U.S. patent application number 10/691462 was filed with the patent office on 2004-07-22 for compounds and methods for stimulating beta-catenin mediated gene expression and differentiation.
This patent application is currently assigned to Adherex Technologies, Inc.. Invention is credited to Blaschuk, Orest W., Byers, Stephen, Gour, Barbara J..
Application Number | 20040142854 10/691462 |
Document ID | / |
Family ID | 23106829 |
Filed Date | 2004-07-22 |
United States Patent
Application |
20040142854 |
Kind Code |
A1 |
Blaschuk, Orest W. ; et
al. |
July 22, 2004 |
Compounds and methods for stimulating beta-catenin mediated gene
expression and differentiation
Abstract
Modulating agents for inhibiting degradation of cytoplasmic
.beta.-catenin are provided. The modulating agents comprise one or
more of: (1) the peptide sequence SYLDS(PO.sub.4)GIHS(PO.sub.4)G
(SEQ ID NO:1) or (2) a peptide analogue or peptidomimetic thereof.
Methods for using such modulating agents for stimulating
.beta.-catenin mediated gene expression and cellular
differentiation are provided.
Inventors: |
Blaschuk, Orest W.;
(Westmount, CA) ; Byers, Stephen; (Washington,
DC) ; Gour, Barbara J.; (Kemptville, CA) |
Correspondence
Address: |
SEED INTELLECTUAL PROPERTY LAW GROUP PLLC
701 FIFTH AVE
SUITE 6300
SEATTLE
WA
98104-7092
US
|
Assignee: |
Adherex Technologies, Inc.
Suite 220 600 Peter Morand Crescent
Ottawa
CA
K1G 5Z3
|
Family ID: |
23106829 |
Appl. No.: |
10/691462 |
Filed: |
October 21, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10691462 |
Oct 21, 2003 |
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09545433 |
Apr 5, 2000 |
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6706685 |
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09545433 |
Apr 5, 2000 |
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09288373 |
Apr 5, 1999 |
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Current U.S.
Class: |
424/134.1 ;
514/1.2; 514/1.4; 514/14.7; 514/16.4; 514/17.6; 514/18.3; 514/19.3;
514/2.4; 514/3.3; 514/3.7; 514/4.6; 514/4.9; 514/7.6; 514/9.7;
514/9.8 |
Current CPC
Class: |
A61P 17/14 20180101;
A61P 25/00 20180101; C07K 14/4705 20130101; A61K 38/00
20130101 |
Class at
Publication: |
514/007 |
International
Class: |
A61K 038/16 |
Claims
What is claimed is:
1. A method for stimulating activation of gene transcription in a
cell, comprising contacting a cell with a modulating agent capable
of inhibiting degradation of cytoplasmic .beta.-catenin, wherein
the agent comprises an internalization moiety and one or more of:
(a) the amino acid sequence SYLDS(PO.sub.4)GIHS(PO.sub.4)G (SEQ ID
NO:1); or (b) a peptide analogue or peptidomimetic of the amino
acid sequence SYLDS(PO.sub.4)GIHS(PO.sub.4)G (SEQ ID NO:1); and
thereby stimulating activation of gene transcription in the
cell.
2. A method according to claim 1, wherein the modulating agent
comprises the linear peptide sequence
SYLDS(PO.sub.4)GIHS(PO.sub.4)G (SEQ ID NO:1).
3. A method according to claim 1, wherein the internalization
moiety is a peptide internalization sequence.
4. A method according to claim 3, wherein the internalization
sequence comprises a sequence selected from the group consisting of
RQIKIWFQNRRMKWKK (SEQ ID NO:9), RQIKIWPQNRRNKWKK (SEQ ID NO:10) and
YGRKKRRQRRR (SEQ ID NO:14).
5. A method according to claim 4, wherein the modulating agent has
the sequence YGRKKRRQRRRGSYLDS(PO.sub.4)GIHS(PO.sub.4)G (SEQ ID
NO:15).
6. A method according to claim 4, wherein the modulating agent has
the sequence SYLDS(PO.sub.4)GIHS(PO.sub.4)GRQIKIWPQNRRNKWKK (SEQ ID
NO:12).
7. A method according to claim 1, wherein the internalization
moiety is a liposome.
8. A method according to claim 1, wherein the internalization
moiety is an antibody or ligand that specifically binds to a cell
surface receptor.
9. A method according to claim 1, wherein the modulating agent is
linked to a targeting agent.
10. A method according to claim 1, wherein the modulating agent is
present within a pharmaceutical composition comprising a
pharmaceutically acceptable carrier.
11. A method according to claim 1, wherein the activation of gene
transcription is mediated by a member of the Wnt signaling
cascade.
12. A method for stimulating differentiation of a cell, comprising
contacting a cell with a modulating agent capable of inhibiting
degradation of cytoplasmic .beta.-catenin, wherein the agent
comprises an internalization moiety and one or more of: (a) the
amino acid sequence SYLDS(PO.sub.4)GIHS(PO.sub.4)G (SEQ ID NO:1);
or (b) a peptide analogue or peptidomimetic of the amino acid
sequence SYLDS(PO.sub.4)GIHS(PO.sub.4- )G (SEQ ID NO:1); and
thereby stimulating differentiation of the cell.
13. A method according to claim 12, wherein the modulating agent
comprises the linear peptide sequence
SYLDS(PO.sub.4)GIHS(PO.sub.4)G (SEQ ID NO:1).
14. A method according to claim 12, wherein the internalization
moiety is an internalization sequence.
15. A method according to claim 14, wherein the internalization
sequence comprises a sequence selected from the group consisting of
RQIKIWFQNRRMKWKK (SEQ ID NO:9), RQIKIWPQNRRNKWKK (SEQ ID NO:10) and
YGRKKRRQRRR (SEQ ID NO:14).
16. A method according to claim 15, wherein the modulating agent
has the sequence YGRKKRRQRRRGSYLDS(PO.sub.4)GIHS(PO.sub.4)G (SEQ ID
NO:15).
17. A method according to claim 15, wherein the modulating agent
has the sequence SYLDS(PO.sub.4)GIHS(PO.sub.4)GRQIKIWPQNRRNKWKK
(SEQ ID NO:12).
18. A method according to claim 12, wherein the internalization
moiety is a liposome.
19. A method according to claim 12, wherein the internalization
moiety is an antibody or ligand that specifically binds to a cell
surface receptor.
20. A method according to claim 12, wherein the modulating agent is
linked to a targeting agent.
21. A method according to claim 12, wherein the modulating agent is
present within a pharmaceutical composition comprising a
pharmaceutically acceptable carrier.
22. A method according to claim 12, wherein the cell is a skin
cell.
23. A method according to claim 12, wherein the cell is a
keratinocyte.
24. A method for stimulating hair growth on a mammal, comprising
administering to a mammal a modulating agent capable of inhibiting
degradation of cytoplasmic .beta.-catenin, wherein the agent
comprises an internalization moiety and one or more of: (a) the
amino acid sequence SYLDS(PO.sub.4)GIHS(PO.sub.4)G (SEQ ID NO:1);
or (b) a peptide analogue or peptidomimetic of the amino acid
sequence SYLDS(PO.sub.4)GIHS(PO.sub.4- )G (SEQ ID NO:1); and
thereby stimulating hair growth or reducing hair loss on the
mammal.
25. A method according to claim 24, wherein the modulating agent
comprises the linear peptide sequence
SYLDS(PO.sub.4)GIHS(PO.sub.4)G (SEQ ID NO:1).
26. A method according to claim 24, wherein the internalization
moiety is an internalization sequence.
27. A method according to claim 26, wherein the internalization
sequence comprises a sequence selected from the group consisting of
RQIKIWFQNRRMKWKK (SEQ ID NO:9), RQIKIWPQNRRNKWKK (SEQ ID NO:10) and
YGRKKRRQRRR (SEQ ID NO:14).
28. A method according to claim 27, wherein the modulating agent
has the sequence YGRKKRRQRRRGSYLDS(PO.sub.4)GIHS(PO.sub.4)G (SEQ ID
NO:15).
29. A method according to claim 27, wherein the modulating agent
has the sequence SYLDS(PO.sub.4)GIHS(PO.sub.4)GRQIKIWPQNRRNKWKK
(SEQ ID NO:12).
30. A method according to claim 24, wherein the internalization
moiety is a liposome.
31. A method according to claim 24, wherein the internalization
moiety is an antibody or ligand that specifically binds to a cell
surface receptor.
32. A method according to claim 24, wherein the modulating agent is
linked to a targeting agent.
33. A method according to claim 24, wherein the modulating agent is
present within a pharmaceutical composition comprising a
pharmaceutically acceptable carrier.
34. A method according to claim 24, wherein the step of
administering comprises contacting skin cells with the modulating
agent.
35. A method according to claim 34, wherein the skin cells are
present on the scalp of the mammal.
36. A method according to claim 34, wherein the skin cells are
present within the ear of the mammal.
37. A method for stimulating exfoliation of skin on a mammal,
comprising administering to a mammal a modulating agent capable of
inhibiting degradation of cytoplasmic .beta.-catenin, wherein the
agent comprises an internalization moiety and one or more of: (a)
the amino acid sequence SYLDS(PO.sub.4)GIHS(PO.sub.4)G (SEQ ID
NO:1); or (b) a peptide analogue or peptidomimetic of the amino
acid sequence SYLDS(PO.sub.4)GIHS(PO.sub.4- )G (SEQ ID NO:1); and
thereby stimulating exfoliation of skin on the mammal.
38. A method according to claim 37, wherein the modulating agent
comprises the linear peptide sequence
SYLDS(PO.sub.4)GIHS(PO.sub.4)G (SEQ ID NO:1).
39. A method according to claim 37, wherein the internalization
moiety is an internalization sequence.
40. A method according to claim 39, wherein the internalization
sequence comprises a sequence selected from the group consisting of
RQIKIWFQNRRMKWKK (SEQ ID NO:9), RQIKIWPQNRRNKWKK (SEQ ID NO:10) and
YGRKKRRQRRR (SEQ ID NO:14).
41. A method according to claim 40, wherein the modulating agent
has the sequence YGRKKRRQRRRGSYLDS(PO.sub.4)GIHS(PO.sub.4)G (SEQ ID
NO:15).
42. A method according to claim 40, wherein the modulating agent
has the sequence SYLDS(PO.sub.4)GIHS(PO.sub.4)GRQIKIWPQNRRNKWKK
(SEQ ID NO:12).
43. A method according to claim 37, wherein the internalization
moiety is a liposome.
44. A method according to claim 37, wherein the internalization
moiety is an antibody or ligand that specifically binds to a cell
surface receptor.
45. A method according to claim 37, wherein the modulating agent is
linked to a targeting agent.
46. A method according to claim 37, wherein the modulating agent is
present within a pharmaceutical composition comprising a
pharmaceutically acceptable carrier.
47. A method according to claim 37, wherein the step of
administering comprises contacting skin cells with the modulating
agent.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
09/545,433, filed Apr. 5, 2000, now allowed; which is a
continuation-in-part of U.S. Ser. No. 09/288,373, filed Apr. 5,
1999, now abandoned, which applications are incorporated herein by
reference in their entireties.
TECHNICAL FIELD
[0002] The present invention relates generally to compounds and
methods for use in stimulating .beta.-catenin mediated gene
expression and cellular differentiation. The invention is more
specifically related to modulating agents capable of increasing the
level of free .beta.-catenin in a cell cytoplasm, and to
therapeutic methods employing such agents.
BACKGROUND OF THE INVENTION
[0003] .beta.-catenin is a cytoplasmic protein that is critical for
classical cadherin-mediated intercellular adhesion. Inside the
cell, a .beta.-catenin/.alpha.-catenin complex interacts with the
second cytoplasmic domain (CP2) of the classical cadherins. In the
absence of this .beta.-catenin/.alpha.-catenin complex, the
classical cadherins cannot promote cell adhesion (see Wheelock et
al., Current Topics in Membranes 43:169-185, 1996).
[0004] In addition to its role in cell adhesion, .beta.-catenin
also appears to be a key component of certain cellular signaling
pathways, leading to activation of gene expression and a variety of
developmental processes, such as differentiation. In particular,
.beta.-catenin functions in Wnt-mediated signaling, associating
with LEF-1/TCF DNA binding proteins to form a transcription factor
(see FIG. 2). The level of signal transduction appears to correlate
with the level of free .beta.-catenin in the cytoplasm of the cell
(see Willert and Nusse, Genetics and Development 8:95-102,1998).
Glycogen synthase kinase 3.beta. (GSK-3.beta.) and adenomatous
polyposis coli tumor suppressor protein (APC) interact with
cytoplasmic .beta.-catenin and facilitate its degradation via the
ubiquitin/proteosome pathway (see FIG. 2).
[0005] Wnt-mediated signaling is involved in a variety of
developmental processes, including cellular differentiation. For
example, skin cells expressing a stabilized form of .beta.-catenin
display increased hair growth (Gat et al., Cell 95:605-614,1998;
Ono et al., Cell 95:575-578,1998). Thus, therapies based on
increasing the level of free .beta.-catenin in the cytoplasm have
potential for stimulating Wnt-mediated signal transduction,
resulting in differentiation and, in certain instances, enhanced
hair growth. However, there are presently no available therapies
for modulating Wnt-mediated signaling.
[0006] Accordingly, there is a need in the art for improved methods
for inducing Wnt-mediated signal transduction and cellular
differentiation. The present invention fulfills this need and
further provides other related advantages.
SUMMARY OF THE INVENTION
[0007] The present invention provides methods for stimulating
.beta.-catenin mediated gene transcription and cellular
differentiation. Within certain aspects, the present invention
provides modulating agents capable of increasing the level of free
.beta.-catenin in a cell. In one such aspect, the modulating agent
comprises an internalization moiety and one or more of: (a) the
amino acid sequence SYLDS(PO.sub.4)GIHS(PO.sub.4)- G (SEQ ID NO:1)
or (b) a peptide analogue or peptidomimetic of the amino acid
sequence SYLDS(PO.sub.4)GIHS(PO.sub.4)G (SEQ ID NO:1). The
internalization moiety may comprise, within certain embodiments, an
internalization sequence covalently linked to the modulating agent,
a liposome that encapsulates the modulating agent or an antibody or
ligand that binds to a cell surface receptor. Within further
embodiments, any of the above modulating agents may be linked to a
targeting agent and/or a drug.
[0008] Within other aspects, the present invention provides
pharmaceutical compositions comprising a modulating agent as
described above, in combination with a pharmaceutically acceptable
carrier.
[0009] The present invention further provides, within other
aspects, methods for increasing the level of .beta.-catenin in a
cell, comprising contacting a cell with a modulating agent as
described above.
[0010] Within further related aspects, the present invention
provides methods for stimulating the activation of .beta.-catenin
mediated gene transcription in a cell, comprising contacting a cell
with a modulating agent as described above.
[0011] Within further related aspects, the present invention
provides methods for stimulating differentiation of a cell,
comprising contacting a cell with a modulating agent as described
above. In certain embodiments, the cell is a skin cell, such as a
keratinocyte.
[0012] In other aspects, methods are provided for stimulating hair
growth or reducing hair loss on a mammal, comprising administering
to a mammal a modulating agent as described above. Such
administration may be topical, and the skin cells may be present,
for example, on the scalp or within the ear of the mammal.
[0013] The present invention further provides, within other
aspects, methods for stimulating exfoliation of skin on a mammal,
comprising administering to a mammal a modulating agent as
described above.
[0014] These and other aspects of the invention will become evident
upon reference to the following detailed description and attached
drawings. All references disclosed herein are hereby incorporated
by reference in their entirety as if each were individually noted
for incorporation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0015] FIG. 1 presents an alignment of representative amino
terminal regions of .beta.-catenin from human, chicken, frog, mouse
and zebrafish (SEQ ID NOs:2 to 6 respectively). Sequences were
aligned using a Clustal W protein sequence alignment. Amino acids
are represented by their IUPAC amino acid codes, where X is any
amino acid and "-" represents a gap. The consensus sequence (60% or
better) is shown in italics (SEQ ID NO:7), and amino acid
capitalized within the consensus sequence represent identity. The
.beta.-catenin phosphorylation region is shown in bold.
[0016] FIG. 2 is a schematic diagram of .beta.-catenin cellular
functions. To enhance intercellular adhesion, .beta.-catenin
(.beta.) forms a complex with .alpha.-catenin (.alpha.),
alpha-actinin (a) and a cell surface cadherin. In the pathway
starting with Wnt, the inactivation of glycogen synthase kinase
3.beta. (GSK-3.beta.) leads to stabilization and accumulation of
cytoplasmic .beta.-catenin, which interacts with the Lef/Tcf
transcription factor, leading to target gene activation. In the
absence of Wnt signal, GSK-3.beta. binds to the adenomatous
polyposis coli tumor suppressor protein (APC)/.beta.-catenin
complex, leading to ubiquitination and degradation.
DETAILED DESCRIPTION OF THE INVENTION
[0017] As noted above, the present invention provides methods for
stimulating .beta.-catenin mediated gene transcription and cellular
differentiation. The present invention is based, in part, on the
discovery that agents comprising the sequence
SYLDS(PO.sub.4)GIHS(PO.sub.- 4)G (SEQ ID NO:1) and an
internalization moiety are capable of entering a cell and
inhibiting the degradation of cytoplasmic .beta.-catenin. The
resulting accumulation of .beta.-catenin in the cytoplasm functions
as a transcriptional activator, leading to responses such as
cellular differentiation. Modulating agents that comprise the
sequence SYLDS(PO.sub.4)GIHS(PO.sub.4)G (SEQ ID NO:1) as provided
herein may be used to stimulate .beta.-catenin mediated gene
transcription within a variety of contexts. For example, such
agents may be used to stimulate hair growth or to stimulate
exfoliation of skin.
[0018] Modulating Agents
[0019] As noted above, the term "modulating agent," as used herein,
refers to a molecule comprising one or more of (1) the peptide
sequence SYLDS(PO.sub.4)GIHS(PO.sub.4)G (SEQ ID NO:1) or (2) a
peptide analogue or peptidomimetic thereof. The above peptide
sequence is derived from .beta.-catenin (residues 29-38), and the
serine residues at positions 33 and 37 are phosphorylated, as
indicated by the (PO.sub.4) in the peptide sequence (i.e., the --OH
present within the serine side chains of the designated residues is
replaced by --O--PO.sub.3). A modulating agent is further capable
of inhibiting degradation of cytoplasmic .beta.-catenin, as
described herein. Within preferred embodiments, a modulating agent
further comprises an internalization moiety, which is associated
(covalently or noncovalently) with one or more of the above
components.
[0020] Peptide agents as described herein may, but need not,
contain additional amino acid residues from .beta.-catenin. Such
additional residues may flank the SYLDS(PO.sub.4)GIHS(PO.sub.4)G
(SEQ ID NO:1) sequence in a native .beta.-catenin molecule (i.e.,
may be adjacent to that sequence in a native .beta.-catenin
molecule). Flanking sequences for .beta.-catenin of a variety of
organisms are shown in SEQ ID NOs:2 to 7, and FIG. 1. Flanking
residue(s) may be present on the N-terminal and/or C-terminal side
of the above peptide sequence, preferably on both sides. A
modulating agent may consist entirely of a .beta.-catenin sequence,
or may additionally comprise further peptide and/or non-peptide
regions, such as regions that facilitate cyclization, purification
or other manipulation, and/or residues having a targeting or other
function. Agents comprising derivatives of
SYLDS(PO.sub.4)GIHS(PO.sub.4)G (SEQ ID NO:1) having one or more
side chain modifications are also contemplated. Modulating agents
may further be associated (covalently or noncovalently) with a
targeting agent, drug, solid support and/or detectable marker.
[0021] Modulating agents, or peptide portions thereof, may be
linear or cyclic peptides. A "linear" peptide is a peptide or salt
thereof that does not contain an intramolecular covalent bond
between two non-adjacent residues. Within preferred embodiments,
linear peptide modulating agents typically comprise from 10 to
about 20 amino acid residues derived from .beta.-catenin,
preferably from 10 to 16 amino acid residues derived from
.beta.-catenin.
[0022] The term "cyclic peptide," as used herein, refers to a
peptide or salt thereof that comprises an intramolecular covalent
bond between two non-adjacent residues, forming a cyclic peptide
ring that comprises the SYLDS(PO.sub.4)GIHS(PO.sub.4)G (SEQ ID
NO:1) sequence. The intramolecular bond may be a backbone to
backbone, side-chain to backbone or side-chain to side-chain bond
(i.e., terminal functional groups of a linear peptide and/or side
chain functional groups of a terminal or interior residue may be
linked to achieve cyclization). Preferred intramolecular bonds
include, but are not limited to, disulfide bonds; amide bonds
between terminal functional groups, between residue side chains or
between one terminal functional groups and one residue side chain;
thioether bonds and .delta..sub.1,.delta..sub.1-ditryptophan or a
derivative thereof. Preferred cyclic peptide modulating agents
generally contain 10 to 15 residues within the cyclic peptide
ring.
[0023] As noted above, modulating agents may be polypeptides or
salts thereof, containing only amino acid residues linked by
peptide bonds, or may additionally comprise non-peptide regions,
such as linkers. Peptide regions of a modulating agent may comprise
residues of L-amino acids, D-amino acids, or any combination
thereof. Amino acids may be from natural or non-natural sources,
provided that at least one amino group and at least one carboxyl
group are present in the molecule; .alpha.- and .beta.-amino acids
are generally preferred. The 20 L-amino acids commonly found in
proteins are identified herein by the conventional three-letter or
one-letter abbreviations shown in Table 1.
1TABLE 1 AMINO ACID ONE-LETTER AND THREE-LETTER ABBREVIATIONS A Ala
Alanine R Arg Arginine D Asp Aspartic acid N Asn Asparagine C Cys
Cysteine Q Gln Glutamine E Glu Glutamic acid G Gly Glycine H His
Histidine I Ile Isoleucine L Leu Leucine K Lys Lysine M Met
Methionine F Phe Phenylalanine P Pro Proline S Ser Serine T Thr
Threonine W Trp Tryptophan Y Tyr Tyrosine V Val Valine
[0024] A modulating agent may also contain rare amino acids (such
as 4-hydroxyproline or hydroxylysine), organic acids or amides
and/or derivatives of common amino acids, such as amino acids
having the C-terminal carboxylate esterified (e.g., benzyl, methyl
or ethyl ester) or amidated and/or having modifications of the
N-terminal amino group (e.g., acetylation or alkoxycarbonylation),
with or without any of a wide variety of side-chain modifications
and/or substitutions (e.g., methylation, benzylation, t-butylation,
tosylation, alkoxycarbonylation, and the like). Preferred
derivatives include amino acids having a C-terminal amide group.
Residues other than common amino acids that may be present with a
modulating agent include, but are not limited to,
2-mercaptoaniline, 2-mercaptoproline, omithine, diaminobutyric
acid, .alpha.-aminoadipic acid, m-aminomethylbenzoic acid and
.alpha.,.beta.-diaminopropionic acid.
[0025] As noted above, a modulating agent may comprise a peptide
analogue or a non-peptide peptidomimetic of a
SYLDS(PO.sub.4)GIHS(PO.sub.4)G (SEQ ID NO:1) sequence, provided
that the analogue or peptidomimetic retains the ability to
stimulate a .beta.-catenin mediated response. In general, a peptide
analogue may contain conservative substitutions such that the
ability to stimulate a .beta.-catenin mediated response is not
substantially diminished. A "conservative substitution" is one in
which an amino acid is substituted for another amino acid that has
similar properties, such that one skilled in the art of peptide
chemistry would expect the secondary structure and hydropathic
nature of the polypeptide to be substantially unchanged. Amino acid
substitutions may generally be made on the basis of similarity in
polarity, charge, solubility, hydrophobicity, hydrophilicity and/or
the amphipathic nature of the residues. For example, negatively
charged amino acids include aspartic acid and glutamic acid;
positively charged amino acids include lysine and arginine; and
amino acids with uncharged polar head groups having similar
hydrophilicity values include leucine, isoleucine and valine;
glycine and alanine; asparagine and glutamine; and serine,
threonine, phenylalanine and tyrosine. Other groups of amino acids
that may represent conservative changes include: (1) ala, pro, gly,
glu, asp, gin, asn, ser, thr; (2) cys, ser, tyr, thr; (3) val, ile,
leu, met, ala, phe; (4) lys, arg, his; and (5) phe, tyr, trp, his.
The critical determining feature of a peptide analogue is the
ability to inhibit degradation of cytosolic .beta.-catenin. Such an
ability may be evaluated using the representative assays provided
herein.
[0026] A peptidomimetic is a non-peptide compound that is
structurally similar to the peptide sequence
SYLDS(PO.sub.4)GIHS(PO.sub.4)G (SEQ ID NO:1), such that the
peptidomimetic retains the ability to stimulate a .beta.-catenin
mediated response, as described below. Peptidomimetics are organic
compounds that mimic the three-dimensional shape of the peptide
sequence SYLDS(PO.sub.4)GIHS(PO.sub.4)G (SEQ ID NO:1).
Peptidomimetics may be designed based on techniques that evaluate
the three dimensional shape, such as nuclear magnetic resonance
(NMR) and computational techniques. NMR is widely used for
structural analysis of molecules. Cross-peak intensities in nuclear
Overhauser enhancement (NOE) spectra, coupling constants and
chemical shifts depend on the conformation of a compound. NOE data
provide the interproton distance between protons through space.
This information may be used to facilitate calculation of the
lowest energy conformation for the peptide sequence
SYLDS(PO.sub.4)GIHS(PO.sub.4)G (SEQ ID NO:1). Once the lowest
energy conformation is known, the three-dimensional shape to be
mimicked is known. It should be understood that, within embodiments
described herein, an analogue or peptidomimetic may be substituted
for the sequence SYLDS(PO.sub.4)GIHS(PO.sub.4)G (SEQ ID NO:1).
[0027] Peptide modulating agents (and peptide portions of
modulating agents) as described herein may be synthesized by
methods well known in the art, including chemical synthesis and
recombinant DNA methods. For modulating agents up to about 50
residues in length, chemical synthesis may be performed using
solution phase or solid phase peptide synthesis techniques, in
which a peptide linkage occurs through the direct condensation of
the .alpha.-amino group of one amino acid with the .alpha.-carboxy
group of the other amino acid with the elimination of a water
molecule. Peptide bond synthesis by direct condensation, as
formulated above, requires suppression of the reactive character of
the amino group of the first and of the carboxyl group of the
second amino acid. The masking substituents must permit their ready
removal, without inducing breakdown of the labile peptide
molecule.
[0028] In solution phase synthesis, a wide variety of coupling
methods and protecting groups may be used (see Gross and
Meienhofer, eds., "The Peptides: Analysis, Synthesis, Biology,"
Vol. 1-4 (Academic Press, 1979); Bodansky and Bodansky, "The
Practice of Peptide Synthesis," 2d ed. (Springer Verlag, 1994)). In
addition, intermediate purification and linear scale up are
possible. Those of ordinary skill in the art will appreciate that
solution synthesis requires consideration of main chain and side
chain protecting groups and activation method. In addition, careful
segment selection is necessary to minimize racemization during
segment condensation. Solubility considerations are also a
factor.
[0029] Solid phase peptide synthesis uses an insoluble polymer for
support during organic synthesis. The polymer-supported peptide
chain permits the use of simple washing and filtration steps
instead of laborious purifications at intermediate steps.
Solid-phase peptide synthesis may generally be performed according
to the method of Merrifield et al., J. Am. Chem. Soc. 85:2149,
1963, which involves assembling a linear peptide chain on a resin
support using protected amino acids. Solid phase peptide synthesis
typically utilizes either the Boc or Fmoc strategy. The Boc
strategy uses a 1% cross-linked polystyrene resin. The standard
protecting group for .alpha.-amino functions is the
tert-butyloxycarbonyl (Boc) group. This group can be removed with
dilute solutions of strong acids such as 25% trifluoroacetic acid
(TFA). The next Boc-amino acid is typically coupled to the amino
acyl resin using dicyclohexylcarbodiimide (DCC). Following
completion of the assembly, the peptide-resin is treated with
anhydrous HF to cleave the benzyl ester link and liberate the free
peptide. Side-chain functional groups are usually blocked during
synthesis by benzyl-derived blocking groups, which are also cleaved
by HF. The free peptide is then extracted from the resin with a
suitable solvent, purified and characterized. Newly synthesized
peptides can be purified, for example, by gel filtration, HPLC,
partition chromatography and/or ion-exchange chromatography, and
may be characterized by, for example, mass spectrometry or amino
acid sequence analysis. In the Boc strategy, C-terminal amidated
peptides can be obtained using benzhydrylamine or
methylbenzhydrylamine resins, which yield peptide amides directly
upon cleavage with HF.
[0030] In the procedures discussed above, the selectivity of the
side-chain blocking groups and of the peptide-resin link depends
upon the differences in the rate of acidolytic cleavage. Orthoganol
systems have been introduced in which the side-chain blocking
groups and the peptide-resin link are completely stable to the
reagent used to remove the .alpha.-protecting group at each step of
the synthesis. The most common of these methods involves the
9-fluorenylmethyloxycarbonyl (Fmoc) approach. Within this method,
the side-chain protecting groups and the peptide-resin link are
completely stable to the secondary amines used for cleaving the
N-.alpha.-Fmoc group. The side-chain protection and the
peptide-resin link are cleaved by mild acidolysis. The repeated
contact with base makes the Merrifield resin unsuitable for Fmoc
chemistry, and p-alkoxybenzyl esters linked to the resin are
generally used. Deprotection and cleavage are generally
accomplished using TFA.
[0031] Within preferred synthesis methods, a phosphorylated peptide
modulating agent such as
N-Ac-SYLDS(PO.sub.4)GIHS(PO.sub.4)G-NH.sub.2 (SEQ ID NO:1) may be
prepared using solid phase peptide synthesis techniques that allow
selective phosphorylation of hydroxy-containing residues of the
peptide. Such a peptide may be assembled using Boc or Fmoc-amino
acid-OPfp (pentafluorophenyl) and amino-acid-ODhbt
(3,4-Dihydro-3-hydroxy-4-oxo-1,2,3-benztriazine) activated esters.
Their high reactivity and selectivity towards amino groups allows
the introduction of serine, threonine, and tyrosine without
side-chain protection. This strategy allows the incorporation of
O-unprotected serine residues in the peptide chain at specific
positions for later phosphorylation and incorporation of protected
serine residues when no phosphorylation is needed. Phosphorylation
of the unprotected amino acid side-chain hydroxyl groups may be
carried out on the solid support after the assembly of the linear
peptide is complete. Alternatively, the peptide is prepared by
phosphorylating the serine residues of cleaved, purified peptides
or using appropriately protected, phosphorylated serine residues
that can be incorporated into the peptide like any regular amino
acid.
[0032] For protection, t-Butyloxycarbonyl (Boc) may be used as the
protecting group used for the histidine side-chain, and the t-butyl
ester may be used to protect the side chains of aspartic acid,
tyrosine and the serine residues which are not to be
phosphorylated. The appropriate pentafluorophenyl ester of the
unprotected serine may be made in situ from Fmoc-serine and
pentaflurophenol with DIC in DMF. The N-terminal amino acid can be
protected with Boc when the free amine is required on the
N-terminus or with an acetyl group if the N-acetylated peptide is
required after cleavage. Upon completion of the peptide chain
assembly, the free hydroxyl groups of the serine residues may be
phosphorylated with dibenzylphosphochloridate. Cleavage of
protecting groups may be carried out by suspending the resin in a
mixture of TFA, phenol and anisole.
[0033] Another method to prepare such peptides includes
phosphorylating the serine residues of cleaved, purified peptides
while other amino acids are protected. For example, a linear
peptide can be assembled in solution, using Boc amino acids as
activated esters (hydroxybenztriazole ester). The Boc group can be
removed with saturated HCl in ether. The side-chain of the serine
to be phosphorylated can be protected with a benzyl group and then
introduced into the sequence. In order to phosphorylate the
peptide, the serine side chain may be deprotected by hydrogenolysis
using palladium black in methanol and acetic acid, followed by
phosphitylation using diethylaminobenzylphosphoramidite in the
presence of tetrazole. The resulting phosphite is immediately
oxidized with excess tert-butylhydroperoxide. Final deprotection is
achieved by acidolysis with trifluromethanesulfonic acid in
trifluoroacetic acid to remove all protecting groups from the
peptide.
[0034] Yet another method for preparation of phosphorylated
peptides involves the use of appropriately protected phosphorylated
serine residues that can be incorporated into the peptide, as would
be a regular amino acid. A protected phosphoester serine that is
stable under the conditions of solid phase peptide synthesis has to
be prepared. This can be done by different phosphorylating agents
such as aryl or alkyl phosphorochloridate, phosphorochloridite, and
N,N-dialkyl phosphoramidite. The last two methods require
subsequent in situ oxidation of phosphite triester intermediates
into phosphate triesters. The choice of phosphate protecting groups
is crucial for the synthesis of phosphopeptides. Phenyl and benzyl
groups are widely used and both are easily removed by catalytic
hydrogenation. Both liquid and solid-phase Boc methodologies can be
utilized to prepare the phosphopeptide using Merrifield resin.
These may require repetitive treatments with TFA to remove Boc
protecting groups and with HF for the final cleavage, followed by
catalytic hydrogenation to remove phosphate protecting groups.
[0035] It will be apparent to those of ordinary skill in the art
that modifications may be made to the synthesis procedures
described herein, provided that deprotection and coupling reactions
go to completion and the side-chain blocking groups are stable
throughout the entire synthesis. In addition, solid phase synthesis
is generally most suitable when peptides are to be made on a small
scale.
[0036] Acetylation of the N-terminus can be accomplished by
reacting the final peptide with acetic anhydride before cleavage
from the resin. C-amidation may be accomplished using an
appropriate resin such as methylbenzhydrylamine resin using the Boc
technology.
[0037] Following synthesis of a linear peptide, cyclization may be
achieved if desired by any of a variety of techniques well known in
the art. Within one embodiment, a bond may be generated between
reactive amino acid side chains. For example, a disulfide bridge
may be formed from a linear peptide comprising two thiol-containing
residues by oxidizing the peptide using any of a variety of
methods. Within one such method, air oxidation of thiols can
generate disulfide linkages over a period of several days using
either basic or neutral aqueous media. The peptide is used in high
dilution to minimize aggregation and intermolecular side reactions.
This method suffers from the disadvantage of being slow but has the
advantage of only producing H.sub.2O as a side product.
Alternatively, strong oxidizing agents such as 12 and
K.sub.3Fe(CN).sub.6 can be used to form disulfide linkages. Those
of ordinary skill in the art will recognize that care must be taken
not to oxidize the sensitive side chains of Met, Tyr, Trp or His.
Cyclic peptides produced by this method require purification using
standard techniques, but this oxidation is applicable at acid pHs.
Oxidizing agents also allow concurrent deprotection/oxidation of
suitable S-protected linear precursors to avoid premature,
nonspecific oxidation of free cysteine.
[0038] DMSO, unlike I.sub.2 and K.sub.3Fe(CN).sub.6, is a mild
oxidizing agent which does not cause oxidative side reactions of
the nucleophilic amino acids mentioned above. DMSO is miscible with
H.sub.2O at all concentrations, and oxidations can be performed at
acidic to neutral pHs with harmless byproducts.
Methyltrichlorosilane-diphenylsulfoxide may alternatively be used
as an oxidizing agent, for concurrent deprotection/oxidation of
S-Acm, S-Tacm or S-t-Bu of cysteine without affecting other
nucleophilic amino acids. There are no polymeric products resulting
from intermolecular disulfide bond formation. Suitable
thiol-containing residues for use in such oxidation methods
include, but are not limited to, cysteine, .beta.,.beta.-dimethyl
cysteine (penicillamine or Pen), .beta.,.beta.-tetramethylene
cysteine (Tmc), .beta.,.beta.-pentamethylene cysteine (Pmc),
.beta.-mercaptopropionic acid (Mpr),
.beta.,.beta.-pentamethylene-.beta.-mercaptopropionic acid (Pmp),
2-mercaptobenzene, 2-mercaptoaniline and 2-mercaptoproline.
[0039] Within another embodiment, cyclization may be achieved by
amide bond formation. For example, a peptide bond may be formed
between terminal functional groups (i.e., the amino and carboxy
termini of a linear peptide prior to cyclization), with or without
an N-terminal acetyl group and/or a C-terminal amide. Within
another such embodiment, the linear peptide comprises a D-amino
acid. Alternatively, cyclization may be accomplished by linking one
terminus and a residue side chain or using two side chains, with or
without an N-terminal acetyl group and/or a C-terminal amide.
Residues capable of forming a lactam bond include lysine, omithine
(Orn), amino adipic acid, m-aminomethylbenzoic acid,
.alpha.,.beta.-diaminopropionic acid, glutamate or aspartate.
[0040] Methods for forming amide bonds are well known in the art
and are based on well established principles of chemical
reactivity. Within one such method, carbodiimide-mediated lactam
formation can be accomplished by reaction of the carboxylic acid
with DCC, DIC, EDAC or DCCI, resulting in the formation of an
O-acylurea that can be reacted immediately with the free amino
group to complete the cyclization. The formation of the inactive
N-acylurea, resulting from O.fwdarw.N migration, can be
circumvented by converting the O-acylurea to an active ester by
reaction with an N-hydroxy compound such as 1-hydroxybenzotriazole,
1-hydroxysuccinimide, 1-hydroxynorbornene carboxamide or ethyl
2-hydroximino-2-cyanoacetate. In addition to minimizing O.fwdarw.N
migration, these additives also serve as catalysts during
cyclization and assist in lowering racemization. Alternatively,
cyclization can be performed using the azide method, in which a
reactive azide intermediate is generated from an alkyl ester via a
hydrazide. Hydrazinolysis of the terminal ester necessitates the
use of a t-butyl group for the protection of side chain carboxyl
functions in the acylating component. This limitation can be
overcome by using diphenylphosphoryl acid (DPPA), which furnishes
an azide directly upon reaction with a carboxyl group. The slow
reactivity of azides and the formation of isocyanates by their
disproportionation restrict the usefulness of this method. The
mixed anhydride method of lactam formation is widely used because
of the facile removal of reaction by-products. The anhydride is
formed upon reaction of the carboxylate anion with an alkyl
chloroformate or pivaloyl chloride. The attack of the amino
component is then guided to the carbonyl carbon of the acylating
component by the electron donating effect of the alkoxy group or by
the steric bulk of the pivaloyl chloride t-butyl group, which
obstructs attack on the wrong carbonyl group. Mixed anhydrides with
phosphoric acid derivatives have also been successfully used.
Alternatively, cyclization can be accomplished using activated
esters. The presence of electron withdrawing substituents on the
alkoxy carbon of esters increases their susceptibility to
aminolysis. The high reactivity of esters of p-nitrophenol,
N-hydroxy compounds and polyhalogenated phenols has made these
"active esters" useful in the synthesis of amide bonds. The last
few years have witnessed the development of
benzotriazolyloxytris-(dimethylamino)phosphonium
hexafluorophosphonate (BOP) and its congeners as advantageous
coupling reagents. Their performance is generally superior to that
of the well established carbodiimide amide bond formation
reactions.
[0041] Within a further embodiment, a thioether linkage may be
formed between the side chain of a thiol-containing residue and an
appropriately derivatized .alpha.-amino acid. By way of example, a
lysine side chain can be coupled to bromoacetic acid through the
carbodiimide coupling method (DCC, EDAC) and then reacted with the
side chain of any of the thiol containing residues mentioned above
to form a thioether linkage. In order to form dithioethers, any two
thiol containing side-chains can be reacted with dibromoethane and
diisopropylamine in DMF. Cyclization may also be achieved using
.delta..sub.1,.delta..sub.1-ditryptophan.
[0042] For longer peptide modulating agents, recombinant methods
are preferred for synthesis. Within such methods, all or part of a
modulating agent can be synthesized in living cells, using any of a
variety of expression vectors known to those of ordinary skill in
the art to be appropriate for the particular host cell. Suitable
host cells may include bacteria, yeast cells, mammalian cells,
insect cells, plant cells, algae and other animal cells (e.g.,
hybridoma, CHO, myeloma). The DNA sequences expressed in this
manner may encode portions of an endogenous .beta.-catenin and/or
other sequences. Endogenous .beta.-catenin sequences may be
prepared based on known cDNA or genomic sequences (see Wheelock et
al., Current Topics in Membranes 43:169-185,1996), which may be
isolated by screening an appropriate library with probes designed
based on such known sequences. Screens may generally be performed
as described in Sambrook et al., Molecular Cloning: A Laboratory
Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y.,
1989 (and references cited therein). Polymerase chain reaction
(PCR) may also be employed, using oligonucleotide primers in
methods well known in the art, to isolate nucleic acid molecules
encoding all or a portion of an endogenous .beta.-catenin. To
generate a nucleic acid molecule encoding a desired modulating
agent, an endogenous .beta.-catenin sequence may be modified using
well known techniques. Alternatively, portions of the desired
nucleic acid sequences may be synthesized using well known
techniques, and then ligated together to form a sequence encoding
the modulating agent.
[0043] Within preferred embodiments, a modulating agent comprises
an internalization moiety. An internalization moiety is any moiety
(such as a compound, liposome or particle) that can be used to
improve the ability of an agent to penetrate the lipid bilayer of
the cellular plasma membrane, thus enabling the agent to readily
enter the cytoplasm. As used herein, the term "associated with"
refers to covalent attachment or a non-covalent interaction
mediated by, for example, ionic bonds, hydrogen bonds, van der
waals forces and/or hydrophobic interactions, such that the
internalization moiety and modulating agent remain in close
proximity under physiological conditions.
[0044] Within certain embodiments, an internalization moiety is a
peptide internalization sequence. An internalization sequence may
be any sequence (generally a peptide sequence) that is capable of
facilitating entry of the modulating agent into the cytosol of a
living cell. One suitable internalization sequence is a 16 amino
acid peptide derived from the third helix of the Antennapedia
protein, and having the sequence RQIKIWFQNRRMKWKK (SEQ ID NO:9; see
Prochiantz, Curr. Op. Neurobiol. 6:629-34, 1996) or
RQIKIWPQNRRNKWKK (SEQ ID NO:10). Analogues of this sequence (i.e.,
sequences having at least 25% sequence identity, such that the
ability to facilitate entry into the cytosol is not diminished) may
also be employed. One such analogue is KKWKKWWKKWWKKWKK (SEQ ID
NO:11). One preferred modulating agent that comprises a covalently
linked Antennapedia internalization sequence has the sequence
SYLDS(PO.sub.4)GIHS(PO.sub.4)GRQIKIWFQNRRNKWKK (SEQ ID NO:12).
[0045] Alternatively, an internalization sequence may be unrelated
to the Antennapedia sequence. Any sequence that facilitates entry
to the cell, via a cell surface receptor or other means, may be
employed. Protein-derived helical peptide sequences that may be
used as internalization sequences include, but are not limited to,
KLALKLALKLAKAALKLA (SEQ ID NO:13; see Oehike et al., Biochim.
Biophys. Acta 1414:127-139,1998, and references cited therein).
Other internalization sequences include the 11 amino acid TAT
protein transduction domain YGRKKRRQRRR (SEQ ID NO:14; see Nagahara
et al., Nature Medicine 4:1449-1452,1998) and the transduction
domain of HSV VP22 (see Elliot and O'Hare, Cell 88:223-244, 1997).
One preferred modulating agent comprising the TAT protein
transduction domain is YGRKKRRQRRRGSYLDS(PO.sub.4)GIHS(PO.sub.4)G
(SEQ ID NO:15).
[0046] In general, the ability of a sequence to facilitate entry
into the cytosol may be evaluated in any of a variety of ways. For
example, a candidate internalization sequence may be covalently
linked to the sequence SYLDS(PO.sub.4)GIHS(PO.sub.4)G (SEQ ID NO:1)
and contacted with cells. The ability of such a construct to
stimulate a .alpha.-catenin mediated response, as described herein,
may then be assessed. Alternatively, the ability of a candidate
internalization sequence to cross the plasma membrane may be
assessed directly using any assay known in the art. Within such any
assay, an internalization sequence should result in a response that
is statistically greater than that observed in the absence of
internalization sequence. Preferably, an internalization sequence
incorporated into a modulating agent results in a response that is
comparable to, or greater than, that observed for the modulating
agent comprising an internalization sequence derived from TAT, as
described above.
[0047] An internalization sequence may be covalently linked to the
remainder of a modulating agent. Such linkage may be generated
using any of a variety of means well known in the art, either
directly or by way of a spacer. In general, spacers may be amino
acid residues (e.g., amino hexanoic acid) or peptides, or may be
other bi- or multi-functional compounds that can be covalently
linked to at least two peptide sequences. Covalent linkage may be
achieved via direct condensation or other well known
techniques.
[0048] Other internalization moieties may be covalently or
noncovalently linked to the remainder of the modulating agent. For
example, the .beta.-catenin derived portion of the modulating agent
may be encapsulated by the liposome (i.e., an artificial membrane
vesicle), using well known technology. Other internalization
moieties include, but are not limited to, antibodies and ligands
that bind to cell surface receptors. Alternatively, a
polynucleotide encoding a modulating agent may be incorporated into
an appropriate viral vector, such that the modulating agent is
generated within the target cell. Various particle-mediated
delivery systems are also available, and their use is well known to
those of ordinary skill in the art.
[0049] Evaluation of Modulating Agent Activity
[0050] As noted above, modulating agents are capable of inhibiting
degradation of cytoplasmic .beta.-catenin. This ability may
generally be evaluated using any suitable assay known to those of
ordinary skill in the art. For example, an immunoprecipitation
assay as described herein may be employed. Within such an assay,
decreased degradation of .beta.-catenin is measured by assessing
the ability of an antibody directed against .beta.-catenin to
immunoprecipitate full length .beta.-catenin from cell lysates in
the presence and absence of the modulating agent. For example,
mammalian cells that express .beta.-catenin (see Pishvaian et al.,
Cancer Res. 59:947-952, 1999) may be homogenized in the presence
and absence of modulating agent. An antibody directed against
.beta.-catenin is used to immunoprecipitate .beta.-catenin from the
homogenate, and a Western blot analysis of immunoprecipitated
protein is used to evaluate the level of full length
.beta.-catenin. In general, a modulating agent should increase the
level of cytoplasmic .beta.-catenin by at least 50%.
[0051] Alternatively, or in addition, the effect of a modulating
agent on any of a variety of .beta.-catenin-mediated processes may
be evaluated. The effect of a modulating agent on .beta.-catenin
mediated gene transcription may be determined using any appropriate
assay, such as the keratinocyte differentiation assay provided
herein. Briefly, keratinocytes may be treated with a candidate
modulating agent (e.g., 1 mg/ml for 48 hours). Treated and
untreated cells are then photographed. At a concentration of 1
mg/ml, a modulating agent should detectably induce the formation of
terminally differentiated cells known as squams, which may be
identified based on detachment from the substratum, and
morphological alterations that are well known to those of ordinary
skill in the art.
[0052] Other suitable assays are those designed to detect changes
in hair growth. Such assays may be performed using plucked hair or
hair follicles cultured in vitro. Such assays are described, for
example, within U.S. Pat. Nos. 5,527,772 and 5,739,111. For assays
using hair, the effect of a modulating agent may be determined
based on DNA content in the hair. Increased DNA content should be
observed in hair cultured for 48 hours in the presence of 1 mg/ml
modulating agent, relative to hair cultured in the absence of
modulating agent. In vivo assays may be performed, for example, by
application of a modulating agent to shaved skin on a mouse, in
which a modulating agent results in increased hair density and/or
hair length.
[0053] Modulating Agent Modification and Formulations
[0054] A modulating agent as described herein may, but need not, be
linked to one or more additional molecules. Although modulating
agents as described herein may preferentially bind to specific
tissues or cells, and thus may be sufficient to target a desired
site in vivo, it may be beneficial for certain applications to
include an additional targeting agent. Accordingly, a targeting
agent may be associated with a modulating agent to facilitate
targeting to one or more specific tissues. As used herein, a
"targeting agent" may be any substance (such as a compound or cell)
that, when associated with a modulating agent enhances the
transport of the modulating agent to a target tissue, thereby
increasing the local concentration of the modulating agent.
Targeting agents include antibodies or fragments thereof,
receptors, ligands and other molecules that bind to cells of, or in
the vicinity of, the target tissue. Known targeting agents include
serum hormones, antibodies against cell surface antigens, lectins,
adhesion molecules, tumor cell surface binding ligands, steroids,
cholesterol, lymphokines, fibrinolytic enzymes and those drugs and
proteins that bind to a desired target site. Among the many
monoclonal antibodies that may serve as targeting agents are
anti-TAC, or other interleukin-2 receptor antibodies; 9.2.27 and
NR-ML-05, reactive with the 250 kilodalton human
melanoma-associated proteoglycan; and NR-LU-10, reactive with a
pancarcinoma glycoprotein. An antibody targeting agent may be an
intact (whole) molecule, a fragment thereof, or a functional
equivalent thereof. Examples of antibody fragments are
F(ab').sub.2, -Fab', Fab and F[v] fragments, which may be produced
by conventional methods or by genetic or protein engineering.
Linkage is generally covalent and may be achieved by, for example,
direct condensation or other reactions, or by way of bi- or
multi-functional linkers. Within other embodiments, it may also be
possible to target a polynucleotide encoding a modulating agent to
a target tissue, thereby increasing the local concentration of
modulating agent. Such targeting may be achieved using well known
techniques, including retroviral and adenoviral infection.
[0055] For certain embodiments, it may be beneficial to also, or
alternatively, link a drug to a modulating agent. As used herein,
the term "drug" refers to any bioactive agent intended for
administration to a mammal to prevent or treat a disease or other
undesirable condition. Drugs include hormones, growth factors,
proteins, peptides and other compounds. The use of certain specific
drugs within the context of the present invention is discussed
below.
[0056] Within certain aspects of the present invention, one or more
modulating agents as described herein may be present within a
pharmaceutical composition. A pharmaceutical composition comprises
one or more modulating agents in combination with one or more
pharmaceutically or physiologically acceptable carriers, diluents
or excipients. Such compositions may comprise buffers (e.g.,
neutral buffered saline or phosphate buffered saline),
carbohydrates (e.g., glucose, mannose, sucrose or dextrans),
mannitol, proteins, polypeptides or amino acids such as glycine,
antioxidants, chelating agents such as EDTA or glutathione,
adjuvants (e.g., aluminum hydroxide) and/or preservatives. Within
yet other embodiments, compositions of the present invention may be
formulated as a lyophilizate. One or more modulating agents (alone
or in combination with a targeting agent and/or drug) may, but need
not, be encapsulated within liposomes using well known technology.
Compositions of the present invention may be formulated for any
appropriate manner of administration, including for example,
topical, oral, nasal, intravenous, intracranial, intraperitoneal,
subcutaneous, or intramuscular administration.
[0057] A pharmaceutical composition may also, or alternatively,
contain one or more drugs, which may be linked to a modulating
agent or may be free within the composition. Virtually any drug may
be administered in combination with a modulating agent as described
herein, for a variety of purposes as described below. Examples of
types of drugs that may be administered with a modulating agent
include analgesics, anesthetics, antianginals, antifungals,
antibiotics, anticancer drugs (e.g., taxol or mitomycin C),
antiinflammatories (e.g., ibuprofen and indomethacin),
anthelmintics, antidepressants, antidotes, antiemetics,
antihistamines, antihypertensives, antimalarials, antimicrotubule
agents (e.g., colchicine or vinca alkaloids), antimigraine agents,
antimicrobials, antiphsychotics, antipyretics, antiseptics,
anti-signaling agents (e.g., protein kinase C inhibitors or
inhibitors of intracellular calcium mobilization), antiarthritics,
antithrombin agents, antituberculotics, antitussives, antivirals,
appetite suppressants, cardioactive drugs, chemical dependency
drugs, cathartics, chemotherapeutic agents, coronary, cerebral or
peripheral vasodilators, contraceptive agents, depressants,
diuretics, expectorants, growth factors, hormonal agents,
hypnotics, immunosuppression agents, narcotic antagonists,
parasympathomimetics, sedatives, stimulants, sympathomimetics,
toxins (e.g., cholera toxin), tranquilizers and urinary
antiinfectives.
[0058] The compositions described herein may be administered as
part of a sustained release formulation (i.e., a formulation such
as a capsule or sponge that effects a slow release of modulating
agent following administration). Such formulations may generally be
prepared using well known technology and administered by, for
example, oral, rectal or subcutaneous implantation, or by
implantation at the desired target site. Sustained-release
formulations may contain a modulating agent dispersed in a carrier
matrix and/or contained within a reservoir surrounded by a rate
controlling membrane (see, e.g., European Patent Application
710,491 A). Carriers for use within such formulations are
biocompatible, and may also be biodegradable; preferably the
formulation provides a relatively constant level of modulating
agent release. The amount of modulating agent contained within a
sustained release formulation depends upon the site of
implantation, the rate and expected duration of release and the
nature of the condition to be treated or prevented.
[0059] Pharmaceutical compositions of the present invention may be
administered in a manner appropriate to the disease to be treated
(or prevented). Appropriate dosages and a suitable duration and
frequency of administration will be determined by such factors as
the condition of the patient, the type and severity of the
patient's disease and the method of administration. In general, an
appropriate dosage and treatment regimen provides the modulating
agent(s) in an amount sufficient to provide therapeutic and/or
prophylactic benefit. Within particularly preferred embodiments of
the invention, a modulating agent or pharmaceutical composition as
described herein may be administered at a dosage ranging from 0.001
to 50 mg/kg body weight, preferably from 0.1 to 20 mg/kg, on a
regimen of single or multiple daily doses. For topical
administration, a cream typically comprises an amount of modulating
agent ranging from 0.00001% to 1%, preferably 0.0001% to 0.002%.
Fluid compositions typically contain about 10 ng/ml to 5 mg/ml,
preferably from about 10 .mu.g to 2 mg/mL modulating agent.
Appropriate dosages may generally be determined using experimental
models and/or clinical trials. In general, the use of the minimum
dosage that is sufficient to provide effective therapy is
preferred. Patients may generally be monitored for therapeutic
effectiveness using assays suitable for the condition being treated
or prevented, which will be familiar to those of ordinary skill in
the art.
[0060] Modulating Agent Methods of Use
[0061] In general, the modulating agents and compositions described
herein may be used for stimulating .beta.-catenin mediated gene
transcription. Such stimulation may be performed in vitro and/or in
vivo, preferably in a mammal such as a human. Within each of the
methods described herein, one or more modulating agents may
generally be administered alone, or within a pharmaceutical
composition. In each specific method described herein, as noted
above, a targeting agent may be employed to increase the local
concentration of modulating agent at the target site.
[0062] .beta.-catenin mediated gene transcription may be stimulated
in any of a variety of contexts. As used herein, the phrase
".beta.-catenin mediated gene transcription" refers to the
transcription of any gene that increases in the presence of
increased levels of cytosolic .beta.-catenin. Such genes include,
but are not limited to, genes that are activated by the
Wnt-mediated signaling pathway, such as c-myc (see He et al.,
Science 281:1509-12, 1998).
[0063] Within certain aspects, the present invention provides
methods for increasing the level of cytoplasmic .beta.-catenin in a
cell. Such methods comprise the step of contacting a cell with a
modulating agent as described herein. The step of contacting may be
performed using any method that is suitable for the particular cell
type. In vitro, for example, contacting may be achieved by adding
modulating agent to the growth medium. In vivo, contact may be
achieved by administration, as described herein. For administration
to skin cells, topical administration is generally preferred.
Contact is performed using an amount of agent and for a sufficient
duration to result in a detectable increase in the level of
.beta.-catenin in the cell. Such an increase may be detected
directly (e.g., using immunohistochemical methods), or indirectly,
based on a detection of cellular differentiation, as described
herein.
[0064] Contact with a modulating agent as described above further
results in enhanced activation of .beta.-catenin mediated gene
transcription in the cell. Such activation may be readily detected
using any standard method for detecting changes in transcription,
such as hybridization techniques and amplification techniques
involving polymerase chain reaction (PCR). Alternatively,
downstream effects of such transcription may be detected. Such
downstream effects may include, but are not limited to, terminal
differentiation and hair growth.
[0065] As noted above, contact of a cell with a modulating agent as
described herein may stimulate terminal differentiation of the
cell. Accordingly, the present invention provides methods for using
a modulating agent to stimulate differentiation in a cell. Cells in
which differentiation may be stimulated include, but are not
limited to, skin cells, such as keratinocytes. Terminal
differentiation may be detected by photographic methods, based on
standard criteria that are well known in the art. For example, one
sign of terminal differentiation is the loss of intermediate
filament bundles.
[0066] Contact of a skin cell with a modulating agent may further
stimulate hair growth. For such applications, administration is
preferably achieved by direct contact with the scalp of the mammal
(e.g., by topical application or cutaneous injection). Enhanced
hair growth may be detected based on increased hair density and/or
rate of growth.
[0067] It has been found, within the context of the present
invention, that a modulating agent can induce keratinocytes to
terminally differentiate into squams. Accordingly, a modulating
agent may be used to cause the shedding (exfoliation) of old skin.
For such uses, administration is preferably topical, with direct
application to the skin of a mammal. Enhancement of exfoliation may
be beneficial, for example, in plastic surgery, for improvement of
photodamaged skin and for minimization of wrinkles. Such modulating
agents may represent an improvement over harsh chemical exfoliants
presently in use. Enhancement of exfoliation may generally be
detected based on the appearance of new skin, which may be
identified visually or using any of a variety of well known assays
for detecting sloughing of skin cells.
[0068] A modulating agent may further be used to ameliorate hearing
loss resulting from a variety of inner ear disorders, such as
hyperacusis and tinnitus. Regeneration of hair cells of the inner
ear, by contact with a modulating agent as described herein, may
result in improvement in such ear disorders and lessened hearing
loss.
[0069] Modulating agents as provided herein may also be used to
inhibit the development of Alzheimer's disease. Within such
methods, a modulating agent may be administered to a patient that
is at risk for developing Alzheimer's disease (but without
detectable symptoms), or may be administered following diagnosis of
the disease, based on clinical parameters that are accepted by
those skilled in the art. Modulating agents may be administered to
a patient alone or in combination with other therapeutic agents. In
general, a modulating agent is administered in an amount sufficient
to delay the onset, slow the progression or effect an improvement
in symptoms of the disease.
[0070] The following Example is offered by way of illustration and
not by way of limitation.
EXAMPLE
Preparation of Representative Modulating Agents
[0071] This Example illustrates the solid phase synthesis of a
representative phosphorylated peptide modulating agent.
[0072] N-Ac-SYLDS(PO.sub.4)GIHS(PO.sub.4)G-NH.sub.2 (SEQ ID NO:1)
is prepared using solid phase peptide synthesis techniques that
allow selective phosphorylation of hydroxy-containing residues of
the peptide. The peptide is assembled using Boc or Fmoc-amino
acid-OPfp (pentafluorophenyl) and amino-acid-ODhbt
(3,4-Dihydro-3-hydroxy-4-oxo-1,2- ,3-benztriazine) activated
esters. When phosphorylating on the resin, the peptide is assembled
on Rink Amide AM resin (4-(2',4'-Dimethoxyphenyl-Fmo-
c-aminomethyl)-phenoxyacetamidoaminomethyl, 0.65 meq/g, 1% DVB
Grain size 200-400 mesh, available from CHEM-IMPEX, Wood Dale,
Ill.) (0.150 g) using the Fmoc procedure. In each cycle the
N-protecting Fmoc-group is removed with piperidine-DMF solution
(20% v/v) 20 minutes. After washing two times with DMF, a six fold
molar excess of the next Fmoc-amino acid pentafluorophenyl ester in
DMF is added. After monitoring the coupling for completion, the
resin is washed 2 times with DMF. Couplings are carried out over a
4-6 hour period. t-Butyloxycarbonyl (Boc) is the protecting group
used for the histidine side-chain, and the t-butyl ester is used to
protect the side chains of aspartic acid, tyrosine and the serine
residues which are not to be phosphorylated. The appropriate
pentafluorophenyl ester of the unprotected serine is made in situ
from Fmoc-serine and pentaflurophenol with DIC in DMF. The
N-terminal amino acid can be protected with Boc when the free amine
is required on the N-terminus or with an acetyl group if the
N-acetylated peptide is required after cleavage.
[0073] Upon completion of the peptide chain assembly, the free
hydroxyl groups of the serine residues are phosphorylated with
dibenzylphosphochloridate (1.2 M), prepared in situ from
dibenzylphosphite and N-chlorosuccinimide in dry toluene. The resin
(0.15 g, peptide content: 0.075 mmol for the acetylated peptide) is
suspended in a dry solution containing toluene:pyridine at a ratio
of 1:2 respectively (1 mL). The mixture is cooled to -40.degree. C.
and 0.3 mL of the dibenzylphosphochloridate solution is added. The
reaction mixture is brought to -20.degree. C. and stirred for 3
hours. The same procedure is repeated, and a third coupling is
performed overnight at room temperature. The resin is washed once
with dry pyridine, twice with dry toluene and finally with DCM. The
cleavage is carried out by suspending the resin in a cleavage
cocktail (consisting of TFA: phenol: anisole, (96 mL:2 g: 2 mL
respectively)) and shaking for 4 hours. The resin is filtered and
washed with dichloromethane. The solvent volume is reduced under
vacuum (water aspirator) to approximately 2 mL and the crude
product is precipitated with the addition of cold ether and
lyophilized from 0.1% TFA. This cleavage procedure removes all
protecting groups. Peptides can be purified on a reverse phase HPLC
using a linear gradient of acetonitrile in 0.1% aqueous TFA
solution.
[0074] As an alternative, phosphorylated peptides can be prepared
using the commercially available Wang resin (CHEM-IMPEX, Wood Dale,
Ill.) with Fmoc-protected glycine as the first amino acid
(Fmoc-Gly-p-benzyloxybenzy- l alcohol resin, 0.55 mmol/g). The
peptide may be assembled on the solid support using
Fmoc-amino-acid-ODhbt (3,4-Dihydro-3-hydroxy-4-oxo-1,2,3-be-
nztriazine) activated esters, without side-chain protection on the
serine residues which are to be phosphorylated, and with the serine
side chain protected as the t-butyl ester for the serine residues
which are not to be phosphorylated (N-Ac-Ser (OtBu)-Odhbt).
Coupling reactions are monitored using the ninhydrin test and are
generally completed in less than 20 minutes. After each coupling
step the Fmoc group is removed using a 20% solution of piperidine
in DMF.
[0075] The phosphorylation reaction is carried out directly on the
peptide-resin (0.3 g), which is washed with DMF, DCM and THF,
followed by the addition of di-tert
butyl-N,N-diethylphosphoramidite (10 eqv/OH) and 1H-tetrazole (30
eqv/OH) in THF (6 mL; 1 hour). After removal of excess reagent,
oxidation of the di-tert butyl-phosphate ester is carried out by
adding t-butyl hydroperoxide (70%, aqueous, 20 eqv/OH) in DCM (4
mL) to the resin and shaking for 1 hour. The resin is washed with
DCM, tert amyl alcohol, and diethylether, and dried. The cleavage
is carried out by suspending the resin in a cleavage cocktail
(consisted of TFA: phenol: anisole, (96 mL: 2 g: 2 mL
respectively)) and shaking for 4 hours. The resin is filtered and
washed with dichloromethane. The solvent volume is reduced under
vacuum (water aspirator) to approximately 2 mL and the crude
product precipitated with the addition of cold ether and finally
lyophilized from 0.1% aqueous TFA. Alternatively, the linear
peptide is phosphorylated on resin using
N,N-diisopropyl-bis(4-chlorobenzyl) phosphoramidate (10 eq) and
1H-tetrazole (50 eq) for 1 hour, followed by oxidation with
t-butylhydroperoxide (20 eq., 1 hour). The peptide is then cleaved
from the resin and purified as described above.
[0076] From the foregoing, it will be evident that although
specific embodiments of the invention have been described herein
for the purpose of illustrating the invention, various
modifications may be made without deviating from the spirit and
scope of the invention. Accordingly, the present invention is not
limited except as by the appended claims.
* * * * *