1 or human PK2 described below.

A prokineticin receptor that contains minor modifications with respect to a naturally-occurring PKR can contain one or more additions, deletions, or substitutions of natural or non-natural amino acids relative to the naturally-occurring polypeptide sequence. Such a modification can be, for example, a conservative change, wherein a substituted amino acid has similar structural or chemical properties, for example, substitution of an apolar amino acid with another apolar amino acid, substitution fo a charged amino acid with another amino acid of similar charge, and the like. Such a modification can also be a non-conservative change, wherein a substituted amino acid has different but sufficiently similar structural or chemical properties so as to not adversely affect the desired biological activity. Further, a minor modification can be the substitution of an L-configuration amino acid with the corresponding D-configuration amino acid with a non-natural amino acid.

In addition, a minor modification can be a chemical or enzymatic modification to the polypeptide, such as replacement of hydrogen by an alkyl, acyl, or amino group; esterification of a carboxyl group with a suitable alkyl or aryl moiety; alkylation of a hydroxyl group to form an ether derivative; phosphorylation or dephosphorylation of a serine, threonine or tyrosine residue; or N- or O-linked glycosylation.

Those skilled in the art can determine whether minor modifications to a naturally occurring prokineticin receptor sequence is advantageous in a method of the invention. Minor modifications are useful, for example, to enhance the stability, selectivity, bioactivity of a receptor. Modified PKR polypeptides can be prepared, for example, by recombinant methods, by synthetic methods, by post-synthesis chemical or enzymatic methods, or by a combination of these methods, and tested for ability to bind a prokineticin or signal through a G-protein coupled signal transduction pathway.

Those skilled in the art can readily predict regions in a prokineticin receptor amino acid sequence that can be modified without abolishing binding or signaling. Such prediction can be based on sequence analysis, structure-function studies, and computer algorithms. For example, comparisons of amino acid sequences of PKR2 and PKR1, and of PKRs across species, can provide guidance in determining amino acid residues that are tolerant of modification. Further, a large number of published GPCR-structure-function studies have indicated regions of GPCRs involved in ligand interaction, G-protein coupling and in forming transmembrane regions, and indicate regions of GPCRs tolerant of modification (see, for example, Burstein et al., J. Biol. Chem., 273:24322-24327 (1998) and Burstein et al., Biochemistry, 37(12):4052-4058 (1998)). In addition, computer programs known in the art can be used to determine which amino acid residues of a polypeptide can be modified without abolishing activity (see, for example, Eroshkin et al., Comput. Appl. Biosci. 9:491-497 (1993); Gueros et al., J. Mol. Biol. 320:369-387(2002)). These methods can likewise be applied to PKRs.

For certain applications, a PKR has at least 80%, such as at least 85%, 90%, 95%, 97%, 99% or greater identity with either human PKR1 (SEQ ID NO:1) or with human PKR2 (SEQ ID NO:2).

That a presumptive PKR, such as a PKR from another species or a modified PKR is actually a PKR can be confirmed by prokineticin binding assays and G-protein coupled receptor signaling assays known in the art (see, for example, U.S. published application 20020115610A1, Lin et al., supra (2002) and Masuda et al., supra (2002)), and described further below.

Depending on the intended application, the skilled person can determine an appropriate form for the PKR, such as in a live animal, a tissue, a tissue extract, a cell, a cell extract, or in substantially purified form. For example, for receptor binding or signaling assays, the PKR will typically be either endogenously expressed or recombinantly expressed at the surface of a cell.

Cells that endogenously express a PKR are well known in the art, and include, for example, M2A7 melanoma cells (available from American Type Culture Collection as ATCC CRL-2500), M2 melanoma cells (Cunningham et al., Science 255;325-327 (1992)) and RC-4B/C pituitary tumor cells (ATCC CRL-1903)(see U.S. 20020115610A1). Other cells that endogenously express a PKR include, for example, ileal and other gastrointestinal cells (see U.S. 20020115610A1), endothelial cells such as BACE cells (Masuda et al., supra (2002)), and endocrine cells (Lin et al., supra (2002)). As disclosed herein, other cells that express PKR include neural stem and progenitor cells, including such cells in the subventricular zone of the lateral ventricle, the olfactory bulb/olfactory ventricle, the dentate gyrus of the hippocampus, and the inner nuclear layer of the retina.

Methods of recombinantly expressing a PKR are also well known in the art (see, for example, Lin et al., supra (2002) and Masuda et al., supra (2002)). Host cells suitable for recombinantly expressing polypeptides are well known in the art, and include bacterial cells (e.g. E. coli), insect cells (e.g. Drosophila), yeast cells (e.g. S. cerevisiae, S. pombe, or Pichia pastoris), and vertebrate cells (e.g. mammalian primary cells and established cell lines; and amphibian cells, such as Xenopus embryos and oocytes). Vectors appropriate for expressing polypeptides in the particular host cell are also well known in the art, and include, for example, vectors derived from a virus, such as a bacteriophage, a baculovirus or a retrovirus, and vectors derived from bacteria or a combination of bacterial sequences and sequences from other organisms, such as a cosmid or a plasmid. Suitable expression vectors generally contain elements such as an origin of replication compatible with the intended host cells; constitutive or inducible promoter sequences; transcription termination and RNA processing signals; one or more selectable markers compatible with the intended host cells; one or more multiple cloning sites; and optionally contain tag sequences that facilitate expression or purification of the encoded polypeptide. Methods of recombinantly expressing polypeptides are well known in the art and described, for example, in Sambrook et al., supra (2001) and Ausubel et al. supra (current supplement).

As used herein, the term "prokineticin" or "PK" refers to a peptide that binds to a prokineticin receptor and elicits signaling by the receptor through a G-protein coupled signal transduction pathway.

A prokineticin can have the naturally-occurring amino acid sequence of a PK from any species, or can contain minor modifications with respect to the naturally-occurring sequence. For example, a PK can be a mammalian PK, such as human PK1 (SEQ ID NO:5; GenBank Accession No. P58294; also known as endocrine-gland-derived endothelial growth factor or EG-VEGF, TANGO 266, PRO 1186 and Zven2; Li et al., supra (2001), LeCouter et al., Nature 412: 877-884 (2001), WO 01/36465, WO 99/63088 and WO 00/52022; human PK2 (GenBank Accession No. Q9HC23; isoform 1, SEQ ID NO:6, Wechselberger et al., FEBS Lett. 462:177-181 (1999) or isoform 2, SEQ ID NO:7; also known as Zven1, Li et al., supra (2001)); mouse PK1 (SEQ ID NO:8; GenBank Accession No. AAM49573); mouse PK2 (SEQ ID NO:9; GenBank Accession No. AAM49572); rat PK1 (SEQ ID NO:10; GenBank Accession No. AAM09104; Masuda et al., supra (2002)); rat PK2 (SEQ ID NO:11; GenBank Accession No. AAM09105; Masuda et al., supra (2002)), or a PK of another mammalian species, such as other primate, dog, cat, pig, cow, sheep or goat.

A PK can alternatively be a PK of another vertebrate species, such as a snake, frog or toad. For example, a PK can be black mamba PK (SEQ ID NO:12; GenBank-Accession No. P25687; also known as MIT1; Schweitz et al., FEBS Lett. 461:183-188 (1999)); Bombina variegata frog PK (SEQ ID NO:13; GenBank Accession No. Q9PW66; also known as Bv8; Mollay et al., Eur. J. Pharmacol. 374:189-196 (1999); Bombina maxima toad PK (SEQ ID NO:14; GenBank Accession No. AAN03822), or a PK from another vertebrate species, such as an amphibian, reptile, fish or bird.

A prokineticin can also contain minor modifications with respect to a naturally-occurring PK, so long as the PK retains its ability to bind to, and elicit signaling by, a prokineticin receptor, such as a human PKR1 or PKR2 as described above. Examples of types of suitable minor modifications have been described above with respect to PKRs, and also apply to PKs. Minor modifications to a PK can be useful, for example, for applications in which it is desired to enhance a property of the PK, such as stability, bioavailability, bioactivity or specificity.

Specific examples of prokineticins that are modified from naturally-occurring sequences while retaining activity include human prokineticin chimeras having SEQ ID NO:15 (chimera of PK1 at N-terminus, PK2 at C-terminus) and SEQ ID NO:16 (chimera of PK2 at N-terminus, PK1 at C-terminus). Chimeras between PK1 and PK2 within species, or between prokineticins across species, so long as they retain PK activity, are also considered prokineticins.

As described in U.S. 20020115610A1, prokineticins are not tolerant of additions, deletions or substitutions at the N-terminal 6 amino acids conserved across all species (AVITGA), or at any of the 10 conserved cysteine residues. However, modifications at other internal residues are tolerated, as are modifications at residues C-terminal to the tenth cysteine.

For certain applications, a PK has at least 40%, such as at least 45%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, 99% or greater identity-with either human PK1 (SEQ ID NO:5) or with human PK2 (SEQ ID NO:6).

That a presumptive PK, such as a PK from another species or a modified PK is actually a PK can be confirmed by prokineticin binding assays and G-protein coupled receptor signaling assays known in the art (see, for example, U.S. 20020115610A1, Lin et al., supra (2002) and Masuda et al., supra (2002)), and described further below.

Depending on the intended application, the skilled person can determine an appropriate form for the PK, such as in a live animal, a tissue, a tissue extract, a cell, a cell extract, or in substantially purified form. A prokineticin can be substantially purified from any tissue, cell or body fluid source that endogenously contains PK peptide, or can be produced recombinantly and then substantially purified. Sources of endogenous PK include milk (Masuda et al., supra (2002)), venom (Masuda et al., supra (2002); Mollay et al., Eur. J. Pharmacol. 374:189-196 (1999)), various fetal and adult tissues, including GI tract, liver, spleen, testis and placenta (U.S. 20020115610A1) and, as disclosed herein, neural stem and progenitor cells. Methods of substantially purifying prokineticins from such sources are described in the afore-mentioned references.

Methods of recombinantly expressing prokineticins are also known in the art (see U.S. 20020115610A1 and Masuda et al., supra (2001) for examples of bacterial expression and WO 01/36465 and WO 00/52022 for-examples of eukaryotic expression). Such methods can involve initially expressing the PK as a fusion protein, such as a fusion with a glutathione-S-transferase tag, Fc tag, 6X His tag, myc epitope, or other tag sequences known in the art. Methods of substantially purifying recombinantly expressed prokineticins, and for removing optional tag sequences, are also known in the art. For example, U.S. 20020115610A1 describes conditions for refolding and purifying recombinantly expressed prokineticins that minimize protein aggregation, and also describes methods of confirming correct disulfide bond formation.

In one embodiment, the invention screening and therapeutic methods involve providing a compound that is a prokineticin receptor agonist or antagonist. A PKR agonist or antagonist can optionally be selective for PKR1 or PKR2, or alternatively be equally active with respect to both PKR1 and PKR2.

As used herein, the term "prokineticin receptor, agonist" refers to a compound that promotes or enhances normal G-protein coupled signal transduction through a PKR. A PKR agonist can act by any agonistic mechanism, such as by directly binding a PKR at the normal ligand binding site, thereby promoting receptor signaling. A PKR agonist can also act indirectly, for example, by potentiating the binding activity of the endogenous ligand, or by altering the conformation of the PKR so as to increase its signaling activity.

Examples of PKR agonists include naturally-occurring, chimeric and modified PK peptides, as described above. PKR agonists can also include peptidomimetics of such PK peptides. Methods of preparing peptidomimetics are known in the art and reviewed, for example, in Ripka et al., Curr. Opin. Chem. Biol. 2:441-452 (1998) and al-Obeidi et al., Mol. Biotechnol. 9:205-223 (1998). Other PKR agonists include compounds identified as such by the screening assays described below. An exemplary partial agonist is PK2-insert (insertion of 23 amino acids between exon 2 and exon 3 of PK2) and an exemplary weak agonist is GIL-PK1 (tripeptide Gly-Ile-Leu added to the N-terminus of PK1).

As used herein, the term "prokineticin receptor antagonist" refers to a compound that inhibits or decreases normal G-protein coupled signal transduction through a PKR. A PKR antagonist can act by any antagonistic mechanism, such as by directly binding a PKR at the PK binding site, thereby inhibiting binding between the PKR and its ligand. A PKR antagonist can also act indirectly, for example, by binding a PK, or by altering the conformation or state of phosphorylation or glycosylation of a PKR, thereby affecting its ability to bind or respond to ligand. The term "PKR antagonist" is also intended to include compounds that act as "inverse agonists," meaning that they decrease PKR signaling from a baseline amount of constitutive signaling activity.

Examples of PKR antagonists include peptides with either a single N-terminal residue addition or a single N-terminal residue deletion with respect to human PK1 and PK2 (see U.S. 20020115610A1). Peptides having similar modifications to other prokineticins, or other modifications to the conserved N-terminal six residues of human or other prokineticins, such as more extensive additions or deletions and substitutions, can also be PKR antagonists. An exemplary PKR antagonist is MVITGA.

Other examples of PKR antagonists are antibodies selective for a PKR or a PK. The term "antibody" is intended to include both polyclonal and monoclonal antibodies, as well as antigen binding fragments of such-antibodies (e.g. Fab, F(ab').sub.2, Fd and Fv fragments and the like). In addition, the term "antibody" is intended to encompass non-naturally occurring antibodies, including, for example, single chain antibodies, chimeric antibodies, bifunctional antibodies, CDR-grafted antibodies and humanized antibodies, as well as antigen-binding fragments thereof.

Methods of preparing and isolating antibodies, including polyclonal and monoclonal antibodies, using peptide and polypeptide immunogens, are well known in the art and are described, for example, in Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1988). Non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, can be produced recombinantly or can be obtained, for example, by screening combinatorial libraries consisting of variable heavy chains and variable light chains. Such methods are described, for example, in Huse et al. Science 246:1275-1281 (1989); Winter and Harris, Immunol. Today 14:243-246 (1993); Ward et al., Nature 341:544-546 (1989); Hilyard et al., Protein Engineering: A practical approach (IRL Press 1992); and Borrabeck, Antibody Engineerinq, 2d ed. (Oxford University Press 1995).

A prokineticin receptor agonist will generally have an EC.sub.50 that is no more than 2-fold, 5-fold, 10-fold, 50-fold, 100-fold or 1000-fold higher or lower than the EC.sub.50 for human PK1 or PK2 in the particular assay. For therapeutic applications described below, a prokineticin receptor agonist preferably has an EC.sub.50, and a prokineticin receptor antagonist preferably has an IC.sub.50, of less than about 10.sup.-7 M, such as less than 10.sup.-8 M, and more preferably less than 10.sup.-9 or 10.sub.-10 M. However, depending on the stability, selectivity and toxicity of the compound, a prokineticin receptor agonist with a higher EC.sub.50, or a prokineticin receptor antagonist with a higher IC.sub.50, can also be useful therapeutically.

Signaling assays to identify or confirm the activity of PKR agonists and antagonists are known in the art. Because PKRs are G.alpha.q-coupled receptors, signaling assays typically used with other G.alpha.q-coupled GPCRs can be used to determine PKR signaling activity. G.alpha.q-coupled GPCRs, when bound to ligand, activate phospholipase C (PLC), which cleaves the lipid phosphatidylinositol 4,5-bisphosphate (PIP2) to generate the second messengers inositol 1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). These second messengers increase intracellular Ca.sup.2+ concentration and activate the MAP kinase cascade. The change in activity of PLC, or in abundance of downstream messengers, is a reflection of GPCR activation.

The specificity of G.alpha. subunits for cell-surface receptors is determined by the C-terminal five amino acids of the G.alpha.. Thus, if it is desired to assay a GPCR signaling pathway other than a typical G.alpha.q pathway, a chimeric G.alpha. containing the five C-terminal residues of G.alpha.q and the remainder of the protein corresponding to another G.alpha. can be expressed in a cell such that the PKR is coupled to a different signaling pathway (see, for example, Conklin et al., Nature 363:274-276 (1993), and Komatsuzaki et al., FEBS Letters 406:165-170 (1995)). For example, a PKR can be coupled to a G.alpha.s or G.alpha.i, and adenylate cyclase activation or inhibition assayed by methods known in the art.

Depending on the G.alpha. and the assay system, GPCR signals that can be determined include, but are not limited to, calcium ion mobilization; increased or decreased production or liberation of arachidonic acid, acetylcholine, diacylglycerol, cGMP, cAMP, inositol phosphate and ions; altered cell membrane potential; GTP hydrolysis; influx or efflux of amino acids; increased or decreased phosphorylation of intracellular proteins; and activation of transcription of an endogenous gene or promoter-reporter construct downstream of any of the above-described second messenger pathways.

A variety of cell-based GPCR signaling assays, including assays performed in bacterial, yeast, baculovirus/insect systems and mammalian cells, are reviewed, for example, in Tate et al., Trends in Biotecha. 14:426-430 (1996). More recently developed GPCR signaling assays include, for example, AequoScreen, which is a cellular aequorin-based functional assay that detects calcium mobilization (LePoul et al., J. Biomol. Screen. 7:57-65 (2002)); MAP kinase reporter assays (Rees et al., J. Biomol. Screen. 6:19-27 (2001); and fluorescence resonance energy transfer (FRET) based PLC activation assays (van der Wal, J. Biol. Chem. 276:15337-15344 (2001)). Several examples of PKR signaling assays are described in Lin et al., supra (2002) and in Masuda et al., supra (2002).

Optionally, a compound can initially be tested to determine whether it binds a PKR, such compounds being likely to act as PKR agonists or antagonists. Competitive and non-competitive binding assays for detecting ligand binding to a receptor are described, for example, in Mellentin-Micelotti et al., Anal. Biochem. 272:182-190 (1999); Zuck et al., Proc. Natl. Acad. Sci. USA 96:11122-11127 (1999); and Zhang et al., Anal. Biochem. 268;134-142 (1999). Examples of PKR binding assays are described in Lin et al., supra (2002) and in Masuda et al., supra (2002).

For certain applications of the invention methods it will be advantageous to employ a compound that modulates PKR signaling by a mechanism other than by acting as a prokineticin receptor agonist or antagonist.

Thus, in one embodiment, a compound that modulates PKR signaling is a compound that modulates endogenous prokineticin mRNA expression. A compound that modulates PK expression can be, for example, an expressible nucleic acid molecule that encodes a PK, or that serves to inhibit PK gene expression, such as an antisense nucleic acid molecule, a ribozyme or an RNA interference (RNAi) molecule. Methods of preparing such nucleic acid molecules and using them in ex vivo and in vivo applications are known in the art. For example, viral delivery systems are reviewed in Mah et al., Clin. Pharmacokinet. 41:901-911; non-viral delivery systems in Li et al., Curr. Gene Ther. 1:201-226 (2001); RNAi in Hannon, Nature 418:244-251 (2002); antisense in Galderisi et al., J. Cell Physiol. 181:251-257 (1999) and ribozymes in Lewin et al., Trends Mol. Med. 7:221-228 (2001)).

A compound that enhances endogenous PK mRNA expression can also be a compound identified by a screening assay. In an exemplary screening assay, an animal, tissue or cell that endogenously expresses a PK is contacted with one or more candidate compounds. PK mRNA can be detected and quantitated using methods known in the art, such as hybridization with a detectable probe or primer, or PCR, and the mRNA level compared to a control. Such methods are described, for example, in Sambrook et al., supra (2001) and Ausubel et al. supra (current supplement). Alternatively, PK peptide can be detected and quantitated using methods known in the art, such as by assaying direct or competitive binding to an antibody or receptor. Such methods are described, for example, in Harlow and Lane, supra (1988).

Alternatively, a screening assay to identify a compound that enhances endogenous PK mRNA expression can employ a PK promoter operatively linked to a nucleic acid molecule encoding a reporter gene, such as .beta.-lactamase, luciferase, green fluorescent protein or .beta.-galactosidase. In such assays, a cell transfected with the promoter-reporter construct is contacted with a candidate compound, and the expression of reporter gene product detected by methods known in the art which depend on the particular reporter.

The nucleotide sequences of PK1 and PK2 promoters are known in the art, and human and mouse promoter sequences are set forth herein as SEQ ID NOS:17-21. A smaller portion of a prokineticin promoter sequence can alternatively be used, such as a region of at least about 100 nucleotides, such as at least about 200, 250, 300, 500, 1000, 1500, 2000, 2500 or more nucleotides upstream of the transcriptional start site.

Both the PK1 promoter and the PK2 promoters contain presumptive cyclic-AMP response elements (CREs), in that they contain the core CRE consensus sequence CGTCA. In the human PK2 promoter, for example, the presumptive CRE is about 200 nucleotides upstream of the transcriptional start site. Thus, it is contemplated that compounds that modulate prokineticin mRNA expression include compounds known or expected to modulate cyclic-AMP production and/or MAP kinase activation, such as various growth factors.

In another embodiment, a compound that modulates PKR signaling is a compound that enhances endogenous prokineticin peptide production or secretion. Methods of detecting prokineticin production secretion can include bioassays, binding assays, immunoassays and the like, and can be adapted to prokineticins based on peptide assays known in the art and the guidance provided herein.

In a further embodiment, a compound that modulates PKR signaling can be a compound that modulates PKR mRNA or protein expression. A compound that modulates PKR expression can be, for example, an expressible nucleic acid molecule that encodes a PKR, or that serves to inhibit PKR gene expression, such as an antisense nucleic acid molecule, a ribozyme or an RNA interference (RNAi) molecule. Methods of preparing such nucleic acid molecules and using them are known in the art, as described above.

A compound that modulates PKR expression can also be a compound identified from a screening assay. In an exemplary screening assay, an animal, tissue or cell that endogenously expresses a PKR is contacted with one or more candidate compounds. Expression of PKR mRNA or protein can be detected and the level compared to a control using methods known in the art, such as methods analogous to those described above for detecting increased prokineticin mRNA or peptide expression.

The skilled person will appreciate that the above methods to identify compounds that modulate PKR signaling are exemplary. Alternative methods and mechanisms for modulating GPCR signaling by modulating abundance or activity of a PKR or its ligand are known in the art or can be determined based on the guidance herein.

In the screening assays described above, a candidate compound that is tested for its ability to modulate PKR signaling can be any type of biological or chemical molecule. For example, the candidate compound can be a naturally occurring macromolecule, such as a polypeptide, peptidomimetic, nucleic acid, carbohydrate, lipid, or any combination thereof. A candidate compound also can be a partially or completely synthetic derivative, analog or mimetic of such a macromolecule, or a small organic molecule, such as a molecule prepared by combinatorial chemistry methods. If desired in a particular assay format, a candidate compound can be detectably labeled or attached to a solid support.

Methods for preparing large libraries of compounds, including simple or complex organic molecules, metal-containing compounds, carbohydrates, peptides, proteins, peptidomimetics, glycoproteins, lipoproteins, nucleic acids, antibodies, and the like, are well known in the art and are described, for example, in Huse, U.S. Pat. No. 5,264,563; Francis et al., Curr. Opin. Chem. Biol. 2:422-428 (1998); Tietze et al., Curr. Biol., 2:363-371 (1998); Sofia, Mol. Divers. 3:75-94 (1998); Eichler et al., Med. Res. Rev. 15:481-496 (1995); and the like. Libraries containing large numbers of natural and synthetic compounds also can be obtained from commercial sources.

The number of different candidate compounds to test in the methods of the invention will depend on the application of the method. For example, one or a small number of candidate compounds can be advantageous in manual screening procedures, or when it is desired to compare efficacy among several compounds that are known or predicted to act as PKR modulatory compounds. However, it will be appreciated that the larger the number of candidate compounds, the greater the likelihood of identifying a compound having the desired activity in a screening assay. Additionally, large numbers of compounds can be processed in high-throughput automated screening assays. Therefore, the term "one or more candidate compounds" can refer, for example, to 2 or more, such as 5, 10, 15, 20, 50 or 100 or more different compounds, such as greater than about 10.sup.3, 10.sup.5 or 10.sup.7 different compounds. Such candidate compounds can be assayed simultaneously, such as in pools of compounds or in parallel assays, or sequentially.

Assay methods for identifying compounds that modulate PKR signaling generally involve comparison to a control. One type of a "control" is a preparation that is treated identically to the test preparation, except the control is not exposed to the candidate compound. Another type of "control" is a preparation that is similar to the test preparation, except that the control preparation does not express the receptor, or has been modified so as not to respond to a PK. In this situation, the response of the test preparation to a candidate compound is compared to the response (or lack of response) of the control preparation to the same compound under substantially the same reaction conditions.

The methods of the invention involve contacting a neural stem or progenitor cell with a compound that modulates PKR signaling under conditions in which the compound can modulate neurogenesis.

As used herein, the term "neurogenesis" refers to the process by which neural stem and progenitor cells give rise to more differentiated neural cells. Neurogenesis encompasses proliferation of neural stem and progenitor cells, differentiation of these cells into new neural cell types, as well as migration and survival of the new cells. The term is intended to cover neurogenesis as it occurs during normal development, as well as neural regeneration that occurs following disease, damage or therapeutic intervention. Neurogenesis is reviewed, for example, in Okano et al., J. Neurosci. Res. 69:698-707 (2002).

A number of factors have been shown to modulate neurogenesis in adult mammals. For example, learning and environment enrichment have been shown to enhance survival of neural stem and/or progenitor cells, whereas stress has been shown to diminish proliferation of these cells. In animal models of epilepsy and stroke, neurogenesis is enhanced. At the biochemical level, a number of molecules have been shown to influence neurogenesis. For example, the growth factors EGF, bFGF, VEGF, IGF-1, and the monoamine neurotransmitters have been shown to stimulate neurogenesis, while high levels of corticosterone, glutamate, gamma-aminobutyric acid, and opioid peptides diminish neurogenesis.

As used herein, the term "neural stem cell" refers to a cell of the central nervous system that can self-replicate and can also give rise to cells of a plurality of neural cell lineages, such as astrocytes, oligodendrocytes and neurons.

As used herein, the term "neural progenitor cell" refers to a cell of the central nervous system that can proliferate but is more lineage-restricted in comparison with a neural stem cell. Neural progenitor cells include neuronal progenitor cells, which produce neurons, and glial progenitor cells that produce astroglial and/or oligodendroglial cells.

Neurogenesis has been shown to occur throughout adulthood in several neurogenic areas of the mammalian central nervous system, including the olfactory bulb (OB), the dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ). Low levels of neurogenesis have also been reported in the Ammon's horn. The new neuronal cells in the adult mammalian OB are generated from neural progenitor cells in the anterior part of the SVZ. The SVZ is a narrow zone of tissue in the wall of the lateral ventricle in the forebrain. The neural progenitor cells of the SVZ migrate to the OB via the rostro-migratory stream (RMS), where they differentiate into interneurons of the OB, known as granule cells and periglomerular cells. The neural progenitor cells of the adult DG are generated in the subgranular zone (SGZ) of the DG and differentiate into neuronal and glial cells in the granular layer of the DG. The newly generated neuronal cells extend axons into the CA3 region of the hippocampus as soon as 4-10 days after mitosis (reviewed in Taupin and Gage, J. Neurosci. Res. 69:745-749 (2002)).

Neural stem and/or progenitor cells have been isolated from diverse areas of the adult central nervous system, including the subventricular zone (SVZ), hippocampus, septum, striatum, cortex, olfactory bulb, the rostral extension of the SVZ, in different levels of the spinal cord, including cervical, thoracic, lumbar, and sacral levels. In the spinal cord, neural stem and/or progenitor cells can be isolated from the periventricular area and the parenchyma. Neural stem and/or progenitor cells have also been isolated and cultured from adult postmortem neural tissues (reviewed in Taupin and Gage, supra (2002)), and are present in the inner nuclear layer of the adult retina.

Neural stem cells express a number of selective immunocytochemical markers, including Nestin, an intermediate filament protein (Hockfield and McKay, J. Neurosci. 5:3310-3328 (1985)); Musashil (Msil), an RNA-binding protein (Sakakibara et al., Dev. Biol. 176:230-242 (1996)); and Soxl, a transcription factor (Pevny et al., Development 125:1967-1978 (1998)). These markers are also expressed by neural progenitor cells, albeit at lower levels. Neural stem and progenitor cells are also characterized by their expression of the polysialylated form of the neural cell adhesion molecule (PSA-NCAM; Rousselot et al., J. Comp. Neurol. 351:51-61(1995)) and by their expression of the fibroblast growth factor (FGF) and epidermal growth factor (EGF) receptors (Gritti et al., J. Neurosci. 19:3287-3297 (1999)). Other characteristics of neural stem and progenitor cells are known in the art and reviewed, for example, in Okano et al., supra (2002).

Compounds that modulate neurogenesis can act by enhancing or reducing neural stem and/or progenitor cell proliferation, differentiation, survival or migration. Assays to identify compounds that modulate neurogenesis can be performed either ex vivo or in vivo, as described below.

A compound that modulates neurogenesis will generally promote neurogenesis in a suitable neurogenesis assay, as described below, by at least about 10%, such as at least 25%, 50%, 100%, 500% or more, or alternatively reduce neurogenesis in such an assay by at least about 10%, such as at least 25%, 50%, 90% or more, in comparison to a control compound.

Modulation of neurogenesis can be evidenced following contacting the cells with the compound alone, or with the compound in combination with another neurogenic modulatory factor, such as EGF, FGF, or VEGF. Modulation of neurogenesis can also be evidenced under normal ex vivo or in vivo conditions, or under conditions that mimic neural disease or damage.

Neural stem and/or progenitor cells suitable for use in the assay methods described below can be obtained from mammals, including humans (post-mortem or following surgery) and experimental animals (such as rodents, non-human primates, dogs, cats and the like). Neural stem and/or progenitor cells can also be obtained from other vertebrates, including reptiles, amphibians, fish and birds, or from invertebrates. The human or animal can be male or female, can be fetal, young, adult or old, and can be normal or exhibiting or susceptible to a neural disease or disorder.

For certain applications, tissue explants can be used, whereas for others, dissociated cells or neurospheres can be obtained. Tissue explants or cells can be obtained from any neural tissue that contains neural stem or progenitor cells, including brain and spinal tissues as described above. Exemplary tissues include olfactory bulb (OB), the dentate gyrus (DG) of the hippocampus and the subventricular zone (SVZ) of the lateral ventricles. Example III describes a method of obtaining neural stem and/or progenitor cells from the lateral ventricles of adult mice. Other methods are known in the art (see, for example, U.S. Pat. No. 5,753,506; and Gritti et al., J. Neurosci. 16:1091-1100 (1996)).

In addition to primary cells, neural stem and/or progenitor cell lines can be used in neurogenesis assays, such as MHP36 cells of mouse hippocampal origin (Gray et al., Philos. Trans. Royal Soc. Lond. B. Biol. Sci. 354:1407-1421 (1999)), CSM14.1 cells of rat mesencephalic origin (Haas et al., J. Anat. 201:61-69 (2002)), and embryonic stem cells differentiated along the neural lineage.

In ex vivo neurogenesis assays, either proliferation or differentiation, or both, can be readily assessed by methods known in the art. Proliferation and differentiation assays can also indirectly measure cell survival.

In an exemplary proliferation assay, a cell composition containing neural stem and/or progenitor cells can be cultured under conditions in which clonal spheroid colonies, or "neurospheres," form. These neurospheres can be visualized and counted under a light microscope. Neurospheres can then be dissociated, and the culture continued, such that the number of secondary and subsequent neurospheres can be determined as an indication of proliferation. Conditions for culturing and dissociating neurospheres are described in Example III.

In other exemplary proliferation assays, the cells are assessed for their ability to incorporate .sup.3H thymidine or bromodeoxyurine (BrdU, a thymidine analog), or assessed for their expression of proliferation markers, such as proliferating cell nuclear antigen (PCNA) or cdc2. Such proliferation assays are well known in the art and are described, for example, in Freshney, "Culture of Animal Cells: A Manual of Basic Technique" New York: Wiley-Liss, 4.sup.th ed. (2000).

Other types of proliferation assays suitable for determining whether a compound modulates neurogenesis are known in the art or can be determined using the guidance herein. The total number of cells, or number of labeled cells, observed in the presence of compound and in the absence of compound can be compared to determine if neurogenesis is modulated.

In an exemplary differentiation assay, cells are contacted with suitable primary antibodies specific for markers expressed by the type or types of cells it is desired to detect. Markers expressed by neural-stem and progenitor cells have been described above. As described in Example III, a marker for neurons is .beta.-tubulin, a marker for astrocytes is GFAP, and a marker for oligodendrocytes is O4. Other markers specific for cells of various neural lineages are known in the art, and antibodies thereto are commercially available or can be generated by known methods.

Unless the primary antibody is labeled, the cells are then generally contacted with labeled secondary antibodies, such as enzymatically or fluorescently labeled antibodies, and the cells visualized or sorted. Methods to immunolabel cells, as well as methods to detect and sort immunolabeled cells, are well known in the art and described, for example, in Freshney, supra (2000). The types and numbers of cells of each lineage of interest observed in the presence of compound and in the absence of compound can be compared to determine if neurogenesis is modulated.

Neurogenesis assays can be performed in vivo in a human or other mammal, vertebrate or invertebrate, as described above. Such assays are generally similar to ex vivo assays, except that the tissue of interest is generally fixed and sectioned prior to detection of the label or marker of interest. Example IV describes a method of labeling proliferating cells in vivo with BrdU and subsequently detecting BrdU-labeled cells, and cells expressing neural stage-specific differentiation markers, by immunohistochemistry.

Alternatively, and particularly where the human or animal is not sacrificed, detection of proliferation and differentiation markers can be done using radiolabeled antibodies and non-invasive imaging methods known in the art, such as single photon emission computed tomography (SPECT) and positron emission tomography (PET).

For in vivo applications, various delivery methods can be used to contact neural stem or progenitors within the tissue of interest with a compound, as described in more detail below with respect to therapeutic applications. The delivery method will depend on factors such as the tissue of interest, the nature of the compound (i.e. its stability and ability to cross the blood-brain barrier), and the duration of the experiment. As described in Example IV, for delivery of peptides and BrdU to the brain over a period of several weeks, an osmotic minipump can be implanted into a neurogenic region, such as the lateral ventricle. Alternatively, compounds can be administered by direct injection into the cerebrospinal fluid of the brain or spinal column, or into the eye. Compounds can also be administered into the periphery (such as by intravenous or subcutaneous injection, or oral delivery), and subsequently cross the blood-brain barrier.

Advantageously, in vivo methods allow the effect of compounds to be tested for their effect on neurogenesis both in normal subjects and in subjects having neural damage and disease. Either human subjects or experimental animal models can be used.

Experimental animal models of trauma due to stroke or neural injury are known in the art. One experimental model of stroke involves occluding the right middle cerebral artery and both common carotid arteries of rats for a short period, followed by reperfusion (Moore et al., J. Neurochem. 80:111-118). An experimenal model of CNS injury is the fluid percussion injury (FPI) model, in which moderate impact (1.5-2.0 atm) is applied to the parietal cerebral cortex (Akasu et al., Neurosci. Lett. 329:305-308 (2002). Experimental models of spinal cord injury are also used in the art (Scheifer et al., Neurosci. Lett. 323:117-120 (2002). Suitable models for neural damage due to oxidative stress, hypoxia, radiation and toxins are also known in the art.

Experimental animal models of human neurodegenerative diseases are also known in the art. Various experimental models of Alzheimer's disease are reviewed in Janus, Physiol. Behav. 73:873-886 (2001); models of Parkinson's disease are reviewed in Tolwani et al., Lab. Anim. Sci. 49:363-371 (1999); models of Huntington's disease are reviewed in Menalled et al., Trends Pharmacol. Sci. 23:32-39 (2002); and a SOD-1 transgenic model of amyotrophic lateral sclerosis is described in Ripps et al., Proc. Natl. Acad. Sci. USA 92:689-693 (1995). Other animal models for human neural degenerative diseases, including those described below with respect to therapeutic applications, are known in the art.

Experimental animal models of retinal neurogenesis are described, for example, in Marcus et al., Visual Neurosci. 16:417-424 (1999).

Experimental animal models of demyelinating diseases, such as experimental autoimmune encephalomyelitis (a model of multiple sclerosis), are also known in the art.

In any of the in vivo assays, neurogenesis in the presence of compound and in the absence of compound can be compared to determine if the compound modulates neurogenesis. Such in vivo assays can further provide evidence of safety, toxicity, pharmacokinetics and therapeutic efficacy of the compound of interest in preparation for human therapeutic use.

The compounds of the invention that modulate neurogenesis can be used directly as therapeutic agents to prevent or treat a variety of disorders of the nervous system in which it is beneficial to promote or inhibit neurogenesis. The compounds of the invention can also be used to promote neurogenesis ex vivo, such that a cell composition containing neural stem cells, neural progenitor cells, and/or more differentiated neural cells can subsequently be administered to an individual to prevent or treat the same indications.

Nervous system disorders that can be treated with the compounds of the invention include, but are not limited to, nervous system injuries, and diseases or disorders which result in either a disconnection of axons, a diminution or degeneration of neurons, or demyelination. Such diseases and disorders include, for example, the following lesions of the central nervous system (including spinal cord and brain) or peripheral nervous systems: (1) ischemic lesions, in which a lack of oxygen in a portion of the nervous system results in neuronal injury or death, including cerebral infarction or ischemia, or spinal cord infarction or ischemia; (2) traumatic lesions, including lesions caused by physical injury or associated with surgery, for example, lesions which sever a portion of the nervous system, or compression injuries; (3) malignant lesions, in which a portion of the nervous system is destroyed or injured by malignant tissue which is either a nervous system associated malignancy or a malignancy derived from non-nervous system tissue; (4) infectious lesions, in which a portion of the nervous system is destroyed or injured as a result of infection, for example, by an abscess or associated with infection by human immunodeficiency virus, herpes zoster, or herpes simplex virus or with Lyme disease, tuberculosis, or syphilis; (5) degenerative lesions, in which a portion of the nervous system is destroyed or injured as a result of a degenerative process including but not limited to, degeneration associated with Parkinson's disease, Alzheimer's disease, Huntington's chorea, amyotrophic lateral sclerosis (ALS) and retinal degeneration; (6) lesions associated with nutritional diseases or disorders, in which a portion of the nervous system is destroyed or injured by a nutritional disorder or disorder of metabolism including, but not limited to, vitamin B12 deficiency, folic acid deficiency, Wemicke disease, tobacco-alcohol amblyopia, Marchiafava-Bignami disease (primary degeneration of the corpus callosum), and alcoholic cerebellar degeneration; (7) neurological lesions associated with systemic diseases including, but not limited to, diabetes (diabetic neuropathy, Bell's palsy), systemic lupus erythematosus, carcinoma, or sarcoidosis; (8) lesions caused by toxic substances including alcohol, lead, or particular neurotoxins; and (9) demyelinated lesions in which a portion of the nervous system is destroyed or injured by a demyelinating disease including, but not limited to, multiple sclerosis, human immunodeficiency virus-associated myelopathy, transverse myelopathy or various etiologies, progressive multifocal leukoencephalopathy, and central pontine myelinolysis.

Because neurogenesis is involved in learning and memory, compounds of the invention can also be used in normal individuals to enhance learning and/or memory, or to treat individuals with defects in learning and/or memory.

Other conditions that can be beneficially treated with compounds that modulate neurogenesis are known in the art (see, for example, U.S. published application 20020106731).

It is expected that the compounds identified by the methods described herein as compounds that modulate PKR signaling and further modulate neurogenesis will have beneficial activities in addition to modulating neurogenesis. For example, PKR1 and/or PKR2 are expressed on a variety of tissues apart from the neural tissues described herein. Modulation of PKR signaling has been proposed to be beneficial to treat conditions relating to these tissues, such as disorders of gastrointestinal motility (see U.S. 20020115610A1), circadian rhythm (Cheng et al., Nature 417:405-410 (2002)) and angiogenesis (LeCouter et al., supra (2001)).

Stem cells have the ability to divide for indefinite periods in culture and to give rise to specialized cells. The fertilized egg is totipotent, meaning that its potential is total. After several cycles of cell division, these totipotent cells begin to specialize, forming a hollow sphere of cells, called a blastocyst. The cells of the inner cell mass of the blastocyst are pluripotent, and will go on to form many but not all types of cells necessary for fetal development. The pluripotent stem cells undergo further specialization into stem cells that are committed to give rise to cells that have a particular function. These cells are called multipotent stem cells. Examples of multipotent stem cells include hematopoietic stem cells, which give rise to red blood cells, white blood cells and platelets; hepatic stem cells, which differentiate into hepatocytes and biliary epithelial cells; myogenic stem cells, which differentiate into muscle; and neural stem cells described above. Multipotent stem cells have been identified in many other organs and tissues, including lung, heart, prostate, skin and gastrointestinal tract.

Because neural stem cells share certain features with other types of stem cells, such as certain surface markers (see, for example, Klassen et al., Neurosci. Lett. 312:180-182 (2001); Hunziker et al., Biochem. Biophys. Res. Commun. 271:116-119 (2000)), as well as the ability to self-replicate and differentiate, it is expected that signaling through prokineticin receptors, disclosed herein to be implicated in neurogenesis, is also important in modulating proliferation and differentiation of pluripotent and other multipotent stem cells. Accordingly, the methods described herein for identifying and using compounds that modulate neurogenesis can be readily applied to identifying and using compounds that modulate proliferation and differentiation of other stem cells in ex vivo and in vivo applications known in the art.

Those skilled in the art can determine other useful applications for compounds that modulate PKR signaling.

Compounds of the invention that modulate neurogenesis can be formulated and administered in a manner and in an amount appropriate for the condition to be treated; the weight, gender, age and health of the individual; the biochemical nature, bioactivity, bioavailability and side effects of the particular compound; and in a manner compatible with concurrent treatment regimens. An appropriate amount and formulation for a particular therapeutic application in humans can be extrapolated based on the activity of the compound in the ex vivo and in vivo neurogenesis assays described herein.

The total amount of a compound can be administered as a single dose or by infusion over a relatively short period of time, or can be administered in multiple doses administered over a more prolonged period of time. Additionally, the compound can be administered in a slow-release matrice, which can be implanted for systemic delivery at or near the site of the target tissue. Contemplated matrices useful for controlled release of compounds, including therapeutic compounds, are well known in the art, and include materials such as DepoFoam.TM., biopolymers, micropumps, and the like.

The invention compounds can be administered to a mammal by a variety of routes known in the art including, for example, intracerebrally, intraspinally, intravenously, intramuscularly, subcutaneously, intraorbitally, intracapsularly, intraperitoneally, intracisternally, intra-articularly, orally, intravaginally, rectally, topically, intranasally, or transdermally.

Generally, the invention compounds are administered to an animal as a pharmaceutical composition comprising the compound and a pharmaceutically acceptable carrier. The choice of pharmaceutically acceptable carrier depends on the route of administration of the compound and on its particular physical and chemical characteristics. Pharmaceutically acceptable carriers are well known in the art and include sterile aqueous solvents such as physiologically buffered saline, and other solvents or vehicles such as glycols, glycerol, oils such as olive oil and injectable organic esters. A pharmaceutically acceptable carrier can further contain physiologically acceptable compounds that stabilize the compound, increase its solubility, or increase its absorption. Such physiologically acceptable compounds include carbohydrates such as glucose, sucrose or detrains; antioxidants, such as ascorbic acid or glutathione; chelating agents; and low molecular weight proteins (see for example, "Remington's Pharmaceutical Sciences" 18th ed., Mack Publishing Co. (1990)).

For applications that require the compounds to cross the blood-brain barrier, or to cross cell membranes, formulations that increase the lipophilicity of the compound can be useful. For example, the compounds of the invention can be incorporated into liposomes (Gregoriadis, Liposome Technoloqy, Vols. I to III, 2nd ed. (CRC Press, Boca Raton Fla. (1993)). Liposomes, which consist of phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer. Other approaches for formulating a compound such that it crosses the blood-brain barrier are known in the art and include the use of nanoparticles, which are solid colloidal particles ranging in size from 1 to 1000 nm (Lockman et al., Drug Dev. Ind. Pharm. 28:1-13 (2002)), and peptides and peptidomimetics that serve as transport vectors (Pardridge, Nat. Rev. Drug Discov. 1:131-139 (2002).

Although drug delivery to the central nervous system poses unique challenges, the CNS is also a site of immune privilege. Thus, a compound that could elicit an immune response that precludes its use for many indications can still be an effective therapeutic if delivered into the CNS. Accordingly, compounds that are potentially immunogenic, such as chimeric prokineticin peptides, prokineticins from other species, and certain other PKR modulatory compounds, can be used as effective therapeutics to modulate neurogenesis in humans.

Besides peptides and compounds identified in drug screens, PKR modulatory compounds described herein include nucleic acid molecules that encode prokineticins or prokineticin receptors, as well as ribozymes, antisense molecules, RNAi and the like. Methods of formulating and delivering nucleic acid molecules to individuals for therapeutic use are known in the art. Useful vectors for delivering therapeutic nucleic acid molecules to the CNS include, for example, herpes simplex virus vectors, adenoviral vectors, VSV-G-pseudotyped retroviral vectors and adeno-associated viral vectors. Methods for delivering nucleic acid molecules to the CNS are reviewed, for example, in Hsich et al., Hum. Gene Ther. 13:579-604 (2002).

Cells that have been contacted with PKR modulatory compounds, including neural stem and progenitor cells, more differentiated cells, and genetically modified cells expressing PK, can be used in therapeutic applications described herein. Methods of formulating and delivering cells to individuals for therapeutic use are known in the art (see, for example, reviews by Cao et al., J. Neurosci. Res. 68:501-510 (2002), and Park et al., Gene Ther. 9:613-624 (2002)).

To enhance the modulation of neurogenesis, more than one therapeutic approach or composition can be provided to an individual or used ex vivo. For example, a compound that modulates PKR signaling can be used in conjunction with neurogenic growth factors, such as EGF and FGF, or in conjunction with conventional therapies for the disorder or condition being treated. The skilled clinician will be able to determine appropriate concurrent or sequential therapies for use in conjunction with the compounds-and methods of-the invention.

As described herein, PKR1, PKR2 and PK2 are expressed on cells within regions of the brain and retina that are neurogenically active. Accordingly, molecules that specifically bind these polypeptides can be used in either ex vivo or in vivo applications to label neurogenically active cells or regions. Molecules that specifically bind PKRs and PKs include their cognate ligands and receptors, respectively, as well as antibodies and binding compounds identified in the assays described herein.

Thus, the invention provides a method of labeling neural cells, by contacting a mixed cell population, tissue or animal with a ligand that binds a PKR or a PK, and detecting binding of said ligand to a cell within said population, tissue or animal.

The use of prokineticins and prokineticin receptors as markers is particularly advantageous in cases in which these markers are more abundant or more selective for particular cell types than other markers currently used to identify neural cells, such as the markers described above. The labeling method can be used in a variety of research and clinical applications, such as in identifying and distinguishing different types of cells, in diagnosing neural disorders, and in monitoring the efficacy of therapies with neurogenic agents. The skilled person can determine other useful applications for the compounds and methods described herein.
 

Claim 1 of 9 Claims

1. A method for modulating neurogenesis, comprising contacting a neural stem or progenitor cell with an effective amount of a prokineticin receptor (PKR) agonist compound wherein the PKR agonist is selected from the group consisting of SEQ ID NOS:5-11 and 14-15.

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