Chemistry: natural resins or derivatives; peptides or proteins; – Peptides of 3 to 100 amino acid residues – Cyclic peptides
Reexamination Certificate
1996-05-08
2004-03-30
Wessendorf, T. D. (Department: 1639)
Chemistry: natural resins or derivatives; peptides or proteins;
Peptides of 3 to 100 amino acid residues
Cyclic peptides
C514S011400, C514S009100, C514S002600
Reexamination Certificate
active
06713604
ABSTRACT:
FIELD OF THE INVENTION
This invention relates generally to a process of inhibiting the binding of &agr;
4
&bgr;
1
integrin to proteins such as VCAM-1 or fibronectin. The invention also relates to synthetic cyclic peptides that inhibit that binding.
BACKGROUND OF THE INVENTION
Vascular cell adhesion molecule-1 (VCAM-1) is a protein that is found on the surface of endothelial cells that line the interior wall of capillaries. VCAM-1 recognizes and binds to the integrin &agr;
4
&bgr;
1
(or VLA-4 for very late antigen-4), a beterodimeric protein present on the surface of certain white blood cells. Binding of &agr;
4
&bgr;
1
to VCAM-1 allows white blood cells to adhere to the capillary wall in areas where the tissue surrounding the capillary has been infected or damaged.
When a tissue has been invaded by a microorganism or has been damaged, white blood cells, also called leukocytes, play a major role in the inflammatory response. One of the most important aspects of the inflammatory response involves the cell adhesion event. Generally, white blood cells are found circulating through the bloodstream. However, when a tissue is infected or becomes damaged, the white blood cells recognize the invaded or damaged tissue, bind to the wall of the capillary near the affected tissue and diffuse through the capillary into the affected tissue. VCAM-1 helps certain types of white blood cells recognize the affected sites, bind to the capillary wall, and migrate into the affected tissue.
The are are three main types of white blood cells: granulocytes, monocytes and lymphocytes. VCAM-1 binds to &agr;
4
&bgr;
1
expressed on the surface of monocytes, lymphocytes and two subclasses of granulocytes-eosinophils and basophils.
Monocytes, after leaving the bloodstream through the wall of a capillary, mature into macrophages that phagocytose and digest invading microorganisms, foreign bodies and senescent cells. Lymphocytes produce antibodies and kill infected cells. Eosinophils and basophils secrete mediators of various inflammatory reactions.
Following infection or damage of tissue surrounding a capillary, the endothelial cells that line the capillary express a series of adhesion molecules, including VCAM-1, that are critical for binding white blood cells that are necessary for fighting infection. Prior to binding to VCAM-1, the white blood cells initially bind to another set of adhesion molecules to slow their flow and allow the cells to “roll” along the activated endothelium. Monocytes, lymphocytes, basophils and eosinophils are then able to firmly bind to VCAM-1 on the endothelial cells via the &agr;
4
&bgr;
1
integrin. There is evidence that this interaction is also involved in transmigration of these white blood cells into the damaged tissue.
Although white blood cell migration to the site of injury helps fight infection and destroy foreign material, in many instances this migration can become uncontrolled, with white blood cells flooding to the scene, causing widespread tissue damage. Compounds capable of blocking this process, therefore, would be beneficial as therapeutic agents. Thus, it would be useful to develop inhibitors that would prevent the binding of white blood cells to VCAM-1.
For example, some of the diseases that might be treated by the inhibition of &agr;
4
&bgr;
1
binding include, but are not limited to, atherosclerosis, rheumatoid arthritis, asthma, allergy, multiple sclerosis and type I diabetes. In addition to being found on some white blood cells, &agr;
4
&bgr;
1
is found on various cancer cells, including leukemia, melanoma, lymphoma and sarcoma cells. It has been suggested that cell adhesion involving &agr;
4
&bgr;
1
may be involved in the metastasis of certain cancers. Inhibitors of &agr;
4
&bgr;
1
binding may, therefore, also be useful in the treatment of some forms of cancer.
BRIEF SUMMARY OF THE INVENTION
In one aspect, the present invention provides an isolated and purified cyclic peptide of from 4 to about 13 amino acid residues having (a) an N-terminal amine group, acetyl group (Ac) or a polyethyleneglycol moiety of from about 400 to about 12,000 Daltons average molecular weight (MPEG
X000
) linked through an amide bond to the N-terminal residue; and (b) a C-terminal carboxylic acid group or amide group; the peptide comprising the amino acid residue sequence Xaa
1
-Xaa
2
-Asp-Xaa
3
(SEQ ID NO:15), where Xaa
1
is any L- or D-&agr;-amino acid residue and Xaa
2
and Xaa
3
are any hydrophobic, L-&agr;-amino acid residue with the proviso that when Xaa
1
is Lys or Arg, Xaa
2
cannot be Gly or Cys. In a preferred embodiment, Xaa
1
is Phe, Trp or Ile; Xaa
2
is Leu, Ile, Val, Lys or Met and Xaa
3
is Val, Tyr, Leu, Trp, or Phe. More preferably Xaa
1
is Trp and Xaa
2
is Leu and Xaa
3
is Val.
In one embodiment, a cyclic peptide of the present invention is cyclized via formation of a lactam. Such a peptide comprises the amino acid residue sequence Xaa
4
-Xaa
1
-Xaa
2
-Asp-Xaa
3
(SEQ ID NO:1), where Xaa
1
is any L- or D-&agr;-amino acid residue, Xaa
2
is any hydrophobic, L-&agr;-amino acid residue with the proviso that when Xaa
1
is Lys or Arg, Xaa
2
cannot be Gly or Cys; Xaa
3
is any hydrophobic, L-&agr;-amino acid residue and Xaa
4
is any D- or L-&agr;-amino acid. Preferably, Xaa
1
is Phe or Trp, Xaa
2
is Leu, Xaa
3
is Val and Xaa
4
is Glu. More preferably, such a peptide has the amino acid residue sequence of SEQ ID NO:2-8.
In another embodiment, a cyclic peptide of this invention further comprises a cysteine or modified cysteine residue and a —CH
2
CO— group at the N-terminal position. In accordance with such an embodiment, Xaa
1
is preferably Trp and Xaa
2
is preferably Leu. Exemplary and preferred such peptides have the amino acid residue sequence of SEQ ID NO:9-12.
In another embodiment, a cyclic peptide of this invention further comprises two or more cysteine or modified cysteine residues and is cyclized via a disulfide bond. Preferably, one of the cysteine or modified cysteine residues is located at the N- or C-terminal position. In one embodiment, a cyclic peptide having two cysteine or modified cysteine residues comprises the amino acid residue sequence Xaa
1
-Xaa
2
-Asp-Xaa
3
(SEQ ID NO:15), where Xaa
1
and Xaa
2
and Xaa
3
are as defined above. Preferably, Xaa
1
is Tyr, Phe, Trp, dTrp or Ile; Xaa
2
is Leu, Ile, Val, Lys, Met or Asp; and Xaa
3
is Val, Tyr, Leu, Trp, or Phe.
In yet another embodiment, a cyclic peptide having a cysteine or modified cysteine residue at both the N- and C-terminal positions comprises the amino acid residue sequence of SEQ ID NO:1. Preferably, Xaa
1
is Trp or Phe, Xaa
2
is Leu, Xaa
3
is Val or Tyr and Xaa
4
is Ser or Glu.
Exemplary and preferred cyclic, disulfide peptides of the present invention have the amino acid residue sequence of any of SEQ ID NO:13 or 15-43.
In another aspect, the present invention provides a pharmaceutical composition comprising a physiologically acceptable diluent and a cyclic peptide of the present invention. Preferred cyclic peptides in such a composition are the same as set forth above.
In yet another aspect, the present invention provides a process of selectively inhibiting the binding of &agr;
4
&bgr;
1
integrin to VCAM-1. That process comprises exposing a cell that expresses &agr;
4
&bgr;
1
integrin and a cell that expresses VCAM-1 to an effective inhibiting amount of a cyclic peptide of the present invention. In a preferred embodiment of that process, the VCAM-1 is on the surface of a vascular endothelial cell. In another preferred embodiment, the &agr;
4
&bgr;
1
integrin is on the surface of a white blood cell such as a monocyte, a lymphocyte, a granulocyte (an eosinophil or a basophil), a stem cell or other cell that naturally expresses &agr;
4
&bgr;
1
. Preferred peptides used in that process are the same as set forth above.
Where the cells are located in a living organism, a peptide is preferably administered to the organism in an effective inhibiting amount in a pharmaceutical composition of this invention.
In another aspect, the present invention provides a pro
Beck Pamela J.
Kogan Timothy P.
Ren Kaijun
Vanderslice Peter
Texas Biotechnology Corporation
Wessendorf T. D.
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