CA2103139A1 - Peptide inhibitors of inflammation - Google Patents

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 23-30 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 µM. 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. It has also been found that certain of these modifications can significantly increase the stability of peptides of Formula (I) or (II) against degradation by the enzymes found in human serum. 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.

Description

WO 92J20708 PCI/US92/~40~6 PEPTIDE INHIBITORS OF INFLi~TION

Background of the In~e~tion 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~
The adherence of platelets and leukocytes to vascular surfaces is a critical component of the inflammatory response, and is part o~ a complex series of reactions involving the simultaneous and interrelated activation of the complement, coagulation, and immune systems.
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 ~omplexes, as reviewed by Muller-~berhardl ~.J., Ann. Rev Biochem. 57:321-347 (1988). Central to the complement system are the C3 and C4 proteins, which when~acti~ated coval~ntly attach to nearby targets, marking them for clearance.
In order to help~control~this process, a remarkable family of soluble and membrane-bound regulatory roteins ha~s evolve~d~, each~of; which i~teracts with activated C3:and/or:C4::derivatives. The coagulation and~in~;lammatory~pathways are regulated in a coordinate fashion~in~response to tissue damage. For :example~,~in additi~on~to ~ecoming adh~siv~ for leukQcytes, activated endothelial cells express tisslue actor on the~cell surface and decrease their surface xpression of~ hromb~omodulin, leading to a net facilitation oP~coagulation reactions:on the cell surface.~ In some:cases, a single:receptor can be involved in both~inilammatory~and coagulation processes.
Leukocyte~adherence to~vascular endothelium is a key:initial step :in migration of leukoGytes ~o tissues ~ , ~ 1 3.9 -2-in response to microbial invasion. Although a class of inducible leukocyte receptors, the CDll-CDl8 m~lecules, are thought ~o have some role in adherence to endothelium, mechanisms of equal or even greater importance fox l~ukocyte adherence appear to be due to inducible changes in the endothelium itself.
Activated platelets have also been shown to interact with both neutrophils and monocytes in vitro.
The interaction o~ platelets with monocytes may be mediated in part by the binding of thrombospondinl;o platelets and monocytes, although other mechanisms have not been excluded. The mechanisms for the binding of neu~rophils to activated pl~telets 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.
Platel~ts and other cells may also play an important role~in the recruitment of leukocytes into the wound in order to cont~in microbial invasion.
Endothelium exposed to "rapid" activators such as thrombin and histamine becomes adhesive for neutrophils~within two to ten minutes, while endothelium exposed:to cytoXines such as tumor nearosis factor~ and interleukin-l becomes adhesive a~ter one~to s~ix~:hours. The rapid endothelial-dependent leukocytè~adhesion has been associated with expre~sion of:~the lipid mediator platelet activating fac*or (PAFj~ on the cell surface, and presumab~yt the appearance of other endothelial surface receptors.
The s~:ower cytokine-inducible endothelial adh~sion for leukocytes is media~ed~J at least in part, by an endothe~lial cell;receptor,~ELAM-l, that is synthesized by endothelial cells after exposure to cytokines and :
:~ then transported to~the cell surfaGe, where it ~inds neutrophils. :The~isolation, characterization and cloning o EL~M-1 is reviewed by Bevilacqua, et al~, .

W092/20708 PCT/U~g~/04~1~
2 'i'~J'~139 in Science 243, 1160-1165 (19~9). A peripheral lymph node homing recep~or, also called l'the murine Mel 14 antigen", "Leu 8", the l'Leu 8 antigen" and "LAM-1", is another structure on neutrophils, monocytes, and lymphocytes that binds lymphocytes to high endothelial venules în peripheral lymph nodes. The characterization and cloning of this protein is re~iewed by Lasky, et al., Cell 56, 1045-1055 (1989) (mouse) and Tedder, et al., J. Exp. Med. 170, 123~133 (1989).
GMP-140 (granule membrane protein 140), also known as PADGEM, is a cysteine-rich and hea~ily glycosylated integral membrane glycoprotein with an apparent molecular weight of 140, 000 as assessed by sodium dodecyl sulfate polyacrylamide gel lectrophoresis (SDS P~GE~. GMP-140 was first purified from human platelets by McEver and Martin, J.
Biol. Chem~ 259:97g9-g804 (1~8~). The protein is preæent in alpha granules of resting platelets but is rapidly r~distributed to the plasma membrane following platelet activation, as reported by Stenberg, et al., (1985). The presence of GMP-140 in endothelial cells and its biasynthesis by these cells was reported by ~cEver, et al., Blood 70(5) Suppl. 1:355a, Abstract No. 1274 ~1987). In endothelial cells, GMP-140 is found in ~orage:gr~nules known as the Weibel-Palade bodies. (McEver, et al. J. _ in~ Invest. 84:92-99 (1989) and ~attori,~et al., J. Biol. Chem. ~4:7768-7771 (1989)). GMP-140 (called PADGEM) has also been rPported to me~iate the interaction of activated platalets with neutrophils and monocytes by Larsen, et al,, i~ ~ell 59, 305-312 ~October 1989) and Ham~urger and McEver, Blood 75:550-554 (199Q~.
The cDNA-derived amino acid sequence, reported by Johnston, ~t al., in Cel- 56, 1033-1044 (March 24 1989), and in U.S. Serial No. 07/320,408 filed March 8, 1989/ indicates that it contains a number of ' modular domains that are likely to Eold independently.
Beginning at the N-~erminus, 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 ~orm that appears to result from differential splicing), and a cytoplasmic tail.
When platelets or endothelial cells are activated by mediators such as ~hrombin, the membranes of the storage granules fuse with the plasma membrane, the soluble contents of the granules are released to the external ~nvironment, and membrane bound GMP-140 is presented within seconds on the cell surface. The rapid redistribution of GMP-140 ~o 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 disruptionO
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 ~cEver, Blood 75:550-554 (1990)~, monocytes (Larsen, et al. CeI~ 59:305-312 (1989); Moore, et al., J. Cell Biol. 112:491-499 ~19gl)~, and a subset of lymphocytPs ~Moore, et al. J.~Cell Biol. 112:491-499 (19913 and ~oore, et al., Blood (Suppl 1) 78:439a (1991)). Thus, GMP-140 can ~erve: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 ~tates.
Peptides derived from GMP-I40 are described in U.S. Serial No. 07/554,199 entitled "Functionally Active Selectin-Derived Peptides" filed July 17, 19go by Rodger P. McEver that are useful in diagnostics and W~92~20708 2 1 0 3 1 3 9 PCT/~S92/~4Ot6 in modulating the hemostatic and inflammatory responses in a patien~ wherein a therapeutically effective amount of a peptide capable of blocking leukocyte recognition of GMP-l~O is administered to the patient. U.S. Serial Ns. 07/554,199 filed July 17, 1990 also discloses that peptide sequences within the lectin domain of GMP-14~, having homology with the lectin domains of other proteins, especially ELAM-1 and the homing recep~or, 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 inflamma~ory processes.
EL~M-l, the homing receptor, and GMP-1~0 have been termed "selectins'i, ~ased on their rel~ted structure and ~unction. ELAM-l is not present in unstimulated en~othelium. Howe~er, when endothelium is exposed to cytokines such as tumor necrosis factor or interleukin-1, the gene for ELXM-l lS transcribed, producing RNA which in turn is translated into protei~. The res~lt is that EL~M-1 is expressed on the surface~of:endothelial c~lls one to four hours a~ter exposure to cytokines, as reported by Bevilacqua et alO / Proc.Natl.Acad.Sci.USA 84:9238-9242 (1987) ~in contrast to G~P-140j which is stored in granules and presented on the~ cell surface within seconds after activation). ::ELAM-l has been shown to mediate the adherence of neutrophils to cy~okine~treated endothelium and thus appears to be important in allowing leukocytes to migrate across cytokine-~timula~ed endothelium into tissues. The c~NA-derived primary structure of ~LAM-l indicates that it contains a "lectin" domain, an EGF domain, and six (instead of ' (~ ~ " 3 ~ 6-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.
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 Ro~e, Advances in ImmunoloqY, vol. 44, F.I. Dixon,ed., 313 378 (Academic Press, New York 1989). This binding allows lymphocytes to migrate across the ~n~othelium 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 ~omain, two complement-binding~repeats, a transmambrane domain, and a short cytoplasmic tail. The homing receptor also shares extensive:sequence homology with G~P-140, particularly in the lectin and EGF domains.
Based~on:a comparison of the lectin domains between GNP-14~0,~ ELAM~ :and the homing receptor (LEU-8), it may be possible to select those peptides inhibiting binding:of neutrophils to GMP-140 whlch will inhibit binding~of~ELAM-ll the homing receptor, and other homologaus selectins, to components of the inflammatory process, or, conversely, which will inhibit only GMP-l~40 binding.
The in viYo~ significance of platelet-l~ukocyte intera~ions has~ not been studied carefully. However, in responsè to vascular~injury, platelets are known to adhere ~o subendothelial surfaces, become activated, and support coagula~ion. Platelets and other cells may:also play an important role in the recruitment of leukocytes into the wound in order to contain W~92/20708 PCT/~S92/04016 ~ia3l3s microbial }nvasion. Conversely, leukocytes may recruit platelets into tissues at sites of inflammation, as reported by Issekutz, et al., Lab.
In est. 49:716 (1983).
The coagulation and inflammator~ pathways are regulated in a coordinate ~ashion 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, l~ading to a net facilitation o~ coagulation reactions on the cell surface. In some cases, a single receptor can be invol~ed in ~oth inflammatory and coagulation processes .
Proteins invo~ved 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 quantikies sufficient for administration ~o a patient~ Moreover, there can be a reaction against the protein after it has been administered more than t once to the patient. It is ~herefore desirable to develop peptides~having the same, or ~etter, activity as th~ protein,~which are inexpensive to synthesize, reproducible and relatively innocuous.
It is preferable to~develop peptides whlch can e prepared~synthet~ically, having acti~ity at least e~ual to, or greater than, the peptides derived from t~a protein it~e~lf.
It is therefore ~n object of the present in~ention~to provide~peptides interacting with cells recognized by sel~ctins, including GMP-140, ELAM-1, and lymphocyte homing receptor.
It is another o~ject o~ the present invention to provide methods ~for us~ing these peptides to inhibit leuko~yte adhesion to endothelium or to platelets.

: ~ :

, .
~i~3 ~9 -8-It is a further object of the prPsent invention to provide methods for using these peptides to modulate the immune response and the hemostatic pathway.
It is yet another object of the present invention to provide peptides for use in diagnostic assays relating to G~P-140, ELAM-l, and lymphocyte homing receptor.

~ummary of the Inve~tion Pep~ides derived from three regions of the lectin domain of GMP~1~0 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 ~ollowing formulae:
Rl X-P-Q-S-T-U-V~W-Z-Y-R2 (I) Rl-P-Q-S-T-U-V-W-Z-R2 (II~
or a pharmaceutically acceptable salt thereof, wherein: ~
X in Formula (I) and P in Formula ~II) are the N-terminus amino acids, and Rl is a moiety attached to the function tNHR1~, Y in Formula (I):and ~ in Formula ~II) are the Ct~rminus~amino~acids, and R2 is the moiety attached to the ~ingly--bonded:oxygen in the carboxy function ~C~Q)OR2), ~ ~ P is D or L-tyrosine, D- or L-phenylalanine, D-; j ,or L-lysine, D- or;L-glutamic acid, D- or L-arginine, D- or L-cysteine, D-:or L- O-~3-tyrosine, D- or L-Na-~ R3-~yrosine, D- or~L-4-amino phenylalanine, D- or L-:~: R4-phenylalanine, D- or L~pyridylalanine, D- or L-~; naphthylalanine, or D- or L-tetrahydroisoquinoline : ::

' W~92/20708 PCT/US92/04016 ~1 0~ 1 3~
g carboxylic acid, where R3 is lower alkyl or aryl and R4 is halogen (fluorine, chlorine, bromine or iodine), ~ is D- or L-threonine, D- or L-lysine, D- or L-glutamic acid, D- or L-cysteine, or glycine, S is D- or ~-aspartic acid, D- or L histidine, D- or L-glutamic acid, D- or L asparagine, D or L-glutamine, D~ or L-alanine, D- or L-phenylalanine, D-or L-lysine, or glycine, T, U, V and W are independently D- or L-leucine, D- or L-isoleucine, ~- or ~-alanine, D- or L-valine, ~- or L-alloisoleucine, glycine, D- or L-glutamic acid, D or L-aspartic acid, D- or L-asparagine, D- or L-glutamine, D- or L-threonine, or desamino acid where desamino acid refers to the deletion of either residues T, U, V, or W from the peptide formulas I or Z is D- or L-glutamine, D- or I.-glutamic acid ; and D- or L-asparagine, Rl i H (signifying a free N-terminal group), formyl, lower alkanoyl, ~royl or desamino (meaning the amino acid adjac~nt to the group Rl, either X in formula~I or P in formula 2 lacks the ~-amino group of :the amino acid, and is~replaced with H), ,~ :
R~ is H ~signifying~in a free C-terminal carboxylio acidj,~O(1:ower:alkyl), O(aryl), NR3R4 where R3 and R4~are independently H or lower alkyl, or : descarboxy (meaning the ~-carboxylic acid group of the : ` amino:acid to which~Rl is adjacent in formula I or 2 YI or Z, respe~tively, is replaced with H), : X and Y are linear chains of from one to ten : ~: amino acids~. :~ ; :
` Peptides of the Formula I and II have as their core region portions of~the 23-30~amino acid sequence of GMP-140, with residue~l defined as the N-terminus of the mature protein after the cleavage of the signal peptide. ~ ~

:: :

W~2/20708 PCT/US92/04016 2 1 0 ~ o-Examples demonstrate the inhibition of the binding of neutrophils ~o ~MP-140 of peptides of Formula I or II in concentrations ranging from 5 to 1500 ~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. It has also been found that certain of these modifications can significantly increase the stability of peptides of Formula I or II
against degradatîon by the enzymes found in human serum.
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 proce ses.
.
Brief De cription of the Dr~wings Figure 1 shows the activity of se~eral peptides of Formulas I and: II in inhi~iting the binding of neutroph~ils to GNP-l40, % inhibition versus concentration of peptide (mM~, (dark squares, Cys-Gln-Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-amide;
open square, Asn-~rg-Tyr-Thr-Asp-~eu-Val-Ala-Ile Gln-am~de; dark diamond,~ Arg-Tyr Thr~sp-Leu-Val-Ala-Ile-Gln-amide; open:~diamond, Tyr-Thr~Asp-Leu-Val-~la-Ile-Gln-amide; dark~triangle, ~cetyl-Tyr-Thr-Asp-Leu-Val-Ala-Ile-~ln-amide; open triangle, Tyr-Thr-Glu-Leu-Val-Ala-Ile-Gln-amide; X,: Tyr-Thr H:is-Leu-Val-Ala-Ile-Gln ami~e; *, Tyr-Thr-Asp-Leu-Va~-Ala-Ile-Gln-A~n-Lys-Asn-G~u-amide;~--, Leu-Gln-Thr-Ala-Tyr-Asp-Val-IIe-amide :(negative control)).
Fi;~ure 2 sh~ws the~significant~increase in stability aga~inst enzymes found in human serum that can be achieved;by the modifications set down in Formula I and II, graphlng percent of peptide W092/2~7~8 PCT/US92/0~016 11- ? i c ~ ~ 3 9 remaining ~ersus time (minutes~ (dark triangle, Ac-YTDLVA.IQ-NH2, O, YTDLVAIQ-NH2).

Detailed Description of the Invention Peptîdes having GMP-140-lik~ activi.ty, therapeutic compositions containing these peptides, methods for the preparation of these pep~ides and methods for usP thereof, are disclosed. These peptides have either of the following formulas:
R1-X-P-Q--S~T-U-V-W-Z-Y-R2 (I) R1-P `Q - S-T-U-V-W- Z ~R2 ( I I ) or a pharmaceutically acceptable salt thereof, wherein:
X in Formula (I) and P in Formula (II) are the N-terminus amino acids, and R1 is a moiety attached to the amine function (NHR1), Y in Formula (I) and Z in Formula (II) are the C-terminus amino acids, and R2 is the moiety attached to the singly-bonded oxygen in the carboxy funct.ion c (o) o~2), ~ , P is D- or L-tyrosine, D- or L-phenylalanine, D-or L lysine, D- or L-glutamic acid, D- or L-arginine, D- or L-cysteine,: D-~or L- O-R3-tyrosine, D- or L-Na-R3-tyro~ine, D- or L-4 amino phenylalanine, D or L-. ,"
R~-phenylalanine, D- or ~-pyridylalanine, D or L
naphthyla~aninel~ or D- or L-tetrahydroisoquinoline . : carboxylic acid, where R3 is lower alkyl or aryl and R4 is halogen (fluorine,:chlorine, bromina or iodine~, Q is;:D- or L-threonine, D- or L-lysine, D or L-~ glutamic acid, D- or L-cysteine,:~r:glycine, : : S is D- or L-aspartic acid, D- or L-histidine, D- or ~-glutamic~acid, D- or L~asparagine, D or L-glutamineJ D- or L-alanine, D~ or L-phenylalanine; D-.: ; or L~lysine, or glycine, . T, U, V and W are lndependently D- or L-leucine, D~ or L-isoleucine, D- or L-alanine, D- or L7valine, W092/2~708 PCT/US92/0~016 ~ 12-D- or L-alloisoleucinet glycine, D- or L-glutamic acid, D- or L-aspartic acid, D- or L-asparagine, D- or L-glutamine, D- or L-threonine, or desamino acid where desamino acid refers to the deletion of either residues T, U, V, or W from the peptid~ formulas I or II, Z is D- or L-glutamine, D- or L-glutamic acid and D- or L-aspar~gine, ~ 1 is H (signifying a free N-terminal group), formyl, lower alkanoyl, aroyl or desamino (meaning the ami~o acid adjacent to the group Rl, either X in formula I or P in formula 2 lacks the ~-amino group ~f the amino acid, and is rep1aced with H~, R2 iS H ~signifying in a free C-terminal carboxylic acid), O(lower alkyl), O(aryl), NR3R4 where ~ ~ ~ R3 and R4 are independently H or lower alkyl, or : ; descarboxy (meaning;the ~-carboxylic acid group of the amino ~cid to which Rl is adjacent in ~ormula I or 2, Y or Z, respectively~, is replaced with H), X and Y:are~linear chains of from one ,to ten amino acids.
Preferred peptides are those of Formula I
wherein~Rl is H.and~R2:is:MR3R4, and Formula II wherein 1 is H or:acety1~àn~ R1~is NR3R~t wherein S is aspartic ~acid,~glutamic acid or histidine.
:Most preferred;;peptides are Tyr-Thr-Asp-Leu-Val-Ala-Ile~Gln-NH2:; Tyr-Thr-His-Leu~Val-Ala-Ile-Gln-NH2; Acetyl-Tyr-Thr-A~p-Leu-Val-Ala-Ile-~ln-NH2; Cys-lnAsn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln~NH2; Asn-Arg~Tyr~Thr-Asp-L u-Val-Ala-Ile-Gln-NH2J Arg-Tyr-Thr-Asp-Leu-Val-A1a-Ile-Gln-NH2;~Tyr-Thr-Glu-Leu-~al-Ala-: I1e-Gln-NH2; Tyr~-Thr-Asp-Leu-Val-Ala-Ile-Gln-Asn Lys-Asn-Glu-NH2; D-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2;
Tyr D Thr-Asp-Leu-Val-Ala-Ile-Gl~-NH2; Tyr-Thr-D-Asp-Leu~Val-Ala-Ile-Gln-NH2;: Phe-Thr-Asp-Leu-Val-P~la-Ile-: : :

WOg2/20708 PCT/US92/04016 -13- 21~ 3 Gln-NH2; Tyr-Thr-D-Asp-Leu-Val-Ala~Ile-Gln-NH2; Tyr-Thr-Asp-Leu-Val-Ala-D-Ile~Gln-NH2; Tyr-Thr-Asp-Ala-Val-Ala~Ile-Gln-NH2; Tyr-Thr-~la-Leu-Val-Ala-Ile-Gln-NH2; Tyr-Thr-Phe-Leu-Val-Ala~Ile-Gln-NH2; Tyr-Thr-Lys-Leu Val Ala-Ile-Gln-NH2; Lys-Thr-Asp-Leu-Val-Ala-Ile~
Gln-NH2; Gln Asn-Arg-Tyr-~hr~Asp-Leu-Val-Ala-Ile-Gln-NH2; Asn-Arg-Tyr-Thr-Asp-Leu-Val Ala-Ile Gln-NH2; Arg-Gly-His~Leu-Val-~la-Ile-Gln-NH2; and ~rg-Gly Asp~Leu-Val-Ala-Ile-G1n-NH2.
~ s used herein, the term "lower alkyl" incl~des branched, straight-chain, and cyclic saturated hydrocarbons having ~rom one to six carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, neopentyl, and hexyl. The ~erm "lower alkanoyl" means RC(O), wherein R a lower alkyl group. The term aroyl means where ArC(O), wherein Ar i5 an~aryl group, 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 (~
The peptid~s of formula I can be used in the .
form of the ~ree peptide or a pharmaceutically : aGc~ptable~sa1t. ~:Amine:salts ~an be prepared by ~: : mixing the pepti~de~with an acid according to known methods. ~Suitab;le ac~ids include inorganic acids such as hyd~ochlori~a id, hydrobromic acid, perchloric acid, nitric aciid, thiocyanic acid~ sulfuric acid, and : ~: phosphoric acid,;~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~, n~phthalenesulfonic acid, and sulfanilic acid.~

`: :
.

W~92J2070~ PCT/~Sg2~401~
) 1 3 ~

Carboxylic acid groups in the peptide can be converted to a salt by mixing the peptide with a base according to known methods. Suitable bases include inorganic bases such as sodium hydroxide, ammonium hy~roxide, and potassium hydroxide, and organic bases such as mono-, di-, and tri-alkyl and aryl amines (e.g., triethylamine, disopropylamine, methylamine, and di~ethylamine and optionally substituted mono-, di, and tri-ethanolamines.
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:

.

W~ g2/207~8 PCr/USg2/040~6 21û31~J

.. . . . .
~mino Acid Abbreviations L-alanine Ala D-alanine D-Ala a L-allosoleucine AIle P-alloisoleucine D-AIle L-argi~ine ~rg R
D-arginine D-Arg r D-asparagine D-Asn N
L-asparagine L-Asn n L-aspartic acid Asp D-aspartic acid D-Asp d L-cysteine Cys D-cysteine D-Cys c L-glutamic acid ~lu E
D-glutamic acid D-Glu e L-glutamine Gln K
D-glutamine D--Gln k glycine ~ly G
L-histidine His H
D-histidine D-His h L-i~olelucine Ile D-isoleucine D-Ile leucine Leu L
D-leucine D-Leu L-ly~ine 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 5 ~-threonine: L-Thr T
D-threonine :: ~ D-Thr t L-tyr~ine~ ; L-Tyr Y
~-tyrosine ~ D-Tyr . y L-tryp~ophan Trp W
D tryptophan: ~ D-Trp w L-valine Val V
D-Yaline: : D-Val v : - ~ :

: ~ ' ~ ; :

:
-W0~2/2070~ PC~/US9t/04~16 3 ~ -16-Reagents Abbreviations _ Trif luoroacetic acid TFA
Methylene chloride CH2Cl2 N,N-Dii~opropylethylamine DIEA
~-Methylpyrrolidone NMP
l~Hydroxybenzotriazole HOBT
Dimethylsulfoxide DMSO
Acetic anhydride Ac2O
_ _ _ Methods o~ Pr~paration of Peptide~
The peptides can generally be prepared following ~nown techniques, as described for example in the ci~ed publiations, the teachings of which are speci~ically incorporated herein. ~n a preferred method, the peptides are prepared following the solid-phase synthetic technique initially described by Merrifield in J.Amer.Chem~Soc., 85, 21~9-~154 ~1963).
Oth~r tech~iques may be found, for ~xample, 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.
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 , (Plenum Press, New York, 1973). The common protective groups used herein are t-buty~oxyc~rbonyl ~Boc),~fluorenylmethoxycarbonyl (FMOC),~ benzyl (Bzl), tosyl tTos), o-bromo-phenylme~hoxycarbonyl ~BrCBZ~, phenylmethoxycarbonyl Bæ) ~ 2-chlloro-phenylmethoxycarbonyl, (2-Cl-CBZ), 4-methoxy-2,3,6 trimethylbenzenesul~onyl ~Mtr), trityl (Trt), formyl (C~O), and tertiary butyl (t-Bu).
General synthetic procedures for the synthesis of peptides of Formula I and II by svlid phase methodology are as follows:
: -~:

.

W~92/2070~ PCT/~S92/~4016 ~10t-~1J~

A. General ~yn~hetic Pxocedures ~or Solid Pha~e Peptide Synth~sis Using Na-Boc Protection~

REPETITIONS TIME
1. 25% TFA in CH2Cl2 1 3 min 2. 50% TFA in CH~C12 1 1~ min 3~ C~2Cl2 5 3 min 4. 5% DIEA in NMP ~ 4 min 5. NMP 6 5 min 6. Coupling step 1 57 min a. Preformed BOC~mino Acid- 36 min HOBT active ester in NMP
b. DMSO 16 min c. DI~A 5 min 7. 10% ~c2O,5% DIEA in NMP 1 9 min ~- ~HzC12 . 3 min B. Gen~ral Synth~tic Procedure ~or ~olid Ph~e Pepti~e 5y~thesi~ U~i~g N~- FMOC
Protectio~
.
REPETITIONS TIME
1. 20~ piperdine in N~P ~ 3 min :2~ 20% piperdine in NMP 1~15 min 3. NMP 6 9 min 4. Coupling 171 min Preformed FMOC-Amino Acid-HOBT active ester in NMP
: 5. NMP 6 7 min N-t~rminal acetylation on the deprotected Na-amino group o~ peptides synthesized using ~ither Boc ~i o~ FMOC strategies i-s accomplished with 10% Ac2O and 5~ DIEA in NMP, followed by washing of the peptide resin with NMP and/or ~H2C12.
The peptides can also be prepared using standard genetir engineering techniques known to those skilled in the art. For ex~mple, the peptide can be produced enzymatically by inserting nucleic acid encoding the peptide into an expression vector, expressing the DNA, 2~3~3~ -18-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 fu~ed to, and subsequently cleaved from, the peptide by inserting into the expression vector in phase with the peptide encoding se~uence 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 ~ia an antibody to the carrier protein~. The peptide can be cleaved using chemical methodology or enzymatically, as by, fox example, hydrolasesO
~tbods o~ Prepar~tion o~ Pharmaceutica~ Compositions To prepare:the pharmaceutical compositions containing these peptides, a peptide of Formula I or II or a base or acid addikion 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 sn the~form of preparation desired for admini~stration, e~.g;., sublingual, rectal, nasal, oral, or parenteral. In prepariny the compositions in oral dosag~ form,:any ~of:the usual pharmaceutical media may be employed, for~example, water, oils, alcohols, flavo~ing agents~,~preservatives~ and colo~ing agents, to make~an~:oral~ l~quid preparation ~e.g~, suspension, e~ixir, or solution);or with carriers such as starches,~sugars,:~diluents, granulating agents, lubricants,:binders,~and disintegra~in~ agents, to make an oraI solid preparation (e.g., powder, capsule, or tablet~
~ Controlled release forms or en~ancers to increase bioavailabIlity may also be used. Because of their ease in administration, tab:lets and capsules represent the most: advantageous oral dosage unit form, .

: ' :

W092/20708 PCT/US9~/04016 2~a~

in which case solid pharmaceutical carriers are employed. If desired, tablets may be sugar coated or enteric coated by standard techniques.
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.
The peptides can also be administered locally at a wound or inflammatory site by topical application of a solution or cream.
Alternati~ely, 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 th~ ~rt. U.S. Patent No. 4,789,734 describe methods for e~capsulating 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
good review o~ known methods is by G. Gregoriadis, Chapter 14. "Lipo~omes'l, ruq Carriers in Bioloqy and edicine pp. 287:-341 (Academic Press, 1979).
Micro pheres formed of polymers or proteins are well known to those skilled in the art, and can be t~ilored for passage through the gastrointestinal tract dir~ctl~ into the bloodstream. Alternatively, the peptide can be incorporated and the microspheres, or composite of microspheres, implanted for slow release over a period o;~ time, ranging from days to months~
Se~, for ex~mple, U.S. Patent No. 4,906,474, 4,925,673, a~d 3,625,214.
~t~Oa~ for D~mo~trati~g Bi~ g Peptides that are biologically active are those which inhibit binding of neutrophils, monocytes, ~ c~ 20-subsets of lymphocytes or other cells to GMP-140, or which inhibit leukocyte adhesion to endothelium that is mediated by ELAM-1 and/or ~he homing receptor.
Peptides can be screened for their abili~y to inhibit adhesion to cells, for example, neutrophil adhesion to purified GMP-1~0 immobilized on plastic wel l ., using the assay d~scribed by Geng, et al., Nature 343, 757 760 (1990).
Human neutrophils are isolated from heparinlzed whole blo~d by density gradient centrifugation on~
Mono-Poly resolving media, Flow Laboratories.
Neutrophil suspensions are greater than 98% pure and great~r than 95% viable by trypan blue exclusion. For adhesion assays, neutrophils are suspended at a concentration of 2 x 1o6 cellslml in Hanks' balanced salt solution containing 1.26 mM Ca2+ and 0.81 mM Mg2+
(HBSS, Gibco) with 5~mg/ml human serum albu~in ~HBSSjHSA). Adhesion assays are conducted in triplicate in 96-well microtiter plates, Corning, incubated at 4C overnight with 50 microliters of various protein:solutions.
GMP-140 is isolated from human platelet lysates by immunoaffinity~chromatography on antîbody Sl2-SepharoseT~ and;~ion-exohange chromatography on a Mono-~ column (FLP~ Pharmacia;Fine Chemicals), as f~oll~ws.
Outdated~human~p~latel~et packs (100 units~
obtained:from a blood~bank and stored at 4C are pooled, adjusted to s mM EDTA at pH 7~5, centrifuged at 4,000 rpm for:~3~ min ;in 1 liter bottles, then washed thxee times with~l`lit~r of 0.1 M NaCl, 20 mM
Tris pH 7 . 5: (TB5) ,~ ~5 ~M ~ EDTA, 5 mM benzamidine.
The p~lle s~are~then;resuspended in a minimum amount of w~sh buf~er and~made 1 mM: in DIFP, then ~rozen in 50 ml scrèwtop~tubes at -80C~ The froæen platelets are thawed and resuspend~ed in 50 ml TBS, 5 ::

2 iC3133 mM benzamidine, 5 mM EDTA pH 7.5, 100 M leupeptin.
The ~u~pension is frozen and thawed two times in a dry ice-acetone bath using a 600 ml lyophilizing flask, the~ 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 ~ Na~l.
After stirring the suspension at 4C, it is centrifuged in polycarbonate tubes at 33,000 rpm for 60 min at 4~C. The supernatant (0.5 M NaCl wash) is remo~ed and saved; this supernatant contains the soluble form of GMP-140. Care is taken not to remove the top part o~ the pellet with the supernatant. The pellets are then homogenized in extraction buffer ~TBS, 5 mM benzamidine, 5 mM EDTA, pH 7.5, 100 ~M
leupeptin, 2~ Triton X-100). ~fter centri~ugation at 19,500 rpm for 25 min at 4C, 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 m~mbrane form of GMP-140, are adjusted to 0.5 M NaCl.
The soluble fraction (0.5 M NaCl wash~ and the membrane extract~(also adjusted to 0.5 M NaCl) are abso~bed with:separate pools of the monoclonal antibody $12 (directed to human GMP-14Q) previously :
coupl~d to Affigel:~(:Biorad) at s mg~ml for 2 h at 40C.
After letting:the resins settle, the supernatants are removed. The~S1~2~Affigel containing bound GMP-140 is then loaded into a column~and washed oYernight at ~C
wlth 400 ml of 0~.5 M;NaCl, ~0 mM Tris pH 7.5, 0.01%
Lubrol PXr ~ ~ ~
Bound GMP-1~0 is eIuted from the S12 Affigel with 100 ml of 80%~ ethylene glycol, 1 mM MES pH 6.0, 0.01~ Lubrol P~. Peak fractions with absorbance at :: :
280 ~m are pooled. Eluates are dialy~ed against TBS
: :~ with 0.05% Lubrol, then applied to a Mono Q column (FPLC frvm Pharmacia). The concentrated protein is ~ t~ ~ 3 t9 -22-step eluted with ~ 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).
GMP-l40 is plated at 5 micrograms/ml and the control proteins: human serum albumin (Alb), platelet glycoprotein IIb/IIIa (IIb), von Willebrand factor (~WF)~ ~ibrinogen ~IB), thrombomodulin (TM), gelatin (GEL) or human serum (HS), are added at 50 micrograms/ml. All wells are blocked for 2 h at 22C
with 300 microliters HBSS containing lO mg/ml HSA, then washed three times with HB~S containing 0.1%
Tween-20 and once with ~BSS. Cells (2 x 105 per well ~re added to the wells and incubated at 22C for 20 min. The wells are then filled with HBSS/HS~, sealed with acetate tape (Dynatech), and centrifuged inverted at I50 g for 5 min. A~ter discarding nonadherent cells and supernates, the contents of each well are solu~ilized with 200 ~icroliters 0~5%
: ~ hexadecyltrimethylammonium bromide, Sigma, in ~0 mM
po~assium phosphate, pH 6~0, and assayed for :~ : :
. ~ myel~peroxidase activity, Leyj et al., Bloo.d 73, 1324 1330 (1~989). Ths~number a~ cells bound is derived rom a standard~ curve of myeloperoxidase ac~ivity : versus numbers~o~f~cells.~ Under all assay onditions, the cells release less~than 5~ of total : myeloperoxidase~and lactate dehydrogenase. Inhibition : is read as a lower per:cent adhesion, so tha~ a ~alue of:5~ means that:95%~of tha ~pecific adhesion was inhibited.i ~
Cli~ical Applicatio~s.
: .~ The subject peptides:are generally active when :~ administered parenterally in amounts above about l ~g peptide/kg of body weight.: For tr~atment to pre~ent ~ : organ injury in cases involving reperfusion, the : peptides may be administered paren~erally from about ~ O.Ol to about 10 mg peptide/kg body weight.
:~ :
:

W~92/20708 PCT/US9~/04016 ~ ~ a ~ 1 3 ~

~enerally, the same range of dosage amounts may be used in treatment of the oth~r 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-~40.
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. --For example, the pep~ides can be used tocompetitively inhibit leukocyte adherence by competitively bindiny to GMP-140 receptors on the surface of leukocytes. This kind of therapy would be particularly useful in acu~e situations where effective, but transient, inhibi~ion of leukocyte-mediated inflammation is desirable. Chronic therapy by infusion of the peptides may also be feasible in :some circumstances.
An inf lammatory re~ponse may cause damage to the host if unchecked, because leukocytes relezse many toxic molecules that:can:damage normal tissues. These moleules 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, adul~
respiratory distress~syndrome, tumor metastasis rheumatoid arthritis and~atherosclerosis.
Reper~usion injury is a major~ problem in clinical cardiology. Therapeutic agents that reduce leukocyte adherence in isch:emic myocardium can significantly enhance the therapeutic efficacy of , ~ 1 3~ -24-thrombolytic agents. Thrombolytic therapy with agents such as tissu~ plasminogen activator or streptokinase can relieve coronary artery obstruction in many patients with severe myocardial ischemia prior to irreversible myocardial cell death. However, many ~uch patients still suf~er myocardial neurosi5 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 adh~sive for leukocytes (Romson et al., Circulation 67: 1016 1023, ~g83). These adherent leukocytes can mi~rate through the endothelium and destroy ischemic myocardium just as it is beiny rescued by restoration of blood flow.
There are:a number of other common clinical disorders in which ischemia and reperfusion results in organ inju~y mediated by adherence of leukocytes to vascular surfaces, i~cluding strok~s; mesenteric and peripheral vascular disease; organ transplantation;
and ~irculatory shock (in this case many organs might be damaged following restoration of blood flow)~
~ Bacterial sepsis and~disseminated intravascular coagulation often~exist eoncurren~ly in critically ill patients. :They are~a~ss:ociated with generation Qf thrombin,~cytokines,~and other inflammatory mediators, activation of:;p~latelet~s and endothelium, and adherence of leukocy~es and~ aggregation of platelets throughout the vascular system~ ~ Leukocyte-dependent organ damage is an:important f:eature of these conditions.
Adult respiratory~distress syndrome is a :devastating pulmonary~::disorder occurring in patients with sepsis or~followi~g trauma, which is associated with widespread~adherence:and aggregation of leukocytes i~ the pulmonary circulation. This leads to:extravasation of large amounts of plasma into the W092/207~ PCT/US92/04~1~
~ I O 31 ~9 lungs and destruction of lung tissue, both mediated in large part by leukocyte products.
Two related pulmonary disorders that are often fatal are in immunosuppressed patients undergoing ~llogeneic bone marrow transplantation and in cancer patients su~fering from complications that arise from generalized vascular leakage resulting from treatment with interleukin-2 treated LAK cells ~lymphokine-act~vated 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 n~t known, such cells could potentially release m~lecu~es that activate endothelium and then bind to endothelium by mechanisms similar to those operative in neutrophils.
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 ~ndothelium and their subsequent migration are not well understood, but may be ~imilar to those of leukocytes in at le~s~ some cases. The association of platelets with metastasizing tumor cells has been well described, æugges~ing~a role for platelets in the spread ~f some cancers.~
Plate~et-leukocyte interactions are b~lieved to be important in::atherosclerosis. Platelets might have a role in recruitment o~ monocytes into atherosclerotic~plaques; the accumulation of monocytes is known ~o be one of the earliest detectable events during atherogen~sis.:~Rupture of a fully developed pl~que 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.

-W092/20708 P~T/~S92/04016 2ia3l3s Another area of potential application is in the treatment of rheumatoid arthritis.
The criteria for assessing response to therapeutic modalities employing these peptides are dictated by the specifi~ condition and will generall~
follow standard medical practices. For example, the criteria for the ~ffective dosage to prevent extension of myocardial inf~rction 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, an~ clinical response.
Fox 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 tachypne~. For treatment of patients in shock (low blood pressure), the e~fective dosage would be based on the clinical response and specific measurements of function of vital organs such as the liver and kidney following restoratio~ of blood pressure. Neurologic function would be monitored in p~tients with stroke.
Specific tests are used to monitor the ~unctioning of transplanted organs; for~example,~serum creatinine, urine~ flow, and serum elèctrolytes~in patients ;undergoing kidney transplantation.
Di~gnosti~Reage~ts.
, The peptides~ ca~ also;be used~for the detection o~ human disorders in whioh the ligands for the selec~ins might;be defective. Such disorders would i`~ mos~ likely be~seen in patients with increased susceptibility~to;infections in which leukocytes might ; not~be able to bind~to~ activated platelPts or endothelium. Ce;lls to be~ested, usually leukocytes, are collec~ed by standard medically approved techniques and screened.~ Detection~systems include ELISA procedurest~binding of radiolabeled antibody to immobilized activated cells, flow cytometry, or other :
:

WOg2/20708 PCT/US92/0~016 ~103~39 ~27-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 selectins, such disorder~ 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 radioaoti~ely, with a fluorescent tag, enzymatically, or with electron dense material such as gold for electron micr~scopy. The cells to be examined, usually leukocytes, are incubated with th~ labeled peptides and hinding assessed by methods described abo~e with antibodies to ~MP-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 xadioimmunoassay procedur~es, using labeled GMP-140-derived peptide instead of antibody~as the detecting reagent.
:The following examples are presented to illustrate the invention without intendi~g to J
speci~ically limit the invention thereto. In the examples and throughout the specifications, ~arts are by weight unless:otherwise indicated.
EXAN~E ~: Pr~p~ration of:Tyrosyl-threonyl-hi3tidyl-1 eucyl-Yalyl ~laIlyl-isoleuc:yl gluta~i~e-me peptide was prepared on an ABI model 431A
peptide synthesizer using Version 1.12 of the standard scale Boc software. ~ThP amino acids used were Boc-(BrCBZ~)Tyr, Boc~ Bzl~Thr, Boc-(Tos)His, Boc-Leu, Boc-~al, Boc Ala,;::Boc-Ile~and:Boc~Gln. 4-Methylbenzhydrylamine resin (0.625 g, 0.5 mmol) was used in the synthesis. The final weight of the resin was 1.13 y. The~ peptide was cleaved from the resin (1.03 g) using ll mL of HF and 1.1 mL of anisole for ., .. . .... ~ .. .... . . ...

W092/20708 PCT/~S92/04~16 ~ 31~ -28-60 min at 0 C. The hydrogen fluoride was evaporated using a stream of ni~rogen and thQ resulting mixture triturated with ether. The solids were removed by filtration and extracted with 25 mL of a 50% solution of TFA in methylehe chloride. Removal of the resin by filtration, evaporation of the solvent and trituration of the residue with ether gave 0.~2 g of crude peptide.
The crude peptide (55 mg) was purified on a Vydac C 18 column (lO ~ 2.2 x 25 cm), eluting with a gradient of lO to 20% of acetoni~rile in 0.1% aqueous TFA over 20 minutes at a flow rate of ~ mL per minute~
Fractions were collected, analyzed by HPLC ~nd pure fractions pooled and lyophiIized to give 3.9 mg of purified peptide. Amino acid analysis: Ala o.98 (l.0), Glx 1.02 (l.0), His l.0~ (l.0), Ile 1.07 (l.0), Leu 1.07 (l.0), Thr 0.85 (l.0), Tyr O.B5 (l.0), Val 0~94 (l.0). FABlMS: MH~ 944 (calcd 944).
EXAMP~E 2: Pr~paration o~ Tyrosyl-threonyl-glutamyl leucyl~-valyl-alanyl-isoleuc~l-glutami~e-ami~
The peptide was prepared on a ABI model 431A
.
peptides synthesizex using:Version lv12 of the standard scale Boc software. The amino acids used ~ were:Boc-(BrCBZ)Tyr, Boc-(Bzl)Thr, Boc (Bzl)Glu, Boc : : Leu, Boc-Val,~Boc-Ala,:Boc-Ile, Boc-Gln. 4-:: methylbenzhydrylamine resin (0~625 g, 0.5 mmol) was :: used in~the synthesis.~ Final weight of the resin was .20 g. The peptide was cleaved from the resin (l.lO
: ~ g~ using li mL of HF and~l.l mL of anisole for 60 minutes at 0 C~. The hydrogen fluoride was removed using a s~ream of dry~nitrogen, the residue triturated with ether and the ether removed by filtration. The : remaining solids~were~triturated with 25 mL of a 50%
solution of TFA in m thylene chloride. The resin was removed by filtration, the solution evaporated ~nder reduced pressure and the residue triturated with ether :: :

W092/20708 PCT/US9~/04016 ,,, -2~ 3 1 3 ~

to give 0.40 g of the crude peptide, isolated by filtration. The crude peptide (0.31 g) was purified by HPLC (multiple injections) on a Vydac C-18 column ~10 ~, 2.2 x 25 cm) eluting with 30% acetonitrile in 0.1~ aqueous ~FA over 90 minutes at a flow rate of 8 mL/min. Fractions were collected, analyzed by HPLC
and pure fractions pooled and lyophilized to give ~7 mg of pure peptide. Amino acid analysis: Ala 1.01 (1.0), Glx 2.00 (2~0), Ile 1.02 (1.0), Leu 1.06 (1.0), Thx 0.78 (1.0), Tyr 0.~ ~1.0), Val 0.93 (1.0).
FAR/MS: MH+ ~36 (calcd 936).
EX~MP~ 3: Prep~ra~ion of Acetyl-~yrosyl threonyl-aspart:yl-leucyl-valyl-alanyl-isoleu~yl-glutamine amidg.
The pepti~e was prepared on ABI model 431A
peptide synthesizer using Version 1.12 of the standard scale Boc software mo~ified for N-terminal acetylation according the instrument operations manual. 4-Methylbenzhydrylamine resin (0.625 g, 0.5 mmol) was usad in the synthesis. Final weight of the pepti~e resin was 1.23 g. :The:peptide was cleaved from the resin (1.11 g) with 10 mL of HF and 1 mL o~ anisole for 60 minutes;at~ 0 C. The HF was removed by a nitrogen ~tream~ The resulting solid w~s triturated with ether,~collected by;~filtration and washed with ether. ~The peptide was~extrac~ed f~rom the resin with 50% TFA~and~methylene~chloride (5 x 20 mL). The resin was removed::by filtration~ the solvents removed under reduced~pressureland the~residue triturated with ether to give 0.50 g of crude peptide. The crude peptide was purified by~preparation HPLC using a Vydac C-~8 column:(10 ~ 2~.~2;~x~2~ cm) eluting with a 20 ~o 30~
gradient of acetonitrile and 0.1~ aque~us TFA over 140 minutes~at a flow rate~of 3 mL per minute. Fractions were collected, analyzed:by HPLC and pure fractions : ~ :
: ~~ pooled and lyophilized to give 60~mg of ~he purified ~:: peptide as a white so~lid. Amino:acid analysis: Tyr W092/207a8 PC~`/US92/0~016 ~ ~ 3 1 r3 ~ ~ 30~

0.99 (1.0), Thr 0.91 (1.0), Asx 0.9~ (1.0~, Leu 1.03 (1.0), Val 1.05 (1.0), Ala 1.03 (1.0), Ile 1.00 (1.0), Glx 0.01 (1,0).
EX~MPLE 4: Preparation of Tyro~yl~threonyl-aspartyl-leuayl-valyl-alanyl-isoleucyl~glutamine amide~
The peptide was prepared by manual solid phase synthesis using Boc chemistry. The amino acids used were Boc-(BrCBZ)Ty.r, ~oc-~Bzl)Thr, Boc-(Bzl)Asp, Boc-Leu; Boc~Val, Boc-Ile and Boc-~ln. 4-Methylbenzhydrylamine resin (6.25 g, 5.0 mmol) was used in synthesis. 20 Mmol of each Boc-AA was acti~ated by dicyclohexylcarbodiimide and hydroxybenzotriazole (20 mmol of each) and couple~ to ~he resin. The sequence used was as follows:
WA~ REpErrITIoNs TIME~mins) 25% TFA/C~2Cl2 1 3 50% TFA/~H2Cl2 1 16 5% N~methylmorpholine/C:H2C12 1 CH2~l2 1 3 Coupling step (monitored by ninhydrin testing of a resin ~ample).
'rhe final weight of:the peptide-resin was 11.97 g. The resin-peptide~(11,8 g) was treated with 1~ mL
of anisole and 1~20 mL of HF for one hour at 0 to 4VC.
The HF was removed by nitrogen stream followed by a~piration. The resultant solids were triturated with ether (1 x 100 mL then l x 80 mL), collected by filtration and washed with ether (3 x lO0 mL). The residue was then extracted with 50~ trifluoroacetic acid/methylene chloride (4 x 50 mL), and thP solve~ts removed by vacuum. The residue was triturated with 500 mL of diethyl ether. The scrlids were collected by filtration and air-dried overnight at ambien~

W092/207~8 PCT/US92/04016 2iO3l~3 temperature followed by drying in vacuo at room temperature for 1 hour. The yiel~ of crude peptide was 4.08 g. The crude peptide (106 mg) was purified in two 53 mg runs by reverse phase HPLC using Vydac 22 x ~50 mm C-18, 10 ~ 300 Angstrom pore packed column.
Elution with a gradient of 25% to 40% B over 72 minutes at a flow rate of 6 mL/min was carried out tsolvent A - 0.1% TFA; solvent B = 0.1% TFA in 50%
~cetonitrile/Water~. Fractions were collected and the appropriate fractions pooled to give 56.~ mg of whj.te solid~ Amino Acid Analysis: Asx 1.02 (1.00), Thr 0.89 (l.oo), Glx 0.99(1.00, Ala 1.02 (1.00), Val 0.96 (1.00~, Ile 1.03 (l.OOj, ~eu 1.06 (1.00), Tyr 0.91 (1.00). FAB/MS: MH~ = 922 (calcd-92~).
~XAMPLE 5: Preparation of Arginyl-tyrosyl-threonyl-~partyl-leucyl-valyl-alanyl-isoleucyl-glutamine amide.
The peptide was prepared on a DuPont RAMPS
system using th~ FMOC strategy. The amino acids used ~or the synthesls were~ FMOC-t~tr)Arg, FMOC-(t-Bu~Tyr, FMOC-(t-Bu)Thr, FMOC-(t-Bu)Asp, FMOC-Leu, FMOC-Val, FMOC-Ala, FMOC-Ile and~ FMOC-Gln. DuPont rapid amide r~sin (0.1 mmol)~was used in the synthesis. The peptide was cleaved~rom;the resin using a mixture of phenol (0~25 g~), ethanedithiol (0.083 mL~, thioanisole O.66 mL), water (0.166:mL): and trifluoroacetic acid (3033 mL) for~ hours~or:~0 ~. ~he resin was removed:;by: f~iltration and the peptide precipitated from the filtrate; by:the~a~dition of ether. The s~lids were removed by filtration, extracted with 20%
aoetic acid ~nd lyophilized to give 0.147 g of crude peptide. The;peptidé was~purified by preparative reverse~phase (~ HPLC using an acetonitrile wat~r gradient in 0.1% in~TFA. Fractions were collected and those containing pure peptide were pooled and lyophilized.

W092/20708 PCT/USg2/04016 2 ~q 5313.~

EXAMPL~ 6: Prepar2tion of Asparaginyl-arginyl-tyro~yl-threonyl aspartyl~leucyl-valyl-alanyl-isoleucyl-glutamine amide.
The peptide was prepared using a DuPont RAMPS
system and the FMOC strategy. The amino acids used were FMOC-Asn, FMOC-(~tr)Arg, FMOC-(t-Bu)Tyr, FMOC-(t-Bu)Thr, FMOC-~t-Bu)~sp, FMOC-Leu, FMOC-Val, FMOC-Ala, FMOC-Ile and FMOC-Gln. DuPont rapid amide resin (O.2 mmol) was used in the synthesis. The peptide was cleaved in the resin using a mixture of TFA ~2.~5 mL), thioanisole (0.135 mL) and ethanedithiol (0.015 mL) for 16 hours at ambient temperature. The resin was removed by filtration and the peptide precipitated from the f iltrate by the addition of ether. The peptide was removed by filtration, extracted with 20%
acetic acid and lyophilized to ~ive 50 mg of crude peptide. The crude peptide was purified by preparative reverse phase (C-18) HPLC using a gradient of acetonitrile water in 0.1% TFA. Fractions were collected and those containing the pure peptide were pooled and lyophilized. :
E~AMPL$ 7: Preparation of Cystei~yl-glutami~yl-a~paragi~yl-arginyl-tyro~yl-threo~yl-a3partyl-leucyl-~alyl-ala~yl-i~oleucyl-: ~lutaminyl-a8par~gi~yl lysyl-~sparagi~yl-glut~mi~e.
The peptide was prepared on an ABI model 430A
pepti~es synthesizer:using the standard scale Boc software. The amino acids used were Boc-(4-Me-Bzl)Cys, Bioc-Gln, Boc-Asn, Boc-(Tos)Arg, Boc-(BrCBZ)Tyr, Boc-~Bz:l)Thr, Bo~-(Bzl~Asp, Boc-Leu, Boc-Val, Boc-Ala,~ Boc-Ile, Boc~(Cl-CBz)Lys. Boc-(Bzl)~lu-Pam resin ~0. 5 mmol): was used in the synthesis. The peptide~was cleaved from the resin using 10 mL of HF, 1.0 m~ of anisole, 1.0 mL~of dimethyl sulfide and 0.2 mL p thiocresol for 30 minutes at -10 C followed by 30 minutes at 0C. The hydrogen fluoride was removed under reduced pressure and the residue triturated with ether. Solids were removed by filtration and the peptide extracted from the resin using 20% acetic acidO Removal of the resin by filtration and lyophilization of the filtrate gave 25 mg of crude peptide. The crude peptide was purified by the preparative HPLC using a 2.2 x 25 cm Synchrom C-18 column (6.5 ~), eluting with a gradient of 5 to 25%
acetonitrile in 0.1~ TFA over 20 minutes at a flow rate of 6 mL per minute. Fractions were collected and the fractions containing the pure peptide and pooled lyophilized.
EXAMPLE 8: Prepara~ion of Tyro~yl-threo~yl-D~spartyl-leuayl-Yalyl-alanyl-isoleucyl-glutamine amide.
The peptide was prepared on an ABI Model 431A
peptide synthesizer using Version 1.12 of ~he standard Boa software. The amino acids used were Boc~
(BrCBZ)~yr, Boc-(Bzl)Thr, Boc~D-(Bzl)Asp, Boc-~eu, Boc-Val, Boc-Ala, ~oc-Ile and Boc-Gln. 4-Methylbenzhydrylamine resin (0.63 g, 0.5 mmol) was used in the:synthesis. The final weight of the peptide resln was 1.43 g. The peptide ~as cleaved from:the resin (1.43: g) using lS mL of HF and 1.5 mL
of anisole for 60 min at 0C. The hydrogen fluoride was removed under reduced pressure and the residue tritura~ed with~ether. Solids were removed by filtration:and:~the:~p ptide extracted from the resin using a 50~ solution of trifluoroacetic acid in methylene chlor~ide. Removal of the resin by fil ration and~preclpitation with ether gave l.Og g of crude peptide. ~:The crude:peptide (0.5 g) was purified by preparative HPLC using a Vydac C-18 column (15 ~, 5.0 x 25 cm) eluting with a 0-100% gradient of 50 acetonitrile in 0;.1% TFA over 120 minutes at a flo~
rate of 15 mL per minute. Fractions were c~llected, analyzed by HPLC and pure fractions pooled and 2 i ~ 3 ~ -34- ~

lyophilized to give 200 mg of the desired product.
Amino acid analysis: Ala 0.99 (1.00), Asx 1.01 (1.00), Glx 1.02 (1.00), Ile 0.97 (1.00), Leu 1.02 (1.00), Thr 0.91 (1.00), Tyr 0.96 (1.00), Val 1.04 (1.00). FAB/MS: MH+ = 921 ~X~PLE 9: Preparatio~ of Phenylalanyl-threo~yl-partyl-leucyl-valyl-alanyl-i~oleucyl-glutamine amide.
The peptide was prepared on an ABI Model 43lA
peptide synthesizer using Version 1.12 of the standard Boc software. The amino acids used were ~oc-Phe, Boc-(Bzl)Thr, Boc-(Bzl)Asp, Boc-Leu, Boc-Val, Boc-Ala, Boc~Ile and Boc-Gln. 4-Methylbenzhydrylamine resin (0.625 g, 0.5 mmol) was used in the synthesis. The final weight of the resin was 1.18 g. The peptide was cleaved from the resin (1.10 g) using 11 mL of HF and 1.1 mL of anisole for 60 min at O~C. The hydrogen fluoride was evaporated using a stream of nitrogen and the resul~ing mixture triturated with ether~ Solids wsre removed by filtra~i;on and extracted with ~5 mL of a 50% solution of trifluoroaceti~ acid in methylene chloride. Removal of~the resin by filtration, ~: evaporation of the solvent and trituration of t~e residue~with ether gav 0.52:g of crude peptide. The crude~peptide ~(82 mg):;~:was purified on a Vydac C-18 column (10 ~/~2.~Z:x 25:cm):eluting with a 0-60%
gradient of aaetonitrile and 1% TFA over 60 minutes at a flow rate ~f~8~ mL per;minute. ~ractions were : ollected, analyzed~by: HPLC and pure fractions pooled and lyophilized to:give~27 mg. ~mino acid. analysis:
Ala ~01~, (l.O~ Asx~1.0~ (1.0), Glx 1.00 (1.0), Ile 0.96 (~1.0)~, Leu 1.01 ~ 0)~,~ Phe 0.94 (1.0), Thr 0.85 (1.0), ~àl 1.02 (1.0).~ FAB/MS: MH+ 906.

.
:~ ; ~: :
~: :

.

2 ~ f~ 3 -35~

~X~MPLE 10: Preparation o~ D-Tyrosyl-threonyl-aspartyl-leucyl-valyl-alanyl-i~oleucyl glutami~e amide.
The peptide was prepared on a ABI Model 43lA
peptide ~ynthesizer using Version 1.12 of the standard scale Boc software. The amino acids used were Boc-D-(BrCBZ) Tyr, Boc-(Bzl)Thr, Boc-(Bzl)Asp, Boc-Leu, Boc-Val, Boc-Ala, Boc-Ile, Boc-Gln. 4-Methylbenzhydrylamine resin ~0.665 g, 0.5 mmol) was used in the synthesis. The final weight of the resin was 2.10 g. The pep~ide was cleaved from the resin (2.10 g) using 20 mL of hydrogen fluoride and 2 mL of anisole for 60 minutes at 0C. The hydrogen fluoride was evaporated using a stream of nitrogen and th~
resulting mixture triturated with ether. The solids were removed by filtration and extracked ~ith a 50%
solution ~f trifluoroacetic acid in methylene chloride. Removal of the resin by filtration, evaporation of the solvent and ~rituration of the residue with ether ga~e 1.96 g of crud~ peptide containing residual solvents. The crude peptide (~00 mL) was purified on a Vydac C-18 column (10 ~, 2.2 x 25 cm) eluting with a 0-100% gradient of 50~
acetonitrile and 0.1~ TFA over 120 minutes ~t a flow rate of 15 mL per minute.~ Fractions were collected, ~naly2ed by ~PLC and pure fractures pooled and lyophilized to give 5Z mg of pure product. Amino acid analysis: Ala 0.9~ (1.0~, Asx 1.04 ~1.0), Glx ~.00 (1.0), Ile 0.96 (1.0), Leu 0.~9 (1.0), Thr 0.92 (1.0), Tyr ~.95 (1~0~ Val 1.04 (1~0). FA~/MS: MH~ 921.
EX~P~E 11: Prepara~ion of Tyrosyl-~-threonyl-~spartyl leueyl ~alyl-alanyl-oleucyl glutamine amide.
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-(BrCBZ)Tyr, Boc-D-~Bzl~Thr, Boc-~Bzl)Asp, Boc-~eu, W092/2070~ PCT/US92/04016 ~ j313~ -36-Boc-Val, Boc-~la, Boc-Ile and Boc-Gln. 4-Methyl~enzhydrylamine resin (0.685 g, 0.5 mmol) was used in the synthesis. The final weight of the peptide resin was l.63 g. The peptide was clea~ed from the resin (l.63 g) using 20 mL of hydrogen fluoride and 2 mL of anisole for 60 min at O~C. The hydrogen fluoride was removed under reduced pressure and the residue triturated with ether. The solids were removed by filtration and the peptide extracted from the resin with a 50~ solution of trifluoroacetic acid in methylene chloride. Removal of the resin by ~iltration and precipitation with ether gave 0.52 g of crud~ peptide. The rude peptide (0.50 g) was purified on a-Vydac C-18 column (15 ~/ S.0 x 25 cm) in four in~ctions, eluting with a 0-100% gradient of 50% acetonitrile in 0.1% TFA over 120 minutes at a flow rate of 15 mL per minute. Fractions were aollected, ar.alyzed by HPLC and pure fraGtions pooled and lyophilized to give 254 mg of pure peptide. ~mino acid analysis: Ala l,Ol (l.0), Asx 1.08 (l.0), Glx 0~98 (loO) / Ile 0.98 (~l.Oj, Leu 0.97 (l.0), Thr U.95 (l.0), Tyr 0.98 (l.0), Val loOl (1~0~ FAB/MS: MH~
922.
~XA~P~E 12: I~hibition of ~eutrophil Binding to - BMPl40 Coated Wells~
Binding~of various~peptides to GMP-140 coated wells, as de~cribed~above,~were compared. The results are shown~ in; Figure 1. ~
Binding of the pep~ides at various oncentratio~s,~ranging from 0 to 1~5 mM, were comparedO~ The~peptide~5 tested were Cys-Gln-Asn-Arg-Tyr Thr-Asp-Leu-Val-Ala-Ile-Gln-amide; Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile Gln-amide; Arg-Tyr-Thr-Asp-Leu~Val-Ala-Ile-Gln-amide;~ Tyr~Thr-Asp-Leu-Val-Ala-Ile-Gln-amide; Acetyl-Tyr-Thr-~sp~Leu~Val-Ala-Ile-Gln-amide; Tyr-Thr-Glu-Leu-Val-Ala-Ile-Gln-amide; Tyr-Thr-His-Leu-Val-Ala-Ile-Gln-amide; Tyr-Thr-Asp-Leu-W092/20708 PCTfUS92/04016 ., . ~ .
_37_ 21 0 31 3 g Val-Ala-Ile-Gln-Asn-Lys-~sn~Glu-amide; Leu-Gln-Thr-Ala-Tyr-Asp-Val-Ile-amide tnegative control).
In subsPquent studies/ additional peptides were tested for activity in inhibiting nPutrophil addition to immobilized P-selectin. The ICso data is presented in mM concentration. The results are shown below in Table l.

WOgZ/20708 PCT/~S92/~4016 2 i 0 3 1 ~ ~ r3 T~BLE 1: INHIBI~ION OF BINDING OF ~EUTROPHILS ~Y
PEPTIDES
~TRUCTURE ICso~m~) CRGDLVAIQ-NH2 .132 QNRYTDLVAIQ-NH2 .034 NRYTDLVAIQ-NH2 .039 NRYTDLVAIQNKNE-NH2 .615 CYTDLVAIQ-NH2 .364 YTDLVAIQ-NH2 .263 Formyl-Y~DLV~IQ-NH2 .936 4-Br-Phe-TDLV~IQ-NH2 .0~0 4-NH2-Phe~TDLVAIQ-NH2 .383 Ac-RGDLVAIQ-NH2 .179 YTALVAIQ-NH2 .184 YTDA~AIQ-NH2 .347 RGHLVAIQ-NH2 .033 RGDLVAIQ-NH2 . .041 RTDL~AIQ-NH2 .622 YTNLVAIQ-NH2 .271 YTDLVAIN-NH2 .187 CGDLVAIQ-NH2 ~304 YTDLVaIQ-NH2 .420 YTdLVAIQ-NH2 ~ .385 YTDL~AIq-NH2: .146 YTDLVAiQ-NH2~ .162 YTDlVAIQ-NH2 ~ .869 YtDLVAIQ-NH2~ . .271 y~dLVAIQ-NH~ ~ .216 y~DL~AIQ-NH2 .316 YTDLVIQ-NH2 .463 YTDVAIQ-NHz .296 desamino-Arg-TDLVAIQ-NH2 ~707 desamino-Tyr-TDVAIQ-NH2 .416 YTDLVAI-descarboxy-Gln-NH2 .391 YTQLVAIQ~NH2 .097 21(~31~9 . -3~-STR~CT~RE IC~O~mM) YTDLVAI~-NH-n-Bu .151 YEDLVAIQ-NH2 .267 YTELVATQ-NH2 .389 YTDEVAIQ-NH2 .668 YGDLVAIQ-NHz 977 YTHLVAIQ--NH2 . 081 KTDLVAIQ-NH2 . 2 Z 6 YKDI.VAIQ-NH2 . 210 YTKLVAIQ-NH2 . 0 6 0 N-Me-Tyr-TDLVAIQ-NH2 .336 Nal-TDLVAIQ NH2 .192 O-Me-Tyr-TDLVAIQ-NH2 .103 FTDLvAIQ-NH2 .384 YTFLVAI ;2--NH2 . 2 3 4 Pya-TDLVAIQ-NH2 .346 YSDLVAIQ-NH2 .202 YTDLT~IQ-NH2 : : .359 ~ Tic-TDLVAIQ-NH2 ~ ~ ~ : .062 : ~YTD~AAIQ-NH~ 893 YTGL~AIQ-NH2 ~ o134 Ac-YtH~VAIq-NH-n-Bu~ ~ : .366 c-YtDL~AIQ-NH-n-8u~ .67 YTDLV~IQN-NH2 :~ ~ .153 Y~DLVAIQNK~NH2 ~ . .199 : Abbrevia ions: Nal - Naphthylalanine O-Me-Tyr~:~- O-methyltyrosine N-Me-Tyr :- N~-methyltyrosine : ; Pya~ Pyridylalanine : : ~ n~Bu :::: - n butyI
;Tic : ~ - tetrahydroisoquinoline carboxylic acid ~: ` The results~emons~rate that, with the exception of the negative ~control, the peptides all inhibit neutrophil binding: to immobilized GMP-140.

W092/2070~ PCT/U~92/~4~16 2 i ~ 3 ~ ~40-EXAMPLE 13: Modification of peptide s~a~ility in ~erum~
Figure 2 shows the signi~icant increase in stability against enzymes found in human serum that can be achieved by the modifications set down in Formula I and II, graphing percent of peptide remaining versus time, for two peptides:
Ac-Y-T--D-L-V-A-I-Q-NH~ and y_T_D-~~v~~ Q~NH2 1'he half--lie in serum increased from Z0 minutes ~or the unmodified peptide to four hours, 37 minutes for the modified peptide.
Modifications and variations of the present invenkion, synthetic peptides and methods for modulating binding reactions involving selectins, will be obvious to those skilled in the art from the ~oregoing detailed description. Such modifications and Yariations are intended to come within the scope of the appended claims.

:~:

2:10~

SEQUENCE LISTING

(1~ GENERAL INFORMATION:
(i) ~PPLICANT: Heavner, George A.
McEver, Rodger P.
Geng, Jian-Guo (ii) TITLE OF INVENTION: Peptide Inhibitors of Inflammation (iii) NUMBER OF SEQUENCES: 45 (iv) CORRESPONDENCE A~DRESS:
(A) ADDRESSEE: Kilpiatrick & Cody (B) STREET: 100 Peachtree Street (C) CITY: Atlanta (D) STATE: Georgia (E) COUNTRY: U~
(F) ZIP: 30303 (v) COMPUTER READABLE FORM:
: : (A) MEDIUM TYPE: Floppy disk (B~ COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS
(D) SOFTWARE: PatentIn Release #1.0, Version #1.25 (vi) CURRENT APPL~CATION DA~A:
: : (A~::APP~ICATION N~MBER: US 07/69969 :~ ~ : (B) FILING DATE: 14~MAY-1991 :~ ~: (C:)~CLASSIFICATION:
(viii):ATToRNEY~GENT IN~ORMATIO~o t~ NAME: Pabst, Patrea L.
: (B) REGIST~ATION:NUMBER: 31,284 (C)~ ~EFERENCE/DOCKET NUMBER: ONRF-CTC100 (ix): TELECQMNUNICATION INFORMATIQN:
: (A)~TELEPHONE: 404-572-6508 B)~ TELEFAX::40~4-658-6555:

(2~ INFOR~ATION :FOR SEQ ID NO:1:
i) SEQUENCE~C~ARACTERISTICS:
: (A) LENGTH: 8 amino acids ~: : (B)~: TYP : am.ino acid : (C) STRANDEDNESS: single D~ TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO

W~92/207~g PCT/~9~/~4016 ~42-(iv) ANTI SENSE: NO
(v) FRAGMENT TYPE: N-terminal ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:l:
Tyr Thr Asp Leu Val Ala Ile Gln l 5 (2) INFORMATION FOR SEQ ID NO:2:
(i) SEQUENCE CHARACTERISTICS:
~A) },ENGTH: ~ amino acids (B) TYPE: amino acid (C) ST~ANDEDNESS: single (D) TOPOLOG~: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTXON: SEQ ID NO:2:
Tyr Thr His Leu Val ~la ~le Gln l 5 (~ INFORNATION FOR SEQ ID NO:3:
ti) SEQUEN~E CH~RACTERISTICS:
(A) LENGT~: 8~amino acids : : (B) TYPE: amino acid ~C)~STRANDEDNESS: single : (D~ TOPOLOGY: linear (ii) MOLECULE TYPE: peptide : (iii) HYPOTHETICAL: NO
: ~ ( iY) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal 8 2 i 0 31 ~ 9 PCI/U$92/0401 (xi~ SEQUENCE DESCRIPTION: SEQ ID NO: 3:
Tyr Thr Asp Leu Val Ala I le Gln ( 2 ) INFORMATION FOR SEQ ID NO: 4:
(i) SEQUENCE CHARACTERISTICS:
(A~) I,ENGTH: 12 amino acids ~B) TYPE: amino acid (C) STRANDEDNESS: single ( D ) TOPOLOGY: l inear ( ii ) MOLECULE TYPE: peptide ( i i i ) HYPOTHETI CAL: NO
( iv) ANTI--SE~SE: NO
(v) FRAGMENT TYPE: N-terminal (xi) SE;aUENCE DESCRIPTION: SE;2 ID NO: 4:
Cys Gln Asn Arg Tyr Thr Asp Leu Val Ala Ile Gln ~, ( 2 ~ INFORMATION FOR SEQ ID ~O: 5:
(i) SEQUENCE CHARACTERISTICS:
(A) I.ENt;TH: lû amino acids (B) TYPE: aminc~ acid (C~ STRANDEDNESS: single (D) TOPOLOGY :~ linear : ~ ~ii) MOLECULE TYPE: peptide ( iii ) HYPOTHETICAL: NO
( iv) ANTI--SElNSE: NO
1 i , I , ~
(v) FRAGMENT TYPE: N-terminal (xi~ SEQUENCE DESCRIPTION: SEQ ID NO:5:
~sn Arg Tyr Thr Asp Leu Val ~la Ile Gln W092/20708 PCT/U~92/~4016 ~ l ii . l 3 !~ _44 (2) INFORMATION FOR SEQ ID NO:6:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 9 amino acids ~B) TYPE: amino acid (C) STRANDEDNESS: sinyle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO-6:
Arg Tyr Thr Asp Leu Val Ala Ile Gln l . 5 (2) INFO~MATION FOR SEQ ID NO:7:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 8 amino acids (B) TYPE: amino acid (Cj STRANDEDNESS: single :~ ~D) TOPOLOGY: linear .
(ii) MOLECULE TYPE: peptide (iii3 HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v~ FRAGMENT ~TYPE: N-terminal (xi)~sEQUENCE DESCRIPTION: SEQ ID NO:7:
Tyr Thr Glu Leu Val Ala Ile Gln l 5 (2~ INFOR~TION~FOR SEQ ID NO:8:
SEQUENCE CHARACTERISTICS:
~: (A) L~NGTH: 12:amino acids ~: : (B):TYPE:: amino:acid :;; (C) STRANDEDNESS: single : (D) TOPOLOGY: linear :: ~
:~ (ii3 MOLECULE~TYPE: peptide : ~ .

WO 92/2070X ~ 1 0 ~ 1 3 3 PCl/US92/04~16 ~45--( i i i ) HYPOTHETI CAL: NO
( iv) ANTI-SENSE: NO
(v) FR~GMENT T~PE: N-terminal (xi) SEQIJ:ENCE DESCRIPTION: SE:52 ID NO: 8:
Tyr Thr ~sp Leu Val Ala Ile Gln Asn Lys Asn Glu ( Z ) INFO~TION FOR SEQ ID NO: 9:
i ~ SEQUENCE CHARACTERI STI CS:
(A) L:ENG~H-: 8 amino acids (B) TYPE: amino acid ( C) ST~NDEDNESS: s ingle (D) TOPOLOGY: linear (ii~ MOLECULE TYPE: peptide ( i i i ) HYPOTHETI CAL: NO
( iv) ~NTI-SENSE: NO
(v) FR~GMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTIO~: SEQ ID NO: 9:
Tyr q~hr Asp IJeu Val Ala Ile Gln ~2) INFs:~RMATIc)N~ ~FOR SEQ ID NO:l0:
( i ) SEQUENCE CHARAC: TERI STI CS:
~A~ I,ENGTH: 8 amino acids 3) TYPE: amino acid ~C3 STRANI)EDNESS: s.ingle : ~ (D~ TOP~LOGY: linear (ii~ MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
( i~7) A~TI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal W~2/2070X P~T/~S92/04016 ~ ., 1 3 3 -46- .

(xi~ SEQUENCE DESCRIPTION: SEQ ID NO:10:
Phe Thr ~sp Leu Val Ala Ile Gln (2) INFORMATION FOR SEQ ID NO:11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii~ HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCR~PTION: SEQ ID NO:11:
Tyr Thr Asp Leu Val Ala Ile Gln : 1 5 ~23 INFORMATION FOR SEQ ID NO:12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids (B) TYPE: amino acid : (C) STRA~DEDNESS: single : (~) TOPOLOGY: lineax (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(1~) ANTI~SENSE NO
~v) FRAGMENT TYPE: N-terminal (xi3 SEQUENCE DESCRIPTION: SEQ ID NO:12:
Tyr Thr Asp Leu Val Ala Ile Gln .

210~1~3~

(2) INFORMATION EOR SEQ ID NO:13:
~i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRAND~DNESS: single (D~ TOPOLO~Y: linear ~ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Tyr Thr ~he Leu Val Ala Ile Gln t2~ INFORMATION FOR SEQ ID NO:14:
(i~ SEQUENCE CHARACTERISTICS:
~A~ LENGTH: 8 amino acids ~B) TYPE: amino acid ~C) STRANDEDNESS~ single (D) TOPOLOGY. linear (ii~ MOLECULE TYPE: peptide (iii~ HYPOTHETICAL: NO
(iv) A~TI-SENSE: NO
(v~ FRAGMENT TYPE: N-kerminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:I4:
Tyr ~hr Lys Leu Val Aia Ile Gln : : ~2) INFORMATION F~R SEQ ID NO:15:
(i) SEQUENCE CHARACTERISTICS:
(R) LENGTH: 8 amino acids ~B) TYPE: amino acid : (C~ STRANDEDNESS: single : (D~ TOPOLOGY: linear - (ii3 MOLECULE TYPE: peptide W092/207~8 PCT/U~9~ 016 ~ 48-(iii) HYPOTHETICAL: NO
(i~) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
Lys Thr Asp Leu Val Ala Ile Gln l 5 (2) INFORMATION FOR SEQ ID NO:l6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids ~B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii~ MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTI SENSE: NO
(v) FRA~MENT TYP~: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: t Tyr Thr Ala ~eu Val Ala Ile Gln ~ 5 (2) INFORMATION FOR SEQ ID NO:17:
~i) SEQUENCE ~H~R~CTERISTICS:
(A~ LENGTH:: B amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal WO92/2070B PCT~US92/04016 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
Phe Thr Asp Leu Val Ala Ile Gln (2) INFORMATION FOR SEQ ID NO:18:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids (B) ~YPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: unknown (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ~NTI SENSE: NO
(v) FRAGMENT TYPE: N-terminal (xi) SEQ~ENCE DESCRIPTION: SEQ ID NO:18:
Tyr Thr Asp Ala Val Ala Ile Gln (2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 7 amino acids ~B) TYPE: amino acid : (C3 STRANDEDNESS: single (D) TOPOLO-~Y: unknown ~ OLECULE TYPE:~peptide (iii) HYPOTHETICAL: NO
~iv) ANTI-SENSE: NO
(v~ FRAGMENT TYPE: ~-terminal :~ . . ` !

(xi) SEQU2NCE ~ESCRIPTION: SEQ ID NO:l9:
Tyr Asp Leu Val Ala Ile Gln (2) INFO~MATXON FOR SEQ ID NO:20:
~i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 9 amino acids WO g2/20708 PCr/US92/OqO~

~,,.. ~c~ 50-(B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii~ MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT ~YPE: C terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:
Cys Arg ~ly Asp ~eu Val Ala Ile ~ly l 5 (2) INFORMATION FOR SEQ ID NO:21:
(i) SEQUENCE CHARACTERISTICS:
(A~ LENGTH: 11 amino acids (B~ TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
~iv) ANTI SENSE: NO
(v) FRAGMENT TYPE: N-terminal ~xi) SEQUENCE DESCRIPTION: SEQ I~ NO:21:
Gln Asn Arg Tyr Thr ~sp Leu Val Ala Ile Gln ~2) INFORMATION FOR SEQ ID NO:22:
1 ~ I
(i~ SEQUENCE CHARACTERISTICS:
~A) LENGTH: 14 amino acids (B~ TYPE: amino acid ~C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii~ MOLECULE TYPE: peptlde (iii) HYPOTHETICAL: NO
(iv~ ANTI-SENSE: NO

WO~2/2070B PCT/US92/04016 2i~31~
-Sl-(v~ FRAGMENT TYPE: N terminal ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
Asn Arg Tyr Thr Asp Leu Val Ala Ile Gln Asn Lys Asn Glu l 5 lO

(2) INFORMAT~ON FOR SEQ ID NO:23:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: g amino acids ~B) TYPE: amino acid (C~ ST~ANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTI-5ENSE: NO
~; (v) FRA~MENT TYPE: N-terminal ~xi3 SEQUENCE D S~RIPTION: SEQ ID NO:23:
Cys Tyr Thr Asp Leu Val Ala Ile Gln ~ 5 : (2) INFO~MATION FOR SEQ ID NO:24:
~i) SEQUENCE ~HARACT~RISTICS:
(A) LENGTH: 8 amino acids SB) TYPE: amino acid : ~C) S~RANDEDNESS: single (D) TOPOLOGY: 1inear (ii) MOLECULE TYPE: peptide (iii~ HYPOTHETICAL: NO
~iv) ANTI;-SENSE: NO
(v) FRAGMENT TYPE: N-terminal W092/207~ PCT/US~2/04016 ~ ~ 03139 (xi~ SEQUENCE DESCRIPTION: SEQ ID NO:24:
Arg Gly His Leu Val Ala Ile Gly l 5 (2) IMFORMATION FOR SEQ ID NO:25:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino ~cids (B) TYPE: amino acid (C~ STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(i~) ~NTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
Arg Gly ~5p Leu Val Ala Ile Gln l 5 :
(2) INFORMATION FOR SEQ ID NO:26:
;~ (i) SEQUENCE CHAR~CTERISTICS:
: (A~ LENGTH: 8 amino acids (B~ TYPE: amino acid (C) STRANDEDNESS: single (D~ TOPOLOGY: linear (ii) MOLECULE TYPE:~ peptide ~; ~iii) HYPOTHETICAL: NQ
(iv) ANTI-SENSE::NO
, ~v) FRAGME~T TYPE: N-terminal ::
: ::
; ~ ~ (xi) SEQUENCE~DESCRIPT~ON: SEQ ID NO:26 : Arg Thr Asp Leu Val Ala Ile Gln ~: ~ l : 5 ~ (2) INFORMATION FOR SEQ ID NO:27:
: : (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids : ~ .

W092/2070~ PCr/US92/~016 _53~ 3 1 ~ 9 (B~ TYPE: amino acid (C) STRANDED~ESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii~ HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:
Tyr Thr Asn Leu Val Ala Ile Gln (2) INFORM~TION FOR SEQ ID NO:2~:
(i) SEQUENCE CHARACTERISTICS:
~A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHÆTICAL: NO
(iv~ ANTI~SENSE: NO
~v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ I~ NO:28:
T~r Thr Asp Leu Val Ala Ile Asn l 5 ~2) INFO~MATION FOR 5EQ ID NO:29:
1 ,: , I ~ I ! :
SEQUENCE CH~RACTERISTICS:
(A)~LENGTH: 8 amino acids (B~: TYPE: amino acid (C3 STRANDEDNESS: single ~D~ TOPOLOGY: linear gii) MOLECULE ~YPE: peptide (iii) HYPOTHETICAL: NO
: (iv) ANTI-SENSE: NO

W~92/2070~ PCT/U~2/0401~
21 03i3~
~5~-(v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:
Cys Gly Asp Leu Val Ala Ile Gln ~ 5 (2) INFORM~TION FOR SEQ ID NO:30:
(i) SEQ~ENCE CHARACTERISTICS:
~A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE ~YPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal txi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
Tyr Thr Asp Leu Val Ile Gln ~Z) INFORMATION FOR SEQ ID NO:31:
(i) SEQUEN OE ~HARACTERISTICS:
(A):~ENGTH: 7 amino acids : (B) TYPE: amino acid (C) STRANDEDNESS: single : (~) TOPO~OGY: linear (ii) MOLECULE TYPE: peptide YPOTHETICAL: NO
(iv) ANTI-SEN~E:~ NO
: (V) FRAGMENT TYPE:~N terminal : (Xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
Tyr Thr Asp Val Ala Ile Gln : l 5 :

WO 92/2071D8 P~/USg2/04016 2 ~ 03139 (2) INFORMATION FOR SEQ ID NO:32:
(i) SEQU~NCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECIJLE TYPE: peptide (iii~ HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) F~AGMENT TYPE: N-terminal ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:
Tyr Thr Gln Leu Val Ala Ile Gln : 1 5 ~2) INFORMATION FOR SEQ ID NO:33:
(i) SEQUENCE C~ARACTERISTICS:
tA) LENGTH: 8 amino acids (B) TYPE: a~ino acid ~C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii~ MOLECULE TYPE: peptide :~ ~iii) HYPOTHETICAL: NO
:
(iv) AN~-SENSE: NO
: Iv) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
Glu Thr Asp Leu Val Ala Ile Gln (2) INFORMATION FOR SEQ ID NO:34:
(i) SEQUENCE CH~RACTERISTICS:
~ ~) LENGTH: 8 amino acids : (B) TYPE: amino acid (C~ STRANDEDNESS: single (D) ~OPOLOGY: linear WO 92/2~70~ PCI`/I~S92/04016 ,~ ~ !J 3 .1. 3 9 --5 6--(ii) MOLECULE TYPE: peptide i i i ) HYPOTHET I CAL: NO
( iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminai (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 34:
Tyr Glu Asp Leu Val Ala Ile Gln (2) INFORMATION FOR SEQ Il) NO: 35:
( i ) Sl :QUENCE CH~CTERI STI CS:
~A) ~ENGTH: 8 amino acids ~B3 T~PE: amino acid (C) STRANDEDNESS: single (D) TOPQLO~;Y: linear ( i i ~ MOLECULE TYPE: pep~ ide ( i i i ) HYPOTHETI CAL: NO
iv~ ANTI-SEMSE: NO
~v) FRAt;MENT TYPE: N-terminal .
(xi) SEQUENC:IS DESRIPTION: SEQ ID NO: 35:
, Tyr Thr Asp Glu Val Ala Ile Gln ( 2 ) INFORMATION FOR SEQ ID N0: 3 6:
( i) SEQUENCE C~ACTERISTICS:
(Aj LENGT~I: $~ amino acids YPE: amino acid C) STE~ANDEDNESS: ~ single ~D~ TOPOLOGY: linear : (ii) MOLECULE TYPE: peptide (iii~ HYPOTHETICAL: NO
( iv) ANTI-sENsE NO
(v) FRAGMENT TYPE: N-terminal WO 92/20708 PCr~US92/04016 ~57- 2i~13~

(xi) SEQUENCE DESCRIPTION: SEQ ID NO 36:
Tyr Gly Asp Leu Val Ala Ile ~:ln (2~ INFOR~TIC)N FOR SEQ ID MO: 37:
( i ) SEQUENCE CHARACTERI STI CS:
(A) LENGTH: 8 amino acids (B~ TYPE: amino acid (C) ST~NDEDNESS: single ( D ) TOPOLOGY: l inear (ii) MC)LECULE TYPE- peptide (iii) HYPOTHETICAL: NO
( iv) ANTI-SEMSE: NO
(~r) FRAGMENT TYPE: N-terminal ( xi ~ SEQUENCE D:E:SCRIPTION: SEQ ID NO: 3 7:
Lys Thr ~sp Leu Val Ala Ile Gln (2) INFO~TION FOR SEQ ID NO: 38:
i ) SEQUENCE CHARACTER~ STI CS:
~A) LENGTH- 8 arnino acids (B~ TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear : ~ ~ (ii) MOLECULE TYPE: peptide ( iii) HYPOTHETICAL: NO
- ~iv)~ ANTI-SENSE: NO
; I (V) FR~GMENT TYPE : N-termirlal ~xi) SEQUENCE DESCRIPTION: SEQ ID NC):3~:
, Tyr Lys ~sp Leu Val Ala Ile Gln ( 2 ~ INFORMATION FOR SEQ ID NO: 3 9:

W092/~07~8 PCT/U592/0~Q~6 ~ 58-(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single 5D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:
Tyr Thr Lys Leu Val Ala Ile Gln (2) INFORMATION FOR SEQ ID NO:40:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids (B) TYPE: amino acid (C3 STRANDEDNESS: single ~D~ TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
~iv) ~NTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal (xi~ SEQUENCE DESCRIPTION: SEQ ID NO:40:
: . Tyr Ser Asp Leu Val Ala Ile Gln (2) INFORMATION FOR SEQ ID NO:41:
(i) SEQU~NCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids 5B) TYPE:: amino acid (C) STRANDEDNESS: s ing 1 e (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii~ ~YPOT~ETICAL: NO
.

W~92/20708 PCT/U~92/04016 ~ C 3 1 ~ 9 (iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4l:
Tyr Thr ASp Leu Thr Ala Ile Gln l 5 (2) INFORMATION FOR SEQ ID NO:4Z:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: sin~le (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii~ HYPOTHETICAL: NO
(iv) ANTI-SENSE: NO
(v) FRAGMENT TYPE: N-terminal (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:
Tyr Thr Asp Val Ala Ala Ile Gln 5 ~
(2) INFORMATION FOR SEQ ID NO:43:
(i) SEQUENCE ~HA~ACTERISTICS:
(A) LENGTH: 8 amino acids ~B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MO~ECULE TYPE: peptide (iii) HYPOT~ TICAL: NO
(iv) ANTI-SENSE: NO~
(v) FRAGMENT TYPE: N-terminal :~

: , .

W092/20708 PCT/US92/04~16 2 i 0 3 1 3 9 -60-(xi~ SEQUENCE DESCRIPTION: SEQ ID NO:43:
Tyr Thr Gly Leu Val Ala Ile Gln (2~ INFO~MATION FOR SEQ ID NO:44:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGT~: 9 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) M~LECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv? ~NTI-SENSE: NO
(v) F~GMENT TYP~: N terminal ~xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:
Tyr Thr Asp Leu Val Ala Ile Gln Asn l 5 : (2) INFORMATION FOR SEQ ID NO:45:
(i3 SEQUENCE CHARACTERISTICS:
(A) LENGTH: l0 amino acids ~B) TYPE: amino acid ~C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO
(iv) ANT~-SENSE: NO
(v~ FRAGMENT TYPE: N-terminal .
xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:
Tyr Thr Asp Leu Val Ala Ile Gln Asn Lys ~ 5 l0

Claims (20)

We claim:

1. A peptide derived from selectins selected from the group consisting of the structures:
R1-X-P-Q-S-T-U-V-W-Z-Y-R2 (I) R1-P-Q-S-T-U-V-W-Z-R2 (II) or a pharmaceutically acceptable salt thereof, wherein:
X in Formula (I) and P in Formula (II) are the N-terminus amino acids, and R1 is a moiety attached to the amine function (NHR1), Y in Formula (I) and Z in Formula (II) are the C-terminus amino acids, and R2 is the moiety attached to the singly-bonded oxygen in the carboxy function (C(O)OR2), P is D- or L-tyrosine, D- or L-phenylalanine, D
or L-lysine, D- or L-glutamic acid, D or L-arginine, D- or L-cysteine, D- or L- O-R3-tyrosine, D- or L-N.alpha.-R3-tyrosine, D- or L-4-amino phenylalanine, D- or L-R4-phenylalanine, D- or L-pyridylalanine, D- or L-naphthylalanine, or D- or L-tetrahydroisoquinoline carboxylic acid, where R3 is lower alkyl or aryl and R4 is halogen (fluorine, chlorine, bromine or iodine), Q is D- or L-threonine, D- or L-lysine, D- or L-glutamic acid, D- or L-cysteine, or glycine, S is D- or L-aspartic acid, D- or L-histidine, D- or L-glutamic acid, D- or L-asparagine, D or L-glutamine, D- or L-alanine, D- or L-phenylalanine, D-or L-lysine, or glycine, T, U, V and W are independently D- or L-leucine, D- or L-isoleucine, D- or L-alanine, D- or L-valine, D- or L-alloisoleucine, glycine, D- or L-glutamic acid, D- or L-aspartic acid, D- or L-asparagine, D- or L-glutamine, D- or L-threonine, or desamino acid where desamino acid refers to the deletion of either residues T, U, V, or W from the peptide formulas I or II, Z is D- or L-glutamine, D- or L-glutamic acid and D- or L-asparagine, R1 is H (signifying a free N-terminal group), formyl, lower alkanoyl, aroyl or desamino (meaning the amino acid adjacent to the group R1, either X in formula I or P in formula 2 lacks the .alpha.-amino group of the amino acid, and is replaced with H), R2 is H (signifying in a free C-terminal carboxylic acid), O(lower alkyl), O(aryl), NR3R4 where R3 and R4 are independently H or lower alkyl, or descarboxy (meaning the .alpha.-carboxylic acid group of the amino acid to which R1 is adjacent in formula I or 2, Y or Z, respectively, is replaced with H), and X and Y are linear chains of from one to ten amino acids.

2. The peptide of claim 1 having the structure R1-X-P-Q-S-T-U-V-W-Z-Y-R2, wherein R1 is H, X is Cys-Xxx-Xxx-Xxx, P is Tyr, T is Leu, U is Val, V is Ala, W is Ile, and Y is Asn-Lys-Xxx-Glu, where Xxx is any amino acid, and R2 is not OH.

3. The peptide of claim 1 of the structure R1-X-P-Q-S T U-V-W-Z-Y-R2; wherein the peptide is selected from the group consisting of a peptide wherein X is Cys-Gln-Asn-Arg and S is Asp, Lys, His, Asn, Gln, or Ala; a peptide wherein X is Gln-Asn-Arg, and S is Asp, Lys, His, Asn, Gln, or Ala; a peptide wherein X is pGlu-Asn-Arg and S is Asp, Lys, His, Asn, Gln, or Ala; a peptide wherein X is Asn-Arg, and S is Asp, Lys, His, Asn, Gln, or Ala; a peptide wherein X
is Arg and S is Asp, Lys, His, Asn, Gln, or Ala; a peptide wherein S is Asp, Lys, His, Asn, Gln, or Ala and Y is Asn; a peptide wherein S is Asp, Lys, His, Asn, Gln, or Ala and Y is Asn-Lys; a peptide wherein S
is Asp, Lys, His, Asn, Gln, or Ala and Y is Asn-Lys-Asn; a peptide wherein S is Asp, Lys, His, Asn, Gln, or Ala and Y is Asn-Lys-Asn-Glu; a peptide wherein X
is Cys-Gln-Asp-Arg, S is Asp, Lys, His, Asn, Gln, or Ala and Y is Asn-Lys-Asn-Glu; a peptide wherein X is Glu-Asn-Arg, P is Arg or Tyr, S is Asp, Lys, His, Asn, Gln, or Ala; and a peptide wherein X is Asn-Arg, P is Tyr or Arg, Q is Gly, and S is Asp, Lys, His, Asn, Gln, or Ala.

4. The peptide of claim 1 having the structure R1-P-Q-S-T-U-V-W-Z-R2 wherein S is Asp or His.

5. The peptide of claim 1 selected from the group consisting of peptides having the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-His-Leu-Val-Ala-Ile-Gln NH2; the formula Acetyl-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2;
the formula Cys-Gln-Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Arg-Tyr-Thr-Asp-Leu-Val-Ala Ile-Gln-NN2; the formula Tyr-Thr-Glu-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-Asn-Lys-Asn-Glu-NH2; the formula D-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-D-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-D Asp-Leu-Val-Ala Ile-Gln-NH2; the formula Phe-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ala-D-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Ala-Val-Ala-Ile-Gln-NH2 the formula Tyr-Thr-Ala-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Phe-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Lys-Leu-Val-Ala-Ile-Gln-NH2; the formula Lys-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2;
the formula Cys-Arg-Gly-Asp-Leu-Val-Ala-Ile-Gln-NH2;
the formula Gln-Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-Asn-Lys-Asn-Glu-NH2; the formula Cys-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Formyl-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula 4-Br-Phe-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula 4-NH2-Phe-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Ac-Arg-Gly-Asp-Leu-Val-Ala-Ile Gln-NH2;the formula Tyr-Thr-Asp-Ala-Val-Ala-Ile-Gln-NH2; the formula Arg-Gly-His-Leu-Val-Ala-Ile-Gln-NH2; the formula Arg-Gly-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Arg-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asn-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-Asn-NH2; the formula Cys-Gly-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-D-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-D-Gln-NH2; the formula Tyr-Thr-Asp-D-Leu-Val-Ala-Ile-Gln-NH2; the formula D-Tyr-Thr-D-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula D-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Val-Ala-Ile-Gln-NH2; the formula desamino-Arg-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2;
the formula desamino-Tyr-Thr-Asp-Val-Ala-Ile-Gln-NH2;
the formula Tyr-Thr-Asp-Leu-Val-Ala-lle-descarboxy-Gln-NH2; the formula Tyr-Thr-Gln-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2;
n-Bu; the formula GIu-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Glu-Asp-Leu-Val-Ala-Ile-Gln-NH2;
the formula Tyr-Thr-Glu-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Glu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Gly-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Lys-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Lys-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Lys-Leu-Val-Ala-Ile-Gln-NH2; the formula N-Me-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Nal-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula O-Me-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Phe-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Phe-Leu-Val-Ala-Ile-Gln-NH2; the formula Pya-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Ser-Asp Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Thr-Ala-Ile-Gln-NH2; the formula Tic-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Val-Ala-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Gly-Leu-Val-Ala-Ile-Gln-NH2; the formula Ac-Tyr-D-Thr-His-Leu-Val-Ala-Ile-D-Gln-NH-n-Bu; the formula Ac-Tyr-D-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH-n-Bu; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-GLn-Asn-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-Asn-Lys-NH2; and pharmaceutically acceptable acid- or base-addition salts thereof.

6. 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.

7. A method for the preparation of peptides of the structure R1-X-P-Q-S-T-U-V-W-Z-Y-R2 (I) R1-P-Q-S-T-U-V-W-Z-R2 (II) or a pharmaceutlcally acceptable salt thereof, wherein:
X in Formula (I) and P in Formula (II) are the N-terminus amino acids, and R1 is a moiety attached to the amine function (NHR1), Y in Formula (I) and Z in Formula (II) are the C-terminus amino acids, and R2 is the moiety attached to the singly-bonded oxygen in the carboxy function (C(O)OR2), P is D- or L-tyrosine, D- or L-phenylalanine, D-or L-lysine, D- or L-glutamic acid, D- or L-arginine, D- or L-cysteine, D- or L- O-R3-tyrosine, D- or L-N.alpha.-R3-tyrosine, D- or L-4-amino phenylalanine, D- or L-R4-phenylalanine, D- or L-pyridylalanine, D- or L-naphthylalanine, or D- or L-tetrahydroisoquinoline carboxylic acid, where R3 is lower alkyl or aryl and R4 is halogen (fluorine, chlorine, bromine or iodine), Q is D or L-threonine, D- or L-lysine, D- or L-glutamic acid, D- or L-cysteine, or glycine, S is D- or L-aspartic acid, D- or L-histidine, D- or L-glutamic acid, D- or L-asparagine, D or L-glutamine, D- or L-alanine, D- or L-phenylalanine, D-or L-lysine, or glycine, T, U, V and W are independently D- or L-leucine, D- or L-isoleucine, D- or L-alanine, D- or L-valine, D- or L-alloisoleucine, glycine, D- or L-glutamic acid, D- or L-aspartic acid, D- or L-asparagine, D- or L-glutamine, D- or L-threonine, or desamino acid where desamino acid refers to the deletion of either residues T, U, V, or W from the peptide formulas I or II, Z is D or L-glutamine, D- or L-glutamic acid and D- or L-asparagine, R1 is H (signifying a free N terminal group), formyl, lower alkanoyl, aroyl or desamino (meaning the amino acid adjacent to the group R1, either X in formula I or P in formula 2 lacks the .alpha.-amino group of the amino acid, and is replaced with H), R2 is H (signifying in a free C-terminal carboxylic acid), O(lower alkyl), O(aryl), NR3R4 where R3 and R4 are independently H or lower alkyl, or descarboxy (meaning the .alpha.-carboxylic acid group of the amino acid to which R1 is adjacent in formula I or 2, Y or Z, respectively, is replaced with H), and X and Y are linear chains of from one to ten amino acids, whereby the amino acids are added either singly or in preformed blocks of amino acids to an appropriately functionalized solid support.

8. The method of claim 7, wherein the peptide has the structure R1-X-P-Q-S-T-U-V-W-Z-Y-R2, wherein R1 is H, X is Cys-Xxx-Xxx-Xxx, P is Tyr, T is Leu, U is Val, V is Ala, W is Ile, and Y is Asn-Lys-Xxx-Glu, where Xxx is any amino acid, and R2 is not OH.

9. The peptide of claim 7 of the structure R1-X-P-Q-S-T-U-V-W-Z-Y-R2; wherein the peptide is selected from the group consisting of a peptide wherein X is Cys-Gln-Asn-Arg and S is Asp, Lys, His, Asn, Gln, or Ala; a peptide wherein X is Gln-Asn-Arg, and S is Asp, Lys, His, Asn, Gln, or Ala; a peptide wherein X is pGlu-Asn-Arg and S is Asp, Lys, His, Asn, Gln, or Ala; a peptide wherein X is Asn-Arg, and S is Asp, Lys, His, Asn, Gln, or Ala; a peptide wherein X
is Arg and S is Asp, Lys, His, Asn, Gln, or Ala; a peptide wherein S is Asp, Lys, His, Asn, Gln, or Ala and Y is Asn; a peptide wherein S is Asp, Lys, His, Asn, Gln, or Ala and Y is Asn-Lys; a peptide wherein S
is Asp, Lys, His, Asn, Gln, or Ala and Y is Asn-Lys-Asn; a peptide wherein S is Asp, Lys, His, Asn, Gln, or Ala and Y is Asn-Lys-Asn-Glu; a peptide wherein X
is Cys-Gln-Asp-Arg, S is Asp, Lys, His, Asn, Gln, or Ala and Y is Asn-Lys-Asn-Glu; a peptide wherein X is Glu-Asn-Arg, P is Arg or Tyr, S is Asp, Lys, His, Asn, Gln, or Ala; and a peptide wherein X is Asn-Arg, P is Tyr or Arg, Q is Gly, and S is Asp, Lys, His, Asn, Gln, or Ala.

10. The method of claim 7 wherein the peptide has the structure R1-P-Q-S-T-U-V-W-Z-R2 wherein S is Asp or His.
:

11. The method of claim 7 wherein the peptide is selected from the group consisting of peptides having the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-His-Leu-Val-Ala-Ile-Gln-NH2;
the formula Acetyl-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2;
the formula Cys-Gln-Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln NH2; the formula Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Glu-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-Asn-Lys-Asn-Glu-NH2; the formula D-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-D-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-D-Asp-Leu-Val-Ala-Ile-Gln-Nh2; the formula Phe-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ala-D-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Ala-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Ala-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Phe-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Lys-Leu-Val-Ala-Ile-Gln-NH2; the formula Lys-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2;
the formula Cys Arg-Gly-Asp-Leu-Val-Ala-Ile-Gln-NH2;
the formula Gln-Asn-Arg Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Asn-Arg-Tyr-Thr Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Asn-Arg-Tyr-Thr Asp-Leu-Val-Ala-Ile-Gln-Asn-Lys-Asn-Glu-NH2; the formula Cys-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Formyl-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula 4-Br-Phe-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula 4-NH2-Phe-Thr-Asp Leu-Val-Ala-Ile-Gln-NH2; the formula Ac-Arg-Gly-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Ala-Val-Ala-Ile-Gln-NH2; the formula Arg-Gly-His-Leu-Val-Ala-Ile-Gln-NH2; the formula Arg-Gly Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Arg-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asn Leu-Val Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-Asn-NH2; the formula Cys-Gly-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-D-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-D-Gln-NH2; the formula Tyr-Thr-Asp-D-Leu-Val-Ala-Ile-Gln-NH2; the formula D-Tyr-Thr-D-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula D-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ile-Gln-NH2;
the formula Tyr-Thr-Asp-Val-Ala-Ile-Gln-NH2; the formula desamino-Arg-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2;
the formula desamino-Tyr-Thr-Asp-Val-Ala-Ile-Gln-NH2;
the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-descarboxy-Gln-NH2; the formula Tyr-Thr-Gln-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH-n Bu; the formula Glu-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Glu-Asp-Leu-Val-Ala-Ile-Gln-NH2;
the formula Tyr-Thr-Glu-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Glu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Gly-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Lys-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Lys-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Lys-Leu-Val-Ala-Ile-Gln-NH2; the formula N-Me-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Nal-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula O-Me-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Phe-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Phe-Leu-Val-Ala-Ile-Gln-NH2; the formula Pya-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formuIa Tyr-Ser-Asp-Leu-Val-Ala-Ile-Gln NH2; the formula Tyr-Thr-Asp-Leu-Thr-Ala-Ile-Gln-NH2; the formula Tic-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Val-Ala-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Gly-Leu-Val-Ala-Ile-Gln-NH2; the formula Ac-Tyr-D-Thr-His-Leu-Val-Ala-Ile-D-Gln-NH-n-Bu; the formula Ac-Tyr-D-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH-n-Bu; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-Asn-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-Asn-Lys-NH2; and pharmaceutically acceptable acid- or base-addition salts thereof.

12. The method for preparation of the peptide of claim 7 whereby the amino acids are assembled either singly or in preformed blocks in solution or suspension by chemical ligation techniques.

13. The method for preparation of a peptide of claim 7 whereby the amino acids are assembled either singly or in preformed blocks in solution or suspension by enzymatic ligation techniques.

14. The method for preparation of a peptide of claim 13 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.

15. A method for modifying binding of a selectin comprising providing a peptide selected from the group consisting of the structures R1-X-P-Q-S-T-U-V-W-Z-Y-R2 (I) R1-P-Q-S-T-U-V-W-Z-R2 (II) or a pharmaceutically acceptable salt thereof, wherein:
X in Formula (I) and P in Formula (II) are the N-terminus amino acids, and R1 is a moiety attached to the amine function (NHR1), Y in Formula (I) and Z in Formula (II) are the C-terminus amino acids, and R2 is the moiety attached to the singly-bonded oxygen in the carboxy function (C(O)OR2), P is D- or L-tyrosine, D- or L-phenylalanine, D-or L-lysine, D- or L-glutamic acid, D- or L-arginine, D- or L-cysteine, D- or L- O-R3-tyrosine, D- or L-N.alpha.-R3-tyrosine, D- or L-4-amino phenylalanine, D- or L-R4-phenylalanine, D- or L-pyridylalanine, D- or L-naphthylalanine, or D- or L-tetrahydroisoquinoline carboxylic acid, where R3 is lower alkyl or aryl and R4 is halogen (fluorine, chlorine, bromine or iodine), Q is D- or L-threonine, D- or L-lysine, D- or L-glutamic acid, D- or L-cysteine, or glycine, S is D- or L-aspartic acid, D- or L-histidine, D- or L-glutamic acid, D- or L-asparagine, D or L-glutamine, D- or L-alanine, D- or L-phenylalanine, D-or L-lysine, or glycine, T, U, V and W are independently D- or L-leucine, D- or L-isoleucine, D or L-alanine, D- or L-valine, D- or L-alloisoleucine, glycine, D- or L-glutamic acid, D- or L-aspartic acid, D- or L-asparagine, D- or L-glutamine, D- or L-threonine, or desamino acid where desamino acid refers to the deletion of either residues T, U, V, or W from the peptide formulas I or II, Z is D- or L-glutamine, D- or L-glutamic acid and D- or L-asparagine, R1 is H (signifying a free N-terminal group), formyl, lower alkanoyl, aroyl or desamino (meaning the amino acid adjacent to the group R1, either X in formula I or P in formula 2 lacks the .alpha.-amino group of the amino acid, and is replaced with H), R2 is H (signifying in a free C-terminal carboxylic acid), O(lower alkyl), O(aryl), NR3R4 where R3 and R4 are independently H or lower alkyl, or descarboxy (meaning the .alpha.-carboxylic acid group of the amino acid to which R1 is adjacent in formula I or 2, Y or Z, respectively, is replaced with H), and X and Y are linear chains of from one to ten amino acids, in combination with a pharmaceutically acceptable carrier.

16. The method of claim 15 wherein the peptide is of the structure R1-X-P-Q-S-T-U-V-W-Z-Y-R2, wherein R1 is H, X is Cys-Xxx-Xxx-Xxx, P is Tyr, T is Leu, U is Val, V is Ala, W is Ile, and Y is Asn-Lys-Xxx-Glu, where Xxx is any amino acid, and R2 is not OH.

17. The method of claim 15 wherein the peptide is of the structure R1-X-P-Q-S-T-U-V-W-Z-Y-R2 and is selected from the group consisting of a peptide wherein X is Cys-Gln-Asn-Arg and S is Asp, Lys, His, Asn, Gln, or Ala; a peptide wherein X is Gln-Asn-Arg, and S is Asp, Lys, His, Asn, Gln, or Ala; a peptide wherein X is pGlu-Asn-Arg and S is Asp, Lys, His, Asn, Gln, or Ala; a peptide wherein X is Asn-Arg, and S is Asp, Lys, His, Asn, Gln, or Ala; a peptide wherein X
is Arg and S is Asp, Lys, His, Asn, Gln, or Ala; a peptide wherein S is Asp, Lys, His, Asn, Gln, or Ala and Y is Asn; a peptide wherein S is Asp, Lys, His, Asn, Gln, or Ala and Y is Asn-Lys; a peptide wherein S
is Asp, Lys, His, Asn, Gln, or Ala and Y is Asn-Lys-Asn; a peptide wherein S is Asp, Lys, His, Asn, Gln, or Ala and Y is Asn-Lys-Asn-Glu; a peptide wherein X
is Cys-Gln-Asp-Arg, S is Asp, Lys, His, Asn, Gln, or Ala and Y is Asn-Lys-Asn-Glu; a peptide wherein X is Glu-Asn-Arg, P is Arg or Tyr, S is Asp, Lys, His, Asn, Gln, or Ala; and a peptide wherein X is Asn-Arg, P is Tyr or Arg, Q is Gly, and S is Asp, Lys, His, Asn, Gln, or Ala.

18. The method of claim 15 wherein the peptide is of the structure R1-P-Q-S-T-U-V-W-Z-R2 wherein S is Asp or His.

19. The method of claim 15 wherein the peptide is selected from the group consisting of peptides having the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-His-Leu-Val-Ala-Ile-Gln-NH2;
the formula Acetyl-Tyr-Thr-Asp Leu-Val-Ala-Ile-Gln-NH2; the formula Cys-Gln-Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Glu-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-Asn-Lys-Asn-Glu-NH2; the formula D-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-D-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-D-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Phe-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ala-D-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Ala-Val-Ala-Ile-Gln NH2; the formula Tyr-Thr-Ala-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Phe-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Lys-Leu-Val-Ala-Ile-Gln-NH2; the formula Lys-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Cys-Arg-Gly-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Gln-Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Asn-Arg-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Asn-Arg-Tyr-Thr-Asp-Leu-Val-AIa-Ile-Gln-Asn-Lys-Asn-Glu-NH2; the formula Cys-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Formyl-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula 4-Br-Phe-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula 4-NH2-Phe-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Ac-Arg-Gly-Asp-Leu-Val-Ala-Ile-Gln-NH2;the formula Tyr-Thr-Asp-Ala-Val-Ala-Ile-Gln-NH2; the formula Arg-Gly-His-Leu-Val-Ala-Ile-Gln-NH2; the formula Arg-Gly-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Arg-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asn-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr Thr-Asp-Leu-Val-Ala-Ile-Asn-NH2; the formula Cys-Gly-Asp-Leu-Val-Ala-Ile-Gln NH2; the formula Tyr-Thr-Asp-Leu-Val-D-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-D-Gln-NH2; the formula Tyr-Thr-Asp-D-Leu-Val-Ala-Ile-Gln-NH2; the formula D-Tyr-Thr-D-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula D-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Val-Ala-Ile Gln-NH2; the formula desamino-Arg-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula desamino-Tyr-Thr-Asp-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-descarboxy-Gln-NH2; the formula Tyr-Thr-Gln-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH-n-Bu; the formula Glu-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Glu-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Glu-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Glu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Gly-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Lys-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Lys-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Lys-Leu-Val-Ala-Ile-Gln-NH2; the formula N-Me-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Nal-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula O-Me-Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Phe-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Phe-Leu-Val-Ala-Ile-Gln-NH2; the formula Pya-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Ser-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Leu-Thr-Ala-Ile-Gln-NH2; the formula Tic-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Asp-Val-Ala-Ala-Ile-Gln-NH2; the formula Tyr-Thr-Gly-Leu-Yal-Ala-Ile-Gln-NH2; the formula Ac-Tyr-D-Thr-His-Leu-Val-Ala-Ile-D-Gln-NH-n-Bu; the formula Ac-Tyr-D-Thr-Asp-Leu-Val-Ala-Ile-Gln-NH-n-Bu; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-Asn-NH2; the formula Tyr-Thr-Asp-Leu-Val-Ala-Ile-Gln-Asn-Lys-NH2; and pharmaceutically acceptable acid- or base-addition salts thereof.

20. The method of claim 15 wherein the pharmaceutical carrier is selected from the group consisting of carriers suitable for parenteral administration, oral administration, topical administration, and controlled release formulations.