U.S. patent application number 10/319340 was filed with the patent office on 2003-07-31 for peptide inhibitors of inflammation mediated by selectins.
This patent application is currently assigned to Centocor, Inc.. Invention is credited to Geng, Jian-Guo, Heavner, George A., McEver, Rodger P..
Application Number | 20030144211 10/319340 |
Document ID | / |
Family ID | 25046495 |
Filed Date | 2003-07-31 |
United States Patent
Application |
20030144211 |
Kind Code |
A1 |
Heavner, George A. ; et
al. |
July 31, 2003 |
Peptide inhibitors of inflammation mediated by selectins
Abstract
Peptides derived from three regions of the lectin domain of
GMP-140 and the related selectins, ELAM-1 and the lymphocyte homing
receptor, have been found to inhibit neutrophil adhesion to
GMP-140. These and additional peptides have been synthesized,
having as their core region portions of the 56-60 amino acid
sequence of GMP-140, with residue 1 defined as the N-terminus of
the mature protein after the cleavage of the signal peptide.
Examples demonstrate the inhibition of the binding of neutrophils
to GMP-140 of peptides in concentrations ranging from 5 to 1500
.mu.mol. It has been found that alterations within the core
sequence, as well as N-terminal and C-terminal flanking regions, do
not result in loss of biological activity. The peptides are useful
as diagnostics and, in combination with a suitable pharmaceutical
carrier, for clinical applications in the modulation or inhibition
of coagulation processes or inflammatory processes.
Inventors: |
Heavner, George A.;
(Malvern, PA) ; McEver, Rodger P.; (Oklahoma City,
OK) ; Geng, Jian-Guo; (Portage, MI) |
Correspondence
Address: |
PATREA L. PABST
HOLLAND & KNIGHT LLP
SUITE 2000, ONE ATLANTIC CENTER
1201 WEST PEACHTREE STREET, N.E.
ATLANTA
GA
30309-3400
US
|
Assignee: |
Centocor, Inc.
|
Family ID: |
25046495 |
Appl. No.: |
10/319340 |
Filed: |
December 13, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10319340 |
Dec 13, 2002 |
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08135319 |
Oct 12, 1993 |
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6528487 |
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08135319 |
Oct 12, 1993 |
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07757131 |
Sep 10, 1991 |
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Current U.S.
Class: |
514/19.1 ;
514/12.2; 514/21.5; 530/326; 530/327; 530/328; 530/329 |
Current CPC
Class: |
C07K 14/70564 20130101;
A61P 7/02 20180101; A61P 29/00 20180101 |
Class at
Publication: |
514/16 ; 514/14;
514/15; 530/326; 530/327; 530/328; 530/329 |
International
Class: |
A61K 038/10; A61K
038/08; C07K 007/08; C07K 007/06 |
Claims
We claim:
1. A peptide inhibiting binding of selectins selected from the
group having the formula: R.sup.1--X-A-B--C-D-E-Y--R.sup.2 (I) or a
pharmaceutically acceptable acid- or based-addition salt thereof
wherein: A is D- or L-asparagine, D- or L-isoleucine or D- or
L-valine; B is D- or L-asparagine or glycine; C is D- or L-lysine,
D- or L-valine or glycine; D is D- or L-valine, D- or L-threonine
or D- or L-isoleucine; E is D- or L-tryptophan; X and Y are linear
chains of from 0 to 10 amino acids; R.sup.1 is H (signifying a free
N-terminal group), formyl, lower alkyl, aryl, lower alkanoyl,
aroyl, alkyloxycarbonyl or aryloxycarbonyl and R.sup.2 is OH
(signifying a free C-terminal group) lower alkyl or aryl esters, or
NR.sup.3R.sup.4 where R.sup.3 and R.sup.4 each selected
independently from H, lower alkyl or aryl.
2. The peptide of claim 1 wherein X is selected from the group
consisting of Arg-Lys, Ile-Arg-Lys, Gly-Ile-Arg-Lys,
Ile-Gly-Ile-Arg-Lys, Ac-Arg-Lys, Cys-Arg-Lys, Arg-Glu, Tyr-Lys,
D-Arg-Lys, Arg-D-Lys, and Cys-Ile-Gly-Ile-Arg-Lys.
3. The peptide of claim 1 wherein Y is selected from the group
consisting of Thr, Val, Thr-Trp, Thr-Trp-Val, Val-Trp, Val-Trp-Val,
Thr-Trp-Val-Gly-Thr-Lys, Thr-Trp-Val-Gly-Thr-Asn,
Thr-Trp-Val-Gly-Thr-Gln- , Val-Trp-Val-Gly-Thr-Gln,
Val-Trp-Val-Gly-Thr-Lys, Val-Trp-Val-Gly-Thr-Asn, Thr-Trp-Glu, and
Thr-Trp-Val-Gly-Thr-Lys-Lys-Ala- -Leu-Thr-Asn-Glu-Cys.
4. The peptide of claim 1 selected from the group consisting of
peptides having the formula: Arg-Lys-Asn-Asn-Lys-Thr-Trp-NH.sub.2;
Cys-Ile-Gly-Ile-Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH.sub.2;
Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-Gly-Thr-Lys-Lys-Ala-Leu-Thr-Asn-G-
lu-Cys-NH.sub.2; Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH.sub.2;
Lys-Asn-Asn-Lys-Thr-Trp-NH.sub.2.
Acetyl-Asn-Asn-Lys-Thr-Trp-NH.sub.2;
Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val;
Arg-Lys-Val-Asn-Asn-Val-Trp-Val-- Trp-Val;
Arg-Lys-Val-Asn-Asn-Val-Trp-Val-Trp-Val-NH.sub.2;
Arg-Lys-Ile-Gly-Gly-Ile-Trp-Thr-Trp-Val;
Arg-Lys-Ile-Gly-Gly-Ile-Trp-Thr-- Trp-Val-NH.sub.2;
Arg-Lys-Ile-Gly-Gly-Ile-Trp-NH.sub.2;
Arg-Lys-Val-Asn-Asn-Val-Trp-NH.sub.2;
Ac-Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-- Trp-Val-NH.sub.2;
Cys-Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH.sub.2;
Arg-Glu-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH.sub.2;
Arg-Lys-Asn-Asn-Lys-Thr- -Trp-Thr-Trp-Glu-NH.sub.2;
Tyr-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH.sub.- 2;
D-Arg-Lys-Ile-Gly-Gly-Ile-Trp-Thr-Trp-Val-NH.sub.2;
Arg-D-Lys-Ile-Gly-Gly-Ile-Trp-Thr-Trp-Val-NH.sub.2;
FMOC-Asn-Asn-Lys-Thr-Trp-NH.sub.2; and pharmaceutically acceptable
acid- or base-addition salts thereof.
5. The peptide of claim 1 in combination with a pharmaceutical
carrier selected from the group consisting of carriers suitable for
parenteral administration, oral administration, topical
administration, and controlled release formulations.
6. A method for the preparation of peptides of the formula:
R.sup.1--X-A-B--C-D-E-Y--R.sup.2 (I) or a pharmaceutically
acceptable acid- or based-addition salt thereof wherein: A is D- or
L-asparagine, D- or L-isoleucine or D- or L-valine; B is D- or
L-asparagine or glycine; C is D- or L-lysine, D- or L-valine or
glycine; D is D- or L-valine, D- or L-threonine or D- or
L-isoleucine; E is D- or L-tryptophan; X and Y are linear chains of
from 0 to 10 amino acids; R.sup.1 is H (signifying a free
N-terminal group), formyl, lower alkyl, aryl, lower alkanoyl,
aroyl, alkyloxycarbonyl or aryloxycarbonyl and R.sup.2 is OH
(signifying a free C-terminal group) lower alkyl or aryl esters, or
NR.sup.3R.sup.4 where R.sup.3 and R.sup.4 each selected
independently from H, lower alkyl or aryl.
7. The method of claim 6 wherein X is selected from the group
consisting of Arg-Lys, Ile-Arg-Lys, Gly-Ile-Arg-Lys,
Ile-Gly-Ile-Arg-Lys, Ac-Arg-Lys, Cys-Arg-Lys, Arg-Glu, Tyr-Lys,
D-Arg-Lys, Arg-D-Lys, and Cys-Ile-Gly-Ile-Arg-Lys.
8. The method of claim 6 wherein Y is selected from the group
consisting of Thr, Val, Thr-Trp, Thr-Trp-Val, Val-Trp, Val-Trp-Val,
Thr-Trp-Val-Gly-Thr-Lys, Thr-Trp-Val-Gly-Thr-Asn,
Thr-Trp-Val-Gly-Thr-Gln- , Val-Trp-Val-Gly-Thr-Gln,
Val-Trp-Val-Gly-Thr-Lys, Val-Trp-Val-Gly-Thr-Asn, Thr-Trp-Glu, and
Thr-Trp-Val-Gly-Thr-Lys-Lys-Ala- -Leu-Thr-Asn-Glu-Cys.
9. The method of claim 6 selected from the group consisting of
peptides having the formula: Arg-Lys-Asn-Asn-Lys-Thr-Trp-NH.sub.2;
Cys-Ile-Gly-Ile-Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH.sub.2;
Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-Gly-Thr-Lys-Lys-Ala-Leu-Thr-Asn-G-
lu-Cys-NH.sub.2; Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH.sub.2;
Lys-Asn-Asn-Lys-Thr-Trp-NH.sub.2;
Acetyl-Asn-Asn-Lys-Thr-Trp-NH.sub.2;
Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val;
Arg-Lys-Val-Asn-Asn-Val-Trp-Val-- Trp-Val;
Arg-Lys-Val-Asn-Asn-Val-Trp-Val-Trp-Val-NH.sub.2;
Arg-Lys-Ile-Gly-Gly-Ile-Trp-Thr-Trp-Val;
Arg-Lys-Ile-Gly-Gly-Ile-Trp-Thr-- Trp-Val-NH.sub.2;
Arg-Lys-Ile-Gly-Gly-Ile-Trp-NH.sub.2;
Arg-Lys-Val-Asn-Asn-Val-Trp-NH.sub.2;
Ac-Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-- Trp-Val-NH.sub.2;
Cys-Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH.sub.2;
Arg-Glu-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH.sub.2;
Arg-Lys-Asn-Asn-Lys-Thr- -Trp-Thr-Trp-Glu-NH.sub.2;
Tyr-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH.sub.- 2;
D-Arg-Lys-Ile-Gly-Gly-Ile-Trp-Thr-Trp-Val-NH.sub.2;
Arg-D-Lys-Ile-Gly-Gly-Ile-Trp-Thr-Trp-Val-NH.sub.2;
FMOC-Asn-Asn-Lys-Thr-Trp-NH.sub.2; and pharmaceutically acceptable
acid- or base-addition salts thereof.
10. The method for preparation of the peptide of claim 6 whereby
the amino acids are assembled either singly or in preformed blocks
in solution or suspension by chemical ligation techniques.
11. The method for preparation of a peptide of claim 6 whereby the
amino acids are assembled either singly or in preformed blocks in
solution or suspension by enzymatic ligation techniques.
12. The method for preparation of a peptide of claim 11 whereby the
peptide is produced enzymatically by inserting nucleic acid
encoding the peptide into an expression vector, expressing the DNA,
and translating the DNA into the peptide.
13. A method for modifying binding of a selectin comprising
providing a peptide having the formula:
R.sup.1--X-A-B--C-D-E-Y--R.sup.2 (I) or a pharmaceutically
acceptable acid- or based-addition salt thereof wherein: A is D- or
L-asparagine, D- or L-isoleucine or D- or L-valine; B is D- or
L-asparagine or-glycine; C is D- or L-lysine, D- or L-valine or
glycine; D is D- or L-valine, D- or L-threonine or D- or
L-isoleucine; E is D- or L-tryptophan; X and Y are linear chains of
from 0 to 10 amino acids; R.sup.1 is H (signifying a free
N-terminal group), formyl, lower alkyl, aryl, lower alkanoyl,
aroyl, alkyloxycarbonyl or aryloxycarbonyl and R.sup.2 is OH
(signifying a free C-terminal group) lower alkyl or aryl esters, or
NR.sup.3R.sup.4 where R.sup.3 and R.sup.4 each selected
independently from H, lower alkyl or aryl.
14. The method of claim 13 wherein X is selected from the group
consisting of Arg-Lys, Ile-Arg-Lys, Gly-Ile-Arg-Lys,
Ile-Gly-Ile-Arg-Lys, Ac-Arg-Lys, Cys-Arg-Lys, Arg-Glu, Tyr-Lys,
D-Arg-Lys, Arg-D-Lys, and Cys-Ile-Gly-Ile-Arg-Lys.
15. The method of claim 13 wherein Y is selected from the group
consisting of Thr, Val, Thr-Trp, Thr-Trp-Val, Val-Trp, Val-Trp-Val,
Thr-Trp-Val-Gly-Thr-Lys, Thr-Trp-Val-Gly-Thr-Asn,
Thr-Trp-Val-Gly-Thr-Gln- , Val-Trp-Val-Gly-Thr-Gln,
Val-Trp-Val-Gly-Thr-Lys, Val-Trp-Val-Gly-Thr-Asn, Thr-Trp-Glu, and
Thr-Trp-Val-Gly-Thr-Lys-Lys-Ala- -Leu-Thr-Asn-Glu-Cys.
16. The method of claim 13 selected from the group consisting of
peptides having the formula: Arg-Lys-Asn-Asn-Lys-Thr-Trp-NH.sub.2;
Cys-Ile-Gly-Ile-Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH.sub.2;
Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-Gly-Thr-Lys-Lys-Ala-Leu-Thr-Asn-G-
lu-Cys-NH.sub.2; Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH.sub.2;
Lys-Asn-Asn-Lys-Thr-Trp-NH.sub.2;
Acetyl-Asn-Asn-Lys-Thr-Trp-NH.sub.2;
Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val;
Arg-Lys-Val-Asn-Asn-Val-Trp-Val-- Trp-Val;
Arg-Lys-Val-Asn-Asn-Val-Trp-Val-Trp-Val-NH.sub.2;
Arg-Lys-Ile-Gly-Gly-Ile-Trp-Thr-Trp-Val;
Arg-Lys-Ile-Gly-Gly-Ile-Trp-Thr-- Trp-Val-NH.sub.2;
Arg-Lys-Ile-Gly-Gly-Ile-Trp-NH.sub.2;
Arg-Lys-Val-Asn-Asn-Val-Trp-NH.sub.2;
Ac-Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-- Trp-Val-NH.sub.2;
Cys-Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH.sub.2;
Arg-Glu-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH.sub.2;
Arg-Lys-Asn-Asn-Lys-Thr- -Trp-Thr-Trp-Glu-NH.sub.2;
Tyr-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH.sub.- 2;
D-Arg-Lys-Ile-Gly-Gly-Ile-Trp-Thr-Trp-Val-NH.sub.2;
Arg-D-Lys-Ile-Gly-Gly-Ile-Trp-Thr-Trp-Val-NH.sub.2;
FMOC-Asn-Asn-Lys-Thr-Trp-NH.sub.2; and pharmaceutically acceptable
acid- or base-addition salts thereof.
17. The method of claim 13 wherein the pharmaceutical carrier is
selected from the group consisting of carriers suitable for
parenteral administration, oral administration, topical
administration, and controlled release formulations.
Description
BACKGROUND OF THE INVENTION
[0001] This invention is generally in the field of methods for the
treatment and prevention of inflammatory responses using peptides
derived from selectins including GMP-140, ELAM-1, and
lymphocyte-homing receptor.
[0002] The adherence of platelets and leukocytes to vascular
surfaces is a critical component of the inflammatory response, and
is part of a complex series of reactions involving the simultaneous
and interrelated activation of the complement, coagulation, and
immune systems.
[0003] The complement proteins collectively play a leading role in
the immune system, both in the identification and in the removal of
foreign substances and immune complexes, as reviewed by
Muller-Eberhard, H. J., Ann. Rev. Biochem. 57:321-347 (1988).
Central to the complement system are the C3 and C4 proteins, which
when activated covalently attach to nearby targets, marking them
for clearance. In order to help control this process, a remarkable
family of soluble and membrane-bound regulatory proteins has
evolved, each of which interacts with activated C3 and/or C4
derivatives. The coagulation and inflammatory pathways are
regulated in a coordinate fashion in response to tissue damage. For
example, in addition to becoming adhesive for leukocytes, activated
endothelial cells express tissue factor on the cell surface and
decrease their surface expression of thrombomodulin, leading to a
net facilitation of coagulation reactions on the cell surface. In
some cases, a single receptor can be involved in both inflammatory
and coagulation processes.
[0004] Leukocyte adherence to vascular endothelium is a key initial
step in migration of leukocytes to tissues in response to microbial
invasion. Although a class of inducible leukocyte receptors, the
CD11-CD18 molecules, are thought to have some role in adherence to
endothelium, mechanisms of equal or even greater importance for
leukocyte adherence appear to be due to inducible changes in the
endothelium itself.
[0005] Activated platelets have also been shown to interact with
both neutrophils and monocytes in vitro. The interaction of
platelets with monocytes may be mediated in part by the binding of
thrombospondin to platelets and monocytes, although other
mechanisms have not been excluded. The mechanisms for the binding
of neutrophils to activated platelets are not well understood,
except that it is known that divalent cations are required. In
response to vascular injury, platelets are known to adhere to
subendothelial surfaces, become activated, and support coagulation.
Platelets and other cells may also play an important role in the
recruitment of leukocytes into the wound in order to contain
microbial invasion.
[0006] Endothelium exposed to "rapid" activators such as thrombin
and histamine becomes adhesive for neutrophils within two to ten
minutes, while endothelium exposed to cytokines such as tumor
necrosis factor and interleukin-1 becomes adhesive after one to six
hours. The rapid endothelial-dependent leukocyte adhesion has been
associated with expression of the lipid mediator platelet
activating factor (PAF) on the cell surface, and presumably, the
appearance of other endothelial surface receptors. The slower
cytokine-inducible endothelial adhesion for leukocytes is mediated,
at least in part, by an endothelial cell receptor, ELAM-1, that is
synthesized by endothelial cells after exposure to cytokines and
then transported to the cell surface, where it binds neutrophils.
The isolation, characterization and cloning of ELAM-1 is reviewed
by Bevilacqua, et al., in Science 243, 1160-1165 (1989). A
peripheral lymph node homing receptor, also called "the murine Mel
14 antigen", "Leu 8", the "Leu 8 antigen" and "LAM-1", is another
structure on neutrophils, monocytes, and lymphocytes that binds
lymphocytes to high endothelial venules in peripheral lymph nodes.
The characterization and cloning of this protein is reviewed by
Lasky, et al., Cell 56, 1045-1055 (1989) (mouse) and Tedder, et
al., J. Exp. Med. 170, 123-133 (1989).
[0007] GMP-140 (granule membrane protein 140), also known as
PADGEM, is a cysteine-rich and heavily glycosylated integral
membrane glycoprotein with an apparent molecular weight of 140,000
as assessed by sodium dodecyl sulfate polyacrylamide gel
electrophoresis (SDS-PAGE). GMP-140 was first purified from human
platelets by McEver and Martin, J. Biol. Chem. 259:9799-9804
(1984). The protein is present in alpha granules of resting
platelets but is rapidly redistributed to the plasma membrane
following platelet activation, as reported by Stenberg, et al.,
(1985). The presence of GMP-140 in endothelial cells and its
biosynthesis by these cells was reported by McEver, et al., Blood
70(5) Suppl. 1:355a, Abstract No. 1274 (1987). In endothelial
cells, GMP-140 is found in storage granules known as the
Weibel-Palade bodies. (McEver, et al. J. Clin. Invest. 84:92-99
(1989) and Hattori, et al., J. Biol. Chem. 264:7768-7771 (1989)).
GMP-140 (called PADGEM) has also been reported to mediate the
interaction of activated platelets with neutrophils and monocytes
by Larsen, et al., in Cell 59, 305-312 (October 1989) and Hamburger
and McEver, Blood 75:550-554 (1990).
[0008] The cDNA-derived amino acid sequence, reported by Johnston,
et al., in Cell 56, 1033-1044 (March 24 1989), and in U.S. Ser. No.
07/320,408 filed Mar. 8, 1989, indicates that it contains a number
of modular domains that are likely to fold independently. Beginning
at the N-terminus, these include a "lectin" domain, an "EGF"
domain, nine tandem consensus repeats similar to those in
complement binding proteins, a transmembrane domain (except in a
soluble form that appears to result from differential splicing),
and a cytoplasmic tail.
[0009] When platelets or endothelial cells are activated by
mediators such as thrombin, the membranes of the storage granules
fuse with the plasma membrane, the soluble contents of the granules
are released to the external environment, and membrane bound
GMP-140 is presented within seconds on the cell surface. The rapid
redistribution of GMP-140 to the surface of platelets and
endothelial cells as a result of activation suggested that this
glycoprotein could play an important role at sites of inflammation
or vascular disruption.
[0010] This important role has been confirmed by the observation
that GMP-140 is a receptor for neutrophils (Geng et al., Nature
343:757-760 (1990); Hamburger and McEver, Blood 75:550-554 (1990)),
monocytes (Larsen, et al. Cell 59:305-312 (1989); Moore, et al., J.
Cell Biol. 112:491-499 (1991)), and perhaps a subset of lymphocytes
(Moore, et al. J. Cell Biol. 112:491-499 (1991)). Thus, GMP-140 can
serve as a receptor for leukocytes following its rapid mobilization
to the surfaces of platelets and endothelial cells stimulated with
agonists such as thrombin. This role in leukocyte recruitment may
be important in hemostatic and inflammatory processes in both
physiologic and pathologic states.
[0011] Peptides derived from GMP-140 are described in U.S. Ser. No.
07/554,199 entitled "Functionally Active Selectin-Derived Peptides"
filed Jul. 17, 1990 by Rodger P. McEver that are useful in
diagnostics and in modulating the hemostatic and inflammatory
responses in a patient wherein a therapeutically effective amount
of a peptide capable of blocking leukocyte recognition of GMP-140
is administered to the patient. U.S. Ser. No. 07/554,199 filed Jul.
17, 1990 also discloses that peptide sequences within the lectin
domain of GMP-140, having homology with the lectin domains of other
proteins, especially ELAM-1 and the homing receptor, selectively
inhibit neutrophil adhesion to purified GMP-140, and can therefore
be used in diagnostic assays of patients and diseases characterized
by altered binding by these molecules, in screening assays for
compounds altering this binding, and in clinical applications to
inhibit or modulate interactions of leukocytes with platelets or
endothelial cells involving coagulation and/or inflammatory
processes.
[0012] ELAM-1, the homing receptor, and GMP-140 have been termed
"selecting", based on their related structure and function. ELAM-1
is not present in unstimulated endothelium. However, when
endothelium is exposed to cytokines such as tumor necrosis factor
or interleukin-1, the gene for ELAM-1 is transcribed, producing RNA
which in turn is translated into protein. The result is that ELAM-1
is expressed on the surface of endothelial cells one to four hours
after exposure to cytokines, as reported by Bevilacqua et al.,
Proc. Natl. Acad. Sci. USA 84:9238-9242 (1987) (in contrast to
GMP-140, which is stored in granules and presented on the cell
surface within seconds after activation). ELAM-1 has been shown to
mediate the adherence of neutrophils to cytokine-treated
endothelium and thus appears to be important in allowing leukocytes
to migrate across cytokine-stimulated endothelium into tissues. The
cDNA-derived primary structure of ELAM-1 indicates that it contains
a "lectin" domain, an EGF domain, and six (instead of the nine in
GMP-140) repeats similar to those of complement-regulatory
proteins, a transmembrane domain, and a short cytoplasmic tail.
There is extensive sequence homology between GMP-140 and ELAM-1
throughout both proteins, but the similarity is particularly
striking in the lectin and EGF domains.
[0013] Homing receptors are lymphocyte surface structures that
allow lymphocytes to bind to specialized endothelial cells in
lymphatic tissues, termed high endothelial cells or high
endothelial venules (reviewed by Yednock and Rose, Advances in
Immunology, vol. 44, F. I. Dixon,ed., 313-378 (Academic Press, New
York 1989). This binding allows lymphocytes to migrate across the
endothelium into the lymphatic tissues where they are exposed to
processed antigens. The lymphocytes then re-enter the blood through
the lymphatic system. The homing receptor contains a lectin domain,
an EGF domain, two complement-binding repeats, a transmembrane
domain, and a short cytoplasmic tail. The homing receptor also
shares extensive sequence homology with GMP-140, particularly in
the lectin and EGF domains.
[0014] Based on a comparison of the lectin domains between GMP-140,
ELAM-1, and the homing receptor (LEU-8), it may be possible to
select those peptides inhibiting binding of neutrophils to GMP-140
which will inhibit binding of ELAM-1, the homing receptor, and
other homologous selectins, to components of the inflammatory
process, or, conversely, which will inhibit only GMP-140
binding.
[0015] The in vivo significance of platelet-leukocyte interactions
has not been studied carefully. However, in response to vascular
injury, platelets are known to adhere to subendothelial surfaces,
become activated, and support coagulation. Platelets and other
cells may also play an important role in the recruitment of
leukocytes into the wound in order to contain microbial invasion.
Conversely, leukocytes may recruit platelets into tissues at sites
of inflammation, as reported by Issekutz, et al., Lab. Invest.
49:716 (1983).
[0016] The coagulation and inflammatory pathways are regulated in a
coordinate fashion in response to tissue damage. For example, in
addition to becoming adhesive for leukocytes, activated endothelial
cells express tissue factor on the cell surface and decrease their
surface expression of thrombomodulin, leading to a net facilitation
of coagulation reactions on the cell surface. In some cases, a
single receptor can be involved in both inflammatory and
coagulation processes.
[0017] Proteins involved in the hemostatic and inflammatory
pathways are of interest for diagnostic purposes and treatment of
human disorders. However, there are many problems using proteins
therapeutically. Proteins are usually expensive to produce in
quantities sufficient for administration to a patient. Moreover,
there can be a reaction against the protein after it has been
administered more than once to the patient. It is therefore
desirable to develop peptides having the same, or better, activity
as the protein, which are inexpensive to synthesize, reproducible
and relatively innocuous.
[0018] It is preferable to develop peptides which can be prepared
synthetically, having activity at least equal to, or greater than,
the peptides derived from the protein itself.
[0019] It is therefore an object of the present invention to
provide peptides interacting with cells recognized by selecting,
including GMP-140, ELAM-1, and lymphocyte homing receptor.
[0020] It is another object of the present invention to provide
methods for using these peptides to inhibit leukocyte adhesion to
endothelium or to platelets.
[0021] It is a further object of the present invention to provide
methods for using these peptides to modulate the immune response
and the hemostatic pathway.
[0022] It is yet another object of the present invention to provide
peptides for use in diagnostic assays relating to GMP-140, ELAM-1,
and lymphocyte homing receptor.
SUMMARY OF THE INVENTION
[0023] Peptides derived from three regions of the lectin domain of
GMP-140 and the related selectins, ELAM-1 and the lymphocyte homing
receptor, have been found to inhibit neutrophil adhesion to
GMP-140. These and additional peptides have been synthesized having
the following formula:
R.sup.1--X-A-B--C-D-E-Y--R.sup.2 (I)
[0024] or a pharmaceutically acceptable acid- or based-addition
salt thereof wherein:
[0025] A is D- or L-asparagine, D- or L-isoleucine or D- or
L-valine;
[0026] B is D- or L-asparagine or glycine;
[0027] C is D- or L-lysine, D- or L-valine or glycine;
[0028] D is D- or L-valine, D- or L-threonine or D- or
L-isoleucine;
[0029] E is D- or L-tryptophan;
[0030] X and Y are linear chains of from 0 to 10 amino acids;
[0031] R.sup.1 is H (signifying a free N-terminal group), formyl,
lower alkyl, aryl, lower alkanoyl, aroyl, alkyloxycarbonyl or
aryloxycarbonyl and
[0032] R.sup.2 is OH (signifying a free C-terminal group) lower
alkyl or aryl esters, or NR.sup.3R.sup.4 where R.sup.3 and R.sup.4
each selected independently from H, lower alkyl or aryl.
[0033] Peptides of Formula I have as their core region portions of
the 56-60 amino acid sequence of GMP-140, with residue 1 defined as
the N-terminus of the mature protein after the cleavage of the
signal peptide. Examples of peptides of Formula I demonstrate the
inhibition of the binding of neutrophils to GMP-140 in
concentrations ranging from 5 to 1500 .mu.M. It has been found that
alterations within the core sequence, as well as N-terminal and
C-terminal flanking regions, do not result in loss of biological
activity.
[0034] The peptides are useful as diagnostics and, in combination
with a suitable pharmaceutical carrier, for clinical applications
in the modulation or inhibition of coagulation processes or
inflammatory processes.
BRIEF DESCRIPTION OF THE DRAWINGS
[0035] FIG. 1 shows the activity of several peptides of Formula I
in inhibiting the binding of neutrophils to GMP-140, % inhibition
versus concentration of peptide (mM): RKNNKTWTWV-NH.sub.2 (closed
squares); RKVNNVWVWV-NH.sub.2 (open squares); RKVNNVWVWV (closed
diamonds); RKIGGIWTWV-NH.sub.2 (open diamonds); rKIGGIWTWV-NH.sub.2
(closed triangles); RkIGGIWTWV-NH.sub.2 (open triangles);
CRKNNKTWTWV-NH.sub.2 (closed circles); YKNNKTWTWV-NH.sub.2 (open
circles); RENNKTWTWV-NH.sub.2 (-X-); RKNNGTWTWV-NH.sub.2
(->.vertline.<-); FMOC-NNKTW-NH.sub.2 (-.vertline.-); and
KWKWNRTNVT-NH.sub.2 (--) (control peptide).
DETAILED DESCRIPTION OF THE INVENTION
[0036] Peptides having GMP-140-like activity, therapeutic
compositions containing these peptides, methods for the preparation
of these peptides, and methods of use thereof are disclosed.
[0037] In their broadest scope, the peptides having the following
formula:
R.sup.1--X-A-B--C-D-E-Y--R.sup.2 (I)
[0038] or a pharmaceutically acceptable salt thereof, wherein:
[0039] A is D- or L-asparagine, D- or L-isoleucine or D- or
L-valine;
[0040] B is D- or L-asparagine or glycine;
[0041] C is D- or L-lysine, D- or L-valine or glycine;
[0042] D is D- or L-valine, D- or L-threonine or D- or
L-isoleucine;
[0043] E is D- or L-tryptophan;
[0044] X and Y are linear chains of from 0 to 10 amino acids;
[0045] R.sup.1-is H (signifying a free N-terminal group), formyl,
lower alkyl, aryl, lower alkanoyl, aroyl, alkyloxycarbonyl or
aryloxycarbonyl and
[0046] R.sup.2 is OH (signifying a free C-terminal group) lower
alkyl or aryl esters, or NR.sup.3R.sup.4 where R.sup.3 and R.sup.4
each selected independently from H, lower alkyl or aryl.
[0047] Preferred peptides are those wherein E is tryptophan,
particularly where R.sup.1 is H and R.sup.2 is NR.sup.3R.sup.4.
[0048] Most preferred peptides are:
[0049] Arg-Lys-Asn-Asn-Lys-Thr-Trp-NH.sub.2;
[0050]
Cys-Ile-Gly-Ile-Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH.sub.2;
[0051]
Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-Gly-Thr-Lys-Lys-Ala-Leu-Thr-
-Asn-Glu-Cys-NH.sub.2;
[0052] Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH.sub.2;
[0053] Lys-Asn-Asn-Lys-Thr-Trp-NH.sub.2;
[0054] Acetyl-Asn-Asn-Lys-Thr-Trp-NH.sub.2;
[0055] Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val;
[0056] Arg-Lys-Val-Asn-Asn-Val-Trp-val-Trp-Val;
[0057] Arg-Lys-Val-Asn-Asn-Val-Trp-Val-Trp-Val-NH.sub.2;
[0058] Arg-Lys-Ile-Gly-Gly-Ile-Trp-Thr-Trp-Val;
[0059] Arg-Lys-Ile-Gly-Gly-Ile-Trp-Thr-Trp-Val-NH.sub.2;
[0060] Arg-Lys-Ile-Gly-Gly-Ile-Trp-NH.sub.2;
[0061] Arg-Lys-Val-Asn-Asn-Val-Trp-NH.sub.2;
[0062] Ac-Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH.sub.2;
[0063] Cys-Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH.sub.2;
[0064] Arg-Glu-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH.sub.2;
[0065] Arg-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Glu-NH.sub.2;
[0066] Tyr-Lys-Asn-Asn-Lys-Thr-Trp-Thr-Trp-Val-NH.sub.2;
[0067] D-Arg-Lys-Ile-Gly-Gly-Ile-Trp-Thr-Trp-Val-NH.sub.2;
[0068] Arg-D-Lys-Ile-Gly-Gly-Ile-Trp-Thr-Trp-Val-NH.sub.2; and
[0069] FMOC-Asn-Asn-Lys-Thr-Trp-NH.sub.2.
[0070] As used herein, the term "lower alkyl" includes branched,
straight-chain, and cyclic saturated hydrocarbons having from one
to six carbon atoms, such as methyl, ethyl, propyl, isopropyl,
butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl,
cyclopentylmethyl and hexyl. The term "lower alkanoyl" means 1
[0071] wherein R.sup.5 is a lower alkyl group. The term aroyl means
2
[0072] wherein R.sup.6 is an aromatic or heteroaromatic structure
having between one and three rings, which may or may not be ring
fused structures, and are optimally substituted with halogens,
carbons, or other heteroatoms such as nitrogen (N), sulfur (S),
phosphorus (P), and boron (B). The term alkoxycarbonyl means 3
[0073] wherein R.sup.7 is a lower alkyl group. The term
aryloxycarbonyl means 4
[0074] wherein R.sup.8 is an aryl or arylmethyl group.
[0075] The peptides of formula I can be used in the form of the
free peptide or a pharmaceutically acceptable salt. Amine salts can
be prepared by treating the peptide with an acid according to known
methods. Suitable acids include inorganic acids such as
hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid;
thiocyanic acid, sulfuric acid, and phosphoric acids and organic
acids such as formic acid, acetic acid, propionic acid, glycolic
acid, lactic acid, pyruvic acid, oxalic acid, malonic acid,
succinic acid, maleic acid, fumaric acid, anthranilic acid,
cinnamic acid, naphthalenesulfonic acid, and sulfanilic acid.
[0076] Carboxylic acid groups in the peptide can be converted to a
salt by treating the peptide with a base according to known
methods. Suitable bases include inorganic bases such as sodium
hydroxide, ammonium hydroxide, and potassium hydroxide, and organic
bases such as mono-, di-, and tri-alkyl and aryl amines (e.g.,
triethylamine, diisopropylamine, methylamine, and dimethylamine and
optionally substituted mono-, di, and tri-ethanolamines.
[0077] As referred to herein, the amino acid components of the
peptides and certain materials used in their preparation are
identified by abbreviations for convenience. These abbreviations
are as follows:
1 Abbreviations Amino Acid L-alanine Ala A D-alanine D-Ala a
L-arginine Arg R D-arginine D-Arg r D-asparagine D-Asn N
L-asparagine L-Asn n L-aspartic acid Asp D D-aspartic acid D-Asp d
L-cysteine Cys C D-cysteine D-Cys c L-glutamic acid Glu E
D-glutamic acid D-Glu e L-glutamine Gln K D-glutamine D-Gln k
glycine Gly G L-histidine His H D-histidine D-His h L-isolelucine
Ile I D-isoleucine D-Ile i L-leucine Leu L D-leucine D-Leu l
L-lysine Lys K D-lysine D-Lys k L-phenylalanine Phe F
D-phenylalanine D-Phe f L-proline Pro P D-proline D-Pro p
L-pyroglutamic acid pGlu D-pyroglutamic acid D-pGlu L-serine L-Ser
S D-serine D-Ser s L-threonine L-Thr T D-threonine D-Thr t
L-tyrosine L-Tyr Y D-tyrosine D-Tyr y L-tryptophan Trp W
D-tryptophan D-Trp w L-valine Val V D-valine D-Val V Reagents
Trifluoroacetic acid TFA Methylene chloride CH.sub.2Cl.sub.2
N,N-Diisopropylethylamine DIEA N-Methylpyrrolidone NMP
1-Hydroxybenzotriazole HOBT Dimethylsulfoxide DMSO Acetic anhydride
Ac.sub.2O
Methods of Preparation of Peptides
[0078] The peptides can generally be prepared following known
techniques, as described for example in the cited publications, the
teachings of which are specifically incorporated herein. In a
preferred method, the peptides are prepared following the
solid-phase synthetic technique initially described by Merrifield
in J. Amer. Chem. Soc., 85, 2149-2154 (1963). Other techniques may
be found, for example, in M. Bodanszky, et al., Peptide Synthesis,
second edition, (John Wiley & Sons, 1976), as well as in other
reference works known to those skilled in the art.
[0079] Appropriate protective groups usable in such syntheses and
their abbreviations will be found in the above text, as well as in
J. F. W. McOmie, Protective Groups in Organic Chemistry, (Plenum
Press, New York, 1973). The common protective groups used herein
are t-butyloxycarbonyl (Boc), fluorenylmethoxycarbonyl (FMOC),
benzyl (Bzl), tosyl (Tos), o-bromo-phenylmethoxycarbonyl (BrCBZ),
phenylmethoxycarbonyl (CBZ), 2-chloro-phenylmethoxycarbonyl,
(2-Cl-CBZ), 4-methoxy-2,3,6-trimethylbenz- enesulfonyl (Mtr),
trityl (Trt), formyl (CHO), and tertiary butyl (t-Bu).
[0080] General synthetic procedures for the synthesis of peptides
of Formula I by solid phase methodology are as follows:
2 A. General Synthetic Procedures for Solid Phase Peptide Synthesis
Using N.sup..alpha.-Boc Protection. REPETITIONS TIME 1. 25% TFA in
CH.sub.2Cl.sub.2 1 3 min 2. 50% TFA in CH.sub.2Cl.sub.2 1 16 min 3.
CH.sub.2Cl.sub.2 5 3 min 4. 5% DIEA in NMP 2 4 min 5. NMP 6 5 min
6. Coupling step 1 57 min a. Preformed BOC-Amino Acid- 36 min HOBT
active ester in NMP b. DMSO 16 min c. DIEA 5 min 7. 10% Ac.sub.2O,
5% DIEA in NMP 1 9 min 8. CH.sub.2Cl.sub.2 5 3 min
[0081]
3 B. General Synthetic Procedure For Solid Phase Peptide Synthesis
Using N.sup..alpha.- FMOC Protection REPETITIONS TIME 1. 20%
piperdine in NMP 1 3 min 2. 20% piperdine in NMP 1 15 min 3. NMP 6
9 min 4. Coupling 1 71 min Preformed FMOC-Amino Acid- HOBT active
ester in NMP 5. NMP 6 7 min
[0082] N-terminal acetylation on the deprotected
N.sup..alpha.-amino group of peptides synthesized using either Boc
or FMOC strategies is accomplished with 10% Ac.sub.2O and 5% DIEA
in NMP, followed by washing of the peptide resin with NMP and/or
CH.sub.2Cl.sub.2.
[0083] The peptides can also be prepared using standard genetic
engineering techniques known to those skilled in the art. For
example, the peptide can be produced enzymatically by inserting
nucleic acid encoding the peptide into an expression vector,
expressing the DNA, and translating the DNA into the peptide in the
presence of the required amino acids. The peptide is then purified
using chromatographic or electrophoretic techniques, or by means of
a carrier protein which can be fused to, and subsequently cleaved
from, the peptide by inserting into the expression vector in phase
with the peptide encoding sequence a nucleic acid sequence encoding
the carrier protein. The fusion protein-peptide may be isolated
using chromatographic, electrophoretic or immunological techniques
(such as binding to a resin via an antibody to the carrier
protein). The peptide can be cleaved using chemical methodology or
enzymatically, as by, for example, hydrolases.
[0084] Methods of Preparation of Pharmaceutical Compositions
[0085] To prepare the pharmaceutical compositions containing these
peptides, a peptide of Formula I or a base or acid addition salt
thereof is combined as the active ingredient with a pharmaceutical
carrier according to conventional pharmaceutical compounding
techniques. This carrier may take a wide variety of forms depending
on the form of preparation desired for administration, e.g.,
sublingual, rectal, nasal, oral, or parenteral. In preparing the
compositions in oral dosage form, any of the usual pharmaceutical
media may be employed, for example, water, oils, alcohols,
flavoring agents, preservatives, and coloring agents, to make an
oral liquid preparation (e.g., suspension, elixir, or solution) or
with carriers such as starches, sugars, diluents, granulating
agents, lubricants, binders, and disintegrating agents, to make an
oral solid preparation (e.g., powder, capsule, or tablet).
[0086] Controlled release forms or enhancers to increase
bioavailability may also be used. Because of their ease in
administration, tablets and capsules represent the most
advantageous oral dosage unit form, in which case solid
pharmaceutical carriers are employed. If desired, tablets may be
sugar coated or enteric coated by standard techniques.
[0087] For parenteral products, the carrier will usually be sterile
water, although other ingredients to aid solubility or as
preservatives may be included. Injectable suspensions may also be
prepared, in which case appropriate liquid carriers and suspending
agents can be employed.
[0088] The peptides can also be administered locally at a wound or
inflammatory site by topical application of a solution or
cream.
[0089] Alternatively, the peptide may be administered in liposomes
or microspheres (or microparticles). Methods for preparing
liposomes and microspheres for administration to a patient are
known to those skilled in the art. U.S. Pat. No. 4,789,734 describe
methods for encapsulating biological materials in liposomes.
Essentially, the material is dissolved in an aqueous solution, the
appropriate phospholipids and lipids added, along with surfactants
if required, and the material dialyzed or sonicated, as necessary.
A review of known methods is by G. Gregoriadis, Chapter 14.
"Liposomes", Drug Carriers in Biology and Medicine pp. 287-341
(Academic Press, 1979). Microspheres formed of polymers or proteins
are well known to those skilled in the art, and can be tailored for
passage through the gastrointestinal tract directly into the
bloodstream. Alternatively, the peptide can be incorporated and the
microspheres, or composite of microspheres, implanted for slow
release over a period of time, ranging from days to months. See,
for example, U.S. Pat. Nos. 4,906,474, 4,925,673, and
3,625,214.
[0090] The peptides are generally active when administered
parenterally in amounts above about 1 .mu.g/kg body weight. For
treatment to prevent organ injury in cases involving reperfusion,
the peptides may be administered parenterally from about 0.01 to
about 10 mg/kg body weight. Generally, the same range of dosage
amounts may be used in treatment of the other diseases or
conditions where inflammation is to be reduced.
Methods for Demonstrating Binding
[0091] Peptides that are biologically active are those which
inhibit binding of neutrophils, monocytes, subsets of lymphocytes
or other cells to GMP-140, or which inhibit leukocyte adhesion to
endothelium that is mediated by ELAM-1 and/or the homing
receptor.
[0092] Peptides can be screened for their ability to inhibit
adhesion to cells, for example, neutrophil adhesion to purified
GMP-140 immobilized on plastic wells, using the assay described by
Geng, et al., Nature 343, 757-760 (1990).
[0093] Human neutrophils are isolated from heparinized whole blood
by density gradient centrifugation on Mono-Poly resolving media,
Flow Laboratories. Neutrophil suspensions are greater than 98% pure
and greater than 95% viable by trypan blue exclusion. For adhesion
assays, neutrophils are suspended at a concentration of
2.times.10.sup.6 cells/mL in Hanks' balanced salt solution
containing 1.26 mM Ca and 0.81 mM Mg (HBSS, Gibco) with 5 mg/mL
human serum albumin (HBSS/HSA). Adhesion assays are conducted in
triplicate in 96-well microtiter plates, Corning, incubated at
4.degree. C. overnight with 50 microliters of various protein
solutions.
[0094] GMP-140 is isolated from human platelet lysates by
immunoaffinity chromatography on antibody S12-Sepharose and
ion-exchange chromatography on a Mono-Q.TM. column (FLPC, Pharmacia
Fine Chemicals), as follows.
[0095] Outdated human platelet packs (100 units) obtained from a
blood bank and stored at 4.degree. C. are pooled, adjusted to 5 mM
EDTA at pH 7.5, centrifuged at 4,000 rpm for 30 min in 1 liter
bottles, then washed three times with 1 liter of 0.1 M NaCl, 20 mM
Tris pH 7.5 (TBS), 5 mM EDTA, 5 mM benzamidine.
[0096] The pellets are then resuspended in a minimum amount of wash
buffer and made 1 mM in DIFP, then frozen in 50 mL screwtop tubes
at -80.degree. C. The frozen platelets are thawed and resuspended
in 50 mL TBS, 5 mM benzamidine, 5 mM EDTA-pH 7.5, 100 M leupeptin.
The suspension is frozen and thawed two times in a dry ice-acetone
bath using a 600 mL lyophilizing flask, then homogenized in a
glass/teflon mortar and pestle and made 1 mM in DIFP. The NaCl
concentration is adjusted to 0.5 M with a stock solution of 4 M
NaCl. After stirring the suspension at 4.degree. C., it is
centrifuged in polycarbonate tubes at 33,000 rpm for 60 min at
4.degree. C. The supernatant (0.5 M NaCl wash) is removed and
saved; this supernatant contains the soluble form of GMP-140. Care
is taken not to remove the top part of the pellet with the
supernatant. The pellets are then homogenized in extraction buffer
(TBS, 5 mM benzamidine, 5 mM EDTA, pH 7.5, 100 .mu.M leupeptin, 2%
Triton X-100). After centrifugation at 19,500 rpm for 25 min at
4.degree. C., the supernatant is removed. The extraction procedure
is repeated with the pellet and the supernatant is combined with
the first supernatant. The combined extracts, which contain the
membrane form of GMP-140, are adjusted to 0.5 M NaCl.
[0097] The soluble fraction (0.5 M NaCl wash) and the membrane
extract (also adjusted to 0.5 M NaCl) are absorbed with separate
pools of the monoclonal antibody S12 (directed to human GMP-140)
previously coupled to Affigel (Biorad) at 5 mg/mL for 2 h at
4.degree. C. After letting the resins settle, the supernatants are
removed. The S12 Affigel containing bound GMP-140 is then loaded
into a column and washed overnight at 4.degree. C. with 400 mL of
0.5 M NaCl, 20 mM Tris pH 7.5, 0.01% Lubrol PX.
[0098] Bound GMP-140 is eluted from the S12 Affigel with 100 mL of
80% ethylene glycol, 1 mM MES pH 6.0, 0.01% Lubrol PX. Peak
fractions with absorbance at 280 nm are pooled. Eluates are
dialyzed against TBS with 0.05% Lubrol, then applied to a Mono Q
column (FPLC from Pharmacia). The concentrated protein is step
eluted with 2 M NaCl, 20 mM Tris pH 7.5 (plus 0.05% Lubrol PX for
the membrane fraction). Peak fractions are dialyzed into TBS pH 7.5
(plus 0.05% Lubrol PX for the membrane fraction).
[0099] GMP-140 is plated at 5 micrograms/mL and the control
proteins: human serum albumin (Alb), platelet glycoprotein IIb/IIIa
(IIb), von Willebrand factor (vWF), fibrinogen (FIB),
thrombomodulin (TM), gelatin (GEL) or human serum (HS), are added
at 50 micrograms/mL. All wells are blocked for 2 h at 22.degree. C.
with 300 microliters HBSS containing 10 mg/mL HSA, then washed
three times with HBSS containing 0.1% Tween-20 and once with HBSS.
Cells (2.times.10.sup.5 per well are added to the wells and
incubated at 22.degree. C. for 20 min. The wells are then filled
with HBSS/HSA, sealed with acetate tape (Dynatech), and centrifuged
inverted at 150 g for 5 min. After discarding nonadherent cells and
supernates, the contents of each well are solubilized with 200
microliters 0.5% hexadecyltrimethylammonium bromide, Sigma, in 50
mM potassium phosphate, pH 6.0, and assayed for myeloperoxidase
activity, Ley, et al., Blood 73, 1324-1330 (1989). The number of
cells bound is derived from a standard curve of myeloperoxidase
activity versus numbers of cells. Under all assay conditions, the
cells release less than 5% of total myeloperoxidase and lactate
dehydrogenase. Inhibition is read as a lower percent adhesion, so
that a value of 5% means that 95% of the specific adhesion was
inhibited.
Clinical Applications
[0100] The peptides are generally active when administered
parenterally in amounts above about 1 .mu.g/kg body weight. For
treatment to prevent organ injury in cases involving reperfusion,
the peptides may be administered parenterally from about 0.01 to
about 10 mg/kg body weight. Generally, the same range of dosage
amounts may be used in treatment of the other diseases or
conditions where inflammation is to be reduced. This dosage will be
dependent, in part, on whether one or more peptides are
administered. A synergistic effect may be seen with combinations of
peptides from different, or overlapping, regions of the lectin
domain, or in combination with peptides derived from the EGF domain
of GMP-140.
[0101] Since the selectins have several functions related to
leukocyte adherence, inflammation, and coagulation, clinically,
compounds which interfere with binding of GMP-140, ELAM-1 or LEU-8
can be used to modulate these responses.
[0102] For example, the peptides can be used to competitively
inhibit leukocyte adherence by competitively binding to GMP-140
receptors on the surface of leukocytes. This kind of therapy would
be particularly useful in acute situations where effective, but
transient, inhibition of leukocyte-mediated inflammation is
desirable. Chronic therapy by infusion of the peptides may also be
feasible in some circumstances.
[0103] An inflammatory response may cause damage to the host if
unchecked, because leukocytes release many toxic molecules that can
damage normal tissues. These molecules include proteolytic enzymes
and free radicals. Examples of pathological situations in which
leukocytes can cause tissue damage include injury from ischemia and
reperfusion, bacterial sepsis and disseminated intravascular
coagulation, adult respiratory distress syndrome, tumor metastasis,
rheumatoid arthritis and atherosclerosis.
[0104] Reperfusion injury is a major problem in clinical
cardiology. Therapeutic agents that reduce leukocyte adherence in
ischemic myocardium can significantly enhance the therapeutic
efficacy of thrombolytic agents. Thrombolytic therapy with agents
such as tissue plasminogen activator or streptokinase can relieve
coronary artery obstruction in many patients with severe myocardial
ischemia prior to irreversible myocardial cell death. However, many
such patients still suffer myocardial neurosis despite restoration
of blood flow. This "reperfusion injury" is known to be associated
with adherence of leukocytes to vascular endothelium in the
ischemic zone, presumably in part because of activation of
platelets and endothelium by thrombin and cytokines that makes them
adhesive for leukocytes (Romson et al., Circulation 67: 1016-1023,
1983). These adherent leukocytes can migrate through the
endothelium and destroy ischemic myocardium just as it is being
rescued by restoration of blood flow.
[0105] There are a number of other common clinical disorders in
which ischemia and reperfusion results in organ injury mediated by
adherence of leukocytes to vascular surfaces, including strokes;
mesenteric and peripheral vascular disease; organ transplantation;
and circulatory shock (in this case many organs might be damaged
following restoration of blood flow).
[0106] Bacterial sepsis and disseminated intravascular coagulation
often exist concurrently in critically ill patients. They are
associated with generation of thrombin, cytokines, and other
inflammatory mediators, activation of platelets and endothelium,
and adherence of leukocytes and aggregation of platelets throughout
the vascular system. Leukocyte-dependent organ damage is an
important feature of these conditions.
[0107] Adult respiratory distress syndrome is a devastating
pulmonary disorder occurring in patients with sepsis or following
trauma, which is associated with widespread adherence and
aggregation of leukocytes in the pulmonary circulation. This leads
to extravasation of large amounts of plasma into the lungs and
destruction of lung tissue, both mediated in large part by
leukocyte products.
[0108] Two related pulmonary disorders that are often fatal are in
immunosuppressed patients undergoing allogeneic bone marrow
transplantation and in cancer patients suffering from complications
that arise from generalized vascular leakage resulting from
treatment with interleukin-2 treated LAK cells
(lymphokine-activated lymphocytes). LAK cells are known to adhere
to vascular walls and release products that are presumably toxic to
endothelium. Although the mechanism by which LAK cells adhere to
endothelium is not known, such cells could potentially release
molecules that activate endothelium and then-bind to endothelium by
mechanisms similar to those operative in neutrophils.
[0109] Tumor cells from many malignancies (including carcinomas,
lymphomas, and sarcomas) can metastasize to distant sites through
the vasculature. The mechanisms for adhesion of tumor cells to
endothelium and their subsequent migration are not well understood,
but may be similar to those of leukocytes in at least some cases.
The association of platelets with metastasizing tumor cells has
been well described, suggesting a role for platelets in the spread
of some cancers.
[0110] Platelet-leukocyte interactions are believed to be important
in atherosclerosis. Platelets might have a role in recruitment of
monocytes into atherosclerotic plaques; the accumulation of
monocytes is known to be one of the earliest detectable events
during atherogenesis. Rupture of a fully developed plaque may not
only lead to platelet deposition and activation and the promotion
of thrombus formation, but also the early recruitment of
neutrophils to an area of ischemia.
[0111] Another area of potential application is in the treatment of
rheumatoid arthritis.
[0112] The criteria for assessing response to therapeutic
modalities employing these peptides are dictated by the specific
condition and will generally follow standard medical practices. For
example, the criteria for the effective dosage to prevent extension
of myocardial infarction would be determined by one skilled in the
art by looking at marker enzymes of myocardial necrosis in the
plasma, by monitoring the electrocardiogram, vital signs, and
clinical response. For treatment of acute respiratory distress
syndrome, one would examine improvements in arterial oxygen,
resolution of pulmonary infiltrates, and clinical improvement as
measured by lessened dyspnea and tachypnea. For treatment of
patients in shock (low blood pressure), the effective dosage would
be based on the clinical response and specific measurements of
function of vital organs such as the liver and kidney following
restoration of blood pressure. Neurologic function would be
monitored in patients with stroke. Specific tests are used to
monitor the functioning of transplanted organs; for example, serum
creatinine, urine flow, and serum electrolytes in patients
undergoing kidney transplantation.
Diagnostic Reagents
[0113] The peptides can also be used for the detection of human
disorders in which the ligands for the selectins might be
defective. Such disorders would most likely be seen in patients
with increased susceptibility to infections in which leukocytes
might not be able to bind to activated platelets or endothelium.
Cells to be tested, usually leukocytes, are collected by standard
medically approved techniques and screened. Detection systems
include ELISA procedures, binding of radiolabeled antibody to
immobilized activated cells, flow cytometry, or other methods known
to those skilled in the arts. Inhibition of binding in the presence
and absence of the lectin domain peptides can be used to detect
defects or alterations in selectin binding. For selecting, such
disorders would most likely be seen in patients with increased
susceptibility to infections in which leukocytes would have
defective binding to platelets and endothelium because of deficient
leukocyte ligands for GMP-140. The peptide is labeled
radioactively, with a fluorescent tag, enzymatically, or with
electron dense material such as gold for electron microscopy. The
cells to be examined, usually leukocytes, are incubated with the
labeled peptides and binding assessed by methods described above
with antibodies to GMP-140, or by other methods known to those
skilled in the art. If ligands for GMP-140 are also found in the
plasma, they can also be measured with standard ELISA or
radioimmunoassay procedures, using labeled GMP-140-derived peptide
instead of antibody as the detecting reagent. The following
examples are presented to illustrate the invention without
intending to specifically limit the invention thereto. In the
examples and throughout the specifications, parts are by weight
unless otherwise indicated.
EXAMPLE 1
Preparation of
Arginyl-lysyl-asparaginyl-asparaginyl-lysyl-threonyl-trypto-
phyl-threonyl-tryptophyl-valine-amide
[0114] The peptide was prepared on an ABI model 431A peptide
synthesizer using Version 1.12 of the standard scale Boc software.
The amino acids used were Boc-(Tos)Arg, Boc-(ClZ)Lys, Boc-Asn,
Boc-(Bzl)Thr, Boc-(CHO)Trp, and Boc-Val. 4-Methylbenzhydrylamine
resin (0.625 g, 0.5 mmol) was used in the synthesis. The final
weight of the peptide resin was 1.63 g.
[0115] The peptide was cleaved from the resin (1.49 g) using 15 mL
of HF and 1.5 mL of anisole for 60 min at 0.degree. C. The hydrogen
fluoride was evaporated using a stream of nitrogen, the resulting
mixture triturated with ether, and the ether removed by filtration.
The resulting solids were extracted with cold 0.1 M piperidine
(5.times.10 mL). The extracts were combined and stirred for three
hours at 0.degree. C. 1 mL of acetic acid was added. The resultant
slurry was lyophilized to yield 0.83 g of crude peptide. The crude
peptide was purified by HPLC (multiple injections) on a Vydac C-18
(10.mu., 2.2.times.25 cm) column eluted with a gradient of 15-30%
acetonitrile in 0.1% TFA over 30 min at a flow rate of 3 mL per
min. Fractions were collected, analyzed by HPLC and pure fractions
pooled and lyophilized to yield 35 mg of pure peptide. Amino acid
analysis: Arg 0.96 (1.0); Asx 1.95 (2.0); Lys 2.16 (2.0); Thr 1.76
(2.0); Trp 1.45 (2.0); Val 0.98 (1.0). FAB/MS:MH.sup. +1332.
EXAMPLE 2
Preparation of
Arginyl-lysyl-valyl-asparaginyl-asparaginyl-valyl-tryptophy-
l-valyl-tryptophyl-valine-amide
[0116] The peptide was prepared on a ABI model 431A peptides
synthesizer using Version 1.12 of the standard scale Boc software.
The amino acids used were Boc-(Tos)Arg, Boc-(ClZ)Lys, Boc-Val,
Boc-Asn, and Boc-(CHO)Trp. 4-methylbenzhydrylamine resin (0.625 g,
0.5 mmol) was used in the synthesis. The resin peptide was treated
with 1 M ethanolamine in DMF with 5% water (2.times.30 min) to
deformylate the tryptophan. After washing and drying the final
weight of the resin was 1.3 g. The peptide was cleaved from the
resin (1.25 g) using 15 mL of HF and 0.75 g p-cresol and 0.75 g
p-thiocresol for 60 minutes at 0.degree. C. The hydrogen fluoride
was removed using a stream of dry nitrogen, the residue triturated
with ether and the ether removed by filtration. The remaining
solids were triturated with a 50% solution of TFA in methylene
chloride. The resin was removed by filtration, the solution
evaporated under reduced pressure and the residue triturated with
ether to give 0.79 g of the crude peptide, isolated by filtration.
The crude peptide (0.3 g) was purified by HPLC (multiple
injections) on a Vydac C-18 column (10.mu., 2.2.times.25 cm)
eluting with a gradient of 10-30% acetonitrile in 0.1% aqueous TFA
over 180 minutes at a flow rate of 3 mL/min. Fractions were
collected, analyzed by HPLC and pure fractions pooled and
lyophilized to give 21 mg of pure peptide. Amino acid analysis: Asx
2.01 (2.0); Val 4.07 (4.0); Lys 0.96 (1.0); Trp 1.93 (2.0); Arg
1.02 (1.0). FAB/MS: MH.sup.+ 1299.
EXAMPLE 3
Preparation of
Arginyl-lysyl-asparaginyl-asparaginyl-lysyl-threonyl-trypto-
phyl-threonyl-tryptophyl-valyl-glycyl-threonyl-lysyl-lysyl-alanyl-leucyl-c-
ysteine-amide
[0117] The peptide was prepared on ABI model 431A peptide
synthesizer using Version 1.12 of the standard scale Boc software.
The amino acids used in the synthesis were Boc-(Tos)Arg,
Boc-(ClZ)Lys, Boc-Asn, Boc-(Bzl)Thr, Boc-(CHO)Trp, Boc-Val,
Boc-Gly, Boc-Ala, Boc-Leu, and Boc-(4-Me-Bzl)Cys.
4-Methylbenzhydrylamine resin (0.625 g, 0.5 mmol) was used in the
synthesis. Final weight of the peptide resin was 1.85 g. The
tryptophan residues on the resin peptide were deformylated using a
solution of 1 M ethanolamine in dimethylformamide with 5% water
(2.times.30 min). The resin was washed with DMF, ethanol and dried
to a constant weight of 1.85 g. The peptide was cleaved from the
resin (1.75 g) with 15 mL of HF, 1 mL of p-cresol, and 1 mL of
p-thiocresole for 60 minutes at 0.degree. C. The HF was removed by
a nitrogen stream. The resulting solids were triturated with ether,
collected by filtration and washed with ether. The peptide was
extracted from the resin with 50% TFA in methylene chloride
(5.times.20 mL). Precipitation with ether gave 0.96 g of crude
peptide. The crude peptide (0.40 g) was purified on a Vydac C-18
column (15.mu., 5.times.25 cm) eluting with a 20 to 30% gradient of
acetonitrile in 0.1% aqueous TFA over 120 minutes at a flow rate of
15 mL per minute. Fractions were collected, analyzed by HPLC and
pure fractions pooled and lyophilized to give 42 mg of the peptide.
A second purification was done by HPLC on a Vydac C-18 column
(10.mu., 2.2.times.25 cm) using a 10-20% gradient of acetonitrile
in 0.1% TFA over 120 min at a flow rate of 3 mL per min. Fractions
were collected, analyzed by HPLC and pure fractions pooled and
lyophilized to give 10 mg of the pure peptide. Amino acid analysis:
Asx 2.04 (2.0), Thr 2.84 (3.0), Gly 1.03 (1.0), Ala 0.97 (1.0), Cys
N.D. (1.0), Val 1.00 (1.0), Leu 1.02 (1.0), Lys 4.04 (4.0), Trp
N.D. (2.0), Arg 0.95 (1.0). FAB/MS: MH.sup.+ 2035.
EXAMPLE 4
Preparation of
Arginyl-lysyl-isoleucyl-glycyl-glycyl-isoleucyl-tryptophyl--
threonyl-tryptophyl-valine-amide
[0118] The peptide was prepared by manual solid phase synthesis
using Boc chemistry. The amino acids used were Boc-(Tos)Arg,
Boc-(ClZ)Lys, Boc-Ile, Boc-Gly, Boc-(Bzl)Thr, Boc-(CHO)Trp, and
Boc-Val. 4-Methylbenzhydrylamine resin (6.25 g, 5.0 mmol) was used
in synthesis. The final weight of the peptide resin was 1.35 g. The
peptide was cleaved from the resin (1.21 g) using 16 mL of HF, 1.2
mL anisole and 0.4 mL thiophenol for one hour at 0.degree. C. The
HF was removed by a nitrogen stream. The residue was triturated
with ether and the ether removed by filtration. The remaining
solids were extracted with 25 mL of a 50% solution of TFA in
methylene chloride. Removal of the resin by filtration, evaporation
of solution and trituration of the residue gave 0.52 g of crude
peptide. The tryptophan was deformylated with 100 mL 0.1 M aqueous
piperidine for one hour at 0.degree. C. The reaction mixture was
evaporated and the residue dissolved in water and lyophilized. The
crude peptide (0.31 g) was purified by HPLC (multiple injections)
on a Vydac C-18 column, (10.mu., 2.2.times.25 cm) eluting with a
gradient of 25% to 75% acetonitrile in 0.1% TFA over 100 minutes at
a flow rate of 3 mL/min. Fractions were collected, analyzed by HPLC
and pure fractions pooled and lyophilized to give 28 mg of pure
peptide. Amino Acid Analysis: Arg 1.02 (1.0), Gly 2.06 (2.0), Ile
1.92 (2.0), Lys 1.00 (1.0), Thr 0.95 (1.0), Trp 1.42 (2.0), Val
0.98 (1.0). FAB/MS: MH.sup.+=1214.
EXAMPLE 5
Preparation of
Arginyl-lysyl-valyl-asparaginyl-asparaginyl-valyl-tryptophy-
l-valyl-tryptophyl-valine
[0119] The peptide was prepared on an ABI Model 431A peptide
synthesizer using Version 1.12 of the standard scale FMOC software.
The amino acids used for the synthesis were FMOC-(Mtr)Arg,
FMOC-(Boc)Lys, FMOC-Val, FMOC-Asn and FMOC-Trp. Wang resin (0.245
g, 0.25 mmol) was used in the synthesis. The final weight of the
resin was 0.53 g. The peptide was cleaved from the resin using 6 mL
of a mixture of 10 mL TFA, 0.7 g phenol, 0.75 mL ethanedithiol, 0.5
mL anisole and 0.5 mL water at ambient temperature for 1.5 hrs. The
resin was removed by filtration and the peptide precipitated from
the filtrate by the addition of ether. The crude peptide (0.18 g)
was purified by HPLC (2.times.90 mg) on a Vydac C-18 column
(10.mu., 2.2.times.25 cm) eluting with a 20-50% gradient of
acetonitrile in 0.1% TFA over 120 min at a rate of 15 mL/min.
Fractions containing pure peptide were pooled and lyophilized to
yield 30 mg of pure product. Amino acid analysis: Asx 2.14 (2.0),
Val 3.80 (4.0), Lys 0.95 (1.0), Trp 1.02 (2.0), Arg 1.03 (1.0).
FAB:MS: MH.sup.+ 1299.
EXAMPLE 6
Preparation of
Cysteinyl-isoleucyl-glycyl-isoleucyl-arginyl-lysyl-asparagi-
nyl-asparaginyl-lysyl-threonyl-tryptophyl-threonyl-tryptophyl-valine-amide
[0120] The peptide was prepared on an ABI Model 431A peptide
synthesizer using Version 1.12 of the standard scale FMOC software.
The amino acids used for the synthesis were Boc-(4-Me-Bzl)Cys,
Boc-Ile, Boc-Gly, Boc-(Tos)Arg, Boc(ClZ)Lys, Boc-Asn, Boc-(Bzl)Thr,
Boc-(CHO)Trp and Boc-Val. 4-methyl benzhydrylamine resin (0.632 g,
0.5 mmol) was used in the synthesis. The final weight of the resin
was 1.83 g. The typtophans on the peptide resin were deformylated
using 1 M ethanolamine in a mixture of 95% DMF and 5% water. The
peptide resin was isolated by filtration and dried under reduced
pressure. The peptide was cleaved from the resin (1.31 g) using 20
mL of HF, 1.4 mL anisole, and 0.6 mL thiophenol for 60 min at
0.degree. C. The HF removed using a stream of dry nitrogen. The
residue was triturated with ether and the ether removed by
filtration. The remaining solids were extracted with 25 mL of a 50%
solution of TFA in methylene chloride. Removal of the resin by
filtration, evaporation under reduced pressure and trituration of
the residue gave 0.55 g of crude peptide. The crude peptide (0.32
g) was purified by HPLC (multiple injections) on a Vydac C-18
column, (10.mu., 2.2.times.25 cm) eluting with a gradient of 17.5%
to 37.5% acetonitrile in 0.1% TFA over 90 minutes at a flow rate of
6 mL/min. Fractions were collected, analyzed by HPLC and pure
fractions pooled and lyophilized to give 20 mg of pure peptide.
Amino Acid Analysis: Arg 0.91 (1.0), Asx 2.08 (2.0), Cys 0.96
(1.0), Gly 1.00 (1.0), Ile 1.70 (2.0), Lys 2.00 (2.0), Thr 1.83
(2.0), Trp 1.19 (2.0), Val 1.05 (1.0). FAB/MS: MH.sup.+ 1717.
EXAMPLE 7
Preparation of
Arginyl-lysyl-asparaginyl-asparaginyl-lysyl-threonyl-trypto-
phyl-threonyl-tryptophyl-valyl-glycyl-threonyl-lysyl-lysyl-alanyl-leucyl-t-
hreonyl-asparaginyl-glutamyl-cysteine-amide
[0121] The peptide was prepared on an ABI model 431A peptides
synthesizer using Version 1.12 of the standard scale Boc software.
The amino acids used were Boc-(Tos)Arg, Boc-(ClZ)Lys, Boc-Asn,
Boc-(Bzl)Thr, Boc-(CHO)Trp, Boc-Val, Boc-Gly, Boc-Ala, Boc-Leu,
Boc-(Bzl)Glu, and Boc-(Acm)Cys. 4-methyl benzhydrylamine resin
(0.625 g, 0.5 mmol) was used in the synthesis. The tryptophan
residues on the peptide resin were deformylated using 1 M
ethanolamine in a mixture of 95% DMF and 5% water (2.times.20
mL.times.30 min). The final weight of the resin peptide was 2.13 g.
The peptide was cleaved from the resin (2.06 g) using 2 mL of
p-cresol, 0.8 g dithiothreitol and 20 mL HF for one hour at
0.degree. C. The hydrogen fluoride was removed by a stream of dry
nitrogen followed by aspiration. The residue was triturated with
ether, the resulting solids removed by filtration and the peptide
extracted from the resin (5.times.10 mL) with 10% acetic acid. The
extracts were passed through a G-15 gel filtration column
(2.5.times.29 cm) eluting with 1% acetic acid. Appropriate
fractions were combined and lyophilized to give 0.44 g of crude
peptide.
[0122] Analysis showed deformylation of the tryptophan residues to
be incomplete, therefore, 0.39 g of the crude peptide was treated
with 10 mL of 0.1 M aqueous piperidine for 1 hr at 0.degree. C. The
pH was adjusted to between 4 and 5 with 70% acetic acid and the
crude peptide solution purified by HPLC using Vydac C-18 column
(10.mu., 2.2.times.25 cm), eluting with a gradient of 0 to 37.5%
acetonitrile in 0.1% TFA over 42 minutes at a flow rate of 10 mL
per minute. Fractions were collected and the fractions containing
the pure peptide pooled and lyophilized to yield 19 mg of a
yellowish solid. This material was dissolved in 1.2 mL of 30%
acetic acid and treated with 8 mg of mercury (II) acetate for 1 hr
at ambient temperature to remove the Acm protecting group. After
one hour, 12 mg of dithiothreitol was added and stirring continued
for an additional hour. The resulting precipitate was removed by
filtration and the filtrate loaded onto a G-15 column (2.5.times.29
cm) and eluted with 30% acetic acid. Appropriate fractions were
pooled and lyophilized to give 17 mg of the pure peptide as an
off-white solid. Amino acid analysis: Ala 0.99 (1.0), Arg 0.86
(1.0), Asx 2.89 (3.0), Glx 1.06 (1.0), Gly 1.08 (1.0), Leu 1.08
(1.0), Lys 3.87 (4.0), Thr 3.68 (4.0), Trp N.D. (2.0), Val 1.17
(1.0). FAB/MS: MH.sup.+ 2377.
EXAMPLE 8
Preparation of
Arginyl-lysyl-asparaginyl-asparaginyl-lysyl-threonyl-trypto-
phyl-threonyl-tryptophyl-valine
[0123] The peptide was prepared on an ABI Model 431A peptide
synthesizer using Version 1.12 of the standard Boc software. The
amino acids used were Boc-(Tos)Arg, Boc-(ClZ)Lys, Boc-Asn,
Boc-(Bzl)Thr, and Boc-(CHO)Trp. Boc-Valyl-PAM resin (0.83 g, 0.5
mmol) was used in the synthesis. The final weight of the resin
peptide was 1.34 g. The peptide was cleaved from the resin (1.0 g)
using 10 mL of HF and 1.0 mL of anisole for 60 min at 0.degree. C.
The hydrogen fluoride was removed using a stream of nitrogen and
the residue triturated with ether. Solids were removed by
filtration, the peptide extracted from the resin using 1 M acetic
acid, and the extract lyophilized to yield 0.32 g of crude peptide.
The formyl groups from the tryptophan residues were removed using
0.1 M piperidine in a 1:1 mixture of DMF/water for two hours at
ambient temperatures. The crude peptide (0.15 g) was purified by
HPLC (3.times.50 mg) using a Vydac C-18 column (10.mu.,
2.2.times.25 cm) eluting with a 25-50% gradient of 50% acetonitrile
in 0.1% TFA over 120 minutes at a flow rate of 15 mL per minute.
Fractions were collected, analyzed by HPLC and pure fractions
pooled and lyophilized to give 48 mg of the desired product. Amino
acid analysis: Arg 0.99. (1.0), Asx 2.01 (2.0), Lys 1.97 (2.0), Thr
1.85 (2.0), Trp 1.93 (2.0), Val 1.02 (1.0).
EXAMPLE 9
Preparation of
Arginyl-lysyl-asparaginyl-asparaginyl-lysyl-threonyl-trypto-
phyl-threonyl-tryptophan-amide
[0124] The peptide was prepared on an ABI Model 431A peptide
synthesizer using Version 1.12 of the standard Boc software. The
amino acids used were Boc-(Tos)Arg, Boc-(ClZ)Lys, Boc-Asn,
Boc-(Bzl)Thr, and Boc(CHO)Trp. 4-Methylbenzhydrylamine resin (0.625
g, 0.5 mmol) was used in the synthesis. The final weight of the
resin was 1.44 g. The peptide was cleaved from the resin (1.0 g)
using 10 mL of HF and 1.1 mL of anisole for 60 min at 0.degree. C.
The hydrogen fluoride was evaporated using a stream of nitrogen and
the resulting mixture triturated with ether. Solids were removed by
filtration and extracted with 1 M acetic acid to yield 0.38 mg of
crude peptide. The formyl groups on the tryptophan residues were
removed using 0.1 M piperidine in 50% aqueous DMF for two hours at
ambient temperature. The crude peptide (0.2 g) was purified by HPLC
(2.times.0.1 g) on a Vydac C-18 column (15.mu., 5.times.25 cm)
eluting with a 25-50% gradient of acetonitrile in 1% TFA over 120
minutes at a flow rate of 15 mL per minute. Fractions were
collected, analyzed by HPLC and pure fractions pooled and
lyophilized to give 48 mg of pure peptide. Amino acid analysis: Arg
1.00 (1.0), Asx 2.01 (2.0), Lys 1.99 (2.0), Thr 1.87 (2.0), Trp
2.04 (2.0).
EXAMPLE 10
Preparation of
Arginyl-lysyl-asparaginyl-asparaginyl-glycyl-threonyl-trypt-
ophyl-threonyl-tryptophyl-valine-amide
[0125] The peptide was prepared on a ABI Model 431A peptide
synthesizer using Version 1.12 of the standard scale Boc software.
The amino acids used were Boc-(Tos)Arg, Boc-(ClZ)Lys, Boc-Asn,
Boc-Gly, Boc-(Bzl)Thr, Boc-(CHO)Trp and Boc-Val.
4-Methylbenzhydrylamine resin (0.625 g, 0.5 mmol) was used in the
synthesis. The final weight of the resin was 1.19 g. The peptide
was cleaved from the resin (1.0 g) using 10 mL of hydrogen fluoride
and 1 mL of anisole for 60 minutes at 0.degree. C. The hydrogen
fluoride was evaporated using a stream of nitrogen and the
resulting mixture triturated with ether. The solids were removed by
filtration and extracted with 1 M acetic acid to give 0.41 g of
crude peptide after lyophilization. The formyl groups were removed
from the tryptophan using a 0.1 M solution of piperidine in 50%
aqueous DMF for two hours at ambient temperature. The crude peptide
(0.12 g) was purified by HPLC (multiple injections) on a Vydac C-18
column (15.mu., 5.times.25 cm) eluting with a 25-50% gradient of
acetonitrile in 0.1% TFA over 120 minutes at a flow rate of 15 mL
per minute. Fractions were collected, analyzed by HPLC and pure
fractions pooled and lyophilized to give 40 mg of pure product.
Amino acid analysis: Arg 0.98 (1.0), Asx 1.98 (2.0), Gly 1.04
(1.0), Lys 0.98 (1.0), Thr 1.82 (2.0), Trp 1.95 (2.0), Val 1.01
(1.0).
EXAMPLE 11
Inhibition of Neutrophil Binding to GMP140 Coated Wells
[0126] Binding of various peptides to GMP-140 coated wells, as
described above, were compared. The results are shown in FIG.
1.
[0127] Binding of the peptides at various concentrations, ranging
from 0.05 to 1 mM, were compared. The peptides tested and the
percent inhibition of neutrophil binding to GMP-140 is shown in
Table I.
[0128] The testing was conducted as follows:
[0129] A 5 .mu.g/mL solution of GMP-140 in HBSS (buffer) is
prepared. 50 .mu.L is pipetted per well with the exception of three
wells (A10 to A12) and the plate is stored overnight at 0.degree.
C. to 4.degree. C. The liquids are removed by aspiration and 300
.mu.L of 5 mg/mL human serum albumin (HSA)/HBSS is placed in the
wells coated with GMP-140 plus the three non-coated wells to be
used as control wells. The plate is allowed to incubate at room
temperature for two hours. The HSA/HBSS solution is removed and the
wells washed three times with 300 .mu.L/well of HBSS. The third 300
.mu.L wash of HBSS is left in each well and the plate is kept at
room temperature until it is used.
[0130] Eight to ten mL of fresh human blood collected in
heparinized tubes is carefully layered over 5 mL of the Mono-Poly
Resolving Medium in 15 mL polypropylene centrifuge tubes. The tubes
are centrifuged for 30 minutes at room temperature and 1400 to 1600
rpm. The tubes are rotated 1800 and centrifuged for another 30
minutes under the same conditions. The top plasma layer and first
cellular layer of leukocytes are drawn off. The second cellular
layer of neutrophils is harvested (usually 2 to 3 mL per tube) and
is placed in a clean 15 mL centrifuge tube. This is then
underlayered with 1.5 mL of fresh resolving medium and spun at 2000
rpm for 15 minutes at room temperature. This is done to further
purify the neutrophils from residual red blood cells. The
neutrophils in the supernatant are collected, placed in a clean 15
mL centrifuge tube and HSA/HBSS is added to bring the volume to 15
mL. The tube is gently inverted 3 or 4 times and is centrifuged at
1400 rpm for 5 minutes to pellet the neutrophils. The supernatant
is removed by aspiration and the cells resuspended gently in
HSA/HBSS (2 to 5 mL, depending on pellet size) and the cells/mL
determined using a hemacytometer and microscope. The cells are
diluted to 2.times.10.sup.6 cells/mL with HSA/HBSS.
[0131] A 3 mM concentration of the peptide to be tested expressed
in mg/mL is calculated as follows:
molecular weight/percent peptide.times.0.003
[0132] Peptides are accurately weighed into clean 4 mL flint glass
vials with screw caps. The amount weighed should be enough to
produce 0.5 to 1 mL of the 3 mM stock based on the mg/mL value
calculated above. The peptides are dissolved in an amount of 25 mM
HEPES buffered HBSS that is calculated as follows:
(peptide weighed out)/(mg/mL for 3 mM solution)=mL of buffer
[0133] The peptides are incubated with the neutrophils at 1.0, 0.3,
0.1 and 0.03 mM concentrations for 30 to 35 minutes at room
temperature in 1.5 mL flip top polypropylene centrifuge tubes as
follows:
4 Amount of Amount of Amount of 3 mM stock 25 mM HEPES/HBSS
Neutrophil suspension 1.00 mM 200 .mu.L 0 400 .mu.L 0.30 mM 60
.mu.L 140 .mu.L 400 .mu.L 0.10 mM 20 .mu.L 180 .mu.L 400 .mu.L 0.03
mM 6 .mu.L 194 .mu.L 400 .mu.L
[0134] 800 .mu.L of neutrophil suspension is mixed with 400 .mu.L
of the 25 mM HEPES buffered HBSS pH 7.4 and the whole is incubated
for 30 to 35 minutes at room temperature.
[0135] 150 .mu.L of the neutrophil suspension/25 mM HEPES buffered
HBSS pH 7.4 is pipetted into designated GMP-140 coated and HSA
wells. 150 .mu.L of the appropriate peptide/neutrophil suspension
incubate is pipetted onto designated GMP-140 coated wells. All are
incubated on the plate for 20 minutes at room temperature. The
liquids are removed by mild aspiration and the wells are washed
twice with HSA/HBSS and are checked to make sure no liquid remains
after the final aspiration. 200 .mu.L of the 0.5% HTAB buffer is
added to each well. After incubating at room temperature for 20
minutes, an eight channel multi-pipetter fitted with the
appropriate tips is set for 100 .mu.L is used to agitate the
contents of the wells four times.
[0136] The contents of each well is tested in a clean Nunc 96 well
flat-bottomed polystyrene microtitration plate. The reagents are
added as follows:
[0137] 1) 15 .mu.L of sample (0.5% HTAB buffer from plate well)
[0138] 2) 55 .mu.L of 80 mM potassium phosphate buffer pH 5.4
[0139] 3) 20 .mu.L of 3.0 mM H.sub.2O.sub.2 in the 80 mM potasium
phosphate buffer pH 5.4
[0140] 4) 10 .mu.L of 16 mM 3,3',5,5'-tetramethylbenzidine in 50%
dimethylformamide/80 mM potassium phosphate buffer.
[0141] The plate is developed for 5 to 15 minutes at room
temperature and the reaction is stopped by the addition of 100
.mu.L of 1 M phosphoric acid. The plate is read in the single
filter mode with the filter set at 450 nm. Blanking is set against
air.
[0142] The calculations are performed by the BioCalc 1.06 sofware.
The mean and standard deviation for each test peptide
concentration, neutrophil controls (neutrophils on HSA blocked
wells only) and neutrophil standards (neutrophils on GMP-140
coated/HSA blocked wells) are obtained via standard formulas in the
software. The coefficient of variation for each sample, standard or
control is calculated by formulas I created in the BioCalc software
of the format:
C.V.=(standard deviation/mean).times.100
[0143] The % inhibition for each sample is calculated as follows: 1
% inhibition = 1 - ( sample mean - control mean ) ( standard mean -
control mean )
[0144] The results demonstrate that, with the exception of the
negative control, the peptides all inhibit neutrophil binding to
immobilized GMP-140.
[0145] Modifications and variations of the present invention,
synthetic peptides and methods for modulating binding reactions
involving selectins, will be obvious to those skilled in the art
from the foregoing detailed description. Such modifications and
variations are intended to come within the scope of the appended
claims.
5TABLE I PERCENT INHIBITION OF NEUTROPHIL BINDING TO GMP-140 BY
SYNTHETIC PEPTIDES. CONCENTRATION (mM) STRUCTURE 0.1 0.5 1.0
RKVNNVW-NH.sub.2 17% RKVNNVWVWV 82% 98% RKVNNVWVWV-NH.sub.2 94%
102% CRKNNKTWTWV-NH.sub.2 87% 105% RKNNKTW-NH.sub.2 31% 53% 99%
RKNNKTWT-NH.sub.2 26% 23% 34% RKNNKTWTWV 14% 35%
Ac-RKNNKTWTWV-NH.sub.2 35% 50% RENNKTWTWV-NH.sub.2 34% 94% 96%
RKNNKTWTWE-NH.sub.2 17% 20% RKNNGTWTWV-NH.sub.2 46% 99% 99%
RKNNKTHTWV-NH.sub.2 13% 20% 33% YKNNKTWTWV-NH.sub.2 9% 98% 104%
RKNNKTWTWVGTKKALTNEC-NH.sub.2 9% 47% FMOC-NNKTW-NH.sub.2 85% 100%
99% rKIGGIWTWV-NH.sub.2 95% 96% RkIGGIWTWV-NH.sub.2 67% 88% 97%
RKIGGIWTWV-NH.sub.2 96% 103% 105% RKNNKTWTWV-NH.sub.2 14% 30% 87%
KWKWNRTNVT-NH.sub.2 0% 0% 0% (control peptide)
[0146]
Sequence CWU 1
1
36 1 10 PRT artificial sequence Synthetic inhibitory peptide 1 Arg
Lys Asn Asn Lys Thr Trp Thr Trp Val 1 5 10 2 10 PRT artificial
sequence Synthetic inhibitory peptide 2 Arg Lys Val Asn Asn Val Trp
Val Trp Val 1 5 10 3 10 PRT artificial sequence Synthetic
inhibitory peptide 3 Arg Lys Val Asn Asn Val Trp Val Trp Val 1 5 10
4 10 PRT artificial sequence Synthetic inhibitory peptide 4 Arg Lys
Ile Gly Gly Ile Trp Thr Trp Val 1 5 10 5 10 PRT artificial sequence
Synthetic inhibitory peptide 5 Arg Lys Ile Gly Gly Ile Trp Thr Trp
Val 1 5 10 6 10 PRT artificial sequence Synthetic inhibitory
peptide 6 Arg Lys Ile Gly Gly Ile Trp Thr Trp Val 1 5 10 7 11 PRT
artificial sequence Synthetic inhibitory peptide 7 Cys Arg Lys Asn
Asn Lys Thr Trp Thr Trp Val 1 5 10 8 10 PRT artificial sequence
Synthetic inhibitory peptide 8 Tyr Lys Asn Asn Lys Thr Trp Thr Trp
Val 1 5 10 9 10 PRT artificial sequence Synthetic inhibitory
peptide 9 Arg Glu Asn Asn Lys Thr Trp Thr Trp Val 1 5 10 10 10 PRT
artificial sequence Synthetic inhibitory peptide 10 Arg Lys Asn Asn
Gly Thr Trp Thr Trp Val 1 5 10 11 5 PRT artificial sequence
Synthetic inhibitory peptide 11 Asn Asn Lys Thr Trp 1 5 12 10 PRT
artificial sequence Synthetic inhibitory peptide 12 Lys Trp Lys Trp
Asn Arg Thr Asn Val Thr 1 5 10 13 7 PRT artificial sequence
Synthetic inhibitory peptide 13 Arg Lys Asn Asn Lys Thr Trp 1 5 14
14 PRT artificial sequence Synthetic inhibitory peptide 14 Cys Ile
Gly Ile Arg Lys Asn Asn Lys Thr Trp Thr Trp Val 1 5 10 15 20 PRT
artificial sequence Synthetic inhibitory peptide 15 Arg Lys Asn Asn
Lys Thr Trp Thr Trp Val Gly Thr Lys Lys Ala Leu 1 5 10 15 Thr Asn
Glu Cys 20 16 6 PRT artificial sequence Synthetic inhibitory
peptide 16 Lys Asn Asn Lys Thr Trp 1 5 17 5 PRT artificial sequence
Synthetic inhibitory peptide 17 Asn Asn Lys Thr Trp 1 5 18 10 PRT
artificial sequence Synthetic inhibitory peptide 18 Arg Lys Asn Asn
Lys Thr Trp Thr Trp Val 1 5 10 19 10 PRT artificial sequence
Synthetic inhibitory peptide 19 Arg Lys Ile Gly Gly Ile Trp Thr Trp
Val 1 5 10 20 7 PRT artificial sequence Synthetic inhibitory
peptide 20 Arg Lys Ile Gly Gly Ile Trp 1 5 21 7 PRT artificial
sequence Synthetic inhibitory peptide 21 Arg Lys Val Asn Asn Val
Trp 1 5 22 10 PRT artificial sequence Synthetic inhibitory peptide
22 Arg Lys Asn Asn Lys Thr Trp Thr Trp Val 1 5 10 23 10 PRT
artificial sequence Synthetic inhibitory peptide 23 Arg Lys Asn Asn
Lys Thr Trp Thr Trp Glu 1 5 10 24 17 PRT artificial sequence
Synthetic inhibitory peptide 24 Arg Lys Asn Asn Lys Thr Trp Thr Trp
Val Gly Thr Lys Lys Ala Leu 1 5 10 15 Cys 25 9 PRT artificial
sequence Synthetic inhibitory peptide 25 Arg Lys Asn Asn Lys Thr
Trp Thr Trp 1 5 26 8 PRT artificial sequence Synthetic inhibitory
peptide 26 Arg Lys Asn Asn Lys Thr Trp Thr 1 5 27 4 PRT artificial
sequence region of artificial inhibitory peptide 27 Gly Ile Arg Lys
1 28 5 PRT artificial sequence region of artificial inhibitory
peptide 28 Ile Gly Ile Arg Lys 1 5 29 6 PRT artificial sequence
region of artificial inhibitory peptide 29 Cys Ile Gly Ile Arg Lys
1 5 30 6 PRT artificial sequence region of artificial inhibitory
peptide 30 Thr Trp Val Gly Thr Lys 1 5 31 6 PRT artificial sequence
region of artificial inhibitory peptide 31 Thr Trp Val Gly Thr Asn
1 5 32 6 PRT artificial sequence region of artificial inhibitory
peptide 32 Thr Trp Val Gly Thr Gln 1 5 33 6 PRT artificial sequence
region of artificial inhibitory peptide 33 Val Trp Val Gly Thr Gln
1 5 34 6 PRT artificial sequence region of artificial inhibitory
peptide 34 Val Trp Val Gly Thr Lys 1 5 35 6 PRT artificial sequence
region of artificial inhibitory peptide 35 Val Trp Val Gly Thr Asn
1 5 36 13 PRT artificial sequence region of artificial inhibitory
peptide 36 Thr Trp Val Gly Thr Lys Lys Ala Leu Thr Asn Glu Cys 1 5
10
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