CA2193828A1

CA2193828A1 - Process to inhibit binding of the integrin alpha 4 beta 1 to vcam-1 or fibronectin

Info

Publication number: CA2193828A1
Application number: CA002193828A
Authority: CA
Inventors: Timothy P. Kogan; Kaijun Ren; Peter Vanderslice; Pamela J. Beck
Original assignee: Individual
Current assignee: Encysive Pharmaceuticals Inc
Priority date: 1994-06-29
Filing date: 1995-06-27
Publication date: 1996-01-11
Also published as: WO1996000581A1; EP0767674A4; AU2958195A; US6713604B1; JPH10502349A; US6087330A; EP0767674A1

Abstract

The present invention is directed to an isolated and purified cyclic peptide of from 5 to about 13 residues modeled after a portion of the CS1 peptide. A peptide of this invention preferably has the amino acid residue sequence of SEQ ID NO:2-14 or 16-42. The present invention is further directed to a process of selectively inhibiting the binding of .alpha.4.beta.1 integrin to a protein such as VCAM-1, fibronectin or invasin comprising exposing a cell that expresses .alpha.4.beta.1 integrin to that protein in the presence of an effective inhibiting amount of such a peptide. The present invention is still further directed to a pharmaceutical composition comprising a physilogically acceptable carrier and a cyclic peptide of the invention.

Description

WO 961005~1r~
~ ~; ,J I 21 93~2~

PROCESS TO INHIBIT BINDING OF THF INTEGRIN

5 Field of the Invention This invention relates generally to a process of inhibiting the binding of ~1 integrin to proteins such as VCAM-1 or Gl,lu .~i-.. The invention also relates to synthetic cyclic peptides that inhibit that binding.

10 Ba.,k~:,uu.,d of the Invention Vascular cell adhesion molecule-1 ~VCAM-1) is a protein that is found on the surface of l~n~l~,th~ 7,1 cells that line the interior wall of capillaries. VCAM-I recognizes and binds to the integrin ~1 (or VLA-4 for very late antigen-4), a h~L~IUd;I~ C protein present on the surface of 15 certain white blwd cells. Binding of a~ to VCAM-l allows white blood cells to adhere to the capillary wall in areas where the tissue i~UllUU~ldi..l, the capillary has been imfected or damaged.

When a tissue has been invaded by a Illiclwl~;~li -lll or has been 20 damaged, white blwd cells, also called leukocytes, play a major role in the ",n,.,.~ ...y response. One of the most important aspects of the infl y response involves the cell adhesion event. Generally, white blwd cells are found circulating through the bloodstream. However, when a tissue is infected or becomes damaged, the white blood cells recognize the 25 invaded or damaged tissue, bind to the wall of the capillary near the affected tissue and diffuse through the capillary into the affected tissue. VCAM-l helps certain types of white blood cells recogrize the affected sites, bind to the capillary wall, and migrate into the affected tissue.

There are three main types of white blood cells: ~;ldllulo~yt~, monocytes and l~".l,h~.~t~. VCAM-1 binds to c~ l expressed on the surface of monocytes, Iy,u~llouyl~s and two subclasses of ~ ulocyt~
~,c;...,~.l,ilc and basophils.

WO 96100S8 1 1 ~_ I /11.. ,.~ .

2- ,?, ;~3~2~

Monocytes, after leaving the blo~l~., th~ugh the wall of a capillary, mature into ~ v~ ,~ that, ' g ~tu~ and digest invading Illi~,lVUl~ l foreign bodies and senescent cells. L~ l,v~t~,~ produce antibodies and 1~11 infected cells. r~ - and basophils secrete 5 mediators of various '' y reactions.

Following infection or damage of tissue - .v .~ a capillary, the endotheLial cells that line the capillary express a series of adhesion molecules, including VCAM-1, that are critical for binding white blood cells that are necessary for fighting infection. Pnor to binding to VCAM-1, the white biood cells initially bind to another set of adhesion molecules to slow their flow and allow the cells to "roll" along the activated;
Monocytes, Iy , ' yt~, basophils and ~ are then able to fLrmly bind to VCAM-I on the endothelial cells via the ad~, integrin. There is evidence that this interaction is also involved in L ~ ;on of these white blood cells into the damaged tissue.

Although white blood cell migration to the site of mJury helps fight infection and destroy foreign material, in many instances this migration can become, u~ , with white blood cells flooding to the scene, causing wid~L~riad tissue damage. (' . ~ c~pable of blocking this process, therefore, would be beneficial as therapeutic agents. Thus, it would be useful to develop inhibitors that would prevent the binding of white blood cells to VCAM-I.
For example, some of the diseases that might be treated by the inhibition of a4~, binding include, but are not limited to, dt~l~v~,l~v~is, rh~nm~ni~l arthritis, asthma, allergy, multiple sclerosis and type I diabetes.
In addition to being found on some white blood cells, CY4~! is found on 3U various camcer cells, including leukemia, melanoma, Iymphoma and sarcoma cells. It has been suggested that cell adhesion involving a~l may be wo 96/00581 P~

3 2 ~ 93828 involved in the metastasis of certain cancers. Inhibitors of ~, binding may, therefore, also be useful in the treatmen~ of some forms of cancer.

Brief Summary of the Invtonrinn In one aspeGt, the present mvention provides an isolated and purified cyclic peptide of from 4 to about 13 amino acid residues having (a) an N-te~inal amine group, acetyl group (Acj or a ~Jol~,Ll.yl. a~ , vl moiety of from about 400 to about 12,000 Daltons average molecular weight ~IPEGXcoo) linked through an amide bond to the N-terminal residue; and (b) a C-terminal carbo~ylic acid group or amide group; the peptide the amino acid residue sequence Xaal-Xaa2-Asp-Xaa3 (SEQ ID NO:15), where Xaa, is any L- or D-a-amino acid residue and Xaa2 and Xaa3 are any h~ lU~JIlV~ ;, L-~-amino acid residue with the proviso that when Xaa, is Lys or Arg, Xaa2 cannot be Gly or Cys. In a preferred ' " t, Xaat is Phe, Trp or lle; Xaa2 is Leu, lle, Val, Lys or Met and Xaa3 is Val, Tyr, Leu, Trp, or Phe. More preferably Xaa, is Trp and Xaa2 is Leu and Xaa3 is Val.

In one ~ '-. I;. . a a cyclic peptide of the present invention is cyclized via formation of a lactam. Such a peptide comprises the amino acid residue sequence Xaa4-Xaa~-Xaa2-Asp-Xaa3 (SEQ ID NO:l), where Xaa, is any L- or D-~-amino acid residue, Xaa2 is any h~Jlu~hub;c, L-a-amino acid residue with the proviso that when Xaai is Lys or Arg, Xaa2 cannot be Gly or Cys; Xaa3 is any hydluuhub;c~ L-~Y-amino aicd residue and Xaa4 is any D-or L-cY-amino acid. Preferably, Xaa, is Phe or Trp, Xaa2 is Leu, Xaa3 is Val and Xaa4 is Glu. More preferably, such a peptide has the amino acid residue sequence of SEQ ID NO:2-8.

In another ~ o ~ a cyclic peptiàe of this invention further comprises a cysteine or modified cysteine residue and a -CH2CO- group at the N-terminal position. In accul~ with such an ~ udh..~.~L, Xaal is WO 96101~58 1 ~ . I I Ij.. .'.
3 ~ 2 ~

preferably Trp and Xaa~ is preferably Leu. Exemplary and preferred such peptides have the amino acid residue sequence of SEQ ID 'NO:9-12.

In another: ' ' t, a cyclic peptide of this invention further S comprises two or more cysteine or modified cysteine residues and is cyclized via a disulfide bond. Preferably, one of the cysteine or modified cysteine residues is located at the N- or C-terminal position. In one ~ . ~.o.l;.". ..~ acyclic peptide having two cysteine or modified cysteine residues comprises the amino acid residue sequence Xaal-Xaa2-Asp-Xaa3 (SEQ IO NO:15), 10 where Xaa, and Xaa~ and Xaa3 are as defined above. Preferably, Xaal is Tyr, Phe, Trp, dTrp or ne; Xaa2 is Leu, lle, Val, Lys, Met or Asp; and Xaa3 is Val, Tyr, Leu, Trp, or Phe.

In yet another ~ ~ " t, a cyclic peptide having a cysteine or modified cysteine residue at both the N- and C-terminal positions comprises the amino acid residue sequence of SEQ ID NO:1. Preferably, Xaa, is Trp or Phe, Xaa2 is Leu, Xaa3 is Val or Tyr and Xaa~ is Ser or Glu.

Exemplary and preferred cyclic, disulfide peptides of the present 20 invention have the amino acid residue sequence of any of SEQ ID NO:13 or 15~3.

In another aspect, the present invention provides a 1~
a Llh.~ ' 'ly acceptable diluent and a cyclic~5 peptide of the present invention. Preferred cyclic peptides in such a ;.... are the same as set forth above.

In yet another aspect~ the present invention provides a process of selectively inhibiting the binding of or4~1 integrin to VCAM-I. That process cornprises exposing a cell that expresses cr~ integrin and a cell that expresses VCAM-l to an ef~ective inh;biting amount of a cycl;c peptide of ~Y0 ~6/00581 I ~ u,.~
~, r ~ L_ 1 9 3 8 2 ~3 the present invention. ln a preferred ~. ' " of that process, the VCAM-1 is on the surface of a vascular en~ t~ l cell. In anotner preferred ~..,1,, ' t, tne a~, integrin is on ~he surface of a wnite blood cell such as a monocyte, a 1~ , ' J h" a ~ u~ulu~,~ t~ (an eosinophil or a 5 basophil), a stem cell or other cell that naturaUy expresses a4~l. Preferred peptides used in that process are the same as set fortn above.

Where the cells are located in a living organism, a peptide is preferably - ' ~I to the organism in an effective inhibiting amount in 10 a pl,~ ";. -l ", ~ ;",~ of dlis invention.

In another aspect, the present invention provides a process of selectively inhibiting the binding of a4~, integrin to a protein c~ c exposing the integrin to the protein in the presence of an effective inhibiting 15 arnount of a cyclic peptide of the present invention. Preferably, the ~, integrin is expressed on the surface of a cell such as a white blood cell or stem cell and the protein is part of the PYt~Pll ' matrix such as 20 ~Pr~ /i Description of the Invention I. The Invention The present invention provides a process of inhibiting the binding of C~4~1 integrin to proteins such as VCAM-I, ~' and invasin. The invention also provides cyclic peptides that inhibit that binding.
The adhesion of leukocytes to the vascular ~ n~ h 1;.. - and their subsequent c~L~ ~Liull into tissues are critical steps in the ;..n ~
response. Vascular cell adhesion molecule-1 CVCAM-l~, a member of the n~ . superfarnily, is e~.pressed by endothelial cells and a 30 restricted number of other cell types. VCAM-I can be induced by cytokines such as tumor necrosis factor-~,. interleukin-4, and interleukin-l,B and is wo 96100S81 1 ~.,~

6- 21 93~28 therefore h~ ;~ to contribute to leukocyte uA~d~G~;un im ;..n~.. ..y conditions such as ' ' arthritis, asthma, and ~ ,.u~l~u~i,.

One molecular form of VCAM-l contains seven; -- -----,E,I~I, ,I;
5 modules in its: " ' domain VCAM-I is recognized by the integrin receptor a4~l. a~l is expressed p}incipally by leukocytes ~ and B
1~ , ' yt~, monocytes, basophils, and ~,~ ), and is also functional on rnast oells, derivatives of the embryonic neural crest, and in developing muscle.
a4,~ also recognizes the ~ n~. ..11..1~ matrix ~51rw~uL~ill fibronectim Three distinct a~j51-binding sites have been identified wi~in fibronectin and all have been reproduoed in synthetic form. One site (1l ~ ' by the peptide H1) is found in the HepII region and is therefore expressed in all fibronectin isoforms; two others (~ ,t ~I by peptides CSI and CS5) are present in the ' ~"ly spliced type m connecting segment. Of these three the CSl peptide has the higher affinity for a~ll and contains the tripeptide Leu-Asp-Val (LDV) as its minimal active site. Hl contains a related motif, lIe-Asp Ala aDA), while CS5 i..~olL a variant of the prototypic RGD motif, Arg-Glu-Asp-Val.

II. PeDtides In one aspect, the present invention provides cyclic peptides that inhibit binding of the a4~, integrin to VCAM-I. A peptide of the present invention is modeled after the Leu-Asp Val a,DV) domain of the CSl peptide sequence, which domain is preseDted in such a way by the cyclic peptide to produce a potent a~l binding inhibitor.

Peptides are disclosed herein as amino acid residue sequences. Those sequences are written left to right in the direction from the amino (N~ to the carboxyl (C) terminus. Amino acid residue sequences are ~' ' by WO 96100581 1 ~
t ~ 9 3 ~ 2 8 either a single letter or a three letter code. The meanings of those codes as wcll as various ot.'er al~b~ used herein are in ~ with the of the IUPAC-IUB Joint rotTlmic~ n on r ~".. 1 m . r, and are shown below.
A Ala L-alanine Ac acetyl Aic 2 ~ ' 2-carboxylic acid Acm ,.. . 1 .. .1~ h~
C Cys L-cysteine dC dCys D-cysteine C(SO3H) L-cysteic acid tBu tert-butyl D Asp L-aspartic acid dD dAsp D-aspartic acid E Glu L-glutamic acid dE dGlu D-glutarnic acid < E L-~ ,' - acid F Phe L-~ y'' G Gly glycine H His L-histidine lle L-isoleucine L Leu L-leucine K I,ys L-lysine M Met L-r~
N Asn L-asparagine P Pro L-proline WO 9G/00581 r~

3 ~3 2 g dP dPro D-proline dPen D-prni~ili~ ~ini.
Prnc 2,2,5,7,8-I ' '~'' 6-sulphonyl Q Gln L-glutamrne R Arg L-arginine S Ser L-senne T Thr L-threa~ine Trt trityl V Vrl L-valine W Trp L-tryptoplw dW dTrp D-tryptophan Y Tyr L-tyrosine Boc Je~-L ~ UIIJI
DCM methylene chloride Dic N,N'-diisopropyl DIPEA dii~y~v,uJLIhy' EDT 1~2 -~-' ~ 1;1l.;. 1 Fmoc 9-lluu.~ LilvAy carbonyl HOBT l-hydroxy-lH-..,1,;~ lr E~TU O 1, -~
N,N,N',N'-tetra-~ . . .
DMF N.N-dimethyl formamide WO 96/00581 r~
't;'' . '; _9_2 ~ 9382~

MCPBA m-. l.lu.u~lu~y-benzoic ac;d NMM N-~ LI~
T~A i ~~ ~Lic acid M~ and changes can be rnade in the structure of a peptide of the present invention and still obLain a molecule that inhibits the binding of a4~, integrin to VCAM-I. For exarnple, certain amino acids can be 5 substituted for other arnino acids in a sequence without ~,uy~;~le loss of activity; likewise, D- or L- amino acid residues can be used. D-amino acids are indicated herein as d-Xaa, where Xaa is the Liu~ 1~ L~I amino acid code (e.g., dTrp). In fact, certain amino acids can be substituted or added which greatly enhance binding inhibitiom Because it is the interactive capacity and lO nature of a peptide that defines that peptide's biological functional activity, certain amino acid sequence ' can be made in a peptide sequence and ..~.Li.l~ obtain a peptide with like properties, ~Li~ ly inhibition of the binding of a~'~ integrin to VCAM-I. F , ' y such peptides are set forth below.
A peptide r . ' ' by the present invention is cyclic. A cyclic peptide has a ring structure formed between certain amino acid residues of the C~ r~ C. lincar peptide and can be envisioned as a linear peptide that is cyclized by covalent bonding of amino acid residues as described herein.

A Cyclic peptide of the present ;nvention contains from 4 to about 13 amino acid residues. The N-terrninal amino acid residue has a free terminal amine group (NH~), acetyl group (Ac) or a pol~;hyl~ ul moiety of from about 400 to about 12,000 Daltons average molecular we;ght (~IPEGXooo) linked through an ann;de bond to the N-terminal res;due. The C~terminal amino acid residue has a terminal carboxylic acid group (OH) or WO 96~uO5 8 1 . ~, I I IJ ~. .

' ' ' ' - 10 -arnide group. A cyclic peptide comprises the arnino acid residue sequence Xaal-Xaa2-Asp-Xaa3 (SEQ ID NO:15), where Xaal is any L- or D-a-amino acid residuc and Xaa2 and Xaa3 are any hydlu~llvb;c~ L-a-amino acid residue with the proviso that when Xaa, is Lys or Arg, Xaa2 cannot be Gly or Cys.
5 In a preferred, ' " t, Xaa, is Phe, Trp or lle; Xaa2 is Leu, Ile, Val, Lys or Met; and Xaa3 is Val, Tyr, Leu, Trp or Phe. More preferably, Xaa is Trp, Xaa2 is Leu and Xaa3 is Val.

A peptide in accordance with the sequence set forth above can be e~ttended in the N-terrninal direction by the addition of from l to 5 L- or D-a-amino acids and, in the C-terminal direction by the addition of from l to 5 L- or D-cY-amino acids.

A peptide can be cyciized without or with a sulfur containing bridge.
15 Where a cyclic peptide does not comprise a sulfur containin~ bridge, the N-and C-terminal amino acid residues are joined together with an amide bond (formally a lactam in the case of cyclization). Where a cyclic peptide compri~s a sulfur containing bridge, either one or two amino acid residues of the w~ linear peptide is a Cys or modified Cys residue (dCys 20 or dPen). Such a cyclic peptide can comprise a cyclic sulfide, sulfoxide or sulfone (one Cys residue in the ~ c, linear peptide3 or a cyclic disulfide (two Cys residues in the ~ r g linear peptide).

Where cyclization has occurred by forrnation of a lactam by 25 . ' of the N and C terminus, that peptide comprises the amino acid residue sequence:

Xaa4~Xaa,-Xaa2-Asp-Xaa3 (SEQ ID NO:I), where Xaa, is any L- or D-a-amino acid residue; Xaa2 and Xaa3 are j"~ ly any h~dlu~Jl.obic, L-a-amino acid residue with tne proviso that WO 96/00581 I~

t ~ '~1 020')~
- 11 - L l ~ J ~

when Xaal is Arg or Lys, Xaa2 cannot be Gly or Cys; and XaaJ is any D- or L-a-amino acid. In a preferred ~ Xaa~ is Phe or Trp, Xaa2 is Leu, Xaa3 is Val and Xaa~ is Glu or Ser. Exemplary and preferred peptides in acc~ ' with SEQ Il:) N0: I have the sequences Glu-Trp-Leu-Asp-Val-5 Pro (SEQ ID N0:2), Glu-Trp-Leu-Asp-Val-Asp (SEQ ID N0:3), Glu-Trp~
Leu-Asp-Val (SEQ ID N0:4), Glu-Trp-Leu-Asp-Asp (SEQ ID N0:5), Glu-Tr~Leu-As~Val-Pro-Glu-Trp-Leu-Asp-Val (SEQ ID N0:6), Gly-Prt~Glu-Phe-Leu-Asp-Val (SEQ ID N0:7) and Glu-Phe-Leu-Asp-Val ~SEQ ID
N0:8).
Where a peptide of the present invention contains a sulfide, sulfoxide or sulfone bridge, that peptide complises a cysteine or modified cysteine residue at one position and a -CH2C0- group at the N-terminal position. As used herein, the term "modified cysteine~ refers to D-cysteine (dCys) or D-15 pt~nitil' ~ (dPen). The sulfur atom of the cysteine or modified cysteineresidue is at~ched to the CH2 group forming tlhe cyclic peptide.

Such a peptide comprises the amino acid residue sequence Xaal-Xaa2-Asp~Xaa3 (SEQ ID N0:15), whe,n: Xaal, Xaa2 and Xaa3 are as defined 20 above. Preferably, Xaal is Trp, Xaa2 is Leu and Xaa3 is Val or Cys.
Exemplary and preferred peptides have the sequences CH2C0-Ser-Tr~Leu-Asp-Val-Cys-acid (SEQ ID N0:9~1, CH2CO~lu-Tr~Leu-Asp-Val-Cys-acid (SEQ ID N0:10), CH2CO-Glu-Trp-Leu-Asp-Cys-acid (SEQ ID NO:ll), and OEI2C0-Trp-Leu-Asp-Val-Cys-COOH (SEQ ID N0:12).
Where a peptide of the present invention contains a disulfide bridge, that peptide contains two cysteine or modified cystcine residues. Preferably, one of the cysteine or modified cysteine residues is located at the N- or C-terminal position and at least one of those amino acid residues is Cys. Such 30 a disulfde peptide comprises the amino acid residue sequence Xaal-Xaa2-Asp-Xaa3 (SEQ ID N0: 15), where Xaal, Xaa2 and Xaa3 are as defined WO96100S81 r~.,u., 5.

'~ ' ' ~ i) ~ " - 12 - ~ 1 9 3 8 2 ~

above. Preferably, Xaa, is Trp or Cys, Xaa2 is Leu and Xaa3 is Val or Cys.
Exemplary such peptides have the sequence Cys-Leu-Asp-Val-Cys (SEQ ID
NO:13) or Cys-Trp-Leu-Asp-Cys-acid (SEQ ID NO:14~.

S In another preferred _ ' " of SEQ ID NO:lS,Xaalis Tyr, Phe, Trp, dTrp or ILe; Xaa2 is Leu, lle, Val, Lys, Met or Asp; and Xaa3 is Val, Tyr, Leu, Trp, or Phe. More preferably, a disulfide cyclic peptide has the sequence Cys-Ser-Tr~Leu-Asp-Val-Cys (SEQ rD NO:16), Cys-dTrp-Leu-Asp-Val-Cys-acid (SEQ ID NO:17), Ac-Cys-Trp-Leu-Asp-Val-Cys-~;d (SEQ ID NO:18), Cys-Tyr-Leu-Asp-Val-Cys-acid (SEQ ID NO:19), Cys-Trp-Leu-Asp-Phe-Cys-acid ~SEQ IDNO:20), Cys-Phe-Leu-Asp-Val-Cys-acid (SEQ ID NO:21), Cys-Tr~Leu-As~Tr~Cys-acid (SEQ rD NO:22~,Cys-Tr~lle-Asp-Val-Cys-acid (SEQ IDNO:23), Cys-Trp-Met-As~VaL-Cys-acid (SEQ ID NO:24), Cys-Trp-Val-As~V~-Cys-acid (SEQ rD NO:25),Cys-Tr~Lys-Asp-Val-Cys-acid (SEQ rD NO:26), Cys-Tr~Leu-Glu-Val-Cys-acid (SEQ ~ NO:2~, Cys-Tr~Leu-As~Leu-Cys-acid (SEQ ID NO:28),Cys-Trp-Leu-Asp-Tyr-Cys-acid (SEQ ID NO:29), Cys-lLe-Leu-As~Val-Cys-acid (SEQ ID NO:30), Cys-Tr~Leu-Asp-Val-Cys-acid (SEQ ID NO:31), and dCys-Tr~Leu-Asp-VaL-Cys-acid (SEQ ID NO:32).
In another preferred i ' " t, a disulfide cyclic peptide of the present invention comprises the amino acid residue sequence of SEQ ID
NO:l, above. Preferably, Xaa, is Trp or Phe, Xaa2 is Leu, Xaa3 is Val or Tyr and Xaa~ is Ser or Glu. Exemplary and preferrcd such peptides have the sequence of Cys-Glu-Trp-Leu-Asp-Val-Cys-amide ~SEQ rDNO:33), Cys-Glu-Trp-Leu-Asp-VaL-Cys-acid (SEQ ID NO:34), dCys-Glu-Trp Leu-Asp-VaL-Cys-acid (SEQ ID NO:35),Cys-Glu-Trp-Leu-Asp-Tyr-Cys-acid (SEQ ID NO:36), Cys-Ser-Phe-Leu-Asp-Tyr-Cys-acid (SEQ rD NO:37), Cys-Glu-Phe-Leu-Asp-Tyr-Cys-acid (SEQ ID NO:38), dCys-Ser-Trp-Leu-Asp-VaL-dCys-acid (SEQ ID NO:39); Cys-Pro-Glu-Trp-Leu-Asp-Val-Cys-WO 96/00581 1 _11~)1.. .'~

t . . - 13 - ~ l q 3 ~ 2 ~

acid (SEQ ID NO:40), ~PEG500~-Cys-Trp-Leu-Asp-Val-Cys-acid (SEQ ID
NO:41) and dPen-Trp-Leu-Asp-Val-Cys-acid (SEQ ID NO:42).

In another ~ - a cyclic disulfide peptide of the present 5 invention comprises the sequence:

Xaa~-Cys-Tr~Leu-Asp-Val-Cys-acid (SEQ rD NO:43), where Xa~ is any D- or L-~Y-amino acid and n is an integer from 1 to 7.
A peptide of the present invention can be made using standard peptide synthetic procedures well known in the art. Typically, peptides were made with Fmoc-amino acids. Howe~er, peptides can also be made using Boc protecting groups by methods well known to those skilled in the art. Side 15 chain protecting groups of trifunction amino acids used in the synthetic procedure include Arginine (Pmc), Aspartic acid (tBu), Cysteine (Trt), Glutamic acid (tBu), Histidine (13Oc), Lysine (Boc), Serine (tBu), Threonine (tBu), and Tyrosine (tBu). Other protecting groups are specifically described.
The 1~ of the peptides in this invention by solid phase y is well known to those skilled in the art, and can be described as follows. Peptides were s~ ~ ' on an insoluble carrier such as p-b~.L~Iul.~u~yl alcohol resin for the synthesis of C-terminal carboxylic acid 25 peptides ~Wang resin, where normally the resin can be purchased with the first amino acid bound), and 4-~2',4'~1h~ u~yl ' jl F --~.~.~,...~I.~l)-phenoxy resin for C-terrninal arnide peptides (Rink resin). The peptides were prepared by solid phase synthesis using either HBTU or DIC chemistry procedures on a Protein Te, ~ Inc. Symphony peptide synthesizer.

~VO 9611~0581 P~ IIIJ.J _.

14 ~ 3 ~ 2 8 The C-terminal amude peptides were prepared by coupling the C-terminal amino acid of the sequence to the Rink resin using the sarne general method as the other couplings. The C-terrninal carooxylic acid peptides were prepared by purchasing Wang resin to which the C-terminal amino acid S was bound to the resin as a carboxylic ester. Tbe cl-amino protecting group was removed by piperidine treatment, and the next Fmoc-amino acid coupled to the resin by ~ treatment of the resin with the Fmoc-arnino acid, a coupling reagent such as DIC or BTU, and if necessary HOBT.
Such ~h~ v~ and couplings were repeated to afford each desired 10 peptide. In all cases the Fmoc protecting group was removed by treatment with a 20% solution of piperidine in DMF. However, it is understood by those skilled in the art that the exact percentage of piperidine is not criticaland should not be considered limiting in this invention. It is also understood by those sl~lled in the art that piperidine can be replaced by o~her bases, r~ n the coupling reagents and protocols used can be substituted with any of those known in the field of peptidc synthesis (including the use of Boc chemistry based soLid phase synthesis and also solution phase peptide synthesis), and those reagents specifically used in the examples provided should not be considered limiting for this invention. All unnatural arnino 20 acids, D-amino acids and other ~ A ' were coupled by manual addition of the reagent, following the same procedure as for automated operation.

Where cyclic sulfides, sulfoxides, sulfones or disulfides with C-termin~
carboxylic acids are desired, the peptides can be s~ h~;~;i on an insoluble 25 carrier such as p-b~lu~yb~l alcohol resin (Wang resin), whereas the equivalent C-terminaL amides were p~pared on 4-(2'~4'-~ ,lfiu~.yl~h~ yl-Fmoc-.. h.u,l.~Ll.yl)-phenoxy resin (Rink resin). Where two cysteines are present, rnild acid removal of the trityl protecting groups and oxidative cyclization on the resin using DMSO or NIS forrns the disulfide bond, and 3û this compound was cleaved from the resin in the normal way. Alternatively disulfides can be prepared by solution phase cyclization of the linear WO 96/005~1 r~ l/U..
2 1 9382~

sequence in guanidine hydlu~ hluliJc. Where cyc,ic sulfides (and their oxidatiûn products) are desired the N-terminus can be acylated witih b~ulllu~ tfc acid, the cysteir,e trityl group removed and cyclization achieved by NMM in DMF treatmer,t. The head-tail lactams were ~ylfthwi~f on a chlûrotrityl resin which forms a carboxylic ester linkage bet veen the C-termina, amino acid and the resin. The ,ir,ear peptide was cleaved from the resin with acet,c acid ir, DCM and cyc"7ed in solution to form the lactam.

Peptides were cleaved from the resir, witih a TFA cocktail after ti,e remova, of the N-terminal Fmoc protecting group. The exact ~ u ~ . of the TFA cocktail was varied depending on the side chain protectir,g groups present, and is we,'"known to those s,l~lled in the art. The rar,ge of TFA
was from 85 to 95%, and the remainder comprised of a mixture of scaver,gers selected from a ~ --. of anisole, rhin~-licnl~ cresol, thiocresol, phenol, i ~ . l, FDT, i ~;SifGtl~ and water. The time of the cleavage reaction required is ~quence dependant, normar,y being from 1 to 3 hours. After cleavage, the resin was removed by filtration and cold ether added to the solution to give a I The precipitate was coliected and washed a few times with ether to remove residua', TFA and scavengers. The precipitate was .~d,.,,ul~cd in aqueous solution for Iyu~ to give the crude ploduct.

~"irl~lion was carried out by revers~phase lIPLC on a C~ f~i~c column (300A, 21.4 mm x 25 cm, 5 am spherical packing) at a flow rate of lO m,/min. The selection of any other suitable packing known to one skilled in the art is equaliy acceptable. Products were detected by W absorption at 214 nm. Two mobile phases were used in the HPLC system, solution A and B using a gradient elution. Solution A was comprised of Sa~o ~r~rnnitrjlP in deionized water containing 0.15% TFA, while solution B was comprised of 5% deionized water in pret(!nitr~ containing O.la~a of TFA. A gradient of increasing percentage of solution B was used to elute the peptide from the W0 9610/}581 r~ O~

16 - 2 ~ 9 3 ~ 2 8 solid support, however the gradient used is sequence dependant. Other methods of ~..,;1;. -l;.. . known to one skilled in the art are equaUiy acceptable. The purity of the peptide was checked by C13 analytical HPLC
(300 A, 4.6 mm x 25 cm, 5 ~m spherical packing) at a flow rate of 1 S m~lmin.

A detailed description of the synthesis of e~emplary peptides is set forth hereinafter in the Examples.

~ C~ n~
In another aspect, the present invention provides a pl,,... .
;... comprising a peptide of the present invention and a l/h,~iùlul;i~lly tolerable diluent.

The present invention includes one or more peptides as described above formulated into ~ 11 together with one or more non-toxic ,uh,~,,;ùlu~i~lly tolerable or acceptable diluents, carriers, adjuvants or vehicles that are collectively referred to herein as diluents, for parenteral injection, for intranasal delivery, for oral --l .;: u~l... in solid or liquid 20 form, for rectal or topical ' or the like.

The, . can be - ' ' to humans and animals either orally, rectally, parenterally (i .,~u~.ly~ ' 'y or ), 'Iy, ~ i~u~aJily, ;..n~l~ ;s... -lly, locally (powders, ointments or drops), or as a buccal or nasal spray or aerosol.

The ~ can also be ddiveredi through a catheter for local delivery at a target site, via an Ul~iciWlUIl~y stent (a tubular device composed of a Fme wire mesh), or via a b: )~Ir~" W.l- polymer. The cu,. yu,;l;.~c may also be complexed to ligands, such as an~ibodies, for targeted delivery of the . u.. ~ c W096/O~Ar~r8] r~"~,,.,~".
.

17 2 ~ 9 3 ~ 2 ~3 The . l".,~ c are preferably ~J-lu~ a,.r d by catheter, i.v. or L injection, or intranasally via a spray or aerosol.

Co.. ~ suitable for parente~al injection may comprise S ~h~ra;ùlu~ ,ally acceptable sterile aqueous or nl.,A-q~ r ~ solutions, rliqY~r~;nnc, r ' or emulsions and sterile povvders for ~ ;n ~
into sterile injectable solutions or ~ Examples of suitable aqueous a;nd n~r-- ~ J C camers, diluents, solvents or vehicles include water, ethanol, polyols ~-u~jL.~;ly~ul, PUIJ~hYI~negIY~I~ glyc,erol, and the 10 like), suitable mixtures thereof, vegetable oils (such as olive oil) and injectable organic esters such as ethyl oleate.

lAhese ~ , can also contain adjuvants such as preserving, wetting, ' '~,k.~, and dispensing agents. Prevention of the action of ~ .. u.~;a ~ ,c can be ensured by various ~ ;AI and antifungal agents, for example, parabens, ~,LI~lub.~ùl, phenol, sorbic acid, and the like. It may also be desirable to include isotoniic agents, for example sugars, sodium chloride amd tl-ie like. Prolonged absorption of the injectable I form can be brought about by the use of agents delaying 20 ahsnTtinn for example, alumimlm and gelatin.

Besides such inert diluents, the . can also include adjuvants, such as wetting agents, . ' ~ g and ~ agents.

S,~ n.~c, in addition to the active e4.. ~ "''''1', can contain suspending agents, as for example, .liluf.y' ' isostearyl alcohols, polyuA~y~lilrl~..., sorbitol and sorbitan esters, l~ u~lra~ c cellulose, aluminum ul~ JluA~iri~, bentonite, agar-agar and tragacanth, or mixtures of these cnhctAnA"c and the like.

WO 96/005~ r~

2 1 9 3 ~ 2 Dosage forms for topical: ' ~ of a compound of this invention include ointments, powders, sprays and inhalants. The active component is admixed under sterile conditions with a ~h~r ' ~' lly acceptable carrier and any ~ dti~, buffers or ~rr~JelLu~ts as may be required.
S Ophthalmic r, ~ , eye ointments, powders and solutions are also ~ I' ' as being within the scope of this invention.

III. Process of Inhibiting the Binding of c~l In another aspect, the pre~nt invention ~ , ' a process of ~lectively inhibiting the binding of a,~l integrin to VCAM-I. A process of the pre~nt invention can be used h~ or in vivo in a living organism. In ~ ~ o~ with a process of the present invention, a cell expressing a4~, integrin is e~~d to a cell expressing VCAM-1 in the presence of an effective inhibiting amount of a peptide of the present invention. Means for ~' , an effective inhibiting amount are well known in the art.

A cell expressing ~l integrin can be a naturally occurring white blood ceU, mast cell or other cell type that naturally expresses ~r~l on the cell surfaoe, or a cell transfected with an expression vector that contains a - 20 ~ul, ' ' (e.g., genomic DNA or cDNA) that encodes a~ integrin.
In an especially preferred e L t, a~, integrin is present on the surfaoe of a white blood cell such as a monocyte, a 1~ , ' ~ or a y,~allUI~ r t~. (e.g., an eosinophil or a basophil).

A cell that expres~s VCAM-l can be a naturally occurring cell (e.g. an endothelial cell) or a oell transfected with an expression vector containing a L~l~ ' ' that encodes VCAM-l. Methods for prcducing transfected cells that express VCAM-I are well known in the art.

WO~36/00581 r~-,u~
.'- ' f'.~ ,'' 2 t ~ 3 ~ 2 8 - 1'3-Where VCA~L-1 exists on the surface of oeell, the expression of that VCAM-l is preferably induced by ;,.n~ cytokines such as tumor necrosis factor-a, ' ' 4 and ' ' 1,B.

S Where the cells expressing af,~l integrin and VCAM-l are in a iving organism, a peptide is ' ~ ' in an effective amnount to the living organism. Preferably, the peptide is in a I ' f~ of this invention. ,A~IIA; ~ ..illg is preferably ~f ~ .I via illh~V
injection or intranasal - ' A process of the present invention is especially useful in treating diseases associated with uncontrolled migration of white blood cells to damaged tissue. Such diseases include, but are not limited to, asthma, a~ ,scl~,lusis, ' arthritis, allergy, multiple sclerosis, leukemia, and brain cancer.

A process of inhibiting VCA~I-1 and af4~l binding uses a cyclic peptide of the present invention as set forth 1 ~ i. Preferred such pepti~des are the sarne as set forth above. More preferably, a peptide used in a process of the present invention has the arnino acid residue sequence of SE12 ID N 0:2-14 or 16-42. Even more preferably, peptides have the amino acid residue sequence of SF,Q II) NO:3-7, 9-12, 16-26, 28, 29 or 31-42.

The present invention also provides a process of selectively inhibiting the binding of a4,3l integrin to a protein . . ~ exposing the integrin to the protein in the presence of an e~fective inhibiting amount of a cyclic peptide of the present invention. In a preferred ~, hf"l;.. l, the a4,B~
integrin is expressed on the surface of a cell, e;ther naturally occuring or a cell tl~ ,rul~ d to express a4~ integrin.

WO96100~5XI P.IIL~

-20- 2 ~ 93~2~

The protein to which the ~f~31 integrin binds can be expressed on a ceil surface or part of the . .n~ fl' l~r matrix. EspeciaUy preferred proteins are fibronectin or invasin. Preferred peptides for use in such a process are the same as set forth above.
s The ability of peptides of the present invention to inhibil binding are described in detail hereinafter in the Examples.

The following examples illustrate particular ~ .~l.o l.. a~ of the present invention and are not limiting of the ~ ;.,.. and claims in any way.

FXAMPT F.C

EXAMpT F. 1: SrthPcic of Cys-T~Leu-As~Val-Cys-~ri~ F.O Tn NO:31) The Fmoc-anuno acids and an equimolar arnount of HOBT were dissolved in DMF. DIC in DCM was used as the coupling reagent with 1-1.2 hour reaction times. The Fmoc-Cys~Trt) Wang resin (25 nM) was swollen by treatment with DMF (1.5 ml) for 15-20 min, then d~ ut_t~l by treatment with 20% piperidine in DMF (3x, 8 min each~, and the resin was washed with DAlF (6x). The first amide bond was forrned using Fmoc-Val (150 nM) and DIC (150 nM), and this procedure was repeated until all alhino acids were coupled. After l I""~' u~ - of the N-terminal pTntecting group, the peptide was cleaved from t_e resin with a TFA cocktail (containing 5 Yo anisole and 5% EDI~ for 1 hour at RT. The TFA solution was reduced to about 0.5 ml and the product IJ.~;lJil~.t. d with cold ether.
After washing with ether (3x), the peptide was Iyophilized from aqueous solution to give the crude linear peptide (21.3mg). Cyclization was achieved by dissolving in water (20 ml) contàining guanidine-HCI (2.0 g) and amm~ m acetate (1.6 g) at pH 7.8, and the solution stirred at 4~C for 48 hours and then Iyophilized.

wo 96/005X ~

~ , ~ 2 1 2 1 9 3 8 2 ~

~ lifiwtiù~l was carried out by reverse phase HPLC as described above using a gradient of 5-70% B over 60 min, and the pure product isolated as a white powder by lyu~ (10.2 mg, >~8% pure by analytical l~PLC).

FX~MpT F 2: Synthesis of dPen-T~Leu-As~Val-Cys-acid ~,~FO Tn NQ:42!
This peptide was prepared using the same general procedure as in EXAMPLE I for the couplings except using Fmoc-Cys(Acm) Wang resin amd Fmoc-Trp(Boc) and Fmoc-dPen(Acm). Cyclization was achieved on the resin by treatment with NIS (4 equiv. for each thiol) in DCM: DMF (2:1) for 3 hours at room ~ ..u, ~ After washing with DMF and DCM the peptide was cleaved with a TFA cocl~tail and purified by HPLC.

S=O
EXAMPLE 3: Synthesis of C.H~CO-Tr~Leu-As~Val-Cys-acid (SEO ID
NO: 12!
The linear peptide was prepared as described in EXAMPLE I, however Fmoc-Trp(Boc) was used in this case. After d.,~.ut_Lu.. of the N-terminai Fmoc group, blu.. v~c~L~, acid ~200 nM), HOBT (200 nM) and DIC (2b~0 nM) in DCM (1.5 ml) was added and the reaction stirred at RT for 1 hour.
The Trt group on the Cys residue was removed with 297O TFA in DCM (2.5 ml) which contained 5% of trimethylsilane (4~, 10 min) and then the peptide was cyclized by treatment with 570 NMM in DMF overnight. Oxidation of the sulfide to sulfoxide was achieved with NalO~ (2.5 equv.) in DMF/DCM/water for 4 hours. The resin was washed with DCM, and the product cleaved and purified as before. Anaiytical HPLC showed two major peaks c., " ~ ;"~ to the t vo ILu~lCU;~U~

wo 9610058~

- 22 - 2 t 9 3 8 2 ~

SO~
EXAMPLE 4: Synthesis of l'~CO-Trp-l P~-As~o-v~l-cys-~ l SSEO lr3 NO: 12) S Synthesis was calTied out as in I~AMPLE 3, however oxidation was achieved with MCPBA (6 equiv.) in DCM.

EXAMPLE 5: Synthesis of -Glu-Tr~Leu-As~Val-As~
(SEO ID NO:3) This peptide was synthesized on 2-chlorotrityl chloride resin following the sarne procedure as described in EXAMPLE 1. After removal of the N-tern~inal Fmoc group the peptide was cleaved frvm resin using 30% acetic acid in DCM (3x, lOmin). The DCM solution was ~ the residue treated with water and Iyophilized to give the protected linear peptide.
lS Cyclization was achieved with HBTU (1.2 equv.) in DMF-containing 0.4 M
NMM 20-24 hours (Img/ ml) The DMF was er, r ~, and the residue IJIC ', " ' ' with water and washed (3x). Dcl,.vt~liv.. and 1~ . was conducted as described previously.

20 EXAMPI F 6: Binding Assays Peptides were assayed for their ab;lity to inhibit the binding of the integrin 4~ to VCAM. The _pecificity of the most potent peptides as inhibitors Of ~ was detemuned using a fibronectin binding assay. The assays are described below.
VCAM- I /~ Bindin~ A cc~
The assay involves d ~ the ability of cells th~t express ct~l to bind directly to purified VCAM. The illt~i.. C~ t, ceU type used in this assay was HL-oO, a human p~vll~yclv~ Mine.
An expression vector was designed such that a region of VCAM Icnown to bind the integrin was expressed as a fusion protehl with mouse Ig(~ A
cDNA containing the two N-terminal domains of human VCAM was wo 96/0058 ~
23 - ~ 3 8 2 8 , . ; ~, generated by the pOly~ a~ chain reaction (PCR) from a full length VCAM
cDNA. Similarly, a cDNA containing the hinge, CH2 and CH3 regions of mouse IgG2,~ was amplified by PCR from c13NA made from total RNA
isolated from the hybridoma cell line 402C10. The VCAM cDNA was 5 ligated to the mouse ~gG cDNA and cloned into a .- ~ expression vectûr. Transfection of the plasmi.d into COS cells resulted in the expression and secretion of the fusion protein into the ~ ~v ~ l ~v cell culture media.

The meclia was collected and active protein was purified by 10 ' r ', " '- using Dynal magnetic polya~ LIl., beads coated with goat anti-mouse ~gG. Following i~ul~lullu~ iL~viu~l~ the beads bound cells that expresscd the integrin a~l such as Ramos and HL-60. Leads incubated with media from mock transfected COS cells did not bind these cell types and served as a negative control for the assay.
HL-60 cells were " ~ b/ labeled with Calcein AM C-3099 (Molecular Probes) and , ' ~ in 1 m~ of binding buffer (Hank's balanced salt solution, pH 7.4, 1.0 rnM CaCl2, 1.0 mM MgCI~). The beads (10 1~1, 4xlO6 beads/ml) were placed in wells of a 96-well microtiter dish 20 with 10 /LI of peptide at various C~IA.'~ ' The beads were incubated with 10 ~1 of labeled cells (107 oellslml) for 10 min at room t~ ~Am~C
Following j~A nnhiii7~ n of the beads onto the plastic with a magnet, unboumd cells were removed by washing three times with binding buffer.
The remaining bound cells were Iysed in 50 mM Tris, pH 7.4, 5.0 mM
25 EDTA, 1.0~ NP-40 and quantitated by nuulhll~l~y using a Millipore Cytofluor 2350 n.,;, ~ Dose response curves were calculated and IC50 values determined. Alh,llldi~ly~ the percent adhesion was d~L~Illlil.~l at a single peptide ~~o ~ for C~ A d~ ~ - arnong peptides.

Peptides having the arnino acid residue sequence of SEQ ID NO:3-7, 9-12, 16-26, 28, 29, and 31-42 were found to s;g.lir~Lly inhibit the binding WOg6~00581 i ~ IJv.. ,!;,I
r ~ - 24 - 2 1 9 3 g ~ ~

of ~4~, integrin to VCAM-1 at c~..,.. In ,~ c of peptide less than about 10 ~M.

Fibrr nP~ fin/~"R, Rin~ cqy The assay accesses the ability of certain peptides to inhibit cells expressing o~, but not those expressing of ~1, from binding to rb The B cell line, Ramos can bind to fibronectin via the a~ integrin while binding of the ~ tlU~ , ceU line, K562, is dependent on the integrin aS~,.
Human plasma fibronectin was coated onto wells of a 96 weU assay plate. Wells coated with BSA were used as a control for the assay.
Following washing with Tris buffered saline, pH 7.4(TBS), the wells were blocked with TBS containing I % BSA. Ramos and K562 ceUs that had been lluu. 'y labeUed with Calcein AM C-3099 were washed and ' ' in binding buffer (Hank's balanced salt solution, pH 7.4, 1.0 mM CaCI~, 1.0 mM MgCI~ 1.û mM MnCI~). CeUs were mixed with peptide at various ~ and placed in the weUs. The plate was incubated at 37~C for 45 min. Following washing, the remaining bound cells were Iysed with 1 % NP-40 and quantitated by '' ~ y using a Millipore Cytofluor 2350 ~ . Dose response curves were calculated.

The foregoing Exarnples illustrate particular . .ho I ~ of the present invention. One of ordinary skill in the art will readily appreciate that changes, .- -~ . and alterations can be made in those I ...IN~ .U~
without departing from true scope and spirit of the invention.

W 096/005~1 r~lrJ~-l ~ 25 - ~ 1 9 3 8 2 8 SEQUENCE LISTING

~1) GENERAL INFORHATION:
(i) APPLICANT: Kogan, Timothy P.
Ren, Xaijun Vanderr~lice, Peter Beck, Pamela J.
(ii) TITLE OF lhv~ ON: A PRO OE SS TO IN~IBIT BINDING OF TEiE
INTEGRIN a,~, TO VCAM-1 OR rl~KONriu~lN
AND CYCLIC PEPTIDES THEREFOR
(iii) NB~HBER OF RFQrl~r~r~q 43 (iV) uu~n~fiu~J~l~u~ ADDRESS:
~Al Dr~nrrccvrc Dresfiler~ C~l' ith, Shore & Hilnamow, Ltd.
BI STREET: 180 North StetLion, Suite 4700 C CITY: Chicago D STATE: IL
El COUNTRY: USA
IFI ZIP: 60601 (v~ COHPUTER READABLE FORH:
(A1 HEDIUH TYPE: Floppy disk (B) COHPUTER: IBM PC iht~
(C) OPERATING SYSTEH: PC DOS/HS-DOS
(D) SOFTWARE: PatentIn Relea~i- ~1.0, Version ~1.25 (vi) CURRENT APPLICATION DATA:
(A) APPLICATION NUHBER:
~B) FILING DATE:
~C) CLA551rl~:RIlr~w:
viii) ATTORNEY/AGENT lhru ~ lul~:
(A) NAHE: Northrup, Thomafi B.
(B) Rr~r-TcTRDTIoN NUMBER: 33,268 ~ ix ) Tr~r r~ lul~ lW~ lr~
(A) TELEPBONE: (312~616-5400 (B) TELEFAX: (312)616-5460 (2) lNru~ ~l~lUW FOR SEQ ID NO:l:
(i) SEQUENCE rh~AR~rTpoTRTTrc (A) LENGTU: S ~ino acids (B) TYPE: a~ino acld (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix~ FEATURE:
(A) NAHE/ B Y: Hodified-site (B) LOCATION: 1,2 (D) OTEIER lNrl - lUW: /label= Xaa /note= Xaa is any D- or L-~-amino acid."

(ix~ FEATURE:
(Aj NAME/XEY: Modified-site (3) LOCATION: 3 (D) OT}iER INFORHATION: ~label= Xaa ~note= Xaa is any l.~d~ Lic L-a-amino ~cid. n W~ 9C/00~81 P~

~ 26-2 ~ 93~

(ix) FEATURE:
(A~ NAME/KEY: Modlried-sLte (3) LOCATION: 5 (D) OTHER INFOR~ATION: ~label= Xaa jnote= ~Xaa i8 any hydrophob c L-~-~mino acLd.

(xi) SEQUENCE ~UKl~lUN: SEQ ID NO:1:
Xaa Xaa Xaa Asp Xaa (2) INPORMATION FOR SEQ ID NO:2:
(i~ SEQUENCE r~v~vrcTIcs:
~A) LENGTH: 6 amino acids (B) TYPE: amlno acLd ~D) TOPOLOGY: linear (ii) MOLECULE TYPE~ peptide (ixj FEATURE:
(A) NAME/KEY: Modiried-site (B) LOCATION: 1..6 (D) OTHER lNrur~AIlun:
/note= orosalink between Glu at position 1 and Pro at position 6.

(xi) SEQUENCE ~u~l~llun: SEQ ID NO:2:
Glu Trp Leu Asp Val Pro (2) INFORMATION FQR SEQ ID No:3:
(i) SEQUENCE ~Y~V ~ u~:
(A) LENGTH: 6 amino aclds ~3) TYPE: amino acid (D) TOPOLOGY: linear (il) MOLECULE TYPE: peptide (ix) FEATURE:
(A) NAME/XEY: M~ifir 3ite (B) LOCATION: 1..6 (D) OTHER l~run~l lU..:
tnote~ crossllnk between Glu at pcsition 1 and Asp at poaition 6.

(xi) SEQVENCE DE~unlr~lul~: SEQ ID NO:3:
Glu Trp Leu A p Val Asp (2) INFORMATION FOR SEQ ID NO:4:
(i~ SEQUENCE CHARACTERISTICS:
(A) LENGT~: 5 aminc acids (B) TYPE: amino acid (D) TOPOLOGY: linear W 096/00581 r~
~ 27-2 1 9 3 8 2 ~i ~ OLECULE TYPE: peptide (ixj FEATURE:
(Aj NAME/KEY: Modified-site (B) LOCATION: 1..5 (Dj OTHER INFORMATION:
/note- cros31ink between Glu at position 1 and Val at position S.

(xi) SEQUEN OE ~E~K~ uN: SEQ ID NO:4:
Glu Trp Leu Asp Val (2) INFORMATION FOR SEQ ID NO:S:
~i) SEQUENCE r~~'rT~~TqTICS:
(A) LENGT~: S a~ino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FE~TURE:
(A) NAME/XEY: Modified-site (B) LOCATION: l..S
(D) OTUER INFORMATION:
/note- cros31ink between Glu at position 1 and Asp at position S.

(xi) SEQUENCE D~S~K1~ ~ lU~: SEQ ID NO:S:
Glu Trp Leu Agp A8p (2~ INFOKMATION FOR SEQ ID NO:6:
(i~ SEQUENCE ~D~rT~oTq~Tc5:
(A) LENGTM: 11 amino acids (3) TYPE: arino acid (D) TOPOLOGY: linear (11) MOLECULE TYPE: peptide (ix) FEATURE
(Aj NAME/KEY: Modified-site (B) LOCATION: 1..11 ~D) OT~ER 1~r~K~aI1UI~:
/note- crosslink between Glu at position 1 and val at position 11.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
Glu Trp Leu Asp Val Pro Glu Trp Leu Asp Val wo s6/nussl -I ~,1/~. ...

~ ' -28~ 3 ~ 2 8 (2) INFORMATION FOR SEQ ID NO:7:
(i1 SEQUENCE CHARAulG~Isllu~
(A) LENGTH: 7 amino acids IB) TYPE: a~ino acld (D) TOPOLOGY: linear iiij MOLECULE TYPE: peptlde (ix) FEATURE:
(Aj NAUEtFEY: Modified-31te (3) LOCATION: 1..7 (D) OTHER }NFORMATION:
/note= cros~lLnk tetween-Gly at po~itlon 1 and Val at po~ition 7.

(xi) SEQUEN OE u~ nl~.lON: SEQ ID NO:7:
Gly Pro Glu Phe Leu Asp Val (2) lN~unnAlluN FOR SEQ ID NO:8:
~i) SEQUENCE rqa~TFDT~TIcs:
(A) LENGTH: S amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE:
(A) NAME/~EY: Modified-site (B~ LOCATION: l..S
(D) O = lhrU...~llU~:
/note~ crosslink between Glu at po~ition 1 and Val at position S.

(xL) SEQUENCE uh~unlr~ : SEQ ID NO:8:
Glu Phe Leu Asp Val (2) }NFORMATION FOR SEQ }D NO:9:
(i) SEQUENCE ~Y~o~ u5:
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ixl FEATURE:
(A) NAME/FEY: Modified-slte (B) LOCATION: 1 (D) OT~ER lh~uK~AIluh: /labeL= ~aa /note= "Xaa=CH2CO-ser."
(ix) FEATURE:
(A) NAME/XEY: Modified-site (B) LOCATIO~: 6 ... . _, ., . _ _ ..

W 096/00581 r~

(D) OTHER l~ro~ ~Ilua: /label= Xaa /note= "Xaa=Cys-COOH."
~ix) FEATURE:
(A) NAME/ ~ Y: Hodified-site (B) LOCATION: 1..6 (D) OTHER l~rl ~ lUI:
/note= crosslink between Xaa at position 1 and Xaa at position 6.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:
Xaa Trp Leu Asp Val Xaa (2) INFOPMATION FOR SEQ ID NO:10:
(i) SEOUENCE ~D~T~RT.qTICS:
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear ~ii) NOLECULE TYPE: peptide ~ix) FEATURE:
~A) NAME/ ~ Y: Modified-site (B) LOCATION: 1 (D) OTHER INFORMATION: /label= Xaa /note~ "V~ u7r~Glu.
(ix) FEATURE:
(A) NAHE/ICEY: M~ fi~ ' aite B) LOCATION: 6 (D) OTHER lN~ 'lU~: /label= Xaa /note= ~Xaa=Cys-COOH."
(ix) FEATURE:
(A) NANE/XEY: Hodified-~ite (B) LOCATION: 1..6 (D) OTHER INFORMATION:
/noteS crosslink between Xaa at position 1 and Xaa at posltion 6.

(xi) SEQUENCE DE5~Kl~1lu~: SEQ D NO:l~:
Xaa Trp Leu Asp Val Xaa (2) INFORHATION FOR SEQ ID NO:11:
(i) SEQUENCE ~ARA~RTqTICS:
(A) LENGTH: 5 amino acids (B) TYPE: ~ino acid (D) TOPOLOGY: linear ~ii) NOLECULE TYPE: peptide ~ix~ FEATURE:
~A) NAME/~EY: Modified-site ~B) LOCATION: 1 W 09~00581 r~ u~. ~
~ 30- 2 1 9 3 ~ 2 ~

(D) OTHER IUFORMATION: /label= Xaa /note= "Xaa=CH2CO-Glu."
(ix) FEATURE:
~A) NAME/~EY: Modified-site (8) LOCATIOU: S
(D~ OTHER INFORMATION: llabel= Xaa /note= "Xaa=Cy~-CCO~.
(ix~ FEATURE:
(A) NAME/~EY: Modified-site ~B) LOCATION: l..S
~D~ OTHEA INFO~MATION:
/note= crosslink between Xaa at position 1 and Xaa at position S.

(xi) SEQUENCE Dr;~nltllu~: SEQ ID UO:11:
xad Trp Leu Asp Xaa (2) INFORMATION FOR SEQ ID NO:12:
~i) SEQUENCE rV~R~TFRT~TCS:
~A~ LENGTH: S amino 4cids ~B) TYPE: ~mino ~cid ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATUAE:
(A) NAME/~EY: Modified-site ~B) LOCATION: 1 ~D) OTHER INFORMATION: /label= Xaa /note= ~Xaa=C~2CO-Trp.
(ix) FEATURE:
(A) UAME¦~EY: Modlfied-site (B) LOCATION: S
(D) OTHER l~rl LUN: /label= Xaa /note= ~Xaa=Cys-COOH.
(ix) FEATUAE:
~A) UAME/~EY: Modiiied-site ~B) LOCATION: 1..5 (D) OTHER l~r- lUN:
/note= crosolink between Xaa at position 1 and Xaa at posLtLon S.

(xi1 SEQUENCE DESunIrllOd: SEQ ID NO:12:
Xaa Leu Asp Val Xaa (2) INFORMATION FOR SEQ ID NO:13:

(i) SEQUENCE r~A~A~T~TCTICS:
(A) LENGTH: S amino acLds (B) TYPE: amino acid (D) TOPOLOGY: linear WO 96/02~
3 1 -- ,2 1 9 3 ~ 2 8 (ii) MOLECULE TYPE: peptide (ix~ FEATURE:
~A) NAME/REY: Disulfide-bond ~B) LOCATION: 1..5 (xi) SEQUENCE ~L.~l~ lUL~: SEQ }D NO:13:
Cys Leu Asp Val Cys (2~ INFOPI~ATION FOR SEQ ID NO:14:
(i) SEQUENCE ~ ~Da ~ lu~
(A) LENGTH: 5 amino acids (B) TYPE: amino acid (D~ TOPOLOGY: linear (ii) MOLECULE TYPE: peptide ~ix) FEATURE:
(A) NA~E/REY: Modified-site (B) LOCATION: 5 (D) OTHER lNrU~MA}lUI~: /label= Xaa /note= "Xaa=Cys-COOH."
~ix) FEATURE2 (A) NAME/REY: Disulfide-bond (B) LOCATION: 1..5 (Xi) BEgUENCE DESUK~r.lUVN: SEQ ID NO:14:
Cys Trp Leu Asp Xaa (2~ INFORMATION FOR SEO ID NO:15:
(i) SEQUENCE r~aD~TT~RT8~ 2 (A) LENGTH: 4 amino acids (B) TYPE: amino acid ~D) TOPOLOG2Y: linear (ii) MOLECULE TYPE: peptide (ix) FEATUPE:
(A) NAME/REY: Modified-~ite (B) LOCATION: 1 (D) OTHER INFORMATION: /label= Xaa /note= "Xaa i5 any D- or L-a-amino aoid.
(ix) FEATURE:
~A) NAME/REY: Modified-site (B) LOCATION: 2,4 (D) OT~ER INFOP~ATION: /label= Xaa /note= "Xaa is any I~ILV~I~VLiC L-a-amino acid."

(xi) SEOUENCE J~uKlrLluN: SEQ ID NO:15:
Xaa Xaa Asp Xaa W 096/00581 r~
32 ~ 2 1 ~ 3 8 2 8 (2) INFORMATION FOR SEQ ID NO:16:
~i) SEQUENCE rY~
(A) LENGTM: 7 amino ac}d3 (B) TYP~: amlno acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE:
(Al NAME/~EY: Disuliide-bond ~3~ LOCATION: 1..7 (xi) SEQUENCE DEa~l~lud: SEQ ID NO:16:
Cys Ser Trp Leu Asp Val Cy8 } 5 (2) INFORMATION FOR SEQ ID NO:17:
(i) SEQUENCE rY~
(A) LENGTH: 6 amino acids (B) TYPE: amino ac$d (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (Lx) FEATURE:
(A) NAME/KEY: NodlfLed-s$te (B) LOCATION: 2 (D3 OTRER l~r~ -lU#: /label- Yaa /note- Xaa~d-Trp."
(ix~ FEATURE:
(A) NANE/KEY: Modified-3ite (B) LOCATION: 6 (D) OT~ER INFORMATION: /label= XaH
/note~ Yaa-Cy~-COOR. n (ix) FEATURE:
(A) NAME/XEY: D~ fi' b~..1 (B) LOCATION: 1..6 (xi) SEQUENCE DES~L~LUU: SEQ ID NO:17:
Cys Xaa Leu Asp Val Xaa (2) L3rUK~AIlUM FOR SEQ ID NO:1&:
(i) SEQUENCE ~ Lua:
(A) LENGT~: 5 &mino acid8 (B) TYPE: amlno acid (D) TOPOLoGY: linear (ii) NOLECULE TYPE: peptide (ix) FEATURE:
(A; NANE/KEY: Modified-~ite (B) LOCATION: 1 W 096100581 r~
~ 33 ~2 ~ ~3~323 (D) OTEER INFORMATION: /label= Xaa /note= "Xaa=Ac-Cya.
(ix) FEATURE:
(A) NAKE/~EY: Modified-site (B) LOCATION: 6 (D) OTEER InFoRMATIoN: /label= Xaa /note= Xaa=Cys-COO~."
( ix ) FEATUp~E:
(A) NAME/~EY: Disulfide-bond (B) LOCATION: 1..6 (xi) SEQUENCE ~G~uKIP~lu~: SEQ ID No:i8:
Cys Trp Lou Asp Val Xaa s (2) lNrv~T~vl~ FOR SEQ ID NO:19:
(i) SEQUENCE ~5Tl~R~-rTTi'~TCTICS
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (D) TvPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE:
(A) NAKE/~EY: KodLfied-aite (B) LOCATION: 6 (D) OTEER lNrvnnhILvn: /label= Xaa /note= Xaa-Cys-COOH.

(ix) FEATURE:
(A) NAME/REY: Disulfide-bond (B) LOCATION: 1..6 (xi) SEQUENCE DESvnIrllUn: SEQ ID N-O:l9:
Cy9 Tyr Leu Asp Val Xaa s (2) lNru~nallv~ FOR SEQ ID NO:20:
(i) SEQUENCE ry~ TF~TcTIcs:
(A) LENGTH: 6 amino acids (B) TYPE: amino acLd (D~ TOPOLOGY: linear (ii) HOLECULE TYPE: peptide (ix) FEATURE:
(A) NAKE/~EY: Kodifi~d-aite (B) LOCATION: 6 (D) OTEER ln~ u~: /label= Xaa /note= Xaa-Cys-COOH.
(ix) FEATURE:
(A) NAME/EEY: DisulfLde-bond (B) LOCATION: 1..6 W 0'~6/00581 r~1~u~,s,.
~ 34~ 2 1 9 3 ~ 2 8 (xi) SEQUENCE DESuKlrllu1~: SBQ ID NO:20:
Cys Trp Leu Asp Phe Xaa (2j INFORMATION FOR SEQ ID ~0:21:
(i~ SEQUENCE r~DDr~RT~TICS:
tA) LENGTQ: 6 amino acids (B; TYPE: amino acid (D) TopoLaGy: linear (ii~ MOLECULE TYPE: peptide (ix~ FEATURE:
(A) NA~E/XEY: Modified-site (8) LOcATIoy~: 6 (D) aTQER lNr- -T~N ¦la~el= Xaa /notee ~Xaa=Cys-COOQ."

(ixj FEATURE:
(A) NA~E~KEY: Disuliide-kond (B) LOCATION: l..6 (x~ SEQUENCE DESCRIPTION: SEQ ID NO:2l:
Cys Phe Leu Asp Val Xaa l 5 (2~ lNru.__.TluN FOR SEO ID NO:22:
~i~ SEQUENCE r~- R~ 1L~O:
(A) LENGTQ: 6 amino acids (B~ TYPE: ~mlno acid (D~ TOPOLOGY: linear (ii~ MOLECULE TYPE: peptide (ix~ PEATURE:
~A) NAMEt~EY~ ModifLed-site (B~ LocAT}aN: 6 (D) OTQER lNr~K~ArluN: /labels Xaa /note= "Xaa=Cy~-COoQ.
(ix) FEATURE:
(A~ NA~E/XEY: Di~lfi~' ~..
(B) LOCAT}ON: l..6 (xi) SEQUENCE DESCRIPT}ON: SE9 ID NO:22:
Cys Trp Leu A~p Trp Xaa l 5 (2) lNtU~AI~lUN FOR SEQ ID NO:23:
(i~ SEQUENCE r~DAr~RRT.CTTCS:
(A~ LENGT~: 6 amino acids (B1 TYPE: amino acid W 096~581 1~~
~ 35 -~ 1 93~2~

(D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptlde ! ~ iX ) FEATURE:
(A) NAME/KEY: Modified-site (B) LocATIon: 6 (D) OTHER INFORNATION: /l~bel= Xaa /note= "Xaa=Cys-COOH."
(ix) FEATURE:
(A) NAME/KEY: Di3ulfide-bond (B) LOCATION: l..6 (xi) SEOUENCE b~UKlallUN: SEQ ID NO:23:

Cys Trp Ile Asp Val Xaa (2) INFORN.5.TION FOR SEQ ID NO:24:
(i) SEQUENCE ~5rTRDT~TIcs:
(A) LENGTH: 6 amino aclds (B) TYPE: amino acid (D) TOPOLOCY: linear (i.i) NOLECULE TYPE: peptide (ix) FEATURE:
(A) NAME/~EY: Modified-site (B) LOCATION: 6 (D) OTUER l~K~'l'lUN: /label~ Xaa /note= "Xaa=Cys-COOL."
(ix) FEATURE:
(A) NAME/KEY: Di~lf;~ ~..d (B) LOCATION: 1..6 (xi) SEQUENCE D~lr~lUN: SE~ ID NO:24:
Cy5 Trp Net Asp Val Xaa (2) lNruhdhIluN FOR SEQ ID No:2s:
(L) SEQUENCE ~D5rT~TcTIcs:
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lLnear (ii) MOLECULE TYPE: peptide (ix) FEATURE:
(A) NAME/REY: Modified-site (B) LOCATION: 6 (D) OTHER INFORNAT}ON: /label~ Xaa /note= "Yaa=Cys-COOH."
(ix) FEATUP~E:
(A; NAME/KEY: Disulfide-bond (B) LOCATION: l..6 wo s6/nossl r~
~ 36- 2 1 93g2~

(xi~ SEQUENCE ~.a~nlr~luN. SEQ ID NO:25:
Cys Trp Val Asp Val xad ~2) INFOR~ATION FOR SEQ ID NO:26:
~i) SEQUENCE r~D~ ua (A) LENGTH: 6 amino acids (B) TYPE: a~ino acLd (D~ TOPOLOGY: linear OLECULE TYPE: peptide ~ix) FEATURE:
IA) NAHE/XEYI Modified-site (B~ LOCATIONI 6 (D) OTHER INPORHATION: /labelr Yaa /note= "XaarCys-COOH."
(Lx) FEATURE
(A) NAHE/REY: Disulifide-bond (B) LOCATION: 1..6 ~xi) SEQUENCE D~nlr~lON: SEQ ID NO:26:
Cys Trp Lys A~p Val Xaa (2) INFORMATION POR SEQ ID NO:27:
(i) SEQUENCE r~:rTP~T~5TICS
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (D) TOPOLOGY: lin~ar ~il) MOLECULE TYPE: peptide (ix) PEATUPE~
(A) NAME/REY: ~odified-site ( B ) LOCATION: 6 (D) OT~ER lNr~ ~T10N: /label= Xaa /note= ~xda~cy~-cooL~
(ix) FEATURE:
(A) NAME/XEY: Di5ulfide-bond ~B) LOCATION: 1..6 ~xi) SEQUEN OE 5~6U~l~lluN: SEQ ID NO:27:
Cys Trp Leu Glu Val Xaa 2) INFORMATION FOR SEQ ID NO:28:

(i) SEQUENOE CHARACTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: a~ino acid (D) ~TOPOLOGY: linear WO g6/00581 r~
~ 372 1 9 3 8 28 , , ~ MOLECULE TYPE: poptide (ix1 FEATURE:
IA) NAME/REY: Modified-site ! (B) LOcATIoN 6 (D) OTHER INFORMATION: /label= Xaa /note= Xaa=Cys-Coo~..
(ix) FEATURE:
(A) NAME/REY: Disulfide-bond ( B ) LOCATION: 1..6 (xi~ SEQUENCE U~.~Kl~llU~: SEQ ID NO:28:
Cys Trp Leu Asp Leu Xaa (2) INFORMATION FOR SEQ ID NO:29:
(i) SEQUENCE r~A~..r.~T~,l~
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (iij HOLECULE TYPE: peptLde (ix) FEATURE:
(A) NAME/KEY: Hodified-site (8) LOCATION: 6 (D) = R lNrU~IlUN: /label= Xaa /note= "Xaa=CyD-COOH.
(ix) FEATUP~E:
(A~ NAME/REY: Disulfide-bond (B) LOCATION: 1..6 (xi) SEQUENCE D~KI~lu~: SEQ ID NO:29:
Cy~ Trp Leu Asp Tyr Xaa (2) INFORMATION FOR SEQ ID NO:30:
(i) SEQUENCE rU~D~.., ,_ "", ",~.
(A~ LENGTH: 6 ~mino ~cids (B) TYPE: a~ino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE:
(A) NAME/REY: Hodified-site (B) LOCATION: 6 (D) OTHER laruK~Ilu~: /label= Xaa /note= "Xaa=Cys-COO~."
(ix) FEATUP~E:
(A) NAME/REY: Disulfide-bond (B) LOCATION: 1..6 W 096100S81 ~ u~
~ 38- 2 1 9 3 8 2 ~

xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:
Cys Ile Leu Asp Val Xaa ~2) INFORMATION FOR SEQ ID NO:31:
(i) SEQUENCE r~
(A) LENGTH: 6 ~mino acids (B) TYPE: amino acid ~D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix~ FEATURE:
~a) NAME/~EY: Modified-site ~B) LOCATION: 6 (D) OT~ER INF3RMATION: ~label= Xaa /note= "Xaa=Cys-COO~."
~ix) FEATURE:
A) NAME/XEY: Disulfide-bond (B) LOCATION: 1..6 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:
Cy9 Trp Lou Asp Val Xaa (2) INFORMATION FOR SEQ ID NO:32:
(i) SEQUENCE r~P~
A) LENGT8: 6 a~ino acids (B~ TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE:
(a) NAME/~EY: Modified-site ~B) LOCATION: l (D) = R INFORMATION: /label= Xaa /note= "Xaa=d-Cys."
(ixl FEATURE:
(A) NAME/ ~ Y: Modified-site (B) LOCATION: 6 (D) OT~ER lNrl - LUN: /label= Xaa /note= "Xaa=Cys-CCO~."
(ixj FEATUP~E:
(A) NAME/XEY: Disulfide-bond (B) LOCATION: 1..6 (xl) SEQUENCE u~nlrl~N: SEQ ID NO:32:
Xaa Trp Leu Asp Val Xaa W 096/00581 r~l~u~
39 ~ 1 9 3 8 2 ~

(2) INFOBMATION FOR SEQ ID No:33:
~i~ SEOUENCE r~apDr~rpTcTIcs (A) LENGTH: 7 amino aclds (B) TYPE: amino 2cid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ixj FEATURE:
(A~ NAME/REY: Modified-site (Bj LOCAT}ON: 7 (D~ OTNER INFORMATION: /label= Xaa /note= "Xaa=Cys-NH2."
(ix) FEATURE:
(A) NAME/~EY: Diaulfide-bond (B) LO Q TION: 1..7 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
Cys Glu Trp Leu Asp Val Xaa (2) INFORMATION FOR SEQ ID NO:34:
(i) SEQ~ENCE euaDa..~T~,l~-~A~ LENGTU: 7 amino acics (B~ TYPE: amino acid (D~ TOPOLOGY: linear (ii~ MOLECULE TYPE: peptide (ix) FEATURE:
(A) NAME/XEY: Modified-site (B) LO Q TION: 7 (D) OTNER lNru~.l~N: /label= Xaa ~note= Xaa-Cya-COOH.
(ix) FEATURE:
(A) NAME/FEY: Disulfide-bond (B) LO Q TION: 1..7 (xi) SEQUENCE ~e~lr~ : SEQ ID NO:34:
Cys Glu Trp Leu Asp Val Xaa (2~ INFoRMATIon FOR SEQ ID No:35:
(i~ SEQUENCE CHARA~-~I~
(A3 LENGTN: 7 amino acids (B~ TYPE: amino acid (D~ TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE:
(A~ NAME/REY: Modified-site (B) LOCATION: 1 (D) OTNER lNru~ATl~N: /label= Xaa ~VO 96~00581 2 1 93~23 ~note= ~aa=d-Cys. n (ix~ FEATURE:
(A~ NAME/KEY: Modified-site (Bj LOCATION: 7 (D) OTHER IhFoR~ATIoN: /label= Xa~
/note= nXaa=Cys-CCOL.
(Lx) FEATURE:
(A) NAMEtKEY: ni~--lfi~-bond ( B ) LOCATION: 1..7 (xL~ SEQUENCE DESCRIPTION: SEQ ID NO:35:
Xaa Glu Trp Leu Asp Val Xaa ~2) INFOP~ATION FOR SEQ ID NO:36:
(i) SEQUEN OE rR~D~FRTsTIcs (A) LENGTH: 7 a~lno acids (B) TYPE: a~ino acLd (D~ TOPOLOGY: linear (ii~ MOLECULE TYPE: peptide (ix) FEATURE:
(A~ NAMEtKEY: Modified-site (B~ LO Q TION: 7 (D) OTHER lhrl ~Ivn: /ltbel= Xaa tnote~ ~Xaa=Cy8-COOH.
(ix) FBATURE:
(A) NAMEt~EY: Disulfide-bond (B) LOCATION: 1..7 (xL) SEQUENCE U~li.lVh: SEQ ID NO:36:
Cy~ Glu Trp Leu Asp Tyr Xaa (2~ INFORMATION FOR SEQ ID NO:37:
(i) SEQUENCE o~D~r~RRTsTTrs (A) LENGTH: 7 amino aclds (B) TYPE: a~ino acid (D) TOPOLOGY: lin~ar (ii) MOLECULE TYPE: peptide (ix) FEATU~E:
(A) NAMEtKEY: ~odified-site (B) LOcATIoN: 7 (D) OTHER INFOP~ATION: tlabel= Xaa /note= nXaa=Cys-COOH. n (ix) FEATURE:
(A) NAME/KEY: Disul~ide-bond (B) LOCATION: 1..7 W 096/00.~81 r~

~ 41 _2 1 938~8 (xi) SEQUENCE UG5~Kl~.luN: SEQ ID NO:37:
Cys Ser Phe Leu Asp Tyr Xaa (2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE r~3r~TSTICS:
~A) LENGTH: 7 amino acids (B~ TYPE: amino acid (D) TOPOLOGY: linear (ii) HOLECULE TYPE: peptide (ix) FEA = :
(A) NANE/ ~ Y: Nodifiod-site (B) LOCATION: 7 (D~ OTHER INFORMATION: ~label= Xaa /note= "Xaa= CY5_COOH.
(ix) FEATURE:
(A) NA~E/REY: Disulfide-bond (B) LOCATION: 1..7 (xi~ SEQUENCE DESCRIPTION: SEQ ID NO:38:
Cys Glu Phe Leu Asp Tyr Xaa (2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE r~3.~
(A) LENGTH: 7 amino aoids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (ix) FEATURE
(A) NA~E/XEY: Nodified-site (B) LOCATION: l (D~ OT~ER lNr~ ~ : /laoel= Xaa /note= Xaa=d-Cys.
(ix) FEATURE:
(A) NANE/ ~ Y: Nodified-site (B~ LOCATION: 7 (D) OTHER lNru~A~ : /label= Xaa /note= Xaa=d-Cys-COOH.
(ix) FEATURE:
(A) NANE/~EY: Disulfide-ocmd (B) LOCATION: 1..7 (xi) SEQUENCE ~G~nl~luN: SEQ ID NO:39:

Xaa Sor Trp Leu Asp Val Xaa (2) INFO~ATION FOR SEQ ID NO:40:

42 2 ~ 9 3 ~ 2 ~

(i) SEQUENCE rup~ r~r~s~T5TIcss (A) LENGTH: 8 a=ino aclds ~B) TYPE: aoLno acid (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide ~ix) FEATUP~E:
(A~ NAME~KEY: Modified-site (B) LOCATION: 8 (D) OTHER I~ ~ : /label= Xaa /note= ~Xaa=Cya-COOP.
(ix) FEATURE:
(A) NAME/~EY: Disulfide-bond ~B) LO Q TION: 1..8 (xi) SEQUENCE DESCR}PTION: SEQ ID NO:40:

Cys Pro Glu Trp Leu Asp Val Xaa (2~ INFORHATION FOR SEQ ID NO:41:
(i) SEQUENOE r~D~
(A) LENGTH: 6 aQino aclda (B) TYPE: a~ino acld (D) TOPOLOGY: line-r (Li) HOLECULE TYPE: peptide (ix) FEATURE:
(A) NAHE/KEY: Modified-site (B) LOCATION: 1 (D) OT~u-ER ~hrU. .11~1~: /label= Xaa /note= "Xaa is MPEG~coc ~ Cy8, where HPEG is a polyethyleneglycol of 5000 molecular weight.
(ix) FEATURE~
(A) NAME/KEY: Modified-site (B) LOCATION: 6 (D) OT~ER l~r~ -TnN /label= Xaa /note= ~Xaa=Cys-COo~.

~ix) FEATURE:
(A) NAME/~EY: Diaulfide-bond (B) LOCATION: 1..6 (xi) SEQUENCE u~Kl~ll~8: SEQ ID NO:41:
Xaa Trp Leu Asp Val Xaa (2) INFORHATION FOR SEQ ID NO:42:
(i) SEQUENCE Cu~URACTERIST}CS:
(A) LENGTH: 6 a~ino acicds (B) TYPE: a~ino acid (D) TOPOLOGYs linear (ii) MOLECULE TYPE: peptide W 096/0058l P~l/U~ ~h .

~,S ~' -43- 21 9382~

~ix) FEATURE:
(A) NAME/REY: Modified-s1te (~) LOCATION: l (D) OTEER INFORffATION: /laoel= Xaa /note= "Xaa=dPen."
(ix) FEATURE:
(A) NAME/ ~ Y: Modlfied-site (B) LOCATION: 6 (D) OTHER INFORNATION: /label= Xaa /note= ~Xaa=Cys-COOH."
(ix) FEATURE:
(A) NAME/~ Y: D;~ fi~o-bond (B) LOCATION: l..6 (xi) SEQUENCE DE~UK1e~1UI1: SEQ ID NO:42:
Xaa Trp Leu Asp Val Xaa l 5 (2) INFORNATION FOR SEQ ID NO:43:
(i) SEQUENCE ~D~ DT5TICS:
(A) LENaTE: 7 a~ino acids (B) TYPE: amino acid (D~ TOPOLOGY: l1near (ii) MOLECULE TYPE: peptide (ix) FEATURE:
(A) NAME/ ~ Y: Modlfied-site (B) LOCATION: l (D) OTEER 1n~ ~T~: /label= Xaa /note= ~Xaa is any D- or L- ~-ar~ino acid."
(ix) FEATURE:
(A) NA~E/~EY: Modified-~Lte (B) LOCATION: 7 (D) OTEER INFORNATION: /label= Yaa /note- ~Xaa i8 Cy8-COoE.
(ix) FEATUPE:
(A) NANE/ ~Y: D;~lf;~o-bond (B) LOCATION: l..7 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:
Yaa Cys Trp Leu Asp Val Xaa

Claims

WHAT IS CLAIMED IS:

1. An isolated and purified cyclic peptide of from 4 to about 13 amino acid residues having (a) an N-terminal amine group, acetyl group or a polyethyleneglycol moiety of from about 400 to about 12,000 Daltons average molecular weight linked through an amide bond to the N-terminal residue; and (b) a C-terminal carboxylic acid group or amide group; said peptide comprising the amino acid residue sequence Xaa1-Xaa2-Asp-Xaa3 (SEQ ID NO:15), where Xaa1 is any L- or D-.alpha.-amino acid residue and Xaa2 and Xaa3 are any hydrophobic, L-.alpha.-amino acid residue with the proviso thatwhen Xaa1 is Lys or Arg, Xaa2 cannot be Gly or Cys.

2. A peptide of claim 1 wherein Xaa1 is Phe, lle or Trp, Xaa2 is Leu, lle, Val, Lys or Met and Xaa3 is is Val, Tyr, Leu, Trp, or Phe.

3. A peptide of claim 2 wherein Xaa2 is Leu and Xaa3 is Val.

4. A peptide of claim 1 that is cyclized via formation of a lactam and which peptide comprises the amino acid residue sequence:

Xaa4-Xaa1-Xaa2-Asp-Xaa3 (SEQ ID NO:1), where Xaa1, Xaa2 and Xaa3 are as defined in claim i and Xaa4 is any D- or L-.alpha.-amino acid.

5. A peptide of claim 4 wherein Xaa1 is Phe or Trp, Xaa2 is Leu, Xaa3 is Val and Xaa4 is Glu.

6. A peptide of claim 4 having the amino acid residue sequence of SEQ ID NO:2-8.

7. A peptide of claim 1 further comprising a cysteine or modified cysteine residue and a -CH2CO- group at the N-terminal position.

8. A peptide of claim 7 wherein the sulfur atom of the cysteine or modified cysteine residue is attached to the CH2 group of -CH2CO-.

9. A peptide of claim 7 wherein Xaa1 is Trp or Phe, Xaa2 is Leu and Xaa3 is Val or Tyr.

10. A peptide of claim 9 wherein Xaa1 is Trp Xaa2 is Leu and Xaa3 is Val.

11. A peptide of claim 7 having the amino acid residue sequence of SEQ ID NO:9-12.

12. A peptide of claim 1 further comprising at least two cysteine or modified cysteine residues.

13. A peptide of claim 12 wherein at least one of the cysteine or modified cysteine residues is located at the N- or C-terminal position.

14. A peptide of claim 12 wherein Xaa1 is Trp, Xaa2 is Leu. and Xaa3 is Val.

15. A peptide of claim 12 that comprises the amino acid residue sequence:

Xaa1-Xaa2-Asp-Xaa3 (SEQ ID NO:15) where Xaa1, Xaa2 and Xaa3 are as defined in claim 1.

16. A peptide of claim 15 wherein Xaa1 is Tyr, Phe, Trp, dTrp or Ile; Xaa2 is Leu, Ile, Val, Lys, Met or Asp; and Xaa3 is Val, Tyr, Leu, Trp, or Phe.

17. A peptide of claim 15 that comprises the amino acid residue sequence of:

Xaa4-Xaa1-Xaa2-Asp-Xaa3 (SEQ ID NO:1), where Xaa1, Xaa2 and Xaa3 are as defined in claim 15; and Xaa4 is any D- or L-.alpha.-amino acid.

18. A peptide of claim 17 wherein Xaa1 is Trp or Phe, Xaa2 is Leu, Xaa3 is Val or Tyr and Xaa4 is Ser or Glu.

19. A peptide of claim 12 having the amino acid residue sequence of SEQ ID NO:13, 14 or 16-42.

20. A pharmaceutical composition comprising a physiologically acceptable diluent and a cyclic peptide of claim 1.

21. A composition of claim 20 wherein the peptide has the amino acid residue sequence of SEQ ID NO:2-14 or 16-42.

22. A process of selectively inhibiting the binding of .alpha.4.beta.1, integrin to VCAM-1, comprising exposing a cell that expresses .alpha.4.beta.1 integrin to a cell that expresses VCAM-1 in the presence of an effective inhibiting amount of a cyclic peptide of claim 1.

23. A process of claim 22 wherein the cell that expresses .alpha.4.beta.1 integrin is a white blood cell and the cell that expresses VCAM-1 is an endothelial cell.

24. A process of selectively inhibiting the adhesion of a ceil that expresses .alpha.4.beta.1 integrin to a vascular endothelial cell that expresses VCAM-1 comprising exposing the cell or endothelial cell to an effective inhibiting amount of a cyclic peptide of claim 1.

25. A process of claim 24 wherein the cell expressing .alpha.4.beta.1 is a whiteblood cell, a mast cell or a tumor cell.

26. A process of claim 24 wherein the cell expressing .alpha.4.beta.1 and the endothelial cell are located in a living organism.

27. A process of selectively inhibiting the binding of .alpha.4.beta.1 integrin to protein comprising exposing the integrin to the protein in the presence of an effective inhibiting amount of a cyclic peptide of claim 1.

28. A process of claim 27 wherein the .alpha.4.beta.1 integrin is expressed on the surface of a cell.

29. A process of claim 27 wherein the protein is part of the extracellular matrix.

30. A process of claim 27 wherein the protein is fibronectin or invasin.