CA2178927A1 - Peptide inhibitors of cxc intercrine molecules - Google Patents

Abstract

Disclosed are peptide-based compositions and methods for inhibiting and modulating the actions of CXC intercrine molecules. The antileukinate peptides described inhibit IL-8, GRO and MIP2.beta. binding to neutrophils and neutrophil activation. The peptides are particularly advantageous as they inhibit IL-8-induced enzyme release at a 25 fold lower concentration than is required to inhibit chemotaxis, which makes them ideal for treating various inflammatory diseases and disorders including, amongst others, Adult Respiratory Distress Syndrome (ARDS), cystic fibrosis and chronic bronchitis.

Description

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~lo 95116702 2 1 7 8 9 2 7 p ~ 7. ~1~45 iUlSS~:KIit' 1. lVN
PEPTIDE lN~ ORS OF CXC T~T~p~'RTl~ ~C~T.P!.q;

13ACKGRO~ND OF ~IE lNV~l~LlVN
The U. S. government owns rights in the present invention pursuant to grant number ROI-HL 403650 from 10 NHLB I .
1. Field of the Invention The present invention relates generally to the f ield 15 of cytokine actions and more particularly concerns methods and compositions for inhibiting and modulating the actions of CXC intercrine molecules. Disclosed are peptide compositions which inhibit interleukin 8 ( IL- 8 ) and, particularly, which preferentially inhibit IL-8-20 induced ralease of degradative enzymes by neutrophils. -These compositions may be employed to treat various infli tory diseases and disorders including the Adu~t Respiratory Distress Syndrome (ARDS) and cystic fibrosis.
25 2. De~3criPtion of the Related Art IL-8 is a member of the CXC intercrine family of cytokines, so named due to elements of their N-terminal sequences. This family also includes, amongst others, 30 peptide molecules known as growth related oncogene (GRO, or GRO/MGSA) and macrophage infl: tory protein 2~
(MIP2,(~). IL-8 is a peptide of approximately 8 kD, and is about 72 amino acids in length, with this length varying according to the post-translational processing in 35 different cell types (Yoshimura et al., 1989; Hebert et al., 1990; Strieter et al., 1989) . The IL-8 gene was first identified by analyzing the genes transcribed by

2 1 7~927 WO 95116702 ~ ; ~; P~ Yall~L45 human blood i ~lear cells stimulated with Staphylococcal enterotoxin A (Schmid & Weissman, 1987).
IL-8 production is known to be induced by tumor necrosis factor and interleukin 1 (Strieter et al., 1990).
IL- 8 interacts with at least two distinct receptors on neutrophils (Holmes et al., 1991; Murphy & Tiffany, 1991). The receptors are coupled to GTP-binding proteins, allowing transmission of the .IL-8 signal into 10 the cell (Wu et al., 1993). While most of the members of the intercrine family, such as GRO and MIP2~, bind to one of the receptors, IL- 8 binds to both of the IL- 8 receptors (LaRosa et al., 1992; Cerretti et al., 1993).
The three dimensional structure of IL-8 has been 15 elucidated by NMR (Clore et al., 1990) and by X-ray crystallography (Clore ~ Gronenborn, 1992; Baldwin et al_, 1991). A freely movable amino terminal end is followed:by three beta pleated sheets and an alpha helix is located at the carboxyl-terminal end (Dppenheim et 20 al., 1991). Several lines of evidence suggest that both the amino- and carboxyl-terminal ends are involved in binding to its receptors (Clore et al., 1990; Clark-Lewis et al ., 1991; Moser et al ., 1993 ) .
Certain functions of the CXC intercrines have been elucidated by several laboratories (Yoshimura et al., 1989; Schroder et al., 1988; Peveri et al., 1988). For example, the major functions of the IL-8 peptide appear to be related to its ability to stimulate neutrophil chemotaxis and activation (Larsen et al., 1989; Schroder et al., 1988; Peveri et al., 1988; Yoshimura et al., 1987) and to promote angioge~nesis (Koch et al., 1992).
If neutrophils are `primed', e.g., by agents such as surface adherence or 1~. coli endotoxin (also known as lipopolysaccharide or LPS), IL-8 also stimulates the release of neutrophil enzymes such as elastase and myeloperoxidase .

WO95/16702 21 78q27 P ~ i~4~

Although the neutrophil inflammatory response is essential for the destruction of bacteria which are invading the body, inappropriate neutrophil activation causes several problems. For example, if the neutrophils are properly primed when attracted to the lungs, they release destructive enzymes into the lung tissue. This can lead to the development of adult respiratory distress 2Iylldr~ - (ARDS) (Weiland et al., 1986; Idell et al., 1985). ARDS attacks between 150,000 and 200,000 Americans per year, with a mortality rate of 50-809~ in the best clinical facilities (Balk & Bone, 1983). ARDS
is initiated by bacterial infections, sudden severe dropping Qf the blood pressure (shock), and many other insults to the body. Recent studies have demonstrated that IL-8 is the major neutrophil activator in the lungs of patients with ARDS (Miller et al., 1992), and primate models of endotoxin shock also implicate IL-8 as a causative agent (Van Zee et al., 1991).
Xigh ~ nr~ntrations of IL-8 have also been found in inflammatory exudates in other disorders and pathological conditions in which IL- 8 is thought to play an important pathogenic role (Brennan et al., 1990; Miller & Idell, 1993; Miller et al., 1992). For example, IL-8 has also been implicated as a possible mediator of inflammation in rheumatoid arthritis (Brennan et al., 1990; Seitz et al., 1991) and pseudogout (Miller & Brelsford, 1993); and to have a role in cystic fibrosis (McElvaney et al., 1992;
Nakamura et al., 1992; Bedard et al., 1993). Therefore, modulation of I~-8 function appears to be good strategy to control a variety of pathological conditions.
Some progress has recently been made in identifying compounds capable of reducing IL-8 synthesis. Such cornrol1nrl~ include IL-4, oxygen radical scavengers, secretory leukoprotease inhibitor and interferon gamma (Standiford e~ al., 1990; DeForge et al., 1992; McElvaney WO Y5/16702 ~- . t ~ Y~ 4 et al., 1992; Cassatella et al., 1993a; 1993b), however, such studies do not concern~ IL-8 inhibitors . Other diverse compositions, including protein kinase C
inhibitors, IL-4, and anti-IL-8 antibodies, have also 5 been reported to modulate IL-8 actions (Lam et al., 1990;
Standiford et al., 1992; Mulligan et al., 1993).
Unfortunately, these compou~ds are far from ideal a6 candidates for use a~ IL-8 inhibitors in a clinical setting .
Certain progress has also been made in identifying peptide IL-8 inhibitors, however, most of such work has focused on portions of the IL-8 molecule itself (Miller et al., 1990; Gayle et al., 1993) . For example, the 15 present inventors have shown that synthetic peptides, and particularly, IL-8 amino t~rn;n;ll peptides, inhibit IL-8 binding to neutrophils and ~eutrophil chemotaxis (Miller et al., 1990; Miller et al., 1993). An N-terminal pentapeptide IL-8 inhibitor has also been reported 20 (Goodman et al_, 1991). ~:rnfortunately, to date, the .
inhibitory function of IL-8 derived peptides has proven incomplete and insuf f icient .
As particularly effective peptide inhibitors of CXC
25 intercrines such as IL-8 have yet to be identified, it seems to be clear that compositions other than the IL- 8 molecule itself now need to be investigated. The identification of peptide inhibitors capable of preferentially inhibiti3lg neutrophil enzyme release in 30 comparison to chemotaxis would be a particularly advantageous discovery as this would enable neutrophils to enter the lungs and defend against bacterial invasion and yet not cause tissue damage.

~095116702 ~ 21 7~27 r~".J.,7~

S~RY OF l~IE lNVI:.l`l I lU~Y
The present invention seeks to overcome the drawbacks inherent in the prior art by providing new 5 methods and compositions for modulating and inhibiting the actions of CXC intercrine molecules such as IL- 8, GRO
(GRO/MGSA) and MIP2~. The peptides and pharmacological compositions disclosed reduce IL-8, GRO and MIP2~B binding to neutrophils and inhibit IL-8-induced neutrophil 10 activation. These peptide formulations are particularly advantageous as they are capable of inhibiting IL-8-induced enzyme release at significantly lower r~nf-~ntratione than is required to inhibit neutrophil chemotaxis. Also provided are methods for treating 15 various diseases and disorders, particularly inf lammatory diseases, in which the unrestrained actions of CXC
intercrines play a role.
The invention is generally based upon the inventors 20 surprising discovery that relatively small peptides including the amino acid se~uence Arg Arg Trp Trp Cys Xaal (RR~WCX; SBQ ID NO:23), wherein Xaal is any amino acid residue, are potent inhibitors of CXC intercrine molecules such as IL-8. As used herein, the terms "CXC
25 intercrine family molecules" and "CXC intercrines'~ are used collectively to ref er to the group of peptide intercrines which include the CXC sequence motif in their N-terminal regions. CXC intercrines are known to include IL-8, GRO, MIP2~, MIP2,(3 and ENA78, all of which

3 0 molecules, and any other intercrine polypeptides that include the CXC motif, will be understood to fall within this term as used in the present application.
The inhibitory peptides of the present invention may 35 be termed "antileukinates". Certain hexamer peptides of the sequence RRWWCX (SEQ ID NO:23) have been previously shown to have anti-bacterial activity against WO 95116702 . ~ /I~-15 Staphylococcal aureus ~Houghten e~ al., l99l) . However, there was no previously rlnc~ nted information to suggest that any such peptides would have the advantageous anti-cytokine/intercrine, anti-neutrophil and anti-5 inf lammatory activities disclosed herein.
In certain aspects, the present invention theref orernn~-PrnR methods for inhibiting CXC intercrines, such as GRO and MIP2~ or MIP2~, and most particularly, methods lO for inhibiting IL-8. As used herein, the term ~inhibiting CXC intercrines" refers to the processes by which the biological actions of the CXC intercrines are reduced This may be particularly assessed by inhibiting their binding to one of the I3~- 8 receptors on their 15 target cells, such as neutrophil~, although any mode of determining CXC intercrine inhibition may be employed.
The term IL- 8 is used to ref er to the cytokine compositions previously known as neutrophil-activating 20 factor, monocyte-derived neutrophil-acti~ating peptide, monocyte-derived neutrophil-chemotactic factor and neutrophil-activating peptide-l As used herein, the term "inhibiting IL-8" generally refers to the processes by which the biological actionR of IL- 8 are reduced or 25 lessened. This includes the inhibition of any or all of the known actions of IL- 8 . These actions include modulating sub-~ r effects, 3uch as receptor binding or altering cytosolic calcium levels; modulating cellular effects such as granulocyte recruitment and activation;
30 and also affecting phybiological effects, such as inf lammation and angiogenesis .
In preferred embodiments, the inhibition of IL-8 function referred to in this application is the 35 inhibition of IL-8 action on granulocytes such as neutrophils (polymorphonuclear neutrophils, PMN) This may be determined in many cellular and physiological Wo 95116702 ` ~ ` 2 ~ 7 ~ ~ 2 7 ~ _~/U~9~1~5 ways, as disclosed herein. For example, by measuring inhibition of I~- 8 binding to purif ied receptor compositions or neutrophils; by determining the inhibition of IL-8-induced neutrophil chemotaxis or 5 diapedesis; by measuring the inhibition of IL- 8 -stimulated neutrophil enzyme release (e.g., myeloperoxidase, ~-glucuronidase or elastase release) or superoxide production; or by assaying for anti-;nfli tory effects in vivo, e.g., using a rabbit model lO of dermal inflammation.
The preferred manner of detrrmin;n~ IL-8 inhibition, or indeed GRO or MIP2,~ inhibition, is to assay for a reduction in the intercrine binding to neutrophils, which 15 is the most simple and straightforward method. In addition, binding of the particular intercrine to its receptor (s) must precede any other action that it has on neutrophils or other cell types . " Inhibition" of intercrines, as exemplified by the inhibition of I~-8, 20 G~O or MIP2cY or MIP2~ binding to neutrophils, refers to the capacity of a given peptide or composition to inhibit intercrine binding to any detectable degree, i . e . to reduce binding below the levels observed in the absence of the peptide or composition.
The inhibition of CXC intercrine binding to neutrophils may be expressed as a 9~ Binding Inhibition value, with the higher figures representing the more effective inhibitors. The preferred peptides will 30 generally have the higher ~ binding inhibition figures.
Naturally, the 96 binding inhibition calculated will depend upon the precise assay conditions, such as the rrnr~ntration of CXC intercrine and the concentration of the given peptide or composition. Conditions such as 35 those used to generate the data of Tables lA, lB, 5A and 5B, may be employed to determine whether a given peptide has any inhibitory activity. Howev-r, one may choose to WO95116702 ; ~ ~ 2 1 78q27 .~ Y~/1 ~5 . ,.,, , --employ more discriminatory conditions, such as those using lower peptide concentrations, e.g., on- the order of about 20 ~lM (aæ used to generate the data of Figures 1 and 9), where one desires to obtain particularly accurate 5 quantitative or comparative data. In any event, the determination of whether a peptide or analogue is capable of inhibiting a CXC intercrine, such as IL-8, is a straightforward matter readily achieved using assays such as those disclosed herein.

Alt~ough an understanding of the --~h~n; r~ of action of the CXC intercrine ;nh;~;tnrs iB not relevant in terms of their practical utility, it is, however, important to note that the peptide inhibitors of this invention ~are 15 capable of preferentially inhibiting IL-8-induced neutrophil enzyme release at lower r~nne~ntrations than IL-8-induced chemotaxis. In this sense, the term "inhibiting", when used in connection with this inventio~, also means "modulating" in that certain 20 neutrophil functions are more significantly inhibited than others .

The ability of the peptides to inhibit IL-8-induced neutrophil degradative enzyme release at about a 25 times 25 lower concentration than is re~uired to inhibit IL-8-induced neutrophil chemotaxis is an important discovery that could not have been predicted from prior studies.
This means that neutrophils may still be recruited to a site of injury, but that the detrime~tal effects of the 30 enzymes that they would normally release will be aignif icantly reduced. This property, coupled with their small size, renders these type of peptides ideal for use in various treatment protoc~ls and especially in the treatment of lung inj ury .

To achieve CXC intercrine inhibition, such as IL-8, GRO or MIP2 inhibition, or~ to preferentially reduce ~0 9511~702 2 ~ 7 ~ 9 ,~ 7 p~ y~l~4~
neutrophil enzyme release in comparison to neutrophil chemotaxis, in accordance with this invention one would generally contact the CXC intercrine family molecule or or intercrine target cells, such as granulocytes or 5 neutrophils, with a biologically effective amount of a composition comprising a peptide of the family disclosed herein. The "contact" process is the process by which the active peptide or peptides from within the composition contact either the CXC intercrine peptide or lO one of their receptors present on a target cell, or both, and reduce or inhibit their functional interaction.
Although of scientific interest, the mech;lni ! by which the CXC intercrine signals transmitted to a given cell are reduced are not relevant to the practice of the 15 invention.
To contacl: a CXC intercrine or intercrine target cell ~ith a peptide~ nt~;ning composition one may simply add the peptide or composition to target cells, such as 20 neutrophils, and intercrines in vitrc. Alternatively, one may administer a biologically effective amount of a pharmacologically acceptable form of the peptide or composition to an animal , where it will contact , e . g ., neutrophils or macrophages and intercrines in a 25 = biological fluid in vivo. In this context, "contact" is achieved simply by administering the composition to the animal. Virtually any pharmaceutical peptide formulation may be used, including, but not limited to, formulations for parenteral administration, such as for intravenous, 30 intramuscular and subcutaneous administration; inh;~ ntc, aerosols and spray formulations; formulations of peptides for topical use, such as in creams, oin tc and gels;
and other formulations such as peptides with lipid tails, peptides encapsulated in micelles or liposomes and drug 35 release capsules including the active peptides incorporated within a bio~ tihle coating designed for slow-release .

2 1 7~927 Wo 95116702 I ~ Ui~Y.~ 45 Increased levels of IL-8 are known to be present in lung edema fluids in patients with ARDS (Miller et al., 1992) and in the sputum of patients with cystic fibrosis (Richman-Eisenstat et al . , 1993 ); in pleural spaces of 5 patients with pleural effusions (Miller h Idell, 1993);
in joint fluids from patients with several kinds of joint disease (Brennan et al., l990; Miller & Brelsford, 1993), in psoriatic pla~ues and in synovial fluid from arthritic patients (Lam et al, 1990). Inappropriate neutrophil lO activation is connected with all such disorders and with ischemic and reperfusion injuries (DeForge et al., 1992) .
As the inhibition of IL- 8 neutrophil recruitment has been shown to reduce lung 1 nfl iqmrniltion ln vivo (Mulligan et al., 1993), and as the type of in vitro studies employed 15 herein are accepted as being predictive of i~ vivo activity (see U.S. Patent, 5,079,228, incorporated herein by reference), the highly successful inhibition of IL-8-induced neutrophil activation disclosed in the application supports the broad clinical utility of these 2 o p~ptides . . ~ . .
The present i~vention theref ore also provides methods f or treating a wide variety of diseases and disorders in which CXC intercrines, particularly IL- 8, 25 play a role, especially those which have an ;nfl; tory component . This includes treating subj ects with lung injuries and disorders, including bronchial ;nfli tion, such as chronic bronchitis, cystic fibrosis, pleural effusions, asthma, and ARDS; skin disorders such as 30 psoriasis and dermatitis; diseases of the joints, including rheumatoid arthritis; and generally reducing inflammation in other clinical settings, such as in the treatment of pseudogout, inflammatory bowel disease or reperfusion cardiac damage after myocardial infarction.
35 These peptides could even be used as anti-proliferative agents to downregulate lymphocyte proliferation, for example, in the treatment of cancer and other diseases ~V095116702 ; r~ ,3 ~b~ ~ P~.l/tJ~Y~I~JS
~ ~7~927 and disorders associated with increa3ed t-t?l 1l1 Ar proliferation .
To treat any one o~ the above conditions, or any 5 other disorder influenced by neutrophil activity and characterized by inflammation, one would identify a patient having the particular inf~: tt~ry or I~-8-linked disease and then administer to the patient, preferably parenterally, a biologically effective amount of a 10 pharmaceutical composition which includes one or more peptides of the family disclosed herein.
Naturally, one would generally tailor the particular pharmaceutical formulation according to the disease or 15 disorder being treated. For example, in methods to treat skin disorders, a topical cream or gel formulation would be used, whereas in methods to treat pulmonary disorders, injectable formulations, or even a spray, aerosol or ;nh tl ;-nt, may be employed. In methods to re~uce 20 ;nfl tion in other areas of the body, one may use peptides formulated for parenteral administration or peptides incorporated in a bioct _ ~t;hle coating designed for slow-release. T ;r~ Rt~mt~-encapsulation may be employed, which is known to increase the efficacy and 25 significantly prolong t~e half-life of ad~inistered compounds, particularly those of lower m~'ecular weight such as the peptides disclosed herein. ~3rious compositions and technir;ues for preparinr~ all such pharmaceutical formulations will generally be known to 3 0 those of skill in the art in light of the present disclosure. For a detailed listing of suitable - pharmacological compositions and associated administrative techniques one may wish to refer to Re~7tngton'~: Pharmaceutical Sciences, 16th ed., 1980, Mack 35 Publishing Co., incorporated herein by reference.

WO 95116702 2 ~ 7 8 9 2 7 . ~ iY~/i~45 I~-8 or CXC intercrine inhibition is achieved by using a biologically effective amount of the inhibitory peptide or peptides AY used herein, a "biologically effective amount" of a peptide or composition refers to 5 an amount effective to inhibit the actions of I~-8 or the particular intercrine. For~example, in regard to Il,-8 inhibtion, an appropriate amount would be that effective to reduce neutrophil enzyme release, particularly in comparison to chemotaxis As disclosed herein, a variety 10 of different peptide concentrations are very effective in ~ltro, such as those between about lQ0 ,uM and about 20 /lM. Clinical doses which result in similar a local concentration of peptides are therefore contemplated to be particularly useful.
Naturally, in a clinical context, the quantity and volume ~f the peptide composition administered will depend on the host animal and condition to be treated and the route of administration. The precise amounts of 20 ac'cive peptide required to be administered will depend on the judgment of the practitioner and may be peculiar to each individual. ~lowever, in light of the data presented herein, the determination of a suitable dosage range for use in humans will be straightforward. For example, in 25 treating ARDS or cystic fibrosis, doses in the order of about 0 83 mg/kg body weight/hour (mg~kgJhr) to about 16.56 mg/kg/hr, preferably about 0.83 mg/kg/hr to about

4.14 mg/kg/hr, and more preferably about 1 66 mg/kg/hr of active ingredient peptide per individual are 30 contemplated.
The compositions for use in inhibiting CXC
intercrines, such as I~-8, GR0 and MIP2~ or MIP~, in accordance with the present invention will be 35 compositions that contain a relatively small peptide, generally of from 6 to about 14 residues in length/ which includes within its sequence the amino acid sequence Wo9S/16702 ~ 2 1 ~ Y3Ji~
RRWWCX ~SEQ ID N0:23) . The term "a peptide~ in this sense means at least one peptide, and may refer to one or more such peptides which include a sequence in compliance with the general formula RRWWCX (SEQ ID N0:23).
The relatively small peptides Pn~0~3ed by the present invention may be any length between six residues and about 14 or 15 or so residue6 in length, with the precise length not being an important feature of the 10 invention. There are many advantages to using smaller peptides, for example, the cost and relative ease of large scale synthesis, and their improved pharmacological properties, such as the ease with which they can penetrate tissues and their low immunogenicity.
In addition to including an amino sequence in accordance with the sequence RRWWCX (SEQ ID N0:23), the peptides may include other short peptidyl sequences of various amino acids. For example, in certain 20 embodiments, the peptides may include a repeat of the sequence RRWWCX (SEQ ID N0:23) or RRWWCXX (SEQ ID N0:57) .
They may also contain additional sequences including, e.g., short targeting sequences, tags, labelled residues, amino acids contemplated to increase the half life or 25 stability of the peptide, or indeed, any additional residue desired for any purpose, 80 long as they still function to inhibit intercrines such as IL-8 - which can be readily determined by a simple assay such as those described herein.
Amino acids which may incorporated into the peptides include all o :the commonly occurring amino acids. Two designations~ for amino acids are used interchangeably throughout this application, as is common practice in the 35 art: Alanine = Ala (A); Arginine = Arg (R); Aspartic Acid = Asp (D); Asparagine - Asn (N); Cysteine = Cys (C);
Glutamic Acid = Glu (E); Glutamine = Gln (Q); Glycine =

W095/16702 . j - 2 1 78927 1~ Y.~ 45 Gly (G); Xistïdine = His (H); Isoleucine = Ile (I);
Leucine = Leu (L); Lysine - Lys (K); Methionine = Met (M); Phenyl ~1 ~n; nP = Phe (F); Proline = Pro (P); Serine =
Ser (S); Threonine= Thr (T); Tryptophan = Trp (W);

5 Tyrosine = Tyr (Y); Valine= Val (V).
Any of the so-called rare or modified amino acids may also be incorporated into a peptide oi the invention, including the following: 2-Pm;n~ ;ric acid, 10 3 -PnM nr~ ; ric acid, beta-Alanine (beta-Aminopropionic acid), 2-Aminobutyric acid, 4-Aminobutyric acid (piperidinic acid), 6-Aminocaproic acid, 2-7'n~;n~ Prtanoic acid, 2-Aminoisobutyric acid, 3-Aminoisobutyric acid, 2-Aminopimelic acid, 2,4-Diaminobutyric acid, Desmosine, 15 2, 2 ' -Diaminopimelic acid, 2, 3-Diaminopropionic acid, N-Ethylglycine, N-Ethylasparagine, Hydroxylysine, allo-Xydroxylysine, 3-Hydru~y~Luline, 4-Hydroxyproline Isodesmosine, allo=Isoleucine, N-Methylglycine (sarcosine), N-Methylisoleucine, N-Methylvaline, 20 Norvaline, Norleucine and Qrnithine.
The inhibitory compositions of the invention may include a peptide modified ~to render it biologically protected. Biologically protected peptides; have certain 25 advantages over unprotected peptides when administered to human subjects and, as disclosed in U.S. Patent 5,028,592 (incorporated herein by reference), protected peptides often exhibit increased pharmacological activity, as was found to be true in the present case.
The present invention theref ore encompasses compositions comprising an acylated peptide or peptides, and preferably, a peptide acylated at the N-terminus.
Although virtually any acyl group may be employed in this 35 context, the inventors have ~ound that the addition of an acetyl group to the N-termInus of a given peptide also renders the resultant peptide surprisingly effective at wo 95116702 ' 2 ~ 7 8 9 2 7 . ~l,U~YJil~
.

inhibiting intercrines such as IL- 8 . The inhibitory peptide compositions may also include a peptide (s) which iB amidated at the C-terminus, i.e., to which an NH2 group has been added. In particularly preferred 5 embodiments, peptides which have both an acylated N-terminal and an amidated C-terminal residue are preferred as they are believed to most closely mimic natural protein and peptide structure.
Compositions for use in the present invention may also comprise peptides which include all L-amino acids, all D-amino acids or a mixture thereof. The finding that peptides composed entirely of D-amino acids have potent inhibitory activity is particularly important as such peptides are known to be resistant to proteases naturally found within the human body and are less immunogenic and can therefore be expected to have longer biological half lives .
The anti-intercrine and anti-IL-8 compositions of.
the present invention will generally comprise one or more peptides which include an amino acid sequence in accordance with those set forth in SEQ ID NO:l or SEQ ID
NOS: 24 through 42 . In certain embodiments, short hexamer peptides may be preferred. In such cases, the inhibitory compositions will generally comprise one or more peptides which have an amino acid se~uence in accordance with those set iorth in SEQ ID NO: l or SEQ ID NOS: 24 through 42, presented below:
Arg Arg Trp Trp Cys Arg ~ SEQ ID NO: l ) - Arg Arg Trp Trp Cys Ala (SEQ ID NO:24) Arg Arg Trp Trp Cys Cys (SEQ ID NO:25) - Arg Arg Trp Trp Cys Asp (SEQ ID NO:26) Arg Arg Trp Trp Cys Glu (SEQ ID NO:27) Arg Arg Trp Trp Cys Phe ( SEQ ID NO: 2 8 ) Arg Arg Trp Trp Cys Gly ~SEQ ID NO:29) WO9~116702 . .; 2~789~7 ~ ,Y3~ 4~ ~

Arg Arg Trp Trp Cy9 His ( SEQ ID NO: 3 0 ) Arg Arg Trp Trp Cys Ile (SEQ ID NO:31) Arg Arg Trp Trp Cys Lys (SEQ ID NO:32) Arg Arg Trp Trp Cys Leu (SEQ ID NO:33) Arg Arg Trp Trp Cys Met (SEQ ID NO:34) Arg Arg Trp Trp Cys Asn (SEQ ID NO:35) Arg Arg Trp Trp Cys Pro (SEQ ID NO:36) Arg Arg Trp Trp Cys Gln (SEQ ID NO:37) Arg Arg Trp Trp Cys Ser (SEQ ID NO:38) Arg Arg Trp Trp Cys Thr (SEQ ID NO:39) Arg Arg Trp Trp Cys Val (SEQ ID NO:40) Arg Arg Trp Trp Cys Trp (SEQ ID NO:41) Arg Arg Trp Trp Cys Tyr (SEQ ID NO:42) In other: ',o~;~onts, the inhibitory compositions of the invention may include one or more peptides which include a sequence in accordance with the amino acid sequence Arg Arg Trp Trp Cys Arg Xaa2 (SEQ ID NO:2). In 20 these cases one of the variable positions has been defined as arginine and the: L~ in;n~ Xaa2 may be any amino acid residue. Such sequellces are exemplified by those set forth in SEQ ID NOS:3 through 22. Where~short heptamer peptides are preferred, the compositions will generally comprise one or more peptides which have an amino acid sequence in accordan~ with those set f orth below:
Arg Arg Trp Trp Cys Arg Ala ~SEQ ID NO: 3 ) 3 0 Arg Arg Trp Trp Cys Arg Cys (SEQ ID NO: 4 ) Arg Arg Trp Trp Cys Arg Asp (SEQ ID NO:5) Arg Arg Trp Trp Cys Arg Glu (SEQ ID NO:6) Arg Arg Trp Trp Cys Arg Phe (SEQ ID NO:7) Arg Arg Trp Trp Cys Arg Gly (SEQ ID NO: 8 ) Arg Arg Trp Trp Cys Arg His (SEQ ID NO: 9~
Arg Arg Trp Trp Cys Arg Ile (SEQ ID NO:10) Arg Arg Trp Trp Cys Ar~ Lys (SEQ ID NO :11) ~j095116702 ` ' ~ , ~l 7~927 .~ Y~ ~S

Arg Arg Trp Trp Cyb Arg Leu (SEQ ID N0 :12 ) Arg Arg Trp Trp Cys Arg Met (SEQ ID N0 :13 ) Arg Arg Trp Trp Cyb Arg Asn (SEQ ID N0 :14 ) Arg Arg Trp Trp Cyb Arg Pro (SEQ ID N0:15) Arg Arg Trp Trp Cyb Arg Gln ~SEQ ID N0:16) Arg Arg Trp Trp Cyb Arg Arg (SEQ ID N0:17) Arg Arg Trp Trp Cyb Arg Ser (SEQ ID N0:18) Arg Arg Trp Trp Cyb Arg Thr (SEQ ID N0:19) Arg Arg Trp Trp Cyb Arg Val ( SEQ ID N0: 2 0 ) Arg Arg Trp Trp Cyb Arg Trp (S~Q ID N0:21) Arg Arg Trp Trp Cyb Arg Tyr (SEQ ID N0:22) The invention also contemplates the ube of peptideb 15 having the amino acid sequence Gln Ile Pro Arg Arg Ser Trp Cyb Arg Phe Leu Phe (SEQ ID N0:52), either alone, or more preferably, in combination with one or more of the other peptides debcribed above. The successful ube of thib dodecamer illubtrateb both the fact that longer 20 peptideb are buccessful and that certain biologicaily functional equivalent peptides are active. All such active equivalents therefore fall under the scope of the present invention.
The compositions for use in the inhibitory methods debcribed herein may contain only a single active peptidyl species. Alternatively, they may contain more than one peptide, up to and including about 40 or 45 or so distinct peptideb. Any and all of the varioub 3 0 combinationb are contemplated, such as compositions comprising 2, 3, 5, 10, 15, 20, 30 or 45 or so distinct peptides .
Compobitionb compribing peptideb having the amino acid sequence Arg Arg Trp Trp Cys Arg (SEQ ID N0:1) and/or the amino acid sequence Arg Arg Trp Trp Cys Arg Cyb (SEQ ID N0:4) are contemplated to be particularly W095116N2 ` . ``' 2 1 7 ~ 9 2 7 ~ Y~ 245 useful, although the invention is not limited to these peptides in any way. In this regard, it is important to note that considerations other than in vitro activity, such as plasma half life and stability, may be considered 5 in ultimately choosing peptides which are preferred for clinical ~mho~l;r-ntR. The effects of different amino acid substitutions on these parameters may be readily determined and the results used to design the optimum peptide or combination of peptides for use in vivo.
The RRWWCX (SEQ ID N0:23) sequence element is an important feature of the peptides of this invention.
However, this does not exclude certain biological functional equivalents from falling within the scope of 15 the invention. For example, the inventors have discovered that the first tryptophan in RRWWCX (SEQ ID
N0:23) can be exchanged, e.g., by replacing with serine, with only modest loss of activation. Therefore, one example of equivalents ,onl- ~RRed by the inventio~ are 20 peptides of the sequence RRXWCX (SEQ ID NO:58) .
"Equivalent amino acids" may be defined as amino acids whose hydrophilic or hydropathic index are within * 2;
more preferably, within ~ 1, and most preferably, within : 0.5 of each other. Of course, to be a ~functional 25 equivalent", a peptide must still retain its intercrine or IL-8 inhibitory activity, as may be easily determined using assays such as those disclosed herein.
In addition to the peptidyl compounds described 30 herein, the inventors also contemplate that other =
sterically similar compounds, called peptidomimetics, may be formulated to mimic the key portions of the peptide structure. Such compounds may be used in the same manner as the peptides of the invention and hence are also 35 functional equivalents. The generation of a structural functional equivalent may be achieved by the techniques of modelling and chemical design known to those of skill Wo 95/16702 ~ 7 8 9 2 7 ~ IS

in the art. It will be understood that all such sterically similar constructs fall within the scope of the present invention.
The peptides and compositions for use in the invention may be prepared by any one of a variety of different methods. One preferred method for preparing peptides in accordance with the present invention is contemplated to be via automated peptide synthesis. A
synthetic peptide may be straightforwardly prepared using an automated peptide synthesizer, the operation of which will be generally known to those of skill in the art.
This method is one of those generally preferred for preparing large quantities of a given peptide , e . g ., once a particular peptide has been chosen for therapeutic use.
Another preferred method for preparing inhibitory peptides, and the biological functional equivalents thereof, is to use a combinatorial peptide library method, as described by Houghten et al. (l991) and disclosed in International Patent Application PCT
WO 92/09300, the entire disclosure of which is specifically incorporated herein by reference. These methods are particularly useful for preparing and analyzing a plurality of peptides having a substantially predetermined sequence, such as RRWWC, to which is appended a variety of different amino acids at one or more positions. These methods may be used to synthesize a peptide mixture for direct use in the fQrmulation of a composition in accordance with the invention or to identify a particularly active peptide for subsequent indivi dua l synthe s i s .
- If desired, peptides may also by prepared by 35 molecular biological means and the "recombinant" peptide obtained from reco"~h; nilnt host cells which express the peptide. o achieve this, one would prepare a specific W09511670Z ~1 7~27 oligonucleotide, based upon the sequence of the desired peptide, as is known to those of skill in the art, and then insert the oligonucleotide into an expression vector, such as any one of the many expression vectors 5 currently available commercially. One would then transform a prokaryotic or eukaryotic host cell with the vector, where it will direct the expression of the 80- .
called recombinant version of the peptide, which may then be purified from the recombinant host cell This lO methodology is standard practice in the art (see e_g., S ambrook e t al ., 1 9 8 9 ) .
BRIEF DES~.:K~ ~,. OF THE Dl?A~T~
The following drawings~ form part of the present specification and are inrl~ 1 to further:demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of 20 these drawings in combination with the detailed description of specif ic l~mhorl i t c presented herein .
Flgure l. Binding Inhibition by Ac-RRWWCX ~SEQ ID NO:23) Series. Twenty peptides which have structure of Ac-RRWWC
25 (SEQ ID NO:56) plus one of the 20 standard protein amino acids in the sixth position were tested. The notation on the x axis indicates the residue at the carboxy-terminal position (SEQ ID I~O l and 24 through 42) . In this study, neutrophils were incubated with l pM l25I-labeled IL-8 3 0 and 2 0 ~M of each peptides . ~
Figure 2. IL-8 Binding Inhibition by Ac-RRWWCR-NX2 (SEQ
ID NO:l) . Neutrophils (lx106) in 0.2ml of PBS c~nt~;ninr~
0.1% BSA were incubated with lnM l25I-labeled IL-8 and 35 increasing cnnrPntrations of Ac-RRWWCR-NH2 (SEQ ID NO:l) for 90 min at 4C. These data are representative of four studies .

~ O 95/16702 ~ 7 ~ 9 2 7 ~ Y~/1~5 Figure 3A. Saturation Studies in the Presence of Ac-RRWWCR-NH2 (SEQ ID NO:1), Binding Isotherms with Best Fit Curve Calculated Using Lundon I. Binding assays were performed in the absence of the peptide (O) and in the 5 presence o~ lO~LM (--), 20~LM (v), or 40/1M (-) peptide with increasing nnnF~ntrations of 125I-labeled IL-8. Each data point represent specific binding which was computed by subtracting nonæpecif ic binding in the presence of excess unlabeled IL-8 from total binding.
Figure 3B. Saturation Studies in the Presence of Ac-RRWWCR-NH2 (SEQ ID NO:1), Scatchard Plots. Binding assays were perf ormed in the absence of the peptide (O) and in the presence of lO~M (--), 2011M (v), or 4011M (-) 15 peptide with increasing concentrations of 125I-labeled IL-8 .
Figure 4. Binding Inhibition Studies in the Presence of Ac-RRWWCR-NH2 (SEQ ID NO:1). Neutrophils were incubated 20 with lnM of 125I-labeled IL-8 and increasing rAn~-~ntration of unlabeled IJ.-8 in the presence (--) or the absence (O) of lOIlM Ac-RRWWCR-NH2 (SEQ ID NO:1) .
Figure 5. Effect of Ac-RRWWCR-NH2 (SEQ ID NO:1) on 25 Binding of IL-8, C5a, and Leukotriene B4 to Neutrophils.
The bindings assays were performed with lnM 125I-labeled IL8 (--), 0.25nM 125I-labeled C5a (v), or 0.4nM 3H-labeled leukotriene B4 ( ) and increased concentration of Ac-RRWWCR-NH2 (SEQ ID NO:1) . Analysis of variance was used 30 for multiple comparison. When there was significant difference, the differences between binding without the - peptide and those with peptide were tested using Sheffés test; *~, p~0.001.
35 Figure 6. Cytotoxicity Test. Chromium-labeled neutrophils were ;n-llh~te-l with increasing ~nn~ntrations of Ac-RRWWCR-NH2 (SEQ ID NO:1) in PBS containing 0.1~ BSA

WO 95/16702 ~ ~ ~ ; t ~ 2 1 7 8 9 2 7 P~~ Y~i~5 for 90 min at 4C ¦--) or in RPMI-1640 media cnnt~;nin~ 196 BSA for 30 min at 37C (O). Analysis of variance was used for multiple comparison. When there was significant difference, the differences between ~ lysis without the 5 peptide~and those with peptide were tested using Sheffés test; ***, p~0.001~
Figure 7. The Effect of Ac-RRWWCR-NH2 (SEQ ID NO:1) on Neutrophil Chemotaxis. Chemotaxis was performed in the 10 presence of increasing concentration of Ac-RRWWCR-NH2 (SEQ ID NO:1) in both upper and lower chamber. Stimulants added to lower chamber were lOnM IL-8 (--), lOnM fMLP (--), or media alone (O) as control. Analysis of variance was used for multiple comparison. When there was significant 15 difference, the differences between distance migrated without the peptide and those with peptide were tested using Sheffés test; *, O.Ol~pc0.05, ***, p~0.001.
Figure 8. The Effect of Ac-RRWWCR-NH2 (SEQ ID NO:1) on 2 ,~-glucuronidase Release. Neutrophils pretreated with cytochalasin B were incubated with lOOnM IL-8 (--), lOOnM
fMLP (--), lOOnM C5a (v) or lO~nM leukotriene B4 (-) or without any st; m~ nt (O) in the presence of increasing concentration of Ac-RRWWCR=NH2 (SEQ ID NO:1) for 30 min 25 at 37C. ~-glucuronidase activity of supernatants were measured using phenolphthalein-glucuronic acid as substrate. Analysis of variance was used for multiple comparison. When there was significant difference, the differences between distance migrated without the peptide 30 and those with peptide were tested using Sheffés test;
*, p~o.o5, **, p~0.01, ***, pcO.001.
Figure 9. Binding Inhibition by All D-amino Acid Ac-rrwwcrx-NH2 (SEQ ID NO:2) Series. Ac-RRW~CR (SEQ ID
35 NO:1) was synthesi~ed using D-amino acids and added each of the 20 standard protein D-amino acids were added at the seventh position (SEQ ID NO:3 through 22) . The WO95/16702 ~ ` 21 7~ 927 r~ Y~ 4s .

notation on the x axis indicate i:he residue at the carboxyterminal position. The binding 6tudy was performed using 10 IlM of each peptide. Ac-RRWWCR-NH2 (SEQ ID NO:1) made with L-amino acids was used as a 5 control.
Figure 10. Ac-RRWWCX (SEQ ID NO:23) Inhibits Binding of GRO and MIP2~ to human neutrophils. Radioiodinated MIP2,(~
and GRO/MGSA were mixed with various concentration of 10 Ac-RRWWCR-NH2 and incubated at room temperature for 15 minutes. Neutrophil suspension (1 X 106 cells in 160~1 PBS cr nt~;n;ng 0.1~ BSA) was added to 40~1 of the mixture and incubated for 90 minutes on ice. The radioactivity bound to the cells was separated from free 15 radioactivity by centrifugation through an oil layer.
The ~ bLnding inh;h;t;on was calculated as follows:
9~ binding inhibition = ~ 1 _ B NSP ¦ x 100 where B is bound radioactivity in the presence of the 25 peptiae, T is bound radioactivity in the absence of the peptide, and NSP is bound radioactivity in the presence of excess nonlabelled ligand.
3 0 D3T~TT Rn DES~:K~ N OF THE ~ ENBODINEN-TS
CXC Intercrine~, IL-8 Action~ and Inhibitory Peptide~
IL-8 has been identified as a neutrophil activating molecule (Schroder et al., 1988; Peveri et al., 1988;
Yoshimura et al., 1987). It is produced mainly by monocyte-macrophage and endothelial cell by the stimuli such as bacterial lipopolysaccharide (LPS), tumor necrosis factor or interleukin 1, and shares common neutrophil activating properties with chemotactic Wo 95/167~2 ~ i ; 2 ~ ~ 9~ Y~

agonists, such as fM~P, C5a or leukotriene B4 ~Baggiolini et al., 1992) . I~-8 can stimulate chemotaxis of neutrophils as well as enzyme relea3e and respiratory burst. I~-8 is one member Df the family of peptide 5 molecules termed CXC intercrines, which all have the CXC
se~uence motif in their N-terminal region. The CXC
intercrines also include GR0, MIP2~ or MIP2~ and, more recently, ENA78.
The functions of I~-8 ~are~mediated ~y IL-8 re~ceptors on the neutrophil surf ace membrane . Recent studies showed that I~-8 birlds to at least two distinct receptors, whereas most of ~the other members of the intercrine family, e.g., GR0 and MIP2~, bind to one of 15 the receptors with high aff~inity (Holmes et al., 1991;
Murphy & Tiffany, 1991; ~aRosa et a7., 1992; Cerretti et al., 1993). These receptors are different from the receptors for other chemotactic agonists (Dohlman et al., 1987) .
II,-8 has been found in high concentrations in joint fluids from patients with several kinds of joint disease (Brennan et al. , 1990~, in ~pleural spaces of some patients with pleural effusions (Miller & Idell, 1993), 25 and lung edema fluids from patients with the adult respiratory distress ~ylldl~ (ARDS~ (Miller et al., 1992) . Increased IL-8 levels have also been clearly documented in various recent studies of patients with cystic fibrosis (Richman-Eisenstat et al., 1993;
McElvaney et al., 1992; Nakamura et al., 1992; Bedard et aL., 1993 ) .
I~-8 activates ~eutrophils and, although they are powerful antimicrobial cells, neutrophils can also cause 35 considerable tissue damage through the release of certain enzymes. IL-8 is therefore believed to be important in pathogenesis of these and other infl; tory disorders.

WO 95116702 ~ 9 2 7 ~ Y.)~ 45 The inventors therefore hypothesized that modulation of IL-8 function would be a good strategy to control various diseases and pathological conditions, particularly ARDS
and cystic f ibrosis .
Recently, results from various studies attempting to modify IL-8 function have been reported. These include studies showing some success in inhibiting II.-8 synthesis (Standiford et al., 1992; Lam et al., l990) . Also, anti-10 human IL-8 has been reported to ameliorate lung inflammation in rats suffering from an immunologic injury (Mulligan et al., 1993) . IL-8 secretion in airways of patients with cystic fibrosis was reportedly reduced with the secretory leukoprotease inhibitor of neutrophil elastase (McElvaney et al., 1992). In addition, IL-8 production has been shown to be suppressed in LPS-stimulated whole human blood by oxygen radical scavengers (DeForge et al., 1992) . Finally, two cytokines, interferon gamma (Cassatella et al., 1993a; 1993b) and 20 interleukin 4 (Standiford et al., l990) inhibited the synthesis of IL-8. However, such work has yet to yield a particularly effective IL-8 inhibitor, or an inhibitor capable of preferentially inhibiting certain neutrophil responses over others.
Studies have also been conducted on the interaction of IL- 8 with its receptors . This has led to the identification of certain peptide inhibitors with structures corresponding to portions of the IL-8 30 molecule. For example, Gayle and colleagues found that the 44 amino acids at the amino-t~rm;n~l end of the - rabbit IL- 8 receptor was a moderately good inhibitor of IL-8 binding and function (Gayle et al., 1993) . The - present inventors have shown that synthetic peptides, and 35 particularly, IL-8 amino t~ n~l peptides, inhibit IL-8 binding to neutrophils and neutrophil chemotaxis (Miller et al., 1990; Miller et al.. 1993). An N-ter~Linal WO 9iS116702 ~ U~Y~ W5 pentapeptide I~-8 inhikitor has also been reported (Goodman et al., l99l) .
Nonetheless, effective peptide inhibitors have not 5 yet been developed from a knowledge of the structure of the IL-8 molecule. Therefore, in pursuing this aim, the present inventors assayed various other peptide compositions for I~-8 inhikitory activity by screening a library of 400 groups of hexapeptides. In these lO screening a3says, l2~I-labeled interleukin-8 (lO-l2 M) was mixed with lO0 ~lM peptide, then added to neutrophils and incukated at 4OC for 90 min. The bound radioactivity was separated from unbound by centrifugation through a dense cushion of a mixture of paraffin and silicon oils 15 and the l25I bound to neutrophils was counted in a gamma radiation spectrometer, allowing the results to be expressed as the percent of II~- 8 binding which was inhibited .
In these studies, hexamerG of the sequence RRWWCX
(SEQ ID NO:23), where the t~rm;niql position may be any amino acid, were found to be effective I~-8 inhibitors.
Although certain RRWWCX-type peptides had previously been found to exhibit ar~ti-Staphylococcal properties (Houghten et al., l99l), no other functional properties have been reported which would suggest these peptides to have either anti-cytokine or anti-;nfli tory activities.
The present inventors also showed that RRWWCR ef fectively inhibits other CXC intercrines, such as GRO and MIP2, as evidenced by reducing GRO and MIP2~ binding to human neutrophils .
Although all RRWWCX (SEQ ID ~o: 23 ) series peptides have anti-I~-8 activity (Tables lA and lB)V the peptide RRWWCR (SEQ ID NO:l) was found to be particularly effective. A form of this peptide with an amino-terminus acetyl group and a carboxy-terminus amino group Wo95116702 ~ ` 21 7892~ J~l~u~Yall~15 (AC-RRWWCR-NH2; SEQ ID NO:1), thus modified in order to resemble peptides present among longer sequence of protein, became one of the focal points of these studies.
AC-RRWWCR-NH2 (SEQ ID NO:1) was found to inhibit the specific binding of 125I-labeled IL-8 to neutrophils with an apparent KI of approximately 10 IlM, and to be almost twice as effective as the non-acetylated form of the same peptide. A preci3e KI value could not be obtained due to the presence of positive cooperativity. The binding isotherm of IL-8 in the absence of the peptide fitted one-site model best, when it was analyzed using the computer program Lundon I. One possible explanation of the lower IL-8 concentration data in the Scatchard plots is that cooperativity masked the high affinity bindin~ at low IL-8 concentrations. Recent studies, however, have shown that IL- 8 bound to two distinct classes of IL- 8 receptors with almost similar affinity (Lee et al., 1982;
Srh~ rhf~r et al., 1992) . Therefore, it is more likely that the estimated Bmax for this binding site as one-site model represents the total Bmax of two class of receptors and that the estimated Kd is common for these receptors.
The bindin~ isotherms in the presence of the peptide fit two-site model best. The analysis of binding isotherms in the presence of Ac-RRWWCR-NH2 showed that thiE~ peptide suppressed the binding of IL-8 to two classes of receptors dif f erently . The estimated values of binding parameters showed that af f inity of one class of receptors was suppressed by lO~M peptide, which suggested competitive inhibition. Higher r~mr~on~ration of peptide is needed to inhibit the other class of receptor non-competitively.
-The activity of the present inhibitory peptides is specif ic f or IL- 8 . Ac-RRWWCR-NH2 does not inhibit the binding of C5a or leukotriene B4 to neutrophils, WO 95/16702 i .~ 2 ~ 7 ~ 9 2 7 ~ Ya/1~4S

chemotaxis induced by formyl-L-Met-L-Leu-L-Phe (fMhP), or ~-glucuronidase release induced by fMLP, C5a or leukotriene B4. It also has no intrinsic ability to cause neutrophil chemotaxis or enzyme release. These 5 observations suggest that peptides such as Ac-RRWWCR-NH2 can strongly inhibit human neutrophil activations induced by IL- 8 as a result of modulation of its receptor binding .
Fur~h, ~, hexamer and heptamer peptides based upon the Ac-RRWWCR-NX2 structure are contemplated to preferentially inhibit enzyme release over chemotaxis.
Indeed, Ac-RRWWCR-NH2 is herein shown to significantly suppress neutrophil chemotaxis induced by lOnM IL-8 at a 15 concentration of 50 ~lM and ~-glucuronidase release at 2 llM, even though 10 times more IL- 8 is re~uired tQ cause enzyme release. This preferential inhibition of enzyme release at lower conc~nt~;nnq than chemotaxis is a particularly important and surprising discovery that 20 makes these types of peptides ideal for the treatment of lung injury in patients with the adult respiratory distress syndrome.
The above f indings also suggest that distinct 25 neutrophil functions may reS~uire II,-8 binding to different classes of receptors. Although not important to the utility of the invention per se, this also makes the present peptides suitable for use as investigational tools to further elucidate IL-8 receptor and neutrophil 30 functions.
The inventors next examined the inhibitory activity of a second set of peptides which contained Ac-rrwwcrx-NH2 (SEQ ID NO:2), with all D-amino acids. Again, all 35 RRWWCRX (SEQ ID NO:2) series peptides were found to have anti-IL-8 activity (Tables 5A and 53) . However, the peptide Ac-rrwwcrc-N~I2 (SEQ ID NO:4) was found to be the WOg5/16702 ~ ` 2 1 78 927 1 ~IJ~,~Y~i~4S

best inhibitor, being almost four times as potent an inhibitor as Ac-rrwwcr-NH2 (SEQ ID NO:1). This observation is potentially of great signif icance as l; ~n proteages cannot degrade D-amino acid peptides 5 and proteins (Togo et al., 1989) . Therefore, D-amino acid peptide inhibitors are expected to have a longer half life in vivo.
Various other synthetic peptides were also tested lO for their ability to inhibit IL-8 binding to neutrophils in the standard assay. These peptides were either homologues of the amino - terminal end of IL- 8, or were segments of proteins found in the protein data bases (PIR
or Swiss-Prot) which had five of the six residues in 15 RRWWCR (SEQ ID NO:2). The latter peptides were identif ied by searching the PIR and Swiss-Prot databases for RRWWCR (SEQ ID NO:1), using the IGSUITE program to search the databases present on the CR~Y computer at the Center for High Performance Computing in Austin Texas.
20 None of the proteins in the data bases ,-~ntA;npd all si~
of the amino acids.
Previous studies have shown that the residues Glu, Leu and Arg at the 4, 5, and 6 positions of the 72 amino 25 acid of IL-8 are critical for the binding to neutrophils (Clore et al. , 1992; Clark-Lewis et al. , 1991), and that the amino terminal peptide of IL-8 inhibits IL-8 binding to neutrophils and chemotaxi6 ~liller et al., 1993). The inventors therefore ~Y~m; n.~d the IL-8 homologues 30 Ac-RELRCQ-NH2 (SEQ ID NO:54) and ELRCQCIRTY (SEQ ID
NO:49, including the C-X-C motif characteristic of intercrine peptides), along with its two non-Cys-cr nt~;n;n~ analogues, ELRSQSKTY (SEQ ID NO:50) and - ELRMQM};TY (SEQ ID NO:51). None of these peptides had the 35 ability to inhibit IL-8 binding to neutrophils.

WO95/16702 ~ , 2 1 7~ 92~ ,V~Y~I~4~ ~

The inventors next searched the protein databases to determine if RRWWCR (SEQ ID NO:l~ might occur in other peptides which might have relevant physiologic functions in relation to IL- 8 . Two peptides were identif ied and 5 chosen for investigation because they ~nntA;n~ five of the six residues in RRWWCR (SEQ ID NO:1) and were ~
cnntA;n~-l in known proteins. They were ~iw~Rww~ AVLY (SEQ
ID NO:53) and QIP~S~FLF ~SEQ ID NO:52) . The former peptide is contained in "cell surface glycoprotein :CDllc 10 precursor - human leukocyte adhesion receptor pl50, 95 alpha chain" (Corbi et al., 1990; A~c~As;nn number, A36534\A35543\S00864) and the latter is 3' ,5' -cyclic GMP
phosphodiesterase beta chain - bovine (Ovnh;nn;knv et al , 1987; Accession Number, S00251~.
Although one of these homologues, namely QIPRRSWCRFLF (SEQ ID NO:52), inhibited IL-8 binding to neutrophils by about 609~, this activity is less than Ac-RRWWCR-NH2 (SEQ ID NO:1) . It is not thought likely 20 that the protei~ from which this sequence was extracted, bovine retinal 3'5'-cyclic GMP phosphodiesterase, has a physiological role in IL-8 function, but this cannot be totally ruled out. The most important data from this study suggest that the first tryptophan in RRWWCR (SEQ ID
25 NO:l) can be modified with only modest loss of activation. The lack of activity of the other peptide, ~ww~:~AVLY (SEQ ID NO:53), suggests that ch_nging the last Arg in RRWWCR (SEQ ID NO:1) to Asp significantly reduces its ability to inhibit IL-8 binding to 3 0 neutrophils, as supported by the observation that Ac-rrwwcd (SEQ ID NO:26) is only m;n;r-l ly active.
Thus, the examples set forth herei~ detail the identiiication of a new series of peptide inhibitors 35 capable o~ inhibiting IL-8 binding to neutrophils and neutrophil activation. The peptide inhibitors of the RRWWCX (SEQ ID NO:23) and RRWWCRX (SEQ ID NO:2) types _ _ _ _ , , . .. . .. . .. -- -- --Wo95116702 ` 2 1 78 927 1~,1/LI~Y3/1 ~45 have the distinct advantage over previously described inhibitors that they are only six or seven residues long and that the D-amino acid analogues are also active. It is contemplated that this class of inhibitors will be 5 useful in the therapy of di3eases caused by the unre~3trained action Ih- 8, such as the adult respiratory distress syndrome. These inhibitors have the distinct advantage that they will likely permit neutrophils to enter the lungs, via chemotaxis which is not readily lO inhibited, but that they will then preferentially prevent the detrimental enzyme release (McGuire et al., lg82) .
In addition to their many and varied therapeutic uses, the peptides and compositions of the present 15 invention have utility in other embodiments. The3e include, for example, their use in vari~us bioassays, e.g., as positive controls in assays of II-8 inhibitors or neutrophil inhibitors; uses in further ~1P1 ;nP~ting I~-8 receptor interactions and functionsi generating 20 antibodies, and the like.
R;olo~ics~l Functional Equivalentfi Certain biological functional equivalents of the 25 RRWWCXX-type peptides are contemplated within the scope of this invention. The concept of biologically functional equivalent amino acids is well known to those of skill in the art, and iR embodied in the knowledge that modifications and changes may be made in the 30 structure of a protein or peptide and still obtain a molecule having like or otherwise desirable - characteristics.
- However, it is also well understood by skilled 35 artisans that, inherent in the definition of a biologically functional equivalent protein or peptide, is the concept that there is a limit to the number of .

Wo 9~116702 ~ ~ 2 ~ 7 8 9 2 7 A ~ Y~/1~45 changes that may be made within a def ined portion of the molecule and still result in a molecule with an acceptable level of equivalent biological activity and that key active site or structurally vital residues 5 cannot be exchanged. Biologically functional equivalent peptides are therefore defined herein as those peptides in which certain, not most or all, of the amino acids may be substituted. In particular, where hexamer or heptamer peptides are concerned, it is contemplated that only lO about two, or more preferably, a single amino acid change would be made within a given peptide. Of course, a plurality of distinct peptides with different substitutions may easily be made and used in accordance with the invention.
In regard to changing a limited number of residues within a peptide, it is known that certain amino aclds may be substituted for other amino acids without appreciable loss of~ function, as may be measured by the 2 0 interactive binding capacity f or structures such as receptors and cells, or the ability to compete with other molecules for binding to these sites. Since it is the interactive and competitive capacity of a protein or peptide that defines its biological functional activity, 25 certain amino acid substitutions can be made in a I?eptide sequence (or, of course, its underlying DNA coding sequence) and nevertheless obtain a peptide with like, or even improved properties.
Amino acid substitutions are generally based on the relative similarity of the amino acid side-chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. An analysis of the size, shape and type of the amino acid side-chain substituents reveals that arginine, lysine and histidine are all positively charged residues; that alanine, glycine and serine are all a similar size; and that wo95/16702 ` '~ S 2 ~ 78927 r~ Y3/l~4s phenyl ;31 ~n;ns, tryptophan and tyrosine all have a generally similar shape. Therefore, based upon these conEiderations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and 5 tyrosine; are defined herein as biologically functional equivalents .
To effect more quantitative changes, the hydropathic index of amino acids may be considered. Each amino acid 10 has been assigned a hydropathic index on the basis of their hydrophobicity and charge characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8);
phenyli3lAn;n~ (+2.8); cysteine/cystine (+2.5); methionine ( + 1 . 9 ); al anine ( + 1 . 8 ); glycine ( - 0 . 4 ); threonine ( - 0 . 7 );
serine (-0.8); tryptophan ~-0.9); tyrosine (-1.3);
proline ( -1 . 6); histidine (-3 . 2); glutamate (glutamic acid) (-3.5); glutamine (-3.5); aspartate (aspartic acid) ( -3 . 5); asparagine (-3 . 5); lysine ( -3 . 9); and arginine (-4 .5) .
The importance of the hydropathic amino acid index in conferring interactive biological function on a protein is generally understood in the art (Kyte &
Doolittle, 1982, incorporated herein by reference). It 25 is known that certain amino acids may be substituted for other amino acids having a similar hydropathic index or score and still retain a similar biological activity. In making changes based upon the hydropathic index, the substitution of amino acids whose hydropathic indices are 30 within +2 is preferred, those which are within $1 are particularly preferred, and those within $0 . 5 are even - more particularly preferred.
- Substitution of like amino acids can also be made on 3~ the basis of hydrophilicity, as disclosed in U.S. Patent 4,554,101, incorporated herein by reference. In U.S
Patent 4,554,101, the following hydrophilicity values are WO 95/16702 '~ 2 1 7 8 9 2 7 , ~ Y~ ~S

assigned to amino acid residues: arginine (+3 . 0); lysine (+3 . 0); aspartate (aspartic acid) (+3 . 0 _ 1); glutamate (glutamic acid) (+3.0 _ 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); proline (-0.5 + 1); threonine (-0.4); alanine (-0.5); histidine (-0.5);
eysteine (-1.0); methinnin~ (-1.3); valine 1-1.5);
leucine (-1.8); isoleueine (-1.8) j tyrosine (-2.3);
phenyl a l an i ne ( - 2 . 5 ); tryp tophan ( - 3 . 4 ) .
It is well understood ~that an amino acid can be substituted for another having a similar hydrophilicity value and ætill obtain a biologically functional equivalent protein or peptide. In making changeæ baæed upon similar hydrophilicity values, the substitution of amino acids whose hydrophilieity values are within _2 is preferred, those whieh are=within _l are particularly preferred, and those within _0.5 are even more particularly preferred 20 Pl~rr~ eutiCal Formulation~
.

The peptides and eompositions of the invention may be used for treating a yariety of diseases and disorders in whieh CXC intererines, sueh as IL-8, or neutrophils 25 are involved or in whieh there i5 an inappropriate or inereased inf lammatory response . The invention is partieularly suitable for the treatment of lung inflammation sueh as that eonneeted with asthma, bronehitis, eystic f ibrosis and ARDS . To treat disorders 30 such as ARDS and cystic fibrosis, parenteral administration, such as intravenous, intramuscular or subcutaneous inj ection i5 contemplated to be the most preferred route, although one may also use aerosols or inl~ ntf~ Sprays, aerosols and inhalants, are only 35 effeetive if the droplets are sufficiently fine and uniform in size so that the mist reaches the bronchioles.
Particle size iæ of major importance in the WO 95/16702 ~ 2 1 7 8 9 2 7 r~ Y~i~5 -35 ~
administration of therapeutic agents via aerosols or ;n~ ntq. The optimum particle size for penetration into the pulmonary cavity is of the order of 0 . 5 to 7 ILm.
As fine mists are produced by pressurized aerosols, their 5 use is considered advantageous. Such treatment strategies and therapeutic formulations are described in detail hereinbelow in Example VIII.
As the invention may be employed to treat 10 inflammation in various clinical settings, many types of pharmaceutical peptide formulations are contemplated.
Therapeutic or pharmacological compositions of the present invention, whether for pulmonary or other treatments, will generally comprise~ an effective amount 15 of a relatively small intercrine- or IL-8-inhibiting peptide or peptides, dissolved or dispersed in a pharmaceutically acceptable medium. The phrase ~pharmaceutically acceptable~ refers to molecular entities and compositions that do not produce an 20 allergic, tcxic, or otherwise adverse reaction when administered to a human. Pharmaceutically acceptable media or carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the 25 like. The use of such media and agents for pharmaceutical active substances is well known in the art . Except insof ar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated.
Supplementary active ingredients can also be - incorporated i~to the therapeutic compositions of the present invention. ~or example, the intercrine-, IL-8-- and neutrophil-inhibiting peptides may also be combined 35 with other agents such as IL-8-derived N-terminal peptides, IFN-~, oxygen radical scavengers and the like, to create peptide cocktails for treatment.

Wo 95/16702 ; ~ 2 l 7 ~ 9 2 7 ~~ Y3ll~45 The preparation of ~pharmaceutical or pharmacological compositions cnnt~;n;n~ an intercrine-, I~-8- and neutrophil-inhibiting peptide or peptides, including dextrorotatory peptides, as an active ingredients will be 5 known to those of skill in the art in light of the present disclosure. If desired, such compositions may be prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid prior~ to injection; as tablets or 10 other solids for oral administration; as time release capsules; or in any other form currently used, including cremes, lotions and mouthwashes, and the inhalents and aerosols of Example VIII
Solutions of the active peptides and compounds as free base or pharmacologically acceptable salts can be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. ~ Dispersions can also be prepared in glycerol, liquid polyethylene glycols, and 20 mixtures thereof and in oils. IJnder ordinary conditions of storage and use, these preparations contain a pre6ervative to prevent the growth of microorganisms.
Sterile solutions suitable for injection are 25 contemplated to be useful ~ in treating various diseases and may be administered into the blood stream or into the precise site of the inf lammation . The pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the 30 extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be 35 preserved against the rnnt~mi n~ting action of microorganisms, such as bacteria and fungi.

0 95/16702 2 1 7 8 ~ 2 7 A ~ y ~W

A peptide or peptides can be formulated into a composition in a neutral or salt form. Pharmaceutically acceptable salts, include the acid addition salts (formed with the free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or ~hn~phnric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the like.
Salts formed with the free carboxyl groups can also be derived from inorganic bases such as, for example, 10 sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, histidine, procaine, and the like.
The carrier can also be a solvent or disper~ion 15 medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol , and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating, such a~
20 lecithin, by the m-~i nt.on~n~ of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial ad antifungal agents, for example, 25 parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the 30 compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
Sterile inj ectable solutions are prepared by incorporating the active compounds in the required amount 35 in the ~LU~r iate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilization. Generally, dispersions are . ;,, ~ 2 1 78~27 Wo s5/l6702 ~ 9~ 45 . ., --prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of 5 sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are-vacuum-drying and freeze-drying techni~ues which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered 10 601ution thereof.
The preparation of more, or highly, concentrated solutions for intramuscular injection is also contemplated. In this regard, the use of DMS0 as solvent 15 is preferred as this will result in extremely rapid penetration, delivering high concentrations of the active peptide, peptides or agents to a small area.
The formulation of peptides for topical use, such as 20 in creams, Qintments and gels iS also contemplated. The preparation of oleaginous or water-soluble ointment bases is also well known to those~in the art. For example, these compositions may include vegetable oils, animal fats, and more preferably, semisolid hydrocarbons 25 obtained from petroleum. Particular components used may include white ointment, yellow ointment, cetyl esters wax, oleic acid, olive oil, paraffin, petrolatum, white petrolatum, spermaceti, starch glycerite, white wax, yellow wax, lanolin, anhydrous lanolin and glyceryl 30 monostearate. ~7arious water-soluble ointment bases may also be used, including glycol ethers and derivative~, polyethylene glycols, polyoxyl 40 stearate and polysorbate~. Even delivery through the skin may be employed if desired, e.g., by using transdermal patches, 35 iontophoresis or electrotransport.

~ ogs/l6702 2 ~ ~927 1~1~V~Y~/I~5 Buffered orllt~lm;c solutionb aiso fall within the scope of the invention. These may be used in connection with patients suffering from disorders connected with increased retinal angiogenesis. The buffering is 5 nPcf~g~y due to pH changes the peptide may cause.
Orl~tl~l mi c preparations may be created in accordance with conventional pharmaceutical practice, see for example "Remington' 8 Pharmaceutical Sciences" 15th Edition, pages 1488 to 1501 (Mack Publishing Co., Easton, PA) .
Suitable orht~l m; c preparations will generally contain a novel dipeptide, peptide or agent as disclosed herein in a concentration from 'about 0 . 01 to about 19~ by weight, and preferably from about 0.05 to about 0.5%, in 15 a pharmaceutically acceptable solutio~, suspension or ointment. The ophthalmic preparation will preferably be in the form of a sterile buffered solution containing, if desired, additional ingredients, for example preservatives, buffers, tonicity agents, antioxidants and 20 stabilizers, nonanoic wetting or clarifying agents, viscosity-increasing agents and the like Suitable preservatives for use in such a solution include b~on7~lk~n;um chloride, benzethonium chloride, 25 chlorobutanol, thimerosal and the like. Suitable buffers include boric acid, sodium and potassium bicarbonate, sodium and potassium borates, sodium and potassium carbonate, sodium acetate, sodium biphosphate and the like, in amounts sufficient to n~;nt~;n the pH at between 30 about pH 6 and pH 8, and preferably, between about pH 7 and pH 7 . 5 . Suitable tonicity agents are dextran 40, - dextran 70, dextrose, glycerin, potassium chloride, propylene glycol, sodium chloride, and the like, such - that the sodium chloride equivalent of the ophthalmic 35 solution is in the range 0.9 plus or minus 0.2%.
Suitable antioxidants and stabilizers include sodium bisulfite, sodium metablsulfite, sodium thiosulfate, ~ 1 78927 W095/16702 ~ r~ Y~ 15 thiourea and the like. Suitable wetting and clarifying agents include polysorbate 80, polysorbate 20, poloxamer 282 and tyloxapol. Suitable viscosity-increasing agents include dextran 40, dextran 70, gelatin, glycerin, 5 llydL~y~thylcellulose~ hydroxmethylpropylcellulose, lanolin, methylcellulose, petrolatum, polyethylene glycol, polyvinyl alcohol, polyvinylpyrrolidone, carboxymethylcellulose and the like.
Upon formulation, therapeutics will be administered in a manner compatible with the dosage f~ t;on, and in such amount as is pharmacologically effective. The formulations are easily administered in a variety of dosage forms, such as the type of in~ectable solutions 15 described above, but drug release capsules and the like can also be employed.
A minimal volume of a composition required to disperse the peptide is typically utilized. Suitable 20 regimes for administration are also variable, but would be typified by initially administering the compound and monitoring the results and then giving further controlled doses at further intervals. For example, for parenteral administration, a suitably buffered, and if necessary, 25 isotonic aqueous solution would be prepared and used for intravenous, intramuscular, subcutaneous or even intraperitoneal administration. One dosage could be dissolved in 1 mL of isotonic NaCl solution and either added to lOOOmL of hypodermoclysis fluid or injected at 30 the proposed site of infusion, (see for example, "Remington' 8 Pharmaceutical Sclences" 15th Edition, pages 1035-1038 and 1570-1580).
In certain embodiments, active compounds may be 35 administered orally. This is contemplated for agents which are generally resistant, or have been rendered resistant, to proteolysis by digestive enzymes. Such .. . . . . .. . . . .. . .. _ .. _ _ . .... _ _ _ ....

f, ~o 9sll6702 2 t 7 8 9 2 7 PCT/17S93/~224S

compounds are contemplated to include dextrorotatory peptides; chemically designed or modified agents; and peptide and lipo60mal formulations in time release capsules to avoid peptidase and lipase degradation.
oral formulations may include compounds in eombination with an inert diluent or an assimilable edible carrier; those enclosed in hard or soft shell gelatin capsules; those compressed into tablets; or those 10 incorporated directly with the food of the diet. For oral therapeutic administration, the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such 15 compositions and preparations should generally contain at least 0.19~ of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60~ of the weight of the unit. The amount of active compounds 20 in such therapeutically useful compositions is such that a suitable dosage will be obtained.
Ta}alets, troches, pills, capsules and the like may also contain the following: a binder, as gum tragacanth, 25 acacia, cornstarch, or gelatin; excipients, such as dicalcium phosphate; a disintegrating agent, such as corn starch, potato starch, alginic acid and the like; a lubricant, such as magnesium stearate; and a sweetening agent, such as sucrose, lactose or saccharin may be added 3 o or a f lavoring agent, such as peppermint, oil of wintergreen, or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of- the above type, a li~uid carrier. Various - other materials may be present as coatings or to 35 otherwise modify the physical form of the dosage unit.
For instance, tablets, pills, or capsules may be coatèd with shellac, sugar or both. A syrup of elixir may 2 1 ~927 Wo 95116702 ~ Y~ 45 contain the active ~ sucrose as a SwoP~nirl~
agent methyl and propylparab~ens as preservatives, a dye and flavoring, such as cherry or orange flavor. Of course, any material used in preparing any dosage unit 5 form should be pharmaceutically pure and substantially non-toxic in the amounts emE~loyed. In addition, the active compounds may be incorporated into sustained-release preparation and formulations.

The following examples=are included to demonstrate preferred embodiments of the invention. It should be appreciated by those of skill in the art that the techniques disclosed in the examples which follow 15 represent techniques discovered by the inventor to ~
function well in the practice of the invention, and thus can be considered to constitute preferred modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many 20 changes can be made in the specific embodiments which are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

EXANPLE I
HEXAMER PEPTIDE LN~L~l~J~S OF TT-8 A series of studies was f irst carried out to 30 determine whether hexamer peptides of the seS~uence RRWWCX
(SEQ ID NO:23), where X may be any amino acid, would act as inhibitors of II,-8. The=assays of the initial screen are based upon determining the ability of a given peptide to inhibit the binding of IL-8 to human neutrophils.

wossJ~6702 ' 2 1 78 927 ~ Y~ 4$
A. PreParation o~ ~uman Neutrophils The use of human subjects for acquisition of neutrophils was approved by the Institutional Review 5 Board f or human experimentation . For the preparation of neutrophilæ, human blood was anticoagulated with heparin for enzyme release studies and with 0 . 3396 30dium citrate for other studies. For chemotactic and enzyme releaEe studies neutrophils were 6eparated by dextran 10 sedimentation and erythrocyte lysis by the method of Boyum (Kohler & Milstein, 1975). For binding studies and cytotoxic studies, the neutrophils were further purified in gradients of Percoll (Pharmacia Fine Chemicals, Piscataway, NJ) to a purity of 90-9396 (Kohler & Milstein, 1975).
B . IL- 8 Bindincr AssaYs Recombinant human IL-8 (72 amino acids; Pepro Tech 20 Inc., Rocky Hill, NJ) was radioactively labeled with 125I
` ' by the chloramine T method of Xunter and Greenwood (Hunter & Greenwood, 1962) . Binding studies were performed according to Besemer et al. (1989), as follows:
Neutrophils in phosphate buffered saline pH 7.4 (PBS) 25 c~)nt~;n;ng 0.1% bovine serum albumin (BSA) were incubated with labeled ligand in the presence or absence of the peptide being tested for 90 min at 4C to reach equilibrium and then centrifuged at 12, 000 x g for 3 mln in Beckman B microfuge (Beckman Instruments, Fullerton, 3 0 CA), through a cushion consisting of a mixture of paraffin oil (Fisher Scientific, Fair ~awn, NJ) and - silicon oil (Serva Co., N.Y., NY). The pellet and supernatant were then counted in a gamma radiation - spectrometer. The non-specific binding was estimated to 35 measure the binding in the presence of 100-fold excess of non-labeled ligand. The binding constants were WO 95/1 6702 ~ 8 9 ~ 7 ~ Y~ 45 calculated using the computer programs I,undon I and Lundon I I .
C. Pe~tide~a ~
The RRWWCR-type peptides were synthesized by Houghten Pharmaceutical Company in San Diego using tBOC
for protection of the ~-amino group (Stewart & Young, 1969). All synthetic peptides were purified on high lO performance liquid chromatography (HP~C) using a preparative C18 reverse phase column (Waters Co., New Bedford, MA) . Peptides were eluted using a gradient from 0.1~6 trifluoroacetic acid (TFA) to 80g6 acetonitrile in O .19~ TFA. The composition of the peptides was conf irmed 15 by amino acid analysis and sequencing.
D. Stati~tic~
In this and all of the following examples, the data 20 are expressed as the mean and the variation as the standard deviation (S.D.). Significance was determined by the Shef f és test when the variances were e~ual and the pOplll A~ n~ were normaily distributed and only 2 groups were compared. Multiple comparisons were made using the 25 analysis of variance and Sheffe' 8 test.
E. Re~ult~
In these studies, a screening for inhibition was 30 carried out at 100 IlM concentration of the peptide being tested, with a 10 12 M concentration of I~-8. In the first series of studies, twenty peptides were synthesized with the carboxyl-terminal residue of RRWWCX (SEQ ID
NO: 23 ) being changed to each of the standard protein 35 amino acids in turn. In the ~irst set of studies, several of the peptides totally inhibited IL-8 binding, as shown in Table lA a~d Table 1~. The information WO95116702 2 1 7 8 ~ 2 7 P~ Y.~ IS

presented in Table lA and Table lB is the same data, with Table lA being li6ted in order~of 96 inhibition and Table lB being listed in order of SEQ ID N0, to enable straightforward comparisons. It can be clearly seen that 5 all hexamer peptides other than RRWWCD (SEQ ID N0:26);
RRWWCE (SEQ ID N0:27); RRWWCN (SEQ ID N0:35) and RRWWCQ
(SEQ ID N0:37), have very significant inhibitory activity. It should also be noted that even though the preliminary data in Table 1 shows RRWWCN (SEQ ID NO:35) lO not to have inhibitory activity, subsequent studies showed this peptide did indeed exhibit certain inhibitory properties (Pigure l).

2 t 789~7 WO 95/16702 ~ Ya/l~45 TA3I.E lA
PEPTIDE SEQ ID N~: % IN~IIB.
RRWWCR 1 112 . 2 RRWWCK 32 110 . 3 RRWWCT 3 9 8 8 . 4 RRWWCP 3 6 8 8 . 3 RRWWCH 3 0 87 . 9 RRWWC~ 3 3 8 6 . 9 RRWWCG 29 86 . 8 RRWWCV 4 0 81.1 RRWWCF 2 8 8 0 . 2 RRWWCS 3 8 7 9 . 3 RRWWCY 42 77 . 9 RRWWCC 25 75.1 RRWWCM 34 69 . 0 RRWWCW 41 6 6 . 4 RRWWCA 24 51. 9 RRWWCI 31 45.8 RRWWCQ 3 7 19 . 2 RRWWCE 2 7 17 . 1 RRWWCD 26 11. 8 RRWWC~7 3 5 -12 . O

WO95116702 2 1 7 8 9 ? 7 r~ y~l~S

TAB~E lB
PEPTIDE SEQ ID N0: 96 INE~IB.
RRWWCR 1 112 . 2 RRWWCA 24 51 . 9 RRWWCC 2 5 7 5 .1 RRWWCD 26 11. 8 RRWWCE 27 17 . 1 RRWWCF 2 8 8 0 . 2 RRWWCG 2 9 8 6 . 8 RRWWCH 3 0 8 7 . 9 RRWWCI 31 45 . 8 RRWWCK 3 2 110 . 3 RRWWCI. 3 3 8 6 . 9 RRWWCM 34 69 . (3 RRWWCN 3 5 -12 . O
RRWWCP 3 6 8 8 . 3 RRWWCQ 3 7 19 . 2 RRWWCS 3 8 7 9 . 3 RRWWCT 3 9 8 8 . 4 RRWWCV 40 81 . 1 RRWWCW 41 6 6 . 4 RRWWCY 42 77 . 9 2 1 7~927 WO 95116702 ' ~ Y3/i~ 5 As shown in Tables lA and lB, several of the peptides totally inhibited IL-8 binaing in the first studies. To assess relative effectiveness, the group was therefore re-evaluated with lower concentrations of the 5 peptides. In these studies, Ac-RRWWCR-NH2 (SEQ ID NO:1) was f ound to be the most potent inhibitor of binding of IL-8 to neutrophils (Figure 1) . The data presented in Figure 1 shows RRWWCR (SEQ ID NO:1) to be significantly better than the other peptides under these conditions.
In another initial study, the acetylated derivative of RRWWCR (SEQ ID NO:1; Ac-RRWWCR-NH2) emerged to be almost twice as effective as the non-acetylated peptide in inhibiting IL-8 binding to neutrophils.

EI~AMPLE II
RINETICS OF RRWWCR IL-8 BINDING LN~l~ILL~N
In the studies described in this Example, the same methodology as that detailed in Example I was employed.
An average EC50 was detprnlin~l from multiple replicates of the bi~ding inhibition curve to be 13 . 7 i 0 . 6 ~M (a representative curve from these assays is shown in Figure 2 ) . If the mechanism of inhibition were purely competitive with a single site model, the estimated KI
was approximately 10 ~M. However, the mechanism was more complicated. A steeper binding inhibition curve than usual single site model as well as high Hill coefficient value (1.5 - ~.7) suggested the presence of positive cooperativity . The curve did not f it either one-, two -or three-site model well w~en it was analyzed using the Lundon II computer program, although IL-8 is known to react with at least two distinct receptors on neutrophils. Therefore, it was difficult to estimate individual KI values for each class of receptor.

WO95JI6702 ~ 7 8 9 2 7 P~./V~Y~
The kinetics of IL - 8 binding i~hibition by RRWWCR
(S~Q ID N0:1) were determined. IL-8 binding isotherms in the absence of Ac-RRWWCR-NE~2 and in the presence of three concentrations of the peptide are shown in Figure 3a.
5 The Scatchard plot in the absence of the peptide failed to show the IL- 8 binding to two distinct receptors (Figure 3b). The plots in the presence of Ac-RRWWCR-NEI2 (SEQ ID N0:1) were non-linear and were compatible with a two site model when they were analyzed using the Lundon I
10 computer program. The estimated changes in Kd and Bmax are shown in Table 2. The data indicate that one group of IL-8 receptors decreased its affinity at 10 ILM of the peptide and the other group decreased Bmax with increasing concentrations of the peptide.
For further characterization of binding kinetics, a binding inhibition assay using non-labeled IL-8 in the presence of lO~M Ac-RRWWCR (SEQ ID N0:1) was performed (Figure 4). Both the binding inhibition curves in the 20 presence and absence of Ac-RRWWCR (SEQ ID N0:1) were analyzed using Lundon II and showr~ to best fit a two-site model . There was no evidence f or appearance of a low affinity state of the receptors. Estimated changes in Kd and Bmax indlcate that only the high affinity sites 25 decreased both their affinity and Bmax in the presence of lO~LM peptide (Table 3) .

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' m ' m -Wo 95/16702 ~ 2 1 7 9 2 7 PCrNS93/1224 E~AMPLE III
SPECIFICITY OF R~WWCR TO IL - 8 BIN3ING ~HIBITION
To examine the Epecificity of Ac-RRWWCR-NH2 (SEQ ID
5 NO:1) on ligand-receptor binding, the inventors tested whether the peptide reduced the binding of human 125I-C5a or 3H-leukotriene B4 to human neutrophils.
Recombinant human ~ifth component of complement 10 (C5a) (Sigma Chemical Co., St. Louis, MO) was radioiodinated enzymatically using Enzymobead (Bio Rad, Richmond, CA). Tritiated leukotriene B4 was purchased from Du Pont Co., Wilmington, DE. Binding assays for C5a and leukotriene B4 were performed as described for IL-8 15 binding in Example I, except that the buffers used were PBS containing lmM CaC12, O . 5mM MgC12, O . 596 BSA, and XBSS
,~n~ilin;ng 0.1~ ovulabumin=and 10mM HEPES (pH7.3), respectively, as previously described (Braunwalder et a,l., 1992; Sherman et al., 1988). The radioactivity was 20 measured with liquid scintillation counter for the leukotriene B4 assay . The ~binding of both ligands were saturable and inhibited by the non-labeled agonists in dose dependent manners.
In these studies it was found that at 100~LM
Ac-RRWWCR-NH2 (SEQ ID NO:1) did not affect the binding of C5a nor leukotriene B4, but that it suppressed the IL- 8 binding significantly at a concentration of 100nM
(Figure 5) .

El~aMPLE IV
~,~L~JrL`v~lwl~ OF RRWWCR TO N~:UL~U~llJS
The inventors next ~ m; n~l the cytotoxic capacity of Ac-RRWWCR-NH2 (SEQ ID NO:1). This was achievea by Wo95/16702 ` ~ 2 1 7~927 P~ Y~i~5 measuring the amount of 51Cr released from neutrophils, as follows: The neutrophil preparation (2x107 /ml) in RPMI-1640 media containing 1096 donor' s plasma wa6 incubated with 50011Ci of Na251CrO4 (Du Pont Co. ) for 60 min at 37C. The cells were washed 3 times, resuspended in the media at the concentration of lxlO7/ml, and then incubated for 30 min at 37C to allow spontaneous lysis of marginally viable cells. After washing twice, a 100 ~1 aliquot of the 51Cr-labeled neutrophils (5xl06/ml) was mixed with 100 fLl of Ac-RRWWCR-NE~2 (SEQ ID NO:l) in a siliconized microcentrifuge tube. The buffer and the conditions of incubation simulate~ ~ither the binding assay or the chemotactic assay. After the incubation, the tubes were centrifuged at 300xg for 7 min at 4C and the radioactivity in the supernatant was then counted in a gamma radiation spectrometer. Triplicate tubes c~-ntA;n;n~ buffer alone or 29~i SDS were used to determine spontaneous and maximum release, respectively. The percentage lysis was calculated by using the following 2 0 f ormula:
LYSIS = (EXPERIMENTAI CPM - SPONTAIIEOUS CPM) X100 (MAXIMUM CPM - SPONTANEOUS CPM) When chromium-labeled cells were incubated in PBS
,-,.n~;,;n;ng 0.196 BSA for 90 min at 4C, it was found that the percentage of cells lysed remained near control level up to 500~uM of the peptide (~igure 6). Under conditions used in chemotaxis, 10011M of the peptide had no effect, however, 500~M peptide damaged almost 2596 of the cells.
-Wo95/16702 ~ t 2 1 7 ~ 2 7 PCrlUS93/12245 EFFECT OF RRWWCR ON N~:u-.:cOEHlJ J!U_._J1~,_.;j The effect of RRWWCR on neutrophil chemotaxi6 and enzyme release was next ~-~Arrli n~d.
A. Chemotaxis Chemotaxis was performed using the leading front method as described by Zigmond and Hirsh (Zigmond &
Hirsch, 1973). IL-8 or controls were placed in the lower well of a Boyden chamber. ~ five micron pore size, 100 llm thick cellulose nitrate filter (Sartorius Filter, Inc., San Francisco, CA) was placed on the surface~and the chamber was then assembled. A 200 ~Ll aliquot of the neutrophil preparation (lxl06 cells/ml) in RPMI-1640 media containing lg6 BSA was added to the top of the filter and incubated at 37C for 30 min. The filter was then fixed, stained and mounted on a glass microscope slide. The leading front was determined by the position of the leading two cells. The distance that the leading two cells had moved through the ~ilter was measured for six fields on each filter. The measurements were made with four filters for each set of conditions.
B. ~ ~,,hil E~zYme Relea~e Neutrophil enzyme release was studied by a modif ication of the method of Goldstein and colleagues (Goldstein et al., 1973) . Cy~o~~h;31Aq;n B (Sigma Chemical Co. ) was stored in dimethyl sulfoxide at a concentration of 5 mg/ml and was diluted to a concentration of 50 ~g/ml in Hank' s RA1 An~'fi Salt Solution (HBSS) immediately before use. Cyto~h~lAR;n B, 200 Ill, was added to l ml of suspension of neutrophils, 6.25xlO6 cells/ml in HBSS, to achieve a final cytochalasin B concentration of l0 ~Lg/ml.

Wo 95116702 ~ 1 8 9 2 7 ~ Y3/1~5 The solution was then incubated in 96 well plates at room temperature for 10 min. The st; l~nt, 100 ~l, was added, and this cell suspension was incubated for 30 min at 37C. The plates were centrifuged and 100 /11 of 5 8Uplorn~t~nt wag removed. Alir~uots of ~0 ~11 of supernatant were mixed with 101l1 of 0 . OlM
phenolphthalein-glucuronic acid (Sigma Chemical Co. ) and 40~1 of 0 . lM sodium phosphate pH 4 . 6 in 96-well plates for ~-glucuronidase measurement. After 16 hours ;ncllh~tion at 37C, 200~1 of 0.2M Glycine in 0.2M NaCl, pH10 . 4 was added to each well and OD540 was measured as the enzyme activity.
C. Result~
It was found that the Ac-RRWWCR-NH2 (SEQ ID NO:l) had no effect on neutrophil chemotaxis or enzyme release.
A checkerboard analysis of cell v indicated that it was not chemotactic for neutrophils (Table 4). In 20 control studies shown in Figures 7 and 8, the peptide had no effect on rh k;n~is of neutrophils and ~id not stimulate ,~-glucuronidase release from the cells.
To conf irm that the inhibition of IL- 8 binding by 25 Ac-RRWWCR-NH2 is related to suppression of neutrophil activation by IL-8, the inventors ~oY~m;nPri the effects of the peptide on chemotaxis and ~-glucuronidase release.
Ac-RRWWCR-NH2 (SEQ ID NO:1) significantly inhibited chemotaxis of neutrophils stim~lated with 10 nM of IL-8 30 at a r~nrPntration of Ac-RRWWCR-NH2 (SEQ ID NO:1) of - 50~M, whereas it had no effect on chemotaxis induced by formyl-L-Methionyl-L-Leucyl-L-Phenylalanine (fMLP) (Figure 7) . Ac-RRWWCR-NH2 (SEQ ID NO:1) inhibited ~-glucuronidase release sti l i~tPfi by lOOnM of IL-8 at 35 2~LM, a lower concentration than required for inhibition of chemotaxis, however, it did not affect the enzyme W095/16702 ~ ` 7 1 78 927 P~ Y.S/ ~45 relea~e ~timulated by fMLP, C5a or leukotriene B4 (Figure 8) .

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WO 95/16702 ~ ' ^ ^ ~ ~ '` 2 1 7 PJ 9 2 7 ~ Y~1~45 EXaMPLE VI
FIJRT~ER PEPTIDE lN~ ~S OF IL-8 Further series of studies were carried out to 5 determine whether other peptides related to AC-RRWWCR-NH2 (SEQ ID NO:l) would act as inhibitors of Il:,-8, and to determine their relative effectivenees. In the studies described in this Example, the same methodology as that detailed in Example I was employed.
A. ~ePta~ner PePtides In this series of studies, the inventors ~r~m;nF~l D-amino acid analogues of RRWWCR (SEQ ID NO:l) with an 15 added seventh amino acid. In the heptamer studies, twenty peptides were synthesized with the carboxyl-terminal rexidue of RRWWCRX (SEQ ID NO:2) being changed to each of the standard protein amino acids in turn. In the first set of heptamer studies, several of the~
20 peptides exhibited very strong inhibition of I~-8 binding, as shown in Table 5A and Tabie 5B. The information presented in Table 5A and Table 5B is the same data, with Table 5A being listed in order of %
inhibition and Table 5B being listed in order of SEQ ID
25 NO, to enable straightforward comparisons. It can be clearly seen that all heptamer peptides (SEQ ID NOS:3 through 22) have significant inhibitory activity under these con~1~Lon~ ~T~b1e s~1.

WO 95116702 ~ 217 8 9 2 7 r~ Y~

TAB~E 5A
PEPTIDE SEQ ID ~6 INHIB.
N0:
RRWWCR 1 112 . 2 rrwwcrk 11 113 .1 5rrwwcrn 14 110 . 8 rrwwcrg 8 10 8 . 0 rrwwcrc 4 107 . 7 rrwwcrd 5 107 . o rrwwcrh 9 107 . 0 rrwwcrw 21 106 . 3 rrwwcrf 7 105 . 6 rrwwcrr 17 102 . 8 rrwwcre 6 99.1 rrwwcrl 12 99.1 rrwwcrm 13 96 . 2 rrwwcra 3 95 . 3 rrwwcri 10 93 . 9 rrwwcrq 16 89.2 rrwwcr~ 2 0 8 9 . 2 rrwwcrp 15 87.3 rrwwcry 22 85.4 rrwwcr~ 18 70 . 4 rrwwcrt 19 56 . 3 .

WO9~/16702 ~ 2 1 78 927 P~ Y~/I~45 . . ~1 TAB~E 5B
PEPTIDE SEQ ID 96 I~HIB.
N0:
RRWWCR 1 112 . 2 rrwwcra 3 95 . 3 rrwwcrc 4 107 . 7 rrwwerd 5 107 . 0 rrwwcre 6 9 9 .1 rrwwcrf 7 105 . 6 rrwwcrg 8 10 8 . O
rrwwcrh 9 107. 0 rrwwcri 10 93 . 9 rrwwcrk 11 113 rrwwerl 12 99.1 rrwwcrm 13 96 . 2 rrwwern 14 110 . 8 rrwwerp 15 87 . 3 rrwwerq 16 89.2 rrwwerr 17 102 . 8 rr~wcr~ 18 70 . 4 rrwwcrt 19 56 . 3 rrwwcrv 20 89.2 rrwwerw 21 10 6 3 rrwwery 2 2 8 5 . 4 WO95/16702 ~ 61- J~~ Y3/i~4~
In order to determine the relative effectiveness of the heptamers, their inhibitory effects were determined at lower ~ n~ ntrations. The peptide with D-cysteine present at the carboxyl-terminal end (RRWWCRC; SEQ ID
5 NO:4) was found to be almost 56% more effective than the next best peptide in this group and to be more effective than Ac-RRWWCR-Ni~2 (SEQ ID NO:1; Figure 9) .
Ac-rrwwcrc-NH2 (SEQ ID NO:4) prevented 80% of the binding of IL-8 to neutrophils as compared to 20% inhibition by 10 the L=amino acid peptide Ac-RRWWCR-NH2 (SEQ ID NO:1) at 101lM .
B. Other Pe~tide~
The inventors also tested several additional peptides which were either related to the amino terminal portion of IL-8 or were found in other proteins and had five of the six residues in RRWWCR (SEQ ID NO:1) . The peptides ELRCQCIKTY, ELRSQSIKTY, EI.RMQMIKTY, 20 QIPRRSWCRFLF, and ~w~wwwAVLY (SEQ ID NOS:49 through 53, respectively) were synthesized at The University of Texas Health Center at Tyler llt;l;~;n~ an 431 Peptide Synthesizer (Applied Biosystems, Foster City, CA), using the 9-fluorenylmethoxycarbonyl (fMOC) group to protect 25 the o~-amino group as described by Meienhofer and coworkers (Meienhofer et al., 1979) and Arshady et al.
(1979). All synthetic peptides were purified on high performance liquid cllrl t-~raphy (HP~C) using a preparative C18 reverse phase column (Waters Co., New 30 Bedford, MA). Peptides were eluted using a gradient from 0.1~6 trifluoroacetic acid (TFA) to 80% acetonitrile in 0.1% TFA. The composition of the peptides was confirmed by amino acid analysis and sequencing by the Protein Core - facility at UTHC.

Wo 95116702 ~ 2 1 7 8 ~ 2 7 ~ Y.~112,L.~5 In this Rerie~ of studieR, only Ac-RRWWCR-NH2 (SEQ
ID NO:1) and QIPRRSWCRFLF (SEQ ID NO:52) inhibited binding of IL-8 to neutrophil~ (Table 6).

Inhibition of IL-8 Binding to 11__' ~llils by SYnthetic Pel~tides Peptide Tested SEQ ID NO: % Binding Inhibition 10 Ac-KELRCQ 54 . -0.4 i 11.8 QIPRRSWCRFLF 52 61. 5 i 1. O
~iw~ww~AVLY 53 -12 . 9 i 2 . 9 EBRCQCIKTY 49 7 . 6 i 2 .1 ELRSQSIKTY 50 5 . 8 i 3 - 9 15 ELRMQMIKTY 51 -6 . 9 i 3 . O
Ac-RRWWCR 1 98 . 6 i O . 9 RXXXXX 4 3 11 . 5 XXXXXR 44 5 . 8 XRXXXX 45 9 . 9 2 o XXWXXX 4 6 1 . 4 XXXWXX 4 7 1 8 . 5 XXXXCX 4 8 7 . 1 EXAMP~E VII
OF GRO and MIP21g NEurrROPHIL BINDING
AC-RRWWCX-NH2 (SEQ ID~0:23) was also ,=~ o.l for 30 the ability to inhibit other CXC intercrineF~. The pre~ent example demon~trate~ that, in addition to IL- 8 inhibition, Ac-RRWWCX-NH2 ~SEQ ID NO:23) effectively inhibit~ GRO and MIP2~B binding to human neutrophils.

WO gSil6101 ~ ? ~ 2 1 7 8 9 2 7 PcT/rJs93~l~5 .

MIP2~ and GRO/MGSA were radioiodinated using Bolten Hunter reagent. The radioiodinated components were mixed with various concentration of the Ac-RRWWCX-NH2 (SEQ ID
NO:23) peptide and incubated at room temperature for 15 minutes. Neutrophil suspension (1 X 106 cells in 160~L1 PBS cnnt:~;n;n~ 0.196 BSA) was added to 40~1 of the mixture and incubated for 90 minutes on ice. The radioactivity bound to the cells was separated from free radioactivity by centrifugation through an oil layer.
The bound radioactivity is an indication of bound CXC
intercrine peptide. The ~ binding inhibition in the presence of Ac-RRWWCX-NH2 (SEQ ID NO:23) was calculated as ollows:
96 binding inhibition = ¦ 1 _ B NSP ¦ x 100 T-NSP

where B is bound radioactivity in the presence of the peptide, T i8 bound radioactivity in the absence of the peptide; and NSP i8 bound radioactivity in the presence of excess nonlabelled ligand.
In these studies, it was confirmed that Ac-RRWWCX-NH2 (SEQ ID NO:23) inhibited binding of 1 nM IL-3 to neutrophils in a dose dependent manner, with an EC50 of almost 25 llM (Figure 10). Ac-RRWWCX-NH2 (SEQ ID NO:23) was also found to effectively suppress the binding of 1 nM GRO and 1 nM MIP2~ to neutrophils in a simlar manner, as shown in Figure 10.
-3~ EXAMPLE VIII
l~ER~PE~ITIC F0R~U~AT~ONS AND T~ ~ PROTOCOLS
This example is directed to the techniques c~intP~ ted by the inventors for use in further W095/16702 ` ' ~ ~ 21i~9~7 ~ 45 characterizing the in vivo actions of the II.- 8 inhibitors and their use in animal or ~human treatment protocols .
A. ISffects o_ IL-8 Inhibitor~ OD Tnfl tio rn Vivo Prior to animal model ~studies, in vitra stability examinations may be performed on the peptides including, for example, pre-incubation in human serum and plasma;
treatment with various proteases; and also temperature-lO and pH-stability analyses.: It is already known that D-amino acid peptides are active and that these would likely have enhanced stabiIity in vivo.
The inventors propose ~to examine the in vivo ~
15 properties and ef f ects of the I~- 8 peptide inhibitors in animal models prior to moving onto clinical trialfi. The most suitable form, dose and any possible toxicity of the peptides will be determined in animal studies, as is routinely employed in the art. For example, the bio-20 availability and half lives of the peptides administeredin various ways may be determined using radioactively labeled peptides and ~ min;n~ their longevity and tissue distribution. If further stability ,onll~n~ t was desired, the peptides could also be administered in the 25 form of lipid-tailed peptides, surfactant-like micelles, peptide multimers or in semi-permeable drug release .
capsules .
The biological effects of the peptides may be 30 determined in various models of human disease. For example, II.-8 has been shown to cause the accumulation of neutrophils and edema in rabbit skin (Rampart et al., 1989). Therefor~, a rabbit dermal inflammation model will be employed to determine what dose of the peptides 35 can effectively inhibit the neutrophil accumulation and edema. This model is useful because of the ease o~
assessment of i f lammation . The most suitable route of WO Y5/16N2 , ,`.` 2 1 7 8 9 2 7 I ~ Y~I~S

peptide administration may be easily determined by comparative in vivo tests, In one particular example, New Zealand albino 5 rabbits may be injected with 125I-labeled human serum albumin through the lateral ear vein. Certain sites may then be inj ected intradermally with the test compounds, i e., an agonist to attract neutrophils and an IL-8 inhibitor peptide; the agonist and a control peptide; and 10 the agonist alone. About two hours later, full thickness skin samples 1 cm in diameter may be punched out, f ixed and stained with Wright-Giemsa or for myeloperoxidase and the histology ~x;1~1i n,~,1 for neutrophil accumulation and edema or tissue damage. Other skin biopsies may be 15 counted in a gamma counter to assess the amount of albumin f lux into the inj ected skin . Skin inf lammation after administration of the inhibitor can then be compared to the time-matched controls, ideally be performed in the same animal. At least 4 replicates for 2 o each experimental arm are recl n~
B . IL- 8 Inhibitor Trea t o~ A~n~
The best human model of Adult Respiratory Distress 25 Syndrome (ARDS) is probably the introduction of gram negative bacteria into the circulation of minipigs. This model will be employed to determine if II--8 is the major neutrophil activator in this model of ARDS, as it is in human ARDS, using studies similar to those carried out in 30 man. The effects of Ih-8 administration through intravenous and intr~ ry routes in minipigs will be assessed .
- In the fist step, the minipigs will be treated with 35 I~-8 through the most appropriate route to cause neutrophil influx and enzyme release into the lungs as assessed above. The peptides of interest will be .

WO95/16702 2 ' 78927 1~1/.J~Y~11~5 administered to determine appropriate doses for use in impeding these neutrophil functions, especially the dose with which the peptide suppresses enzyme release, but not the neutrophil influx.
In the next step, the acute lung damage model of minipigs caused by gram negative bacteria in the circulation will be employed. The peptides of interest will be administered ~:o th~ animals and the effect: of the lO peptides on the prevention of lung damage will be assessed The number of neutrophils in bronchoalveolar fluids, the amount of enzyme released into lung parenchyma, and the degree of protein leakage f rom circulation to lung will be used as indicators in this 15 study.
If IL-8 causes neutrophil influx, enzyme release into the lungs, and/or ARDS - like tissue damage to the lungs, as expected, the peptides of interest will be 20 administered to determine the appropriate doses for use in impeding these neutrophil functions. In these studies, various intravenous doses of radioactively labelled peptides will be administered initially. Plasma c~noPntrations and forms of the radioactivity will then 25 be determined. From these data, plasma clearance, half life and steady state volume of distribution will be measured and used to determine the most effective do3e ranges .
30 C. Treatment Protocols Due to precautions which are nP~-PAsA~ily attendant to every new pharmaceutical, the I~- 8 peptide inhibitore and compositions of the present invention have not yet 35 been tested in a clinical setting in human subjects.
Xowever, their clear ill vi tro activity in accepted models is believed to demonstrate the utility of the present ~WO 95/167D2 - 6 7 ~ Y~ tS
invention as an anti-inflammatory agent. Clinical trials will be conducted in due course and, naturally, will be in accordance with the FDA procedures. The following embodiments represent the best modes currently 5 contemplated by the present inventors for carrying out the practice of the invention in various clinical settings .
It is believed that pharmaceutical compositions lO which include peptide inhibitors of I~-8 will,prove to be useful in the treatment of various conditions, ;nr~ l;n~
pulmonary disorders such as bronchial inflammation, cystic fibrosis, pleural effusions, asthma, bronchitis and ARDS; skin disorders such as psoriasis and 15 dermatitis; diseases of the joints, including rheumatoid arthritis; and in the treatment of pseudogout, ;n~l. tory bowel disease, reperfusion cardiac damage or even in the treatment of cancer and other diseases and disorders associated with increased cellular 2 0 prol if erat ion .
As these peptides are thought to be particularly suitable for the inhibition of pulmonary inflammation, such as occurs in ARDS, chronic bronchitis and cystic 25 fibrosis, suitable treatment methods for these disorders will be described. For the treatment of ARDS or cystic fibrosis, one would preferably employ parenteral administration, such as by using intravenous, intramuscular or subcutaneous injections. However, one 30 may also use aerosols or inhalants. The preparation of peptide foL li~t;~n~ for parenteral administration, particularly those for~lated as injectables, is described in detail in the Preferred Embodiments section of the present application. The following describes 35 certain inhalant formulations, should one desire to use such methods in connection with the present invention.

WO 95116702 ~ t C 2 1 7 8 9 2 7 PCrlUS93/12245 Inhalations and inh~l~ntc: are pharmaceutical ~
preparations designed for delivering a drug or compound into the respiratory tree of a patient. A vapor or mist is administered and reaches the affected area to give 5 relief from symptoms of bronchial and nasal congestion.
Inhalations may be administered by the nasal or oral respiratory routes. The administration of inhalation solutions is only effective if the droplets are sufficiently fine and uniform in size so that the mist lO reaches the bronchioles.
Another group of products, also known as inhalations, and sometimes called insufflations, consists of f inely powdered or liquid drugs that are carried into 15 the respiratory passages by the use of special delivery systems, such as pharmaceutical aerosols, that hold a solution or suspension of the drug in a liguefied gas propellant. When released through a suitable valve and oral adapter, a metered dose of the inhalation is 20 prbpelled into the respiratory tract of the patient.
Particle size is of major importance in the administration of this type of preparation. It has been reported that the optimum particle size for penetration 25 into the pulmonary cavity is of the order of 0 . 5 to 7 ILm .
Fine mists are produced by pressurized aerosols and hence their use in considered advantageous.
The intravenous administration of one, or a 30 combination, of the anti-I1,-8 peptides described i~ this application is contemplated to be capable of attenuating inflammation in A~DS and cystic fibrosis. The range of doses to be administered is estimated to be in the range of about 500 to about lOOOmg/day, or between about 0 . 83 35 mg/kg body weight/hour (mg/kg/hr) to about 16 . 56 mg/kg/hr.

wo95/16702 - ~ 2 1 78~27 P~ Y.JI~

of course, one must not lose sight of the fact that various other pharmaceutical formulations of the IL-8 inhibitors may be prepared and used to treat many other disorders connected with neutrophil activation and 5 ;n~l. tion.
* * *

All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure.
While the compositions and methods of this invention have 15 been described in terms of preferred c.mhnrl; ~c, it will be apparent to those of skill in the art that variations may be a~plied to the composition, methods and in the steps or in the sequence of steps of the method described herein without departing from the concept, spirit and 20 scope of the invention. More specifically, it will be apparent that certain agents which are both chemically and physiologically related may be substituted for the agents described herein while the same or similar results would be achieved. A11 such similar substitutes and 25 modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the invention as defined b~ the appended claims.

' .

WO 95/16702 2 ~ 7 8 9 2 7 I~ J~ /l2~.15 R~ :S
The following references, to the extent that they provide exemplary procedural or other details supplementary to those set forth herein, are specif ically incorporated herein by reference.
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WO 95/16702 '- ~ 1'! r~ ~' r ~ 2 1 7 8 ~ 2 7 P~ ~S

UUL~ i I,ISTING
( 1 ) GENERAL INFORMATION: ~

(i) APPLICANTS:
(A) NAME: BOl~RD OF REGENTS, THE ~NIVERSITY OF TEXAS
(B) STREET: 201 West 7th Street (C) CITY: Austin ( D ) STATE: TEXAS
(E) COUNTRY: UNITED STATES OF AMERICA
(F) POSTAL CODE: 78701 and (ii) APPLICANT: CO~EN, Allen B.
MI~LER, Ed~und r.
2 0 KURDOWSKA, Anna ~YASHI, Shinichiro TUTTLE, Ronald R.
(iii) TIT~E OF INVENTION: PEPTIDE INHIBITORS OF CXC
2 5 INTERCRINE MO~ECULES
(iv) NUMBER OF ~ ?U~N~:S: 58 (v) CORRESPONDENCE ADDRESS:
(A) ~nn~C~: Arnold, White & Durkee (B) STREET: P. O. Box 4433 (C) CITY: Houston (D) STATE: Texas 3 5 ( E ) COU~TRY: USA
(F) ZIP: 77210 WO 95/16702 ; ~ C 2 1 7 ~ ~ 2 7 P~_l/v~Y~ 15 (vi ) COMPUTER Rr~'An~Rr.r~ FORM:
~A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS/ASCII
(D) SOFTWARE: WordPerfect 5.1 (vii) CURRENT APPLICATION DATA:
(A) APPLICATION Nt~MBER: Unknown (B) FILING DATE: Concurrently herewith (C) CLASSIFICATION: Unknown (viii) ATTORNEY/AGENT INFORMATION:
(A) NAME: KITCHELL, Bar~ara S.
. (B) REGISTRATION NUMBER: 33,928 (C) REFERENCE/DOCKET NUMBER: rJTFNol6pcT
2 0 ( ix ) TELECOMMUNI CATION INFORMAT ION:
(A) TELEPHONE: (512) 320-7200 (B) TELEFAX: (713) 789-2679 (C) TELEX: 79-0924 ( 2 ) INFORMATION FOR SEQ ID NO :1:
(i) SEQrJENCE (~T~R~RT~TIcs:
(A) LENGTH: 6 amino acid~
(B) TYPE: amino acid (C) STRANDEDNESS: ~ingle - (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide 2 ~ 78927 WO 95/16702 . . . ~~ Y.~112245 (xi) SEQUENCE D13SCRIPTIO~: SEQ ID NO:l:
Arg Arg Trp Trp Cys Arg (2) INFORMATION FOR SEQ ID NO:2:
IU~;N~; CHARACTERISTICS:

(A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) sTRhND~nN~c~c: single l S ( D ) TOPOLOGY: l inear (ii) MOLECULE TYPE: peptide (Xi) ~ U~;N~ DESCRIPTIO~: SEQ ID NO:2:
. Arg Arg.Trp Trp Cys Arg Xaa~

25 (2) INFORM~TION FOR SEQ ID NO:3:
;12U~;N~:~; CHARACTERISTICS:
(A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) sTR~ND~nN~s: single (D) TOPO~OGY: linear (ii) MOLECULE TYPE: peptide ~ ~ t~ - ~ r ~ O 9511670Z ~ 1 7 8 9 2 7 PCI~/US93112Z45 (Xi) ~ U~;N~:~; DESCRIPTION: SEQ ID NO:3:
Arg Arg Trp Trp Cys Arg Ala ( 2 ) INFORMATION FOR SEQ ID NO: 4:
( i ) ~;~;5~U~:N~:~ CHARACTERISTICS:
(A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) sTR~l~n~nN~s single (D) TOPOLOGY: lillear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
Arg Arg Trp Trp Cys Arg Cys ( 2 ) INFORMATION FOR SEQ ID NO: 5:
( i ) ~;~5,)U~:N~,'~ CH~RACTERISTICS:
(A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) ~OLECULE TYPE: peptide WO 95/16702 ~ '~ 2 1 7 8 ~ 2 7 PCT/US93/12245 (xi) ~ U~:N(:~: DESCRIPTION: SEQ ID NO:5:
Arg Arg Trp Trp Cys Arg Asp ( 2 ) INFORMATION FOR SEQ ID NO: 6:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGT~: 7 amino acids (B) TYPE: amino acid (C) STR~Nn~nNF~ C: single (D) TOPOLOGY: linear ( ii ) MOI.ECULE TYPE: peptide (Xi) ~;~UU~:N~ DESCRIBTION: SEQ ID NO 6:
Arg Arg Trp Trp Cys Arg Glu (2) INFORMATION FOR SEQ ID NO:7:

(i) ~;~;UU~;N~ RA~'T~RT~TIcs:
(A) ~ENGTH: 7 amino acids (B) TYPE: amino acid (C) STR~Nn~l)N~ : single ( D ) TOPO~OGY: l inear (ii) MO~ECU~E TYPE: peptide W0 9~ 6N2 ; ~ 2 1 7 8 9 2 7 P., llu~73~ 45 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
Arg Arg Trp Trp Cys Arg Phe ( 2 ) INFORMATION FOR SEQ ID NO: 8:
( i ) ~i ~;(,2 U~;N ~ R ~ ~'T~ R T .q T I CS:
(A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) STR~NnFi~nN~q~q: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (Xi) ~ U~;N~ DESCRIPTION: SEQ ID NO:8:
2 0 ~rg Arg Trp Trp Cys Arg Gly ( 2 ) IN~ORMATION FOR SEQ ID NO: 9:
( i ) SEQUENCE ('T~Z~R ~ t'T~R T .qT I CS:
(A) LENGTE~: 7 amino acids (B) TYPE: amino acid (C) sTR~Nn~.nN~c.q: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE- peptide wo 95rl6702 ; ~ ~ - 2 1 7 8 ~ 2 7 ~ YJ/12245 (Xi) ~ ;1.2U~;I\I~ DESCRIPTION: SEQ ID NO:9:
Arg Arg Trp Trp Cys Arg His (2) INFORMATION FOR SEQ ID NO:10:
(i) SEQUENCE CH~RACTERISTICS:
(A) ~ENGTH: 7 amino aclds (B) TYPE: amino acid (C) STRANDEDNESS: slng~e (D) TOPOLOGY: linear ( ii ) MO~.ECU~E TYPE: peptide (xi)~ Y~:UU~N~; DESCRIPTION: SEQ ID NO:10:
20 Arg Arg Trp Trp Cys Arg Ile (2) INFORMATION FOR SEQ ID NO:ll:
U~;N~; CH~RACTERISTICS:
(A) ~ENGTH: 7 amino acids (.3) TYPE: amino acid (C) STRZ~NnRTlNF~ : 8ingle (D) TOPOLOGY: linear (ii) MO~ECULE TYPE: peptide W095ll6~02 ~ 2 ~ 7~92~ ,IIU~Y3/I~ 45 ~xi) SEQUEN-OE DBSCRIPTION: SEQ ID NO:11:
Arg Arg Trp Trp Cys Arg Lys (2) INFORMATION FOR SEQ ID NO:12:
(i) Y~;~U~;N~; (7TARAt~T~RT~qTIcs:
(A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) sTR~Nn~nN~q~q single (D) TOPOLOGY: linea~
(ii) MOLECULE TYPE: peptide (xi) Y~Uu~No:~; DESCRIPTION: SEQ ID NO:12:
2 0 Arg Arg Trp Trp Cy8 Arg Leu (2) INFORMATION FOR SEQ ID NO:13: -~
(i) Y~;~U~N~:~; CHARACTERISTICS:
(A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRANn~nNl~qq: single (D) TOPOLOGY: linear ( ii ) MOLECULE TYPE: peptide WO95/16702 , ~ 2~78927 p ~ Y~ 4s (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
Arg Arg Trp Trp CYB Arg Met (2) INFORMATION FOR SEQ ID ~0:14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) STR~Nn~.nNR.~.~: single (D) TOPOl.OGY: linear (ii) MOLECULE TYPE: peptlde (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
2 0 Arg Arg Trp Trp CYB Arg Asn .

(2) INFORMATION FOR SEQ ID NO:15-( i ) B ~;U U ~:N ~; CHARACTERI ST I CS:
(A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide .

~095116702 ~ ? ~ 2 1 7~ 9 27 r~ Y~ 45 (Xi) ~ U~;N~.:~ DESCRIPTION: SEQ ID NO:15:
Arg Arg Trp Trp Cys Arg Pro (2) INFORMATION FOR SEQ ID NO:16:
(i) SEQUENOE C~RACTERISTICS:
(A) I ENGTH: 7 amino acids (B) TYPE: amino acid (C) STR~NnRnNRss ~ingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENOE DESCRIPTION: SEQ ID NO:16:
2 0 Arg Arg Trp Trp Cy~ Arg Gln (2) INFORMATION FOR SEQ ID NO:17:
( i ) SEQUENCE ~R z~(~TRR T .STI CS:
(A) I-ENGTH: 7 amino acidq (B) TYPE: amino acid (C) STRANDEDNESS: ~ingle ( D ) TOPO~OGY: 1 inear ~ii) MOI,ECULE TYPE: peptide WO 95/16702 '' ` ~ 2 ~ 7 8 9 2 7 PCr/US93112245 (xi) SEQUENCE DESCRIPTION: SEQ rD NO:17:
Arg Arg Trp Trp Cys Arg Arg (2) INFORMATION FOR SEQ ID NO:18:
JU~N~ CHARACTERISTICS:
(A) ~ENGTH: 7 amino acids (B) TYPE: amino acid (C) STR~NDEDNESS: single (D) TOPOLOGY: linear (ii) MO~ECUIE TYPE: peptide (Xi) ~ U~:N-:~ DESCRIPTION: SEQ ID NO:18:
2 0 Arg Arg Trp Trp Cys Arg Ser .
(2) INFORMATION FOR SEQ ID NO:19:
(i) SEQUENCE ~ ARA~TRRTqTICS:
(A) ~ENGTH: 7 amino acids (B) TYPE: amino acid (C) STRAl~n~:nN~.q.q: single ( D ) TOPO~OGY: l inear (ii) MOl.ECU~E TYPE: peptide ~O 9511670~ ' ' 2 1 7 8 9 2 7 r~ 45 (xi~ U~N-:~ DESCRIPTION: SEQ ID NO:19:
- Arg Arg Trp Trp Cys Arg Thr (2) INFORMATION FOR SEQ ID NO:20:
U~;N~:~; CHARACTERISTICS:
(A) ~ENGTH: 7 amino acidæ
(B) TYPE: amino acid (C) STRANl)RnNR.C~.~: single ( D ) TOPO~OGY: 1 inear (ii) MO~ECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION-- SEQ ID NO:20:
2 0 . Arg Arg Trp Trp Cys Arg Val (2) INFORMATION FOR SEQ ID NO:21:
U~;N0:~: CHARACTERISTICS:
(A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOI-ECULE TYPE: peptide WO 95/16702 ~ 7 ~ ~ 2 7 P~ Y~ 45 ~
-so-(Xi) ~ ;s2U~;N(~; DESCRIPTION: SEQ ID NO:21:
Arg Arg Trp Trp Cys Arg Trp 5 .

(2) INFORMATION FOR SEQ ID NO:22:
2U~;N~ CHARACTERISTICS:
(A) LENGTH: 7 amino acids (B) TY~E: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: pep~ide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:
2 0 Arg Arg Trp Trp Cy8 Arg Tyr (2) INFORMATION FOR SEQ ID NO:23:
( i ) SEQUENCE CE~RACTERISTICS:
(A) LENGT~: 6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide WO 95/16702 ~ r~~ 2 1 7 ~3 9 2 7 ~ ~45 (xi) ~ 5.?U~N~:~; DESCRIPTION: SEQ ID NO:23:
Arg Arg Trp Trp Cy3 Xaa (2) INFORMATION FOR SEQ ID NO:24:
(i) Y~:~UI~;N~I:; CHARACTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STR~Nn~nN~cc 3ingle (D) TOPOI.OGY: linear (ii) MOLECULE TYPE: peptide (xi) S~ U~;N~ DESCRIPTION: SEQ ID NO:24:
2 0 Arg Arg Trp Trp Cys Al a (2) INFORMATION FOR SEQ ID NO:25:
(i) SEQUENOE ~R~T~RT~sTIcs (A) ~ENGTH: 6 amino acid3 (B) TYPE: amino acid (C) STR~Nn~nN~clc 3ingle ( D ) TOPOLOGY: l inear (ii) MOLECI~LE TYPE: peptide WO 95/16702 "~ 7 8 9 2 7 PCr/US93/12245 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:
Arg Arg Trp Trp Cy8 Cy8 (2) INFORMATION FOR SEQ ID NO:26~:
( i ) ~;~;U U ~;N ~; CHARACTERI ST I CS:
(A) ~ENGTH: 6 amino~ acids (B) TYPE: amino aci~
(C) STRANnF~nN~ single ~=
(D) TOPO:~OGY: linear (ii) MOLECULE TYPE: peptide (xi) ~ ;yu~:N~: DESCRIPTION: SEQ ID NO:26:
2 0 Arg Arg Trp Trp Cys Asp (2) IN-FORMATION FOR SEQ ID NO:27:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino~ acids (B ) TYPE: amino acid (C) sTR~Nn~nNE~c single (D) TOPO:~OGY: linear (ii) MO~ECUIE TYPE: peptide WO 95/16702 ; ~ ` 2 1 7 8 9 2 7 ~ Y~1~5 (Xi) ~i~;UU~!;N~:~; DESCRIPTION: SEQ ID NO:27:
Arg Arg Trp Trp Cy8 Glu (2) INFORMATION FOR SEQ ID NO:28:
U~:N~ CH~RACTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STR~NnEnN~q~q single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (Xi) S~ U~;N~:~; DESCRIPTION: SEQ ID NO:28:
2 0 Arg Arg Trp Trp Cy9 Phe (2) INFORMATION FOR SEQ ID NO:29:
U~:N~; CHARACTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STR~Nn~nNE.qq: single (D) TOBOLOGY: linear (ii) MOLECULE TYPE: peptide W0 95/l6702 ; . ~ , 2 1 7 8 9 2 7 ~ Y~/12~45 .

(xi) SEQUENOE DESCRIPTION: SEQ ID NO:29:
Arg Arg Trp Trp Cys Gly (2) INFORMATION FOR SEQ ID NO:30:
( i ) SEQUENCE rM~R ~ rT~R T .~T I CS:
(A) I.ENGTH: 6 amino acids (B) TYPE: amino acid (C) STR~ND~nN~ : single (D) TOPOLOGY: linear (ii) MO~ECUIE TYPE: peptide (Xi) ~;~uu~;N~; DESCRIPTION. SEQ ID NO:30:
2 0 . Arg Arg .Trp Trp Cy5 His (2) INFORMATION FOR SEQ ID NO:31:

UJ:~N~:~; rM7~Rz~rTliRT~TIcs (A) ~ENGTH: 6 amino acids (B) TYPE: amino acid (C) sTR~Nn~nN~ single ( D ) TOPOI-OGY: l inear (ii) MOT.ECU~E TYPE: peptide wo95116702 .. ~. ~ " ~ I. 2 1 78927 PCrlUS93112245 _95_ (Xi) ~ U~;N(~ DESCRIPTION: SEQ ID NO:31:
Arg Arg Trp Trp Cys Ile (2) INFORMATION FOR SEQ ID NO:32:
U~;N~ CHARACTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRZ~NT)~nl~cs: single ( D ) TOPOLOGY: l inear 15 .
(ii) MOLECULE TYPE: peptide (xi).SEQUENCE DESCRIPTION: SEQ ID ~0:32:
2 0 Arg Arg Trp Trp CYB Lys (2) INFORMATION FOR SEQ ID NO:33:

;52U~;N~ RZ~l~T~RT.'~TICS
(A) LENGTH: 6 amino acids (B) TYPE: amino acid 3 0 ( C) STR ~Nn~:nN~.~.S: S ingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide WO 95/16702 ' ` '' r ! ~ 2 ~ 7 8 9 2 7 P~ ~ Y~I~s (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:
Arg Arg Trp Trp Cy9 Leu (2) INFORMATION FOR SEQ ID NO:34:
;UU~;NC:~: CHAR~CTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRANn~.T)N~ : single (D) TOPOLOGY: linear (ii) MOI,ECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:
' A~g Arg Trp Trp Cys Met (2) INFORMATION FOR SEQ ID NO:35:

;UU~;N(.I~ R1~'T~RT~cTIcs (A) LENGTE~: 6 amlno acids (B) TYPE: amlno acid (C) STR~Nn~nN~c~s: single (D) TOPOLOGY: linear (ii) MOLECULE TY~E: peptïde ~0 95116702 ;~ t 7 8 q 2 7 . ~ Y~1~5 .

(Xi) Y~;l"U~;N~; DESCRIPTION: SEQ ID NO:35:
Arg Arg Trp Trp Cys Asn ( 2 ) INFORMATION FOR SEQ ID NO: 3 6:
(i) SEQUENOE (~T~RZ~('T~.RT.C~TICS:
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STRhNn~ : single (D) TOPOLOGY: linear (ii) MO~ECULE TYPE: peptide (xi) Y~:~U~:N~; DESCRIPTION: SEQ ID NO:36:
2 O Arg Arg Trp Trp Cy5 Pro (2) INFORMATION FOR SEQ ID NO:37 (i) Y~;~,2U~N-:~; CHARACTERISTICS:
(A) LENGTH: 6 amino acids =
(B) TYPE: amino acid (C) STR~Nn~nNF~: single ( D ) TOPOI.OGY: 1 inear (ii) MOLECULE TYPE: peptide , WO 95/16702 ' ". . 2 i 7 8 q 2 7 1 ~ Y.~/1~45 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:
Arg Arg Trp Trp Cys Gln (2) INFORMATION FOR SEQ ID NO:38:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: amino acia ( C ) STR ;~NnRnNR.~: B lngl e ( D ) TOPOLOGY: l i near (ii) MOLECULE TYPE: peptide (xi). ~;5.?U~;N~'~; DESCRIPTION: SEQ ID NO:38:
,20 Arg Arg Trp Trp Cys Ser~

25 (2) INFORMATION FOR SEQ ID NO:39:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STR;~NnF.nNF.. q.~: single ( D ) TOPOLOGY: l inear ( ii ) MOLECULE TYPE: peptide t .~ 2 ~ 78927 WO ~5/16702 . ' ~ 7J~ 15 _99 _ (xi) ~:Uu~N~:~ DESCRIPTION: SEQ ID NO:39:
Arg Arg Trp Trp Cys Thr (2) INFORMATION FOR SEQ ID NO:40:
(i) ~i~'UU~N(:~; CHARACTERISTICS:
(A) I-ENGTH: 6 amino acids (B) TYPE: amino acid (C) STR~Nn~llN~CS single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (Xi ) ~;~;UU~iN~:~; DESCRIPTION: SEQ ID NO: 4 0:
Arg Arg Trp Trp Cys Val ' 1 5 (2) INFORMATION FOR SEQ ID NO:41:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 arnino acids (B) TYPE: amino acid (C) STRANDEDN-ESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide WO 95/16702 ~ 2 ~ 7 8 ~ 2 7 r~ Y~112245 (xi) SEQUENCE DESCRIPTIO~: SEQ ID NO:41.
Arg Arg Trp Trp Cys Trp (2) INFORMATION FOR SEQ ID N~:42:
(i) SEQUENCE CHA~ACTERISTICS:
(A) LENGTH: 6 amino - acids (B) TYPE: amino acid (C) sT~NnFn~qq single (D) TOPO~OGY: linear (ii) MOLECULE TYPE: peptide (xi) ~ U~N~:~ DESCRIPTIO~: SEQ ID NO:42:
2 0 Arg Arg Trp Trp Cys Tyr (2) INFORMATION FOR SEQ ID NO:43:
U~:N~:~; CHARACTERISTICS:
(A) ~ENGTH: 6 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single ( D ) TOPOLOGY: l inear (ii) MO~ECULE TYPE: peptide O 95116702 ; ~ , 2 1 7 8 9 ~ 7 1 ~.,V~Y~1~4s (xi) SEQUENOE DESCRIPTION: SEQ ID NO:43:
- Arg Xaa Xaa Xaa Xaa Xaa ( 2 ) INFORMATION FOR SEQ ID NO: 44:
;Uu~:N~:~; CXARACTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) STR~NnRnNR.~.~: single (D) TOPO~OGY: linear (ii) MO~ECULE TYPE: pep~ide (xi ) ~;Uu~;N~:~; DESCRIPTION-: SEQ ID N-O: 44:
2 0 Xaa Xaa Xaa Xaa Xaa Arg (2) INFORMATION FOR SEQ ID NO:45:
( i ) SEQJENCE CE~RACTERISTICS:
(A) ~ENGTH: 6 amino acids (B) TYPE: amino acid (C) STR~NnRnNR~S: single (D) TOPOLOGY: linear , (ii) MOl.ECl~E TYPE: pep:ide WO 95116702 ` i ~ 2 1 7 8 9 2 7 ~ Y3/1~45 (xi) ~i~;UU~N~ DESCRIPTION: SBQ ID NO:45:
Xaa Arg Xaa Xaa Xaa Xaa-( 2 ) INFORMATION FOR SEQ ID NO: 4 6:
(i) ~i~;UU~;N~,~; CHPRACTERISTICS:
(A) LENGTH 6 amino- acids (B) TYPE: amino~acid (C) STRA~ n~ qq: single (D) TOPOI-OGY: linear ( ii ) MO~ECU~E TYPE: peptide (xi) ~;UU~N~:~; DESCRIPTION: SEQ ID NO:46:
2 0 Xaa Xaa Trp Xaa Xaa Xaa l 5 ( 2 ) INFORMATION FOR SEQ ID NO: 47:
UU~;N~:~ CHARACTERISTICS:
(A) :~ENGTE~: 6 amino acids (B) TYPE: amino aci~
(C) STRZ~N~ ]N~:qq: slngle (D) TOPO~-OGY: linear (ii) MOl-ECULE TYPE: peptide WO95/16702 . , ~ 2 1 78927 P. ~ s (Xi) ~;~;5,)U~;N(.~; DESCRIPTION: SEQ ID NO:4~:
Xaa Xaa Xaa Trp Xaa Xaa (2) INFORMATION FOR SEQ ID NO:48:
(i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) sTRAN[)~n~qs: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:
2 0. Xaa Xaa Xaa Xaa Cys Xaa (2) INFORMATION FOR SEQ ID NO:49:
( i ) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino acid (C) STR~ m~ : single ( D ) TOPOLOGY: 1 inear (ii) MO~ECULE TYPE: peptide WO 9!;/i6702 1- . 2 17 ~ ~ 2 ~ r~ ~s (Xi) ~ ,2U~;N~:~ DESCRIPTION: SEQ ID NO:49:
Glu Leu Arg Cys Gln Cys Ile ~ys Thr Tyr (2) INFORMATION FOR SEQ ID NO:50:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 10 amino acids (B) TYPE: amino aci~
(C) STRANDEDNESS: single ( D ) TOPOLOGY: l inear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5Q:
Glu Leu Arg Ser Gln Ser Ile Lys Thr Tyr lQ
25 (2) INFORMATION FOR SEQ ID NO:51:
( i ) ~i~;y U~;N ( :~; CH~RACTERIST I CS:
(A) LENGTH: 10 amino acids (B ) TYPE: amino acid (C) STR~NnF.nNF.. ~.c single ( D ) TOPOLOGY: l i near , (ii) MOLECULE TYPE: peptlde WO95116702 ; ~ ;. 21 78q27 .~1/",Y,~ 45 (xi) SEQUENCE DESCRIPTION: SEQ ID l`J0:51:
Glu Leu Arg Met Gln Met Ile Lys Thr Tyr ~2) INFORMATION FOR SEQ ID NO:52:
SyU~;NI_:~; CHA~ACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECIJLE TYPE: peptide (xi) ~;~;yU~;N~; DESCRIPTION- SEQ ID NO:52:
- Gln Ile Pro Arg Arg Ser Trp Cys Arg Phe Leu Phe (2) INFORMATION FOR SEQ ID NO:53:
;yU~;N( :~; CHARACTERISTICS:
(A) LENGTH: 12 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide WO 95/16702 `~ J "~ 2 1 7 8 9 2 7 1 ~ y~ s (Xi) ~ U~N(~ DESCRIPTIQN: SEQ ID NO:53:
Gly Trp Arg Arg Trp Trp Cy9 Asp Ala Val Leu Tyr l Q
~2) INFORMATION FOR SEQ ID NO:54:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 6 amino acids (B) TYPE: amino acid (C) s~R~n~n~r~qq ~ingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (Xi) ~;(,2U~;NO~; DESCRIPTION: SEQ ID NO:54:
20. I,y8 Glu Leu Arg CYE: Gln 25 (2) INFORMATION FOR SEQ ID NO:55:
(i) ~;~;(~U~;N~ R;~TERT~qTIcs:
(A) LENGTH: 13 amino acid~
(B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: line~:r (ii) MOLECULE TYPE: peptide W<)95116702 '~ 2178927 1~ Y.~ 45 .

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:
- Ser Tyr Ser Met Glu His Phe Arg Trp Gly Lys Pro Val (2) INFORMATION FOR SEQ ID NO:56:
(i) iY~;UU~;N~; CH~RACTERISTICS:
(A) LENGTH: 5 amino acids (B) TYPE: amino acid (C) STR~NnFnNFcs gingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) ~;UU~;N~:~; DESCRIPTION: SEQ ID.NO:56:
2 0 ' Arg Arg Trp Trp Cys (2) INFORMATION FOR SEQ ID ~0:57:
(i) SEQUENCE ('T~R~T~RT.~TICS:
(A) LENGTH: 7 amino acids (B) TYPE: amino acid (C) STR~NnFnN~ : single ( D ) TOPOLOGY: l inear (ii) MO~ECULE TYPE: peptide WO 95116702 ; i ~ ~ - 2 1 7 8 9 2 7 ~ Y~ 4~
.

(xi) Y~;UU~;N~:~; DESCRIPTION: SEQ ID NO:57:
Arg Arg Trp Trp Cy8 Xaa Xaa (2) INFORMATION FOR SEQ ID NO:58:
;UU~;N(~ R~(~T~RT~TICS:
(A) LENGTH: 6 amino acids (B) TYPE: anlino acid (C) STRz~Nn~nN~ : ~ingle (D) TOPOLOGY: linear (ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:
- Arg Arg. Xaa Trp Cy8 Xaa

Claims (82)

CLAIMS:

1. A method of inhibiting a CXC intercrine family molecule, comprising contacting the CXC intercrine molecule or intercrine target cells with a biologically effective amount of a composition comprising a peptide of from 6 to about 14 residues in length including the amino acid sequence Arg Arg Trp Trp Cys Xaa1 (SEQ ID NO :23), wherein Xaa1 is any amino acid residue.

2. The method of claim 1, wherein the CXC intercrine family molecule is IL-8.

3 . The method o E claim 1, wherein the CXC intercrine family molecule is GRO.

4. The method of claim 1, wherein the CXC intercrine family molecule is MIP2.beta..

5. The method of claim 1, wherein the composition comprises an acylated peptide.

6. The method of claim 5, wherein the composition comprises a peptide acylated at the N-terminus.

7. The method of claim 1, wherein the composition comprises a peptide amidated at the C-terminus.

8. The method of claim 1, wherein the composition comprises a peptide acetylated at the N-terminus and amidated at the C-terminus.

9. The method of claim 1, wherein the composition comprises a peptide of D-amino acids.

10. The method of claim 1, wherein the composition comprises a peptide of L-amino acids.

11. The method of claim 1, wherein the composition comprises a peptide of L-amino acids and a peptide of D-amino acids.

12. The method of claim 1, wherein the composition comprises a peptide including both L-amino acids and D-amino acids.

13. The method of claim 1, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Arg (SEQ ID NO:1).

14. The method of claim 1, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Ala (SEQ ID NO:24).

15. The method of claim 1, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Cys (SEQ ID NO:25).

16. The method of claim 1, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Asp (SEQ ID NO:26).

17. The method of claim 1, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Glu (SEQ ID NO:27).

18. The method of claim 1, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Phe ( SEQ ID NO:28).

19 The method of claim 1, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Gly (SEQ ID NO:29).

20. The method of claim 1, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys His (SEQ ID NO:30).

21. The method of claim 1, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Ile (SEQ ID NO:31).

22. The method of claim 1, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Lys (SEQ ID NO:32).

23. The method of claim 1, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Leu (SEQ ID NO:33).

24. The method of claim 1, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Met (SEQ ID NO:34).

25. The method of claim 1, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Asn (SEQ ID NO:35).

26. The method of claim 1, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Pro ( SEQ ID NO:36).

27. The method of claim 1, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Gln (SEQ ID NO:37).

28. The method of claim 1, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Ser ( SEQ ID NO:38).

29. The method of claim 1, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Thr ( SEQ ID NO:39).

30. The method of claim 1, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Val (SEQ ID NO:40).

31. The method of claim 1, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Trp (SEQ ID NO:41).

32. The method of claim 1, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Tyr ( SEQ ID NO:42).

33. The method of claim 1, wherein the composition comprises a peptide including the amino acid sequence Arg Arg Trp Trp Cys Arg Xaa2 (SEQ ID NO:2), wherein Xaa2 is any amino acid residue.

34. The method of claim 33, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Arg Ala (SEQ ID NO:3).

35. The method of claim 33, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Arg Cys (SEQ ID NO:4).

36. The method of claim 33, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Arg Asp (SEQ ID NO:5).

37. The method of claim 33, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Arg Glu (SEQ ID NO:6).

38. The method of claim 33, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Arg Phe (SEQ ID NO:7).

39. The method of claim 33, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Arg Gly (SEQ ID NO:8).

40. The method of claim 33, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Arg His (SEQ ID NO:9).

41. The method of claim 33, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Arg Ile (SEQ ID NO:10).

42. The method of claim 33, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Arg Lys (SEQ ID NO:11).

43. The method of claim 33, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Arg Leu (SEQ ID NO:12).

44. The method of claim 33, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Arg Met (SEQ ID NO:13).

45. The method of claim 33, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Arg Asn (SEQ ID NO:14).

46. The method of claim 33, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Arg Pro (SEQ ID NO:15).

47. The method of claim 33, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Arg Gln (SEQ ID NO:16).

48. The method of claim 33, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Arg Arg (SEQ ID NO:17).

49. The method of claim 33, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Arg Ser (SEQ ID NO:18).

50. The method of claim 33, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Arg Thr (SEQ ID NO:19).

51. The method of claim 33, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Arg Val (SEQ ID NO:20).

52. The method of claim 33, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Arg Trp (SEQ ID NO:21).

53. The method of claim 33, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Arg Tyr (SEQ ID NO:22).

54. The method of claim 1, wherein the composition further comprises a peptide having the amino acid sequence Gln Ile Pro Arg Arg Ser Trp Cys Arg Phe Leu Phe (SEQ ID NO:52).

55. The method of claim 1, wherein the composition comprises between two and forty one distinct peptides, the peptides having an amino acid sequence in accordance with any one of the amino acid sequences set forth in SEQ
ID NO:1, SEQ ID NO:3 through SEQ ID NO:22, SEQ ID NO:24 through SEQ ID NO:42 or SEQ ID NO:52.

56. The method of claim 55, wherein the composition comprises two distinct peptides.

57. The method of claim 55, wherein the composition comprises five distinct peptides.

58. The method of claim 55, wherein the composition comprises ten distinct peptides.

59. The method of claim 55, wherein the composition comprises twenty distinct peptides.

60. The method of claim 55, wherein the composition comprises thirty distinct peptides.

61. The method of claim 55, wherein the composition comprises forty one distinct peptides.

62. The method of claim 55, wherein the composition comprises a peptide having the amino acid sequence Arg Arg Trp Trp Cys Arg (SEQ ID N0:1) and a peptide having the amino acid sequence Arg Arg Trp Trp Cys Arg Cys (SEQ
ID NO:4).

63. The method of claim 1, wherein the composition is a pharmaceutical composition in a pharmacologically acceptable form.

64. The method of claim 63, wherein the pharmaceutical composition is formulated as an injectable, a nasal spray, an inhalant, an aerosol, a cream, a gel, a micelle or liposome encapsulated form or is incorporated within a biocompatible drug release capsule.

65. The method of claim 64, wherein the pharmaceutical composition is formulated as an injectable for intravenous, intramuscular or subcutaneous administration.

66. The method of claim 64, wherein the pharmaceutical composition is formulated as a nasal spray, an inhalant or an aerosol.

67. The method of claim 63, wherein the CXC intercrine family molecule or the intercrine target cells are located within an animal and an effective amount of the pharmaceutical composition is administered to said animal.

68. A method for preferentially reducing neutrophil enzyme release in comparison to neutrophil chemotaxis, comprising contacting a composition containing neutrophils and IL-8 with an amount of a composition comprising a peptide of from 6 to about 14 residues in length including the amino acid sequence Arg Arg Trp Trp Cys Xaa1 (SEQ ID NO:23), wherein Xaa1 is any amino acid residue, the amount effective to reduce neutrophil enzyme release in comparison to chemotaxis.

69. The method of claim 68, wherein the neutrophils and IL-8 are located within an animal and an effective amount of the composition is administered to said animal in a pharmacologically acceptable form.

70. A method for reducing inflammation, comprising administering to an animal with inflammation a biologically effective amount of a pharmaceutical composition comprising a peptide of from 6 to about 14 residues in length including the amino acid sequence Arg Arg Trp Trp Cys Xaa1 (SEQ ID NO:23), wherein Xaa1 is any amino acid residue.

71. The method of claim 70, wherein the inflammation is associated with Adult Respiratory Distress Syndrome (ARDS) or cystic fibrosis.

72. A pharmaceutical composition comprising, in a pharmacologically acceptable formulation, a peptide of from 7 to about 14 residues in length, the peptide being capable of inhibiting a CXC intercrine family molecule and including the amino acid sequence Arg Arg Trp Trp Cys Xaa1 Xaa2 (SEQ ID NO:57), wherein Xaa1 and Xaa2 are any amino acid residue.

73: The pharmaceutical composition of claim 72, wherein the composition comprises a peptide capable of inhibiting IL-8.

74. The pharmaceutical composition of claim 72, wherein the composition comprises a peptide capable of inhibiting GRO.

75. The pharmaceutical composition of claim 72, wherein the composition comprises a peptide capable of inhibiting MIP2.beta..

76. The pharmaceutical composition of claim 72, wherein the composition comprises a peptide acetylated at the N-terminus or a peptide amidated at the C-terminus.

77. The pharmaceutical composition of claim 72, wherein the composition comprises a peptide acetylated at the N-terminus and amidated and the C-terminus.

78. The pharmaceutical composition of claim 72, wherein the composition comprises a peptide including the amino acid sequence Arg Arg Trp Trp Cys Arg Xaa2 (SEQ ID NO:2), wherein Xaa2 is any amino acid residue.

79. The pharmaceutical composition of claim 78, wherein the composition comprises a peptide having an amino acid sequence in accordance with any one of the amino acid sequences set forth in SEQ ID NO:3 through SEQ ID NO:22.

80. The pharmaceutical composition of claim 79, wherein the composition comprises between two and twenty distinct peptides, the peptides having an amino acid sequence in accordance with any one of the amino acid sequences set forth in SEQ ID NO:3 through SEQ ID NO:22.

81. The pharmaceutical composition of claim 72, wherein the composition is formulated as an injectable, a nasal spray, an inhalant, an aerosol, a cream, a gel, a micelle or liposome encapsulated form or is incorporated within a biocompatible drug release capsule.

82. A peptide of from 7 to about 14 residues in length including an amino acid sequence in accordance with any one of the amino acid sequences set forth in SEQ ID NO:3 through SEQ ID NO:22, the peptide being capable of inhibiting IL-8, GRO or MIP2.beta..