Drug – bio-affecting and body treating compositions – Designated organic active ingredient containing – Peptide containing doai
Reexamination Certificate
1998-07-14
2001-03-27
Moezie, F. T. (Department: 1653)
Drug, bio-affecting and body treating compositions
Designated organic active ingredient containing
Peptide containing doai
C514S009100, C530S317000
Reexamination Certificate
active
06207639
ABSTRACT:
TECHNICAL FIELD
The present invention relates generally to methods for modulating N-cadherin mediated processes, and more particularly to the use of cyclic peptides comprising a cadherin cell adhesion recognition sequence for inhibiting or enhancing cadherin-mediated neurite outgrowth.
BACKGROUND OF THE INVENTION
Nerve growth is promoted by a wide range of molecules, including the cell surface adhesion molecules (CAMs) NCAM and N-cadherin. In particular, N-cadherin is the predominant mediator of calcium-dependent adhesion in the nervous system. N-cadherin is a member of the classical cadherin family of calcium-dependent CAMs (Munro et al., In.
Cell Adhesion and Invasion in Cancer Metastasis
, P. Brodt, ed., pp. 17-34, RG Landes Co.(Austin Tex., 1996). The classical cadherins (abbreviated CADs) are integral membrane glycoproteins that generally promote cell adhesion through homophilic interactions (a CAD on the surface of one cell binds to an identical CAD on the surface of another cell), although CADs also appear to be capable of forming heterotypic complexes with one another under certain circumstances and with lower affinity. Cadherins have been shown to regulate epithelial, endothelial, neural and cancer cell adhesion, with different CADs expressed on different cell types. N (neural)-cadherin is predominantly expressed by neural cells, endothelial cells and a variety of cancer cell types. A detailed discussion of the classical cadherins is provided in Munro S B et al., 1996, In:
Cell Adhesion and Invasion in Cancer Metastasis
, P. Brodt, ed., pp.17-34 (RG Landes Company, Austin Tex.).
The structures of the CADs are generally similar. As illustrated in
FIG. 1
, CADs are composed of five extracellular domains (EC1-EC5), a single hydrophobic domain (TM) that transverses the plasma membrane (PM), and two cytoplasmic domains (CP1 and CP2). The calcium binding motifs DXNDN (SEQ ID NO:8), DXD and LDRE (SEQ ID NO:9) are interspersed throughout the extracellular domains. The first extracellular domain (EC1) contains the classical cadherin cell adhesion recognition (CAR) sequence, HAV (His-Ala-Val), along with flanking sequences on either side of the CAR sequence that may play a role in conferring specificity. Synthetic peptides containing the CAR sequence and antibodies directed against the CAR sequence have been shown to inhibit CAD-dependent processes (Munro et al., supra; Blaschuk et al.,
J. Mol. Biol
. 211:679-82, 1990; Blaschuk et al.,
Develop. Biol
. 139:227-29, 1990; Alexander et al.,
J. Cell. Physiol
. 156:610-18, 1993). The three-dimensional solution and crystal structures of the EC1 domain have been determined (Overduin et al.,
Science
267:386-389, 1995; Shapiro et al.,
Nature
374:327-337, 1995).
N-cadherin is known to promote neurite outgrowth via a homophilic binding mechanism. N-cadherin is normally found on both the advancing growth cone and on cellular substrates, and the inhibition of N-cadherin function results in diminished neurite outgrowth. Such inhibition may be the result of pathology or injury involving severed neuronal connections and/or spinal cord damage. In such cases, enhancement of N-cadherin mediated neurite outgrowth would be beneficial. However, previous attempts to promote neurite outgrowth have achieved limited success due, in part, to difficulties associated with maintaining continuous growth over a particular defined region.
Accordingly, there is a need in the art for compounds that modulate and/or direct neurite outgrowth without such disadvantages. The present invention fulfills this need and further provides other related advantages.
SUMMARY OF THE INVENTION
The present invention provides methods for modulating cadherin-mediated neurite outgrowth. Within one aspect, the present invention provides methods for enhancing and/or directing neurite outgrowth, comprising contacting a neuron with a cell adhesion modulating agent, wherein the modulating agent enhances cadherin-mediated cell adhesion.
Within a related aspect, methods for treating spinal cord injuries in a mammal are provided, comprising administering to a mammal a cell adhesion modulating agent as described above, wherein the modulating agent enhances cadherin-mediated cell adhesion.
Within the above aspects, cell adhesion modulating agents generally comprise a cyclic peptide in which nonadjacent amino acid residues are covalently linked to form a peptide ring, wherein the peptide ring comprises the sequence His-Ala-Val. Within certain embodiments, the cyclic peptide has the formula:
wherein X
1
, and X
2
are optional, and if present, are independently selected from the group consisting of amino acid residues and combinations thereof in which the residues are linked by peptide bonds, and wherein X
1
and X
2
independently range in size from 0 to 10 residues, such that the sum of residues contained within X
1
and X
2
ranges from 1 to 12; wherein Y
1
and Y
2
are independently selected from the group consisting of amino acid residues, and wherein a covalent bond is formed between residues Y
1
and Y
2
; and wherein Z
1
and Z
2
are optional, and if present, are independently selected from the group consisting of amino acid residues and combinations thereof in which the residues are linked by peptide bonds. Within certain specific embodiments Z
1
is not present and Y
2
comprises an N-acetyl group and/or Z
2
is not present and Y
2
comprises a C-terminal amide group. Linkage of Y
1
and Y
2
may be achieved via, for example, a disulfide bond, an amide bond or a thioether bond. Certain preferred modulating agents comprise a sequence selected from the group consisting of N-Ac-CHAVC-NH
2
(SEQ ID NO:10), N-Ac-CHAVDC-NH
2
(SEQ ID NO:11), N-Ac-CAHAVC-NH
2
(SEQ ID NO:12), N-Ac-CAHAVDC-NH
2
(SEQ ID NO:13), N-Ac-CAHAVDIC-NH
2
(SEQ ID NO:14), N-Ac-CRAHAVDC-NH
2
(SEQ ID NO:15), N-Ac-CLRAHAVDC-NH
2
(SEQ ID NO:16), N-Ac-DHAVK-NH
2
(SEQ ID NO:17), N-Ac-KHAVE-NH
2
(SEQ ID NO:18), N-Ac-AHAVDI-NH
2
(SEQ ID NO:19) and derivatives of the foregoing sequences having one or more C-terminal, N-terminal and/or side chain modifications. Modulating agents may comprise multiple HAV sequences separated by a linker. Modulating agents may further be linked to one or more of a drug, a solid support, a targeting agent, a cell adhesion recognition sequence that is bound by an adhesion molecule other than a cadherin, wherein the cell adhesion recognition sequence is separated from any HAV sequence(s) by a linker; and/or an antibody or antigen-binding fragment thereof that specifically binds to a cell adhesion recognition sequence bound by an adhesion molecule other than a cadherin. A modulating agent may be present within a pharmaceutical composition that comprises a pharmaceutically acceptable carrier and, optionally, may further comprise a drug, a peptide comprising a cell adhesion recognition sequence that is bound by an adhesion molecule other than a cadherin; and/or an antibody or antigen-binding fragment thereof that specifically binds to a cell adhesion recognition sequence bound by an adhesion molecule other than a cadherin.
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Schense et al. “Enzymatic Incorporation of Bioactive Peptides into Fibrin Matrices Enhances Neurite Extension” Nature Biotechnology, vol. 18, pp. 415-419, Apr. 2000.*
Beesley et al., “The post-synaptic density: putative involvement in synapse stabilizaztion vicahderins and covalent modification by ubiquitination,”Biochemical S
Blaschuk Orest W.
Gour Barbara J.
McGill University
Moezie F. T.
Seed IP Law Group
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