JPH09510688A - Peptide inhibitor of CXC intercrine molecule - Google Patents

Abstract

  (57) [Summary] Disclosed are peptide-based compositions and methods for inhibiting and modulating the action of CXC intercrine molecules. The described anti-leukinate peptides inhibit the binding of IL-8, GRO, MIP2β to neutrophils and the activation of neutrophils. These peptides are particularly advantageous because they inhibit IL-8-induced enzyme release at concentrations that are 25-fold lower than those required to inhibit chemotaxis, which is why these peptides are Especially, it is ideal for treating various inflammatory diseases such as adult respiratory distress syndrome (ARDS), cystic fibrosis and chronic bronchitis.

Description

Detailed Description of the Invention                   Peptide inhibitor of CXC intercrine molecule                                    Background technology   The US Government has rights in this invention pursuant to NHLBI grant number ROI-HL. 1.Field of the invention   The present invention relates generally to the field of action of cytokines, and in particular to CXC intercrine molecules. Methods and compositions for inhibiting and modulating effects. Interleukin 8 (IL -8), especially preferentially inhibiting the release of degrading enzymes by neutrophils induced by IL-8 Disclosed are peptide compositions. These compositions are used in adult respiratory distress syndrome (ARDS ) And cystic fibrosis. 2.Description of related technology   IL-8 is a CXC intercrine named after its N-terminal sequence elements. It is a member of the cytokine group. This group includes especially growth-related oncogenes (GRO, or GRO / MGSA) and as macrophage inflammatory protein 2β (MIP2β) Known peptide molecules are also included. IL-8 is a peptide of about 5 kD, about 72 amino acids Length, which is altered by post-translational processing performed in different cell types. (Yoshimura et al., 1989; Hebert et al., 1990; Strieter et al., 1989). The IL-8 gene is a mononuclear cell of human blood stimulated by staphylococcal enterotoxin A. It was first identified by analyzing the genes transcribed by cells (Schim id & Weissman, 1987). IL-8 production is associated with tumor necrosis factor and interleukin 1 It is known to be induced by (Strieter et al., 1990).   IL-8 interacts with at least two different receptors on neutrophils (Holmes et  al., 1991; Murphy & Tiffany, 1991). Receptor binds to GTP-binding protein It becomes possible to transduce the signal of IL-8 into cells (Wu et al., 1993). G Most of the substances belonging to the intercrine group such as RO and MIP2β are One binds, and IL-8 binds to both IL-8 receptors (LaRosa et al., 1992; Cerretti et al., 1993). The three-dimensional structure of IL-8 was determined by NMR (Clore et al., 1990) and By X-ray crystallography (Clore & Gronenborn, 1992; Baldwin et al., 1991) It has been elucidated. Three β-pleated sheet structures at the free amino terminus Then, the α-helix is located at the carboxyl terminus (Oppenheim et al., 199). 1). Several lines of evidence indicate that both the amino and carboxyl termini are It has been suggested that it is involved in the binding of the putter (Clore et al., 1990; Clark -Lewis et al., 1991; Moser et al., 1993).   Specific functions of the CXC interclin are being studied by several laboratories ( Yoshimura et al., 1989; Schoroder et al., 1988; Peveri et al., 1988). An example For example, the IL-8 peptide's main functions are its ability to stimulate neutrophil chemotaxis and activation. (Larsen et al., 1989; Shoroder et al., 1988; Peveri et al., 1988), blood vessels It appears to be related to the ability to promote formation (Koch et al., 1992). example Surface adhesion factor or E. coli endotoxin (also known as lipopolysaccharide or LPS When IL-8 is “supplied” by a substance such as Stimulates the release of neutrophil enzymes such as eloperoxyedase.   The neutrophil inflammatory response, which is essential for the degradation of bacteria that invade the body, is inappropriate Activation of neutrophils causes several problems. For example, if neutrophils are attracted to the lungs When properly fed, neutrophils release degradative enzymes into lung tissue. This Can cause adult respiratory distress syndrome (ARDS) (Weiland et al. ., 1986; Idell et al., 1985). ARDS is caused by a bacterial infection and suddenly causes severe Causes low blood pressure (shock) and many other injuries to the body. According to recent research Demonstrated that IL-8 is the major neutrophil activator in the lungs of ARDS patients (Miller et al., 1992), a model of endotoxin shock in primates It suggests IL-8 as the quality (Van Zee et al., 1991).   Other diseases and conditions in which IL-8 is believed to play an important pathological role High levels of IL-8 have also been found in abnormal inflammatory exudates (Brennan et al., 199). 0; Miller & Idell, 1992; Miller et al., 1992). For example, IL-8 Equine (Brennan et al., 1990; Seitz et al., 1991) and pseudogout (Miller & Bresfo rd, 1993), suggesting a possible mediator of inflammation and cystic For fibrosis (McElvaney et al., Nakamura et al., 1992; Bedard et al., 1993) A role to play is suggested. Therefore, regulation of IL-8 function suppresses various disease states. Seems to be an excellent method.   Recently, some progress has been made in identifying compounds that can reduce the synthesis of IL-8. Such compounds include IL-4, oxygen free radical scavenger, secretory leukocyte progenitor. Rotase inhibitors, interferon gamma, etc. can be mentioned (Standiford et a l., 1990; DeForge et al., 1992; McElvaney et al., 1992; Cassatella et al. ., 1993a; 1993b), such studies are not for IL-8 inhibitors. Prote Various other compositions such as inkinase C inhibitor, IL-4, anti-IL-8 antibody also exert IL-8 action. It has been reported to regulate (Lam et al., 1990; Stanford et al., 1992; Mu. lligan et al., 1993). Unfortunately, these compounds are IL-8 inhibitors in clinical settings. It is far from ideal as a candidate for use as.   Some progress has been made in identifying peptide IL-8 inhibitors, but such research Most of the research focused on a portion of the IL-8 molecule itself (Miller et al. , 1990; Gayle et al., 1993). For example, we have shown that synthetic peptides, and especially I L-8 amino-terminal peptide inhibits IL-8 binding to neutrophils and neutrophil chemotaxis (Miller et al., 1990; Miller et al., 1993). N-terminal A pentapeptide IL-8 inhibitor has also been reported (Goodman et al., 1991). Unfortunately , To date, the inhibitory function of peptides derived from IL-8 has been proven to be incomplete and insufficient. Have been.   It is necessary to identify particularly effective peptide inhibitors against CXC intercrine such as IL-8. Since the need still exists, we are currently investigating compositions other than the IL-8 molecule itself. It is clear that it is necessary. Prefer neutrophil enzyme release over chemotaxis Identification of peptides that inhibit E. coli may be particularly useful. This means that Neutrophils can enter the lungs and protect them from bacterial invasion, as well as cause tissue damage. Because it doesn't happen.                                   Summary of the Invention   The present invention relates to the action of CXC intercrine molecules such as IL-8, GRO (GRO / MGSA) and MIP2β. By providing new methods and compositions for modulating and inhibiting It aims to overcome the shortcomings of the technology. Peptides and drug groups disclosed herein The product reduces the binding of IL-8, GRO, MIP2β to neutrophils and is induced by IL-8. Inhibit the activation of neutrophils. These peptide formulations impair neutrophil chemotaxis Enzymes induced by IL-8 at concentrations significantly lower than those required to inhibit Is particularly useful because it can inhibit the release of Various diseases, especially CXC Also provides methods for treating diseases in which the unrestricted action of phosphorus plays a role Have been.   The present invention generally relates to the amino acid sequence Arg Arg Trp Trp Cys Xaa.₁(RRWWCX; SEQ ID NO: 23) (in this case Xaa₁Is any amino acid residue) The peptide is a potent inhibitor of CXC intercrine molecules such as IL-8. Based on the amazing discoveries of the Tomorrow. As used here "CXC The words "Turclin family of molecules" and "CXC interclins" refer to C in the N-terminal region. Collectively imply a group of peptide intercrine containing XC sequence motifs It is used for IL-8, GRO, MIP2α, MIP2β, EN for CXC intercrine A78 It is known to be included and includes all of those molecules and any other containing CXC motif. These intercrine polypeptides have the same terminology as that used in this application. It will be understood that it falls within the scope.   The peptide having the inhibitory action of the present invention can be referred to as "anti-leukinate". A hexameric peptide of sequence RRWWCX (SEQ ID NO: 23) It has previously been shown to have antibacterial activity against H. (Houghten et al. , 1991). However, such peptides are not suitable for the anticytokines disclosed herein. / Intercrine, information suggesting that it has anti-neutrophil and anti-inflammatory activity It has never been presented.   Therefore, in some respects, the present invention provides a CXC interface for GRO, MIP2α, MIP2β, etc. And to inhibit IL-8. As used here The term "inhibits CXC interclin" refers to the biology of CXC interclin. It means the process of reducing the physical effect. This is the CXC intercrine of neutrophils etc. Inhibits the binding of CXC intercrine to one of the IL-8 receptors on target cells Can be specifically evaluated by measuring the inhibition of CXC intercrine Any method may be used.   The term IL-8 has previously been used to describe the chemotaxis of neutrophils from neutrophil-activator, mononuclear cells. Cytokine composition known as sex factor and neutrophil activating peptide-1 Used to refer to. As used herein, "inhibits IL-8" The term generally means reducing or attenuating the biological activity of IL-8. this Includes inhibition of any or all of the known effects of IL-8. these Actions include subcellular actions such as receptor binding or cytosolic calcium concentration. And regulation of cellular effects such as recruitment or activation of granulocytes, inflammation and blood It also affects physiological actions such as tube formation.   In a preferred embodiment, the inhibition of IL-8 function referred to in the present application is Inhibition of IL-8 action on granulocytes such as spheres (polymorphonuclear neutrophils, PMNs). this Can be determined by many cellular and physiological methods, as disclosed herein. Wear. For example, measuring the inhibition of IL-8 binding to purified receptor composition or neutrophils. Of IL-8-induced neutrophil chemotaxis or extravasation IL-8-stimulated neutrophil enzyme release by measuring inhibition of efflux ( For example, myeloperoxidase, β-glucuronidase or elastase release ) Inhibition or by using the rabbit skin inflammation model It can be determined by assessing the in vivo anti-inflammatory effect.   A preferred method of measuring inhibition of IL-8, or further inhibition of GRO or MIP2β, is: This is a method for measuring the decrease in the amount of intercrine bound to neutrophils. This is the easiest This is a simple and easy method. In addition, specific intercrine to its receptor Binding is preferential over any other action on neutrophils or other cell types I have to do it. Of IL-8, GRO or MIP2α or MIP2β binding to neutrophils “Inhibition” of intercrine, as exemplified by inhibition, refers to a particular peptide or Means the ability of the composition to inhibit intercrine binding to a detectable degree You. That is, below the level observed in the absence of the peptide or composition. , Means the ability to reduce binding.   Inhibition of CXC intercrine binding to neutrophils is expressed as% binding inhibition, The higher the value, the higher the effect of the inhibitor. Preferred peptides are generally , Shows a higher% binding inhibition rate. Of course, the calculated% binding inhibition is calculated as CXC Accurate analytical parameters such as the concentration of intercrine and the concentration of specific peptides or compositions It depends on the matter. Used to obtain the data in Tables 1A, 1B, 5A and 5B Conditions can be used to determine if a particular peptide has inhibitory activity. Wear. However, if you want to get particularly accurate quantitative or comparison data, For example, a lower concentration of the order of approximately 20 μM (used to obtain FIGS. 1 and 9) Petit It is also possible to use conditions with higher discriminating power such as use. Any place Whether the peptide or analog can inhibit CXC intercrine such as IL-8 Whether or not it can be easily determined using an analytical method as disclosed herein.   Understanding the mechanism of action of CXC intercrine inhibitors is their practical use Irrespective of that, at lower concentrations of the peptide inhibitors of the invention, IL-8 Thus, IL-8-induced neutrophil enzyme release rather than chemotaxis-induced It is important to mention that can be preferentially inhibited. In this sense, The word "ru" when used in connection with the present invention means that the It also means "regulate", because noh is more significantly inhibited than other functions. doing.   Approximately 25-fold below the concentration required to inhibit IL-8-induced chemotaxis The concentration of neutrophil-induced release of neutrophil-degrading enzymes can The inhibitory potency of peptide is an important finding expected from previous studies. this thing Is the detrimental effect of normally released enzymes, while still recruiting neutrophils at the lesion site. It means that can be significantly reduced. Due to this characteristic, its small size makes it Therefore, these peptides are used in various therapeutic protocols, especially in the treatment of lung disorders. Would be ideal for   In accordance with the present invention, inhibition of CXC intercrine, such as inhibition of IL-8, GRO or MIP2 To achieve or prefer neutrophil enzyme release over neutrophil chemotaxis To achieve, generally CXC intercrine group of molecules or granulocytes or neutrophils Intercrine target cells such as those containing one peptide of the group disclosed herein. A biologically effective amount of the composition is contacted. The “contact” process is the process of activity within the inhibitor. Peptides to the CXC intercrine peptides or their rela- tions present on the target cells. Contact one or both of the sceptors to reduce their functional interaction or It is a process by inhibiting. Scientifically interesting, but for specific cells Transmission The signal of the CXC intercrine carried is irrelevant to the practice of the invention.   For contacting CXC intercrine or target cells with a composition containing a peptide Bind peptides or compositions to target cells such as neutrophils and intercrine in vitro. Just add to. Alternatively, a biologically effective amount of a pharmaceutically acceptable form May be administered to the animal. In this case, the peptide In body fluids, for example neutrophils or macrophages and intercrine Will be done. In such a situation, "contact" simply administers the composition to the animal. Achieved by: Most peptide drug formulations are also available. To them In addition to parenteral preparations such as intravenous, intramuscular and subcutaneous administration, , Aerosols, spray formulations and topical coatings for creams, ointments, gels, etc. Peptide formulations as well as lipid-terminated peptides, micelles or liposomes and And active peptides in biocompatible coatings designed for controlled release Other formulations encapsulated in sustained release capsules etc. However, the present invention is not limited to these.   Pulmonary edema fluid in ARDS patients (Miller et al., 1992) and sputum in cystic fibrosis patients (Ri chman-Eisenstat et al., 1993), Pleural space in patients with pleural effusion (Miller & Id. ell, 1993), joint fluid in patients with several types of joint disease (Brennan et al., 1990; Miller & Bresford, 1993), IL in psoriatic plaques and synovial fluid of patients with arthritis (Lam et al., 1990). It is known that -8 concentration increases. Inappropriate neutrophil activation can lead to It was associated with all cases and myocardial damage due to ischemia and reperfusion (DeFroge et al., 1992). I am involved. Inhibition of neutrophil recruitment by IL-8 reduces lung inflammation in vivo (Mulligan et al., 1993) and used here That certain types of in vitro studies have been shown to predict in vivo activity From (US Pat. No. 5,079,22, which is hereby incorporated by reference. 8)), IL-8-induced neutrophil activation disclosed in the present application. Inhibition Good results support the wide clinical application of these peptides .   Thus, the present invention has many roles in playing the role of CXC interclins, especially IL-8. Also provided are methods of treating a wide variety of diseases, particularly those with inflammatory components. This is a pride Lungs with bronchial inflammation such as idiopathic bronchitis, cystic fibrosis, lung exudation, asthma and ARDS In addition to disorders and lung diseases, skin diseases such as psoriasis and dermatitis, and rheumatoid arthritis Treat patients with joint disorders and reperfusion after pseudogout, inflammatory bowel disease or myocardial infarction. Generally reduces inflammation in other clinical situations such as treatment of heart failure due to flow It is included. These peptides can be used, for example, in cancers involving cell proliferation and other It can also be used as a growth inhibitor to suppress the proliferation of lymphocytes to treat the diseases of Wear.   It is affected by any one of the above diseases or the activity of neutrophils and causes inflammation. To treat certain other diseases that are associated with specific inflammation or IL-8. Are disclosed herein, and subsequently to the patient, preferably parenterally, as disclosed herein. A biologically effective amount of a pharmaceutical composition comprising one or more peptides of the group is administered.   Of course, a particular pharmaceutical formulation will generally be prepared according to the disease to be treated. An example For example, for methods of treating skin disorders, creams or gel formulations for application are used. Injectable formulations are used in methods of treating lung disease or It is also possible to use rhes, aerosols or inhalants. Of other parts of the body Methods to reduce inflammation include peptides formulated for parenteral administration or sustained release. Use peptides incorporated into a biocompatible coating designed for be able to. It is also possible to use liposome encapsulation, which Known to increase the efficacy of administered compounds and significantly prolong their half-life . Various compositions and techniques for preparing all such pharmaceutical formulations have been found in this discovery. In the light of this, it is generally known to those skilled in the art. With suitable pharmaceutical composition Details of relevant dosing regimens are incorporated herein by reference. of Remington's Pharmaceutical Science, 16th Edition, 1980, Mack Publishing Co. Please refer to.   Inhibition of IL-8 or CXC intercrine results in a biologically effective amount of inhibitory peptide. Can be achieved by using. As used herein, "a biologically effective amount A peptide or composition is one that inhibits the action of IL-8 or certain intercrine Means an effective amount. For example, with respect to the inhibition of IL-8, a suitable amount is especially chemotactic And an effective amount for reducing neutrophil enzyme release. Disclosed here Various concentrations, such as concentrations of about 100 μM to about 20 μM. Petit is extremely effective in vitro. Therefore, the local concentration of peptides is similar The resulting clinical dose is expected to be particularly useful.   Of course, in a clinical setting, the quantity and volume of peptide composition administered will depend on the host. It will depend on the animal, the circumstances being treated and the route of administration. Must be given The exact amount of active peptide may be patient specific, at the discretion of the physician. I However, in light of the data presented here, the appropriate range for use in humans is It's simple. For example, for treatment of ARDS or cystic fibrosis, about 0.83 mg / kg body weight / Hour (mg / kg / hr) to about 16.56 mg / kg / hr, preferably about 0.83 mg / kg / hr to about 4.14 mg / kg / hr, more preferably 1.66 mg / kg / hr of active peptide is administered per patient It is possible that   According to the present invention, CXC intercrine inhibition such as IL-8, GRO, MIP2α or MIP2β The composition generally comprises 6 amino acid sequences RRWWCX (SEQ ID NO: 23) in its sequence. And a relatively small peptide having a length of about 14 residues. What is "peptide" Means at least one peptide according to the general formula RRWWCX (SEQ ID NO: 23) It may mean one or more peptides containing sequences.   The relatively small peptides included in the present invention are 6 to 14 or 15 residues long and The exact length is not an important feature of the invention. Use smaller peptides When It offers many advantages. For example, the cost of large-scale synthesis is relatively low and It has improved pharmacological properties such as ease, easy penetration into tissues, and low immunogenicity. And the like.   In addition to containing the amino acid sequence of sequence RRWWCX (SEQ ID NO: 23) Other short peptidyl sequences of amino acids may be included. For example, in one embodiment The peptide has the sequence RRWWCX (SEQ ID NO: 23) or RRWWCXX (SEQ ID NO: 57). ) Repetitive sequences. These are, for example, short targeting sequences, It is thought to increase the half-life or increase the stability of labels, labeled residues and peptides. As long as it has an inhibitory effect on the amino acid Certain additional sequences can be included that include the additional residues desired for any purpose. these Can be readily determined by the simple analytical methods described herein.   Amino acids that can be included in peptides include all commonly found amino acids. I will. As commonly practiced in the art, there are two amino acid nomenclatures Used interchangeably in this application. Alanine = Ala (A); Arginine = Arg (R); Aspartic acid = Asp (D); Asparagine = Asn (N); Cysteine = Cys (C) Glutamic acid = Glu (E); Glutamine = Gln (Q); Glycine = Gly (G); Histidine = His (H); Isoleucine = Ile (I); Leucine = Leu (L); Lysine = Lys (K); Methio Nin = Met (M); Phenylalanine = Phe (F); Proline = Pro (P); Serine = Ser (S) Threonine = Thr (T); tryptophan = Trp (W); tyrosine = Tyr (Y); valine = Val (V).   The following rare amino acids or modified amino acids are also included in the peptides of the present invention. Can be 2-aminoadipic acid, 3-aminoadipic acid, β-alanine (Β-aminopropionic acid), 2-aminobutyric acid, 4-aminobutyric acid (piperidine acid) , 6-aminocaproic acid, 2-aminoheptanonic acid, 2-aminoisobutyric acid, 3-ami Noisobutyric acid, 2-aminopimelic acid, 2,4-diaminobutyric acid, desmosine, 2,2'-dia Mi Nopimelic acid, 2,3-diaminopropionic acid, N-ethylglycine, N-ethylas Paragine, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline 4-hydroxyproline, isodesmosine, allo-isoleucine, N-methyl group Lysine, isodesmosine, allo-isoleucine, N-methylglycine (sarcosine ), N-methylisoleucine, N-methylvaline, norvalinene, norleucine and And ornithine.   The inhibitory compositions of the present invention include biologically modified peptides. Including peptides. When administered to humans, biologically protected peptides are protected It has several advantages over unidentified peptides and is described in US Pat. No. 5,028,592. Are used to form a part of this specification). Protected peptides often have increased pharmacological activity. This is also the case this time Was recognized by.   Therefore, the present invention relates to an acylated peptide or peptide, preferably N-terminal. Included are compositions that include peptides that are acylated at the ends. In this case, Although any of these acyl groups can be used, the present inventors have found that an acyl group can be added to the N-terminal of a specific peptide. When added, the obtained peptide also has a surprising intercrine inhibitory effect on IL-8 and the like. Found to have. The peptide composition having an inhibitory effect is modified at the C-terminus. Peptide, ie NH₂Also included are peptides with group additions. Especially preferred In the examples, both an acylated N-terminal and an amidated C-terminal residue are included. Peptides are thought to be very close to the structures of natural proteins and peptides. It is preferable because it is used.   The composition used in the present invention comprises L-amino acids, D-amino acids or a mixture thereof. Peptides containing compounds can also be included. Pep composed only of D-amino acids The finding that tid has potent inhibitory activity is of particular importance. Is that Such peptides have resistance to proteases originally found in the human body But Known and less immunogenic and therefore have a longer biological half-life Because it can be expected.   The anti-intercrine and anti-IL-8 compositions of the present invention generally include SEQ ID NO: 1 or sequence. One or more peptides containing the amino acid sequences set forth in SEQ ID NOs: 24 to 42 Including. In certain embodiments, short hexamer peptides may be preferred. In such cases, the inhibitory composition will generally be from SEQ ID NO: 1 or SEQ ID NO: 24. Includes one or more peptides comprising the amino acid sequence presented at 42.   In another embodiment, the inhibitory composition of the invention comprises the amino acid sequence Arg Arg Trp Trp. Cys Arg Xaa₂Can include one or more peptides comprising the sequence (SEQ ID NO: 2) You. In such cases, one of the variable sites is defined as arginine and the remaining Xaa₂Yes Deviated amino acid residues are also possible. Specific examples of such sequences are SEQ ID NO: 3 or Et al. 22 are presented. If short heptamer peptides are preferred, the composition is One or more comprising an amino acid sequence generally according to the amino acid sequences presented below. Contains peptides:   The present invention provides the amino acid sequence Gln Ile Pro Arg Arg Ser Trp Cys Arg Phe Leu Phe A peptide having (SEQ ID NO: 52), alone or more preferably one of the above It is also envisioned to be used in combination with the above other peptides. This dodecama The successful use of dodecamer is said to be successful with longer peptides. The fact that several specific peptides with equivalent biological functions are active It shows the fact that Accordingly, all such active equivalents are within the scope of this invention. .   The composition for use in the method of inhibition described here is the only active peptide. Jill species may be included. Alternatively, up to about 40 or about 45 different peptides Can be included. 2, 3, 5, 10, 15, 20, 30 or 45 different pepti Any variety of combinations are conceivable, such as compositions of matter.   Amino acid sequence Arg Arg Trp Trp Cys Arg (SEQ ID NO: 1) and / or amino Consists of a peptide with the acid sequence Arg Arg Trp Trp Cys Arg Cys (SEQ ID NO: 4) The composition appears to be particularly useful, but the invention is in any case limited to these peptides. It is not specified. From this point, in vitro activities such as plasma half-life and stability are Considerations other than sex should be considered when finally selecting a preferred peptide for clinical practice. It is important to mention that it is worth considering. Substitution with various amino acids The effects of and on these parameters can be measured immediately and used in vivo. Results to design the optimal peptide or peptide combination for Can be used.   The RRWWCX (SEQ ID NO: 23) sequence element is an important feature of the peptides of the invention. However, this excludes substances with similar biological functions from the scope of the present invention. Not. For example, we have found that the first tryptophore of RRWWCX (SEQ ID NO: 23). By replacing the agent with, for example, serine with little loss of activity. I found that I could exchange it. Thus, one example of equivalents included in the present invention is the sequence RR It is a peptide of XWCX (SEQ ID NO: 58). "Equivalent amino acid" means hydrophilic or Have a hydrophobicity index within ± 2 of each other, more preferably within ± 1, and most preferably ± 0. It can be defined as an amino acid within 5. Of course, to be "functionally equivalent" Tide can be easily determined using analytical methods such as those disclosed herein. It must still retain turcurin or amino acid inhibitory activity.   In addition to the peptidyl compounds described herein, the inventors have found that peptidomimetics Another sterically similar compound, called I also think that it can be prepared. Such compounds are similar to the peptides of the invention Since they can be used, they are also functionally equivalent substances. Structurally and functionally equivalent The production of various substances is well known to those familiar with the art with modeling and chemical design. It can be achieved by the technology of All such three-dimensionally similar substances It will be appreciated that the invention is within the scope of the invention.   The peptides and compositions for use in the present invention can be prepared in any one of a variety of ways. Can be prepared. One of the preferred methods for preparing the peptides of the invention is automated It is considered that this is due to the peptide synthesis method. Synthetic peptides are automated peptide synthesis It is easily prepared using the device, and its operation method is common to those skilled in the art. Is known to This method can be used, for example, once a particular peptide has been selected for treatment. Thus, it is one of the generally preferred methods for large scale preparation of a particular peptide.   Another preferred method for preparing a substance having the biological function equivalent to that of the inhibitory peptide Is described by Houghten et al., (1991) and has been filed in International Patent Application PCT Publication WO92 / 093. The method of using the combinatorial peptide library method disclosed in 00. These disclosures are incorporated herein by reference. This These methods, such as RRWWCX, have a substantially predetermined sequence and have a variety of sequences. Specially for preparing and analyzing a large number of peptides in which mino acid is added at one or more sites. Useful for. These methods are suitable for direct use in formulating the compositions of the present invention. For synthesizing peptide mixtures or for use in subsequent individual syntheses Can be used to identify peptides that have particular activity.   If desired, the peptides can also be prepared by molecular biology methods, A "peptide" can be obtained from a recombinant host cell that expresses the peptide. this To achieve the desired peptide distribution known to those skilled in the art. Based on the row, a specific oligonucleotide was prepared and then the oligonucleotide Expression vector, such as any one of the many expression vectors currently on the market. Insert into the target. The vector is then used to transform a prokaryotic or eukaryotic host cell. Once transformed, the vector then directs the expression of the so-called recombinant peptide, which is then assembled. Recombinant peptides can be purified from the recombinant host cells. This method is This is the standard practice in surgery (see Sambrook et al., 1989).                                Brief description of the drawings   The following drawings form part of the present specification and further clarify some aspects of the present invention. Included for clarity. Detailed description of the specific embodiments presented here The invention may be further understood by reference to one or more of these drawings in conjunction with the description. Can be understood.   Figure 1. Binding inhibition by the Ac-RRWWCX (SEQ ID NO: 23) series. Ac-RRWWC (sequence No. 56) and a structure containing any one of the sixth standard protein amino acids The 20 kinds of peptides possessed were examined. The X-axis display shows the residue at the carboxyl terminal position. The groups are shown (SEQ ID NOS: 1 and 24 to 42). In this test, 1p neutrophils M's¹²⁵Incubated with I-labeled IL-8 and 20 μM of each peptide.   FIG. Ac-RRWWCR-NH₂Inhibition of IL-8 binding by (SEQ ID NO: 1). Contains 0.1% BSA M Neutrophils in 0.2 ml PBS (1 x 10⁶), 1 nM¹²⁵I-labeled IL-8 and increasing concentrations of Ac- RRWWCR-NH₂Incubation with (SEQ ID NO: 1) for 90 minutes at 4 ° C. these Data are representative of 4 trials.   FIG. 3A. Ac-RRWWCR-NH₂Saturation test in the presence of (SEQ ID NO: 1). Lundon I Binding isotherm with best fit curve calculated using. Binding analysis If not present (○), 10 μM (●), 20 μM (△), or 40 μM (▲) pep In the presence of Chid,¹²⁵This was carried out by increasing the concentration of I-labeled IL-8. The points of each data are Non-specific binding in the presence of excess unlabeled IL-8 from total binding It represents the specific binding calculated by subtraction.   FIG. 3B. Ac-RRWWCR-NH₂Saturation test in the presence of (SEQ ID NO: 1). Scatter Chard plot. Binding assay is 10 μM (●) when peptide is absent (○) , 20 μM (△), or 40 μM (▲) peptide,¹²⁵I-labeled IL-8 concentration Was increased.   FIG. Ac-RRWWCR-NH₂Binding inhibition test in the presence of (SEQ ID NO: 1). Neutrophils 1 nM¹²⁵Increasing the concentrations of I-labeled IL-8 and unlabeled IL-8 to obtain 10 μM Ac-RRWWCR-NH₂ It was performed under the condition (●) or the condition (○) where (SEQ ID NO: 1) exists. .   FIG. Ac-RRWWCR-NH₂(SEQ ID NO: 1) IL-8, C5a, leukotriene B_FourNeutrophil Action on binding to. Binding analysis is 1 nM¹²⁵I-labeled IL-8 (●), 0.25 nM¹²⁵I Labeled C5a (△), or 0.4 nM^ThreeH-labeled leukotriene B_Four(▲) and increased the concentration Ac-RRWWCR-NH₂It was carried out using (SEQ ID NO: 1). Analysis of variance for multiple comparisons used. If there is a significant difference, binding without peptide and peptide Differences in binding with inclusions were tested using the Sheffes test. *** is p <0.001.   Figure 6. Cytotoxicity test. Chromium-labeled neutrophils were enriched with Ac-RRW WCR-NH₂(SEQ ID NO: 1) in PBS containing 0.1% BSA at 4 ° C for 90 minutes (●) Alternatively, incubate in RPMI-1640 medium containing 1% BSA at 37 ° C for 30 minutes (○). Was. Analysis of variance was used for multiple comparisons. If there is a significant difference, peptides are not included. Sheffes test It was calibrated using. *** is p <0.001.   FIG. Ac-RRWWCR-NH₂Effect of (SEQ ID NO: 1) on neutrophil chemotaxis. Chemistry As for chemotaxis, the concentration of Ac-RRWWCR-NH increased in the upper and lower compartments.₂In the presence of (SEQ ID NO: 1) It was carried out. The stimulants added to the lower chamber were 10nM IL-8 (●) and 10nM fMLP (■). Or, as a control, only the medium (◯) was used. Use analysis of variance for multiple comparisons did. If there is a significant difference, the migration distance without peptide and the peptide The difference in the moving distance in the case of including the code was tested using the Sheffes test. *** is p <0.001 .   FIG. Ac-RRWWCR-NH₂Effect of (SEQ ID NO: 1) on β-glucuronidase release . Neutrophils pretreated with cytochalasin B were treated with 100 nM IL-8 (●), 100 nM fM LP (■), 100nM C5a (△) or 100nM Leukotriene B_Four(▲) or any Ac-RRWWCR-NH does not contain stimulants of (○)₂Increase the concentration of (SEQ ID NO: 1) And incubated at 37 ° C for 30 minutes. Phenolphthalein as substrate Glucuronic acid was used to measure β-glucuronidase activity in the supernatant. Multiple comparison Analysis of variance was used for. If there is a significant difference, the Using the Sheffes test, the difference between the total migration distance and the migration distance when the peptide is included Tested. * Is p <0.05, ** is p <0.01, *** is p <0.001.   FIG. All D-amino acids Ac-rrwwcrx-NH₂(SEQ ID NO: 2) Combined by series Inhibition. Ac-RRWWCR-NH₂(SEQ ID NO: 1) was synthesized using D-amino acid, and 20 kinds of The standard protein D-amino acid was added to the 7th position (SEQ ID NO: 3 or 22). The label on the X-axis indicates the residue at the carboxyl terminal position. 10 μM each Binding studies were performed using peptides. Ac-RR prepared with L-amino acid WWCR-NH₂(SEQ ID NO: 1) was used as a control.   FIG. Ac-RRWWCX-NH₂(SEQ ID NO: 23) shows the binding of GRO and MIP2β to human neutrophils. Hinder the combination. Radioactive iodine labeled MIP2β and GRO / MGSA at various concentrations of Ac-R RWWCR-NH₂And incubated at room temperature for 15 minutes. Neutrophil suspension (0 1 × I0 in 160 μl PBS containing 1% BSA⁶Individual cells) to 40 μl of the mixture and on ice And incubated for 90 minutes. It is bound to the cells from the oil layer by centrifugation. Radioactivity was separated from free radioactivity. The% binding inhibition value was calculated as follows: .     In this case B is the radioactivity bound in the presence of the peptide and T is the peptide With radioactivity bound in the absence of NSP, NSP is Radioactivity bound in the presence.                           Detailed Description of the Preferred Embodiment CXC intercrine, a peptide with IL-8 action and inhibitory action.   IL-8 has been identified as a molecule that activates neutrophils (Schroder et al., 1988; Peveri et al., 1988; Yoshimura et al., 1987). IL-8 is a bacterial lipopolysaccharide (LP S), tumor necrosis factor or interleukin-1 Produced primarily by rophage and endothelial cells, fMLP, C5a, or leukoto Lien B_FourAnd other chemotactic agonists share the usual neutrophil activation properties (Bag giolini et al., 1992). IL-8 stimulates neutrophil chemotaxis as well as enzyme release and respiration Can stimulate Bath. IL-8 is a group of peptide molecules called CXC intercrine It is a member, and all CXC intercrine has a CXC sequence motif in its terminal region. CXC interclins include GRO, MIP2α or MIP2β and more recently ENA7 8 is also included.   The function of IL-8 is mediated by the IL-8 receptor on the surface membrane of neutrophils. Recent research Studies have shown that IL-8 binds to at least two different receptors, whereas Other substances belonging to the turclin group bind to one of its receptors with high affinity (Holmes et al., 1991; Murphy & Tiffany, 1991; LaRosa)  et al., 1992; Cerretti et al., 1993). These receptors are responsible for other chemotaxis It is distinct from the receptor for sex agonists (Dohlman et al., 1987).   IL-8 was impregnated in the thorax in the synovial fluid of patients with several types of joint diseases (Brennan et al., 1990). In the thoracic cavity of an active patient (Miller & Idell, 1993), adult respiratory distress syndrome (ARDS) High levels were found in the pulmonary edema fluid of patients (Miller et al., 1992). IL-8 concentration Is also shown in recent studies in patients with cystic fibrosis Proven (Richman-Eisenstat et al., 1993; McElvaney et al., 1992; N akamura et al., 1992; Bedard et al., 1993).   IL-8 activates neutrophils, which are powerful antimicrobial cells, but neutrophils Release of this enzyme can cause severe tissue damage. Follow IL-8 is thought to be important in the etiology of these and other inflammatory diseases. I have. Therefore, the present inventors have found that regulating the function of IL-8 is associated with various diseases and pathological conditions, In particular, we hypothesized that it would be an excellent way to control ARDS and cystic fibrosis.   Recently, various research results have been reported which attempt to change the function of IL-8. To these Include several studies showing successful inhibition of IL-8 synthesis (S tandiford et al., 1992; Lam et al., 1990). In addition, anti-human IL-8 causes immune disorders It has been reported to improve lung inflammation in rats (Mullingan et al., 1 993). IL-8 secretion in the respiratory tract of patients with cystic fibrosis is reportedly neutrophil Secreted Leukoprotease Inhibitor of Sphere Elastase Is Reduced (McElv aney et al., 1992). In addition, IL-8 production is affected by oxygen free radical scavengers. It was shown to be inhibited in whole blood stimulated by LPS (DeFo rge et al., 1992). Finally, two cytokines, interferon gamma (Ca ssatella et al., 1993a; 1993b) and interleukin 4 (Standiford et al., 1 990) inhibited the synthesis of IL-8. However, such research has shown that An IL-8 inhibitor or specific neutrophil reaction can be preferentially inhibited over other reactions. Inhibitors have not yet been produced.   Studies on the interaction between IL-8 and its receptor have also been conducted. From now on, IL Specific peptide inhibitors have been identified that have the same structure as part of the -8 molecule. For example , Gayle and coworkers located at the amino terminus of the rabbit IL-8 receptor. Found 4 amino acids to be moderately good inhibitors of IL-8 binding and function (Gayle et al., 1993). We have found that synthetic peptides, especially the amino acids of IL-8. A terminal peptide inhibits amino-terminal neutrophil binding and neutrophil chemotaxis. Found (Miller et al., 1990; Miller et al., 1993). N-terminal penta Peptide IL-8 inhibitors have also been reported (Goodman et al., 1991).   Nonetheless, knowledge of the structure of the IL-8 molecule has shown that it is an effective peptide inhibitor. The drug is not yet developed. Therefore, in order to achieve this object, the present inventors have , By screening a library of 400 groups of hexapeptides Various other peptide compositions were analyzed for IL-8 inhibitory activity. these In the screening analysis of¹²⁵I-labeled interleukin-8 (10^-12M) 100 Mix with μl peptide, add to neutrophils, and incubate at 4 ° C for 90 minutes. Was. For centrifugal separation using a high-density cushion of a mixture of paraffin and silicone oil To separate bound radioactivity from unbound radioactivity and bind it to neutrophils Was done¹²⁵I was counted with a gamma ray spectrometer and the results were inhibited by IL-8 binding. Could be expressed as a percentage of the total.   In these studies, the sequence RRWWCX (SEQ ID NO: 23) (which terminal amino acid is It may be an amino acid. ) Hexamer was found to be a potent IL-8 inhibitor Was. Certain RRWWCX-type peptides have previously shown anti-staphylococcal properties. It has been found (Houghten et al., 1991) that these peptides are antisites. Cain or other functional properties suggesting to have anti-inflammatory activity have not been reported Yes. We found that RRWWCR reduced binding of GRO and MIP2β to human neutrophils The findings revealed that it also effectively inhibits other CXC interclins such as GRO and MIP2. I made it soft.   Although all peptides in the RRWWCX (SEQ ID NO: 23) series have anti-IL-8 activity (Table 1 and 1B), the peptide RRWWCR (SEQ ID NO: 1) was found to be particularly effective. Was. Modified to mimic peptides present in longer protein sequences , This peptide with an acetyl group at the amino terminus and an amino group at the carboxyl terminus One form (Ac-RRWWCR-NH₂SEQ ID NO: 1) is one of the focal points of these trials Was.   Ac-RRWWCR-NH₂(SEQ ID NO: 1) is¹²⁵Preventing specific binding of I-labeled IL-8 to neutrophils Harmful and apparent K_IIs about 10 μM, which is a non-acetylated form of the same peptide. It was about twice as effective. Exact K_IThe value is because there is a positive synergy I couldn't get it. The binding isotherm for IL-8 in the absence of peptide is Best fit to a one-site model when analyzed using the Lundon I tar program did. The lower IL-8 concentration data was obtained in the Scatchard plot. One of the explanations is that low-concentration IL-8 concentration masked high-affinity binding. And the like. However, recent studies have shown that there are two distinct classes of IL-8 receptors. It has been clarified that IL-8 binds to the protein with almost the same affinity (Lee e t al., 1982; Schmacher et al., 1992). Therefore, this result as a one-site model B of joint area_maxEstimates of B for two classes of receptors_maxRepresents the total of , K_dThe estimate of is common to these receptors.   The binding isotherm fits the two-site model very well when the peptide is present . Ac-RRWWCR-NH₂Analysis of the binding isotherm in the presence of Inhibits the binding of 8 and is different to the extent that the two classes of receptors differ Was revealed. From the estimated binding parameters, one class of receptor Since the affinity of the receptor was suppressed by 10 μM of peptide, competitive inhibition was observed. Harm is shown Be inspired. For non-competitive inhibition of another class of receptors, High concentrations of peptide are required.   The activity of this inhibitory peptide is specific to IL-8. Ac-RRWWCR-NH₂Is C5a or Roy Kotrien B_FourBinding to neutrophils, induced by formyl-L-Met-L-Leu-L-Phe (fMLP) Chemotaxis or fMLP, C5a or leukotriene B guided_FourΒ induced by It does not inhibit the release of glucuronidase, nor does it affect neutrophil chemotaxis or enzyme release. It also has no intrinsic ability to provoke. These observations are based on Ac-RRWWCR-NH₂As Peptides alter IL-8 receptor binding, resulting in IL-8-induced It suggests that it may inhibit the activation of neutrophils.   Furthermore, Ac-RRWWCR-NH₂Structure-based hexamer and heptamer peptides are chemically It is thought to preferentially inhibit enzyme release over chemotaxis. Fact Ac-RRWWCR-NH₂ Significantly suppressed neutrophil chemotaxis induced by 10 nM IL-8 at a concentration of 50 μM. Concentration of 2 μM when more than 10 times more IL-8 is required to control and trigger enzyme release Have been shown to significantly and significantly suppress β-glucuronidase release . Inhibiting enzyme release preferentially over chemotaxis at this lower concentration is especially This is an important and surprising finding that makes this kind of peptide a sign of adult respiratory distress. It is ideal for treating lung disorders in patients in this group.   The above findings also indicate that IL-8 has different classes in order to exert different functions of neutrophils. Suggests that it may have to bind to its receptor. Use of the present invention It is not important that the peptides of the present invention show that the IL-8 receptor Suitable for use as a research tool to further elucidate the function of neutrophils and neutrophils Becomes   Next, Ac-rrwwcrx-NH in which all amino acids are D-amino acids₂(SEQ ID NO: 2) The inhibitory activity of the second set of peptides was investigated. RRWWCRX (SEQ ID NO: 2) series Were again found to have anti-IL-8 activity for all the peptides of Table 1 (Tables 5A and 5B) . I Scarecrow, Ac-rrwwcrc-NH₂(SEQ ID NO: 4) is the best inhibitor, Ac-rrwwcr-NH₂( It was found to be almost four times more potent than SEQ ID NO: 1). This observation result Inability of mammalian proteases to degrade D-amino acid peptides and proteins Can have significant implications (Togo et al., 1989). Therefore, D-amino Acid peptide inhibitors are expected to have a longer lifespan in vivo.   A variety of other synthetic peptides were tested for their ability to inhibit IL-8 binding to neutrophils using standard analytical methods. I killed it. These peptides are either amino-terminal analogues of IL-8 or RR Protein database with 5 out of 6 residues of WWCR (SEQ ID NO: 2) (PIR Or a fragment of the protein found in Swiss-Prot) . The latter peptide is a high-performance computing center in Austin, Texas (the Ce nter for High Performance Computing) CRAY computer PIR of RRWWCR (SEQ ID NO: 1) using IGSUITE program to search database And identified by searching the Swiss-Prot database Was done. Data containing all 6 amino acids in proteins in the database There was no.   According to past research, Glu located at the 4th, 5th, and 6th positions of the 72 amino acids of IL-8, Leu and Arg are important for neutrophil binding (Clore et al., 1992; Clark-Lewis et al. al., 1991), an amino-terminal peptide of IL-8 blocks IL-8 binding to neutrophils and chemotaxis. Have been shown to cause harm (Miller et al., 1993). Therefore, the inventors Is an Ac-KELRCQ-NH similar to IL-8₂(SEQ ID NO: 54) and ELRCQCIKTY (SEQ ID NO: 49) , Which contains the C-X-C motif of the interclin peptide), but not its Cys. And two similar analogs, ELRSQSKTY (SEQ ID NO: 50) and ERLMQMKTY (SEQ ID NO: 51). Examined. None of these peptides can inhibit the binding of IL-8 to neutrophils. won.   Next, the inventors have confirmed that RRWWCR (SEQ ID NO: 1) has a physiological function associated with IL-8. In order to clarify whether it is present in another peptide that seems to be , The protein database was searched. Two peptides are RRWWCR (SEQ ID NO: 1) Since it contained 5 of the 6 residues of and was contained in a known protein, Identified and selected for study. Those are GWRRWWCDAVLY (SEQ ID NO: 53) and QI PRRSWCRIt was FLF (SEQ ID NO: 52). The former peptide is "cell surface sugar CD11c precursor-human leukocyte adhesion receptor p150,95 included in alpha chain, Were contained in the 3 ', 5'-cyclic GMP phosphodiesterase beta chain-bovine (Ocvchinnikov et al., 1987; accession number, S00251).   One of these analogs, QIPRRSWCRFLF (SEQ ID NO: 52), is a preferred IL-8. It inhibited the binding to neutrophils by about 60%, but this activity was Ac-RRWWCR-NH.₂(SEQ ID NO: 1) Low. The protein from which this sequence was extracted, bovine 3 ', 5'-cyclic GMP ho Although it is unlikely that schodiesterase plays a physiological role in IL-8 function, , This cannot be ruled out completely. The most important days from this study That RRWWCR (SEQ ID NO: 1) can be modified with only a slight loss of activity. It suggests. Active against another peptide, GRRWWCDAVLY (SEQ ID NO: 53) What does not exist is Ac-rrwcc when the last Arg in RRWWCR (SEQ ID NO: 1) is changed to Asp. Supported by the observation that d (SEQ ID NO: 26) has little activity Thus, it suggests that IL-8 significantly reduces the ability to bind to neutrophils.   Therefore, the examples presented here demonstrate the binding of IL-8 to neutrophils and the activity of neutrophils. Detailed identification of a series of new peptide inhibitors that can inhibit sexualization . RRWWCX (SEQ ID NO: 23) and RRWWCRX (SEQ ID NO: 1) type peptide inhibitors It has been reported previously that it has a length of 6 to 7 residues and that D-amino acids also have activation. It has clear advantages over existing inhibitors. This class of inhibitors Effective in treating diseases caused by the uncontrolled effects of IL-8 such as distress syndrome It seems to be for use. These inhibitors are neutrophil due to chemotaxis that is not easily inhibited. A point that allows the sphere to enter the lungs, but then preferentially inhibits the release of harmful enzymes so, It has clear advantages (McGuire et al., 1982).   In addition to a wide variety of therapeutic applications, the peptides and compositions of the present invention may be used in other embodiments. Is also useful. These include positive controls for the analysis of IL-8 inhibitors, for example. The interaction and function of the IL-8 receptor used in various bioassays It is used for elucidation, and used for antibody production. Substances with equivalent biological functions   It is considered that some biologically equivalent substances fall within the scope of the present invention. Living The concept of amino acids having equivalent physical functions is well known to those skilled in the art. , It is possible to make modifications and changes to the structure of the protein or peptide, The knowledge that anyone can still obtain similar or other desirable properties It is embodied by knowledge.   However, the definition of a protein or peptide with an equivalent biological function is There is a limit to the number of changes that can be made in a defined part, yet biological activity Are acceptably equivalent, with important active sites or structurally essential residues It will be well understood by the skilled technician that the concept of irreplaceability is involved. Follow Thus, peptides with biologically equivalent functions are not considered here for most or all. It is defined as a peptide in which some amino acids have been replaced. In particular, the hexamer For heptamer peptides only about 2 and more preferably 1 amino acid It is believed that only one is substituted within the particular peptide. of course, A large number of different peptides with different substitutions can be generated and used according to the invention. Wear.   From the viewpoint of substituting a limited number of residues in the peptide, the structure of the receptor and cells, etc. Due to their ability to bind to each other or to compete with other molecules that bind to these sites. Specific amino acids without apparent loss of function, as can be measured by potency. It is known that can be replaced with another amino acid. Raw protein or peptide It is its interaction and competitive ability that define the physical function activity. , The peptide sequence (or of course the underlying DNA coding sequence) Substituting certain amino acids and nevertheless similar peptides or Can even obtain improved properties.   Amino acid substitutions are generally based on the relative similarity of amino acid side chain substituents. An example For example, its hydrophobicity, hydrophilicity, charge, size and the like. Amino acid side chain substituent size Based on the analysis of size, shape, and type, arginine, lysine, and histidine were all Charged residues, alanine, glycine, serine are all of similar size, Phenylalanine, tryptophan, and tyrosine all generally have similar forms. Therefore, based on these considerations, arginine, lysine, histidine, and Then, alanine, glycine, serine, phenylalanine, and tryp Tophan and tyrosine are defined here as substances with equivalent biological functions.   To make more quantitative substitutions, the hydrophobicity index of amino acids can be considered. Each net A noic acid was assigned a hydrophobicity index based on its hydrophobicity and charge characteristics. These are the next It is on the street. Isoleucine (+4.5); Valine (+4.2); Leucine (+3.8); Phenylalanine (+2.8); Cysteine / cystine (+2.5); Methionine (+ 1.9); Alanine (+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); Glutami (-3.5); Aspartate (aspartic acid) (-3.5); Asparagine ( -3.5); lysine (-3.9); arginine (-4.5).   The importance of the hydrophobic amino acid index in studying the interaction of protein biological functions The need is generally understood in the art (Kyte & Doolittle, 1982, cited. And form a part of this specification). Similar to certain amino acids Can be replaced with another amino acid having a hydrophobicity index or score of It is known that the biological activity of can be retained. When substituting based on the hydrophobicity index In this case, substitution of amino acids having a hydrophobicity index within ± 2 is preferable, and substitution within ± 1 is particularly preferable. More preferably, it is more preferably within ± 0.5.   US Pat. No. 4,554,101, incorporated herein by reference. Substitution of similar amino acids should be based on hydrophilicity, as disclosed in Is also possible. In U.S. Pat. No. 4,554,101 the following hydrophilicity indices are amino acids: Assigned to residues. Arginine (+3.0); Lysine (+3.0); Asparagus Ginate (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); Cysteine (-1.0); Methionine (-1.3); Bali (-1.5); Leucine (-1.8); Isoleucine (-1.8); Tyrosine (-2.3) Phenylalanine (-2.5); Tryptophan (-3.4).   You can replace a particular amino acid with another amino acid that has a similar hydrophilicity index. However, it is known that they can retain the same biological activity. Based on hydrophilicity index In this case, it is preferable to replace amino acids whose hydrophobicity index is within ± 2, ± 1. Particularly preferred is within, and more preferred is within ± 0.5. Pharmaceutical formulation   The peptides and compositions of the present invention can be administered to CXC interclines such as IL-8 or neutrophils. Involvement of, or inappropriate or enhanced inflammatory reaction It can be used to treat various diseases. The invention is particularly applicable to asthma, bronchitis, cysts. Suitable for the treatment of inflammatory fibrosis and lung inflammation associated with ARDS. ARDS, cystic fibrosis, etc. Parenteral administration such as intravenous injection, intramuscular injection or subcutaneous injection is most preferred for treating the diseases of Although it may be the preferred route of administration, aerosols or inhalants may also be used. Spray And the inhalant, the droplets are fine enough, the size is uniform, and the mist can reach the bronchioles. Come It is effective only when Particle size depends on aerosol or inhalation drug Is very important when administering. The optimal particle size for penetration into the lung cavity is 0. It is on the order of 5 to 7 μm. If a pressurized aerosol produces a fine mist If so, its use seems to be advantageous. Such treatments and treatment requirements are detailed in Example VIII. It is described in detail.   Since the present invention can be used for the treatment of inflammation in various clinical conditions, many types are available. Drug peptide formulations are considered. The therapeutic or pharmaceutical composition of the present invention may comprise lung or Is generally dissolved or dissolved in a pharmaceutically acceptable medium, even when treating other Contains an effective amount of relatively small intercrine or IL-8 inhibitor peptide . The term "pharmaceutically acceptable" means that when administered to the human body, allergies, Refers to whole molecules and compositions that do not cause toxicity or other side effects. Drug A biologically acceptable medium or carrier includes solvents, dispersion media, coatings, antibacterial agents and And antifungal agents, isotonic and absorption delaying agents and the like. This is for pharmaceutically active substances The use of such media and agents is known in the art. Conventional medium Or their use in therapeutic compositions unless the drug is incompatible with the active ingredient. You could think so.   Supplementary active ingredients can also be included in the therapeutic compositions of the present invention. For example, in Peptides that inhibit turculin, IL-8, and neutrophils are IL-8-derived N-terminal peptides, IF In combination with other agents such as N-γ, oxygen free radical scavengers, etc. It is also possible to prepare a peptide mixture.   Intercrine, IL-8, neutrophil inhibitory peptides containing dextrorotatory peptides as active ingredients In the light of the discovery of the present invention, the preparation of a drug or a pharmaceutical composition containing a peptide is disclosed in It is well known to those familiar with. If desired, such a composition may be a liquid solution. A solid suitable for use in a solution or suspension as an injectable liquid or suspension , As liquid before injection, as tablets for oral administration or as other solids, as sustained release hand, Or creams, lotions, mouthwashes, and inhalants and aero of Example VIII It can be prepared as other forms currently in use, including sols.   Of active peptides and compounds as free bases or pharmaceutically acceptable salts Properly mix the solution in water with a surfactant such as hydroxypropyl cellulose. Can be prepared. The dispersion is also glycerol, liquid polyethylene glycol , And mixtures thereof and oils. Under normal storage and use conditions, These preparations contain preservatives which prevent the growth of microorganisms.   A sterile solution suitable for injection may be useful in the treatment of various diseases, either in the blood or Can be administered at the exact site of inflammation. Pharmaceutical forms suitable for injection include sterile aqueous solutions or Contains dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. I will. In all cases the drug form must be sterile and easy to inject It must be a liquid of a certain degree. It must be stable under the conditions of manufacture and storage. , Must be protected from the contaminating action of microorganisms such as bacteria and fungi.   The peptides may be prepared into the composition in neutral or salt form. Pharmaceutically acceptable Active salts include acid-added salts (formed by the free amino group of the peptide) Including, for example, inorganic acids such as hydrochloric acid or phosphoric acid, or acetic acid, oxalic acid, tartaric acid, It is formed by an organic acid such as mandelic acid. Formed by free carboxyl groups The salts also include, for example, sodium hydroxide, potassium hydroxide, ammonium hydroxide, water. Inorganic bases such as calcium oxide or iron hydroxide, and isopropylamine, tris Obtained from organic bases such as methylamine, histidine and procaine.   The carrier may also be, for example, water, ethanol, a polyhydric alcohol (eg, glycerol, alcohol). Propylene glycol, liquid polyethylene glycol etc.) and its suitable Solvents or dispersion media including mixtures and vegetable oils and the like are possible. Lecithin etc. In order to maintain the required particle size in the case of dispersion, use coating To maintain proper fluidity by using a surfactant Can be. Microbiological effects include, for example, paraben, chlorobutanol, phenol, and It can be prevented by various antibacterial agents and antifungal agents such as rubic acid and thimerosal. many In this case, it is preferable to include isotonic agents such as sugar or sodium chloride. Injectable The absorption delay of active compositions is dependent on the absorption of aluminum monostearate and gelatin. It is possible by using a delay agent.   Sterile injectable solutions can be prepared by incorporating the active compound in the required amount with the various other ingredients enumerated above, as required. In a suitable solvent, followed by filter sterilization. Generally, dispersions are Of sterile active ingredient in a sterile medium containing a basic dispersion medium and the required other ingredients above. It is prepared by including. Preferred in the case of sterile powders for the preparation of sterile injectable solutions. The two preparation methods are vacuum drying and freeze drying. Gives the active ingredient and the desired additional ingredients.   Higher concentration solutions for intramuscular injection are also envisioned. From this perspective, very quick It is permeable and allows high concentrations of active peptides or drugs to be administered to small sites. Therefore, it is preferable to use DMSO as a solvent.   Peptide formulations for topical application such as creams, ointments and gels are also conceivable. Oily or Methods for preparing water-soluble ointment bases are also known in the art. For example, these compositions May include vegetable oils, animal lipids, more preferably semi-solid carbon obtained from petroleum Including hydrogen chloride. The individual components used include white ointment, yellow ointment, cetyl esthe Wax, oleic acid, olive oil, paraffin, petrolatum, white petrolatum spermaceti , Glycerin ointment, white wax, yellow wax, lanolin, dehydrated lanolin, monostearin Includes acid glycerides. Glycol ethers and derivatives, polyethylene glycol Various water-soluble ointment bases such as sodium chloride, polyoxyl stearate 40, polysorbate, etc. Available. If desired, eg transdermal patches, iontophoresis or electron transport By the method, transdermal administration is also available.   Ophthalmic buffers are also within the scope of this invention. These are diseases associated with retinal neovascularization. It can be used for affected patients. Peptides can cause changes in pH So buffering is needed. The ophthalmic formulation can be prepared according to conventional formulation methods. An example For example, “Remington's Pharmaceutical Science” No. 15 Edition, pp. 1488-1501 (Mack Publishing Co., Easton, PA) A).   Suitable ophthalmic formulations generally include the novel dipeptides, peptides disclosed herein. Alternatively, the drug is administered in a pharmaceutically acceptable solution, suspension or ointment in an amount of about 0.01 to Contains about 1% by weight. The ophthalmic formulation is preferably stored in the form of a sterile buffer, optionally preserved. Agents, buffers, isotonic agents, antioxidants, stabilizers, nonane wetting or clarifying agents, thickeners Including additional components such as.   Suitable preservatives for use in such solutions include benzalkonium chloride, salt Benzethonium chloride, chlorobutanol, thimerosal, etc. are included. Proper buffer The liquid is sufficient to maintain the pH from approximately pH 6 to pH 8, preferably approximately pH 7 to pH 7.5. Boric acid, sodium and potassium bicarbonate, sodium borate and potassium Including sodium, sodium and potassium carbonate, sodium acetate, sodium phosphate, etc. . Suitable isotonic agents are dextran, dextran 70, dextrose, glycerin. With potassium chloride, potassium chloride, propylene glycol, sodium chloride, etc. Equivalent sodium chloride is in the range of 0.9 ± 0.2%. Suitable for antioxidants and stabilizers Is sodium bisulfite, isomeric sodium bisulfite, sodium thiosulfate, thio Urea and the like are included. Suitable wetting or clarifying agents include Polysorbate 80, Polysodium Includes Rubate 20, Poloxamer 282, Tyloxapol. Suitable Thickener , Dextran 40, dextran 70, gelatin, glycerin, hydroxyethyl Cellulose, hydroxymethylpropyl cellulose, lanolin, methyl cellulose Cotton, petrolatum, polyethylene glycol, polyvinyl alcohol, polyvinyl alcohol Included are loridone, carboxymethyl cellulose and the like.   For formulation, the therapeutic agent should be administered in a pharmacologically effective amount according to the mode of administration. Given. The formulations are easily administered in a variety of dosage forms, such as the injectable solutions above. However, sustained release capsules and the like can also be used.   Typically, the minimum volume of composition required to disperse the peptide is utilized. . There are a variety of suitable dosing regimens, but the compound should be administered first and the results monitored. Therefore, it is typical to administer adjusted doses multiple times. For example, parenteral If given, prepare an isotonic aqueous solution that is appropriately buffered and, if necessary, intravenously or intramuscularly. It is prepared for subcutaneous administration, administered, and prepared and administered until intraperitoneal administration. Throw once Dissolved in 1 mL isotonic NaCl solution and added to 1000 mL subcutaneous infusion solution or Is injected at the proposed injection site (eg, “Remington's Pharmaceutical S cience, 15th Edition, pages 1035-1038 and 1570-1580. Page).   In some embodiments, the active compound may be administered orally. this is, Generally resistant to or conferred resistance to proteolysis by digestive enzymes Thought about the drug. Such compounds include dextrorotatory peptides and chemical Designed or modified drugs and degradation by peptidases and lipases In order to prevent this, a sustained release capsule contains a formulation containing a peptide and a liposome.   Oral formulations should be combined with an inert diluent or an assimilable edible carrier. Compound, in a hard or soft shell capsule, in tablets Included are compounds that have been compressed into, or compounds that have been directly included in food. For oral administration Regarding, it is possible to combine active compounds with excipients, oral tablets, buccal tablets , Troches, capsules, elixirs, suspensions, syrups, cachets, etc. . Such compositions and preparations generally contain at least 0.1% of active compound. Of course Of course, it is possible to vary the composition and formulation proportions, conveniently about 2% of the unit weight. From about 60%. The amount of active compound in such therapeutically useful compositions can be adjusted to an appropriate level. Administration It is the amount that can be achieved.   Tablets, troches, pills, capsules and the like also include: Tragacanth gum, a Binder such as cassia, corn starch or gelatin, and shaping such as calcium phosphate Agents and disintegrating agents such as cornstarch, potato starch, alginic acid, etc. In addition to lubricants such as magnesium argentate, sucrose, lactose or saccharin Sweeteners can be added, or peppermint, wintergreen oil or cherry flavors Fragrances such as can be added. When the dosage unit form is a capsule, in addition to the above substances, A liquid carrier can be added. Other various substances as coating agents or physical It may be included to vary the specific dosage unit form. For example, tablets, pills or Capsules can be coated with shellac, sugar or both. Elixir syrups contain the active compound, sucrose as a sweetener and a preservative. And methyl and propylparaben as a coloring and flavoring agent for cherry or Orange flavors can be included. Of course, what kind of dosage unit form should be adjusted? Even when made, all of the materials used are pharmaceutically pure and poisonous to the dose used. The sex must be virtually unrecognized. In addition, the active compound is released May be included in agents and formulations.   The following examples are provided to present preferred embodiments of the invention. is there. Those who are familiar with the present technology will understand that the technology disclosed in the subsequent examples is the present invention. It is a technique discovered by the inventor, considering that it works well when carrying out And therefore may be considered the preferred method of practicing the present invention. Should be. However, those who are familiar with this technology are not disclosed in light of this discovery. There are many modifications that can be made in a particular embodiment, and yet in the spirit of the present invention. It should be understood that similar results can be obtained without departing from the scope.                                    Example I                        Hexameric peptide inhibitor of IL-8   First, the hexa of the sequence RRWWCX (SEQ ID NO: 23) (X can be any amino acid) In order to clarify whether the mer peptide acts as an inhibitor of IL-8, A series of studies was conducted. The first screening analysis shows that specific peptides It is based on revealing the ability to inhibit binding to human neutrophils. A.Preparation of human neutrophils   Utilizing the human body to obtain neutrophils is a Approved by the review board. For blood neutrophil preparation, human blood enzyme release Anticoagulant with heparin for testing and 0.33% sodium citrate for other testing did. Neutrophil dextrose by Boyum's method for chemotaxis and enzyme release studies Separation was performed by orchid sedimentation and erythrocyte lysis (Kohler & Milstein, 1975). Integration test Percoll (Pharmacia Fine Chemicals, Piscatawe Neutrophils were further purified to 90-93% on a gradient (Bay, NJ) (Kohler & Milstein, 1975). B.IL-8 binding analysis   Recombinant human IL-8 (72 amino acids; Pepro Tech Inc., Rocky Hill, NY -Jersey), Hunter and Green chloramine T method (Hunter & Green) Due to¹²⁵Radiolabeled with I. The method of Besemer et al., (1989) as follows: Binding studies were performed according to: 0.1% bovine in the presence and absence of the test peptide. Neutrophils placed in phosphate buffer pH 7.4 (PBS) containing serum albumin (BSA) Incubate with labeled ligand for 90 minutes at 4 ° C until equilibrium is reached, then With paraffin oil (Fisher Scientific, Fairlawn, NJ) Composed of a mixture of silicone oils (Serve Co., New York, NY) Through the cushion, centrifuge for 3 minutes at 12,000 xg with a Beckman B microcentrifuge. Separated heart. Then count the pellet and supernatant with a gamma-ray spectrometer. Was. Nonspecific binding measures binding in the presence of 100-fold excess of unlabeled ligand Presumed to have Was. Coupling constants were calculated using the computer programs Lundon I and Lundon II. Was. C.peptide   RRWWCR-type peptides use tBOC to protect the α-amino group (S tewart & Young, 1969) by Houghton Pharmaceutical Company of San Diego Was synthesized. All synthetic peptides were preparative C18 reverse phase columns (Waters Co., Ni. High Performance Liquid Chromatograph using a Bedford, Massachusetts) Purified by HPLC (HPLC). 80 in 0.1% TFA from 0.1% trifluoroacetic acid (TFA) The peptide was eluted using a gradient of% acetonitrile. Amino acid analysis and sequencing The composition of the peptide was confirmed by. D.Statistical analysis   In all of this and the following examples, the data are mean and standard deviation (S. D.) variation. Only two groups with equal variance and normal distribution When comparing, the significant difference was determined by the Sheffe test. Variance and Sheffe test Multiple comparisons were performed using the analysis results of. E. FIG.result   In these tests, 10^-12Inhibition was performed at a peptide concentration of 100 μM using M IL-8. Cleaning was carried out. In the first series of tests, X is each standard protein The carboxyl terminal residue of RRWWCX (SEQ ID NO: 23), which was changed one after another, was attached to the mino acid. 20 kinds of peptides were synthesized. The first series of tests, as shown in Table 1A and Table 1B. In, some amino acids completely inhibited IL-8 binding. Shown in Tables 1A and 1B The data shown are the same, and Table 1A shows the inhibition rate (for easy comparison). %), And Table 1B is displayed in the order of sequence numbers. RRWWCD (SEQ ID NO: 26) , RRWWCE (SEQ ID NO: 27), RRWWCN (SEQ ID NO: 35), RRWWCQ (SEQ ID NO: 37) It is clear that all hexamer peptides except Ah You. Preliminary data in Table 1 show that RRWWCN (SEQ ID NO: 35) has no inhibitory activity. However, subsequent studies have shown that this peptide actually has some inhibitory properties. It was clarified that they showed sex (Fig. 1).   As shown in Tables 1A and 1B, in the first test some amino acids were IL-8. Completely inhibited binding. Therefore, in order to assess the relative effectiveness, a low concentration of pep This group was reassessed with Cido. In these tests, Ac-RRWWCR-NH₂(Distribution Column No. 1) is the most potent inhibitor of IL-8 binding to neutrophils It was found (Fig. 1). The data in Fig. 1 shows that RRWWCX (SEQ ID NO: 23) meets this condition. It is shown to be significantly superior to the others.   In another initial study, RRWWCR (SEQ ID NO: 1; Ac-RRWWCR-NH₂) Cylated derivative is not acetylated with respect to inhibition of IL-8 binding to neutrophils It was revealed to be about twice as effective as the peptide.                                    Example II                       Kinetics of RRWWCR inhibitory effect on IL-8 binding   The person described in detail in Example II in the test described in this Example The same method was used. Average EC₅₀Was determined to be 13.7 ± 0.6 μM from multiple binding inhibition curves. Was determined (a representative curve for these analyzes is shown in Figure 2). If the inhibition mechanism is single Estimated K if purely competitive with the site model_IWas about 10 μM. But, The mechanism was more complicated. The binding inhibition curve is steeper than the conventional single-site model Yes, and the high Hill coefficient values (1.5-1.7) indicate the presence of positive synergism. It was suggested. When analyzed using the Lundon II computer program, The curves did not fit well with the 1, 2, or 3 site models, but IL-8 showed neutrophils. Are known to react with at least two different receptors. Therefore, Individual K for each class of receptor_IWas difficult to estimate.   The kinetics of IL-8 binding inhibition by RRWWCR (SEQ ID NO: 1) was determined. Ac-RRWWCR-NH₂ Fig. 3 shows the IL-8 binding isotherm in the case of the absence of H2O3 and the presence of three concentrations of peptides. shown in a. The Scatchard plot in the absence of peptide is 2 for IL-8. It showed no binding to three different receptors (Fig. 3b). Ac-RRWWCR-NH₂(Distribution Row number (1) is present, the plot is non-linear and the Lundon I computer program Analysis with ram was consistent with the two-site model. K_dAnd B_maxEstimation The amount of change is shown in Table 2. Data show that one group of IL-8 receptors was 10 μM peptide. Affinity of B is decreased, and the other group of receptors is_maxBut Dropped.   To further characterize the binding kinetics, 10 μM Ac-RRWWCR-NH₂(Array number Binding inhibition assay was performed using unlabeled IL-8 in the presence of No. 1) (Fig. 4). Ac-RRW WCR-NH₂Two binding inhibitors with and without (SEQ ID NO: 1) The line was analyzed using Lundon II and found to fit well with the two-site model I was killed. No finding was found that a state where the affinity of the receptor was low appeared. K_dAnd B_maxThe estimated change in is the site with high affinity in the presence of 10 μM peptide. Only that affinity and B_maxIt is shown that the decrease of the value occurs (Table 3).                                    Example III                      Specificity of RRWWCR for inhibition of IL-8 binding   Ac-RRWWCR-NH for ligand-receptor binding₂Check the specificity of (SEQ ID NO: 1) To discuss, we found that the peptide was human¹²⁵I-C5a or^ThreeH-Leukotriene B_Four It was investigated whether or not the binding of sucrose to human neutrophils was reduced.   Recombinant human complement fifth component (C5a) (Sigma Chemical Co., St. Louis, Mont. Tana) using Enzimobead (Bio Rad, Richmond, CA) Enzymatically labeled with radioactive iodine. Tritiated leukotriene B_FourIs Du  Purchased from Pont Co., Wilmington, Delaware. The buffer used is 1 mM CaCl each₂, 0.5 mM MgCl₂PBS containing 0.5% BSA and 0.1% ovalbumin And HBSS containing 10 mM HEPES (pH 7.3) except for the IL-8 binding of Example I. As previously mentioned, the method previously reported (Braunwalder et al., 1992; Sherman et al.  al., 1988) C5a and leukotriene B._FourBinding assay was performed. Leukotriene B_Four For analysis, radioactivity was measured using a liquid scintillation counter. The binding of both ligands is saturable and dose-dependent by the unlabeled agonist. It was obstructed.   In these tests, 100 μM Ac-RRWWCR-NH₂(SEQ ID NO: 1) is C5a and Roy Kotrien B_FourDoes not affect the binding of IL-8, but significantly inhibits IL-8 binding at a concentration of 100 nM It was clarified that it did (Fig. 5).                                    Example IV                        Cytotoxicity of RRWWCR on neutrophils   Next, the inventors found that Ac-RRWWCR-NH₂The cytotoxic activity of (SEQ ID NO: 1) was examined. It was released from neutrophils as follows⁵¹Reached by measuring the amount of Cr Made: RPMI-1640 medium with 10% donor plasma in neutrophil samples (2 x 10⁷/ ml) 500 μCi Na₂ ⁵¹CrO_FourIncubate with (Du Pont Co.) for 60 minutes at 37 ℃ did. Wash cells 3 times, 1 x 10⁷Resuspend in medium at a concentration of / ml, followed by 30 min at 37 ° C Incubation was performed to spontaneously lyse cells at the limit of viability. Washed twice After purification, in a silicone treated microcentrifuge tube⁵¹Cr-labeled neutrophils (5 x 10⁶/ ml) 100 A 100 μl aliquot of Ac-RRWWCR-NH₂(SEQ ID NO: 1). With buffer Incubation conditions assume either binding or chemotaxis analysis. Was. After incubation, centrifuge the tubes at 300 xg for 7 minutes at 4 ° C and then The radioactivity of the supernatant was counted with a gamma ray spectrometer. Buffer only, Alternatively, using 3 test tubes containing 2% SDS, spontaneous release and maximum release Was decided respectively. The following formula   Incubate chromium-labeled cells in PBS containing 0.1% BSA for 90 minutes at 4 ° C. Then, the percentage of lysed cells was almost controlled by peptide up to 500 μM. It was found to maintain a low level (Fig. 6). Under the conditions used for chemotaxis Had no effect on 100 μM peptide, but 500 μM peptide Almost 25% of the disability.                                    Example V                       Effects of RRWWCR on the function of neutrophils   Next, the effects of RRWWCR on neutrophil chemotaxis and enzyme release were investigated. A.Chemotaxis   Chemotacticity is reported by Zigmond & Hirsh in the most forward line method (Zigmond & Hi rsh, 1973). Place IL-8 or control under the Boyden chamber. I put it in a box. Cellulose nitrate filter with a pore size of 5 microns and a thickness of 100 μm ( Sartorius Filter, Inc., San Francisco, CA) on its surface Installed and then assembled the chamber. Neutrophil sample in RPMI-1640 with 1% BSA (1 × 10⁶Add 200 μl aliquot of cells / ml) onto the filter and incubate at 37 ° C for 30 minutes. Cubated. Then fix the filter, stain, and slide the microscope glass slide. I put it on the door. The most advanced line was determined from the positions of the two preceding cells. Each filter The distance that the preceding two cells traveled over the filter for the six areas The separation was measured. Measure the four filters for each combination of conditions went. B.Neutrophil enzyme release   By a modified method of Goldstein and co-workers (Goldstein et al., 1973), The enzyme release was examined. Cytochalasin B (Sigma Chemical Co.) Store at a concentration of 5 mg / ml in Hoxide and immediately before use in Hanks' solution at a concentration of 50 μg / ml. Diluted. Cytochalasin B, 200 μl, 6.25 × 10 in HBSS⁶Cells / ml neutrophil suspension In addition to 1 ml of the suspension, the final concentration of cytochalasin B was 10 μg / ml. Then add the solution to 96 Incubated in a well plate for 10 minutes at room temperature. 100 μl of stimulant In addition, this cell suspension was incubated at 37 ° C for 30 minutes. Centrifuge plate Separated and 100 μl of supernatant was removed. 96 wells for β-glucuronidase measurement In a plate, a 40 μl aliquot of the supernatant is added to 10 μl of 0.01 M phenolphthalein- With 0 glucuronic acid (Sigma Chemical Co.) and 40 μl 0.1 M sodium phosphate, pH 4.6 Mixed. After incubation for 16 hours at 37 ° C, 200 μl of 0.2 M glycine, pH 10.4 Was added to each well and OD was added as enzyme activity.₅₄₀Was measured. C.result   Ac-RRWWCR-NH₂(SEQ ID NO: 1) has no effect on neutrophil chemotaxis or enzyme release. It didn't sound. Checkerboard analysis of cell migration, Ac-RRWWCR-NH₂(Distribution Row number 1) showed no chemotaxis for neutrophils (Table 4). Figure In a control study shown in 7 and 8, this peptide was found to affect neutrophil chemotaxis. It had no effect and did not stimulate β-glucuronidase release from cells.   Ac-RRWWCR-NH₂Inhibition of IL-8 binding by IL-2 is related to suppression of neutrophil activation by IL-8. To confirm that they are involved, the inventors have studied chemotaxis and β-glucuronidase. The effect of peptides on release was investigated. Ac-RRWWCR-NH₂(SEQ ID NO: 1) is 50 μM Ac-RRWWCR-NH₂Stimulated with 10 nM IL-8 at the (SEQ ID NO: 1) concentration Formyl-L-methionyl-L-leucyl-L-phene significantly suppresses neutrophil chemotaxis It had no effect on chemotaxis induced by nylalanine (fMLP) (Fig. 7). Ac-RRWWCR-NH₂(SEQ ID NO: 1) is a concentration lower than that required for chemotaxis Release of β-glucuronidase stimulated by 100 nM IL-8 at a concentration of 2 μM Inhibition, but fMLP, C5a, or leukotriene B_FourEnzymes stimulated by It did not affect the release (Fig. 8).                                    Example VI                            Additional IL-8 peptide inhibitors   Ac-RRWWCR-NH₂Other peptides related to (SEQ ID NO: 1) are IL-8 inhibitors To determine whether they work and to determine their relative effectiveness. A series of tests was conducted. The tests described in this example are detailed in Example I. The same method was used as described. A.Heptamer peptide   In this series of tests, we found that RRWWCR (adding the 7th amino acid The D-amino acid analog of SEQ ID NO: 1) was investigated. RRWWC in heptamer test The carboxyl residue of RX (SEQ ID NO: 2) is replaced with each of the standard protein amino acids. Then, 20 kinds of peptides were synthesized. In the first heptamer test, Table 5 As shown in A and Table 5B, some peptides have very potent inhibitory effects on IL-8. Indicated. The data displayed in Tables 5A and 5B are the same data, so you can easily compare As can be seen, Table 5A is displayed in the order of inhibition rate (%), and Table 5B is displayed in the order of SEQ ID NO. ing. All heptamer peptides show significant inhibitory activity under these conditions It is clear to have.   To determine the relative efficacy of heptamers, their inhibitory effect at low concentrations It was measured. Peptide with D-cysteine at the carboxyl terminus (RRWWCRC; SEQ ID NO: No. 4) is almost 56% more effective than the next effective peptide in this group, and Ac-RRW WCR-NH₂It was found to be more effective than (SEQ ID NO: 1; Figure 9). 10 μM concentration Degree L-amino acid peptide Ac-RRWWCR-NH₂20% inhibition by (SEQ ID NO: 1) On the other hand, Ac-rrwwcrc-NH₂(SEQ ID NO: 4) inhibits IL-8 binding to neutrophils by 80% did. B.Other peptides   We are concerned with the amino-terminal portion of IL-8 or in other proteins. , Which has 5 of the 6 residues of RRWWCR (SEQ ID NO: 1). Additional peptides were also investigated. Peptides ELRCQCIKTY, ERLSQSIKTY, ELRMQMIKTY, QIPRRS WCRFLF and GWRRWWCDAVLY (SEQ ID NOS: 49 to 53, respectively) are jointly with Meienhofer Reported by researchers (Meienhofer et al., 1979) and Arshady et al. (1979). As described above, 9-fluorenylmethoxycarbonyl was used to protect the α-amino group. 431 peptide synthesizer (Applied Biosystems, Foster City, California) Tyler University of Texas Hygiene Center , Was synthesized. All synthetic peptides are preparative reverse phase columns (Waters Co., High Performance Liquid Chromatography using New Bedford, Massachusetts) Purified by chromatography (HPLC). 0.1% trifluoroacetic acid (TFA) to 0.1% TFA The peptide was eluted using a gradient of 80% acetonitrile in. The composition of the peptide is , UTHC Protein Core Facility confirmed by amino acid analysis and sequencing.   In this series of tests, Ac-RRWWCR-NH₂(SEQ ID NO: 1) and QIPRRSWCRFLF (distribution Row No. 52) inhibited the binding of IL-8 to neutrophils (Table 6).                                    Example VII                     Inhibition of GRO and MIP2β binding to neutrophils   Regarding other CXC intercrine inhibitory ability, Ac-RRWWCX-NH₂(SEQ ID NO: 23) investigated. This example demonstrates that in addition to IL-8 inhibition, Ac-RRWWCX-NH₂(SEQ ID NO: 23) is , Show that it effectively inhibits the binding of GRO and MIP2β to human neutrophils.   Radioiodinated MIP2β and GRO / MGSA with bolten-Hunter reagent Was. Radioiodine-labeled components were added to various concentrations of Ac-RRWWCX-NH₂(SEQ ID NO: 23) Mix and incubate for 15 minutes at room temperature. Neutrophil suspension (containing 0.1% BSA 1 × I0 in 160 μl PBS⁶Individual cells. ) To 40 μl of the mixture and on ice for 90 minutes Incubated for Binding to cells by centrifugation through an oil layer The radioactivity generated was separated from the free radioactivity. Bound radioactivity bound CX C interface It shows a clean peptide. Ac-RRWWCX-NH₂(SEQ ID NO: 23) exists In this case, the binding inhibition rate (%) was calculated as follows.   Where B is the bound radioactivity when the peptide is present and T is the peptide The bound radioactivity in the absence of NSP results in excess of unlabeled ligand in NSP. The bound radioactivity, if present.   In these tests, Ac-RRWWCX-NH₂(SEQ ID NO: 23) is 1 nM IL-8 neutrophil Binding to E.₅₀Was about 25 μM (FIG. 10). FIG. As shown in 0, Ac-RRWWCX-NH₂(SEQ ID NO: 23) shows 1nM GRO and 1nM MIP2β It has also been found to effectively inhibit binding to neutrophils.                                    Example VIII                           Treatment formulation and treatment protocol   This example is intended to further characterize the in vivo action of IL-8 inhibitors. In order to utilize them in animal or human therapeutic protocols, the inventors have The purpose is to show the technology considered by. A.Effects of IL-8 inhibitors on in vivo inflammation   Pre-incubation in human serum and plasma prior to animal model testing, Treatment with various proteases and stability test against temperature and pH  In vitro stability tests are performed on the peptides. D-amino acid peptide is active It has already been found that it has the following property, and seems to enhance stability in vivo.   The inventors examined the in vivo properties and effects of IL-8 inhibitors before entering clinical trials. Suggest to discuss. Routine methods in the art show that the most appropriate Different forms, doses and potential toxicity are determined in animal studies. For example, a radiolabel Bioavailability of peptides administered by various routes using peptides ー And half-life can be determined and their life span and tissue distribution can be examined. More stable If you want to enhance the property, peptides with lipids at the ends, surfactant-like micelles, Administer peptides in the form of peptide multimers or semipermeable drug release capsules It is also possible.   The biological effects of peptides can be determined in various human disease models. example For example, IL-8 was found to cause neutrophil accumulation and edema in rabbit skin. (Rampart et al., 1989). Therefore, using the rabbit skin inflammation model, The dose of peptide that can effectively inhibit neutrophil accumulation and edema is determined. This model Is useful because it is easy to assess inflammation. The most suitable route of peptide administration is in v Easy to determine in an ivo comparative test.   In a particular embodiment, the New Zealand albino rabbit is placed in the lateral ear vein. La¹²⁵I-labeled human serum albumin can be injected. Next, test compounds and Agonists that attract neutrophils and IL-8 inhibitory peptides, agonists and control peptides Tide, and agonist alone, can be injected transdermally. After 2 hours, the full thickness of the skin Punch out a sample with a diameter of 1 cm, remove it, fix it, and stain it with Wright-Giemsa. Studies on Ruper or Myeloperoxidase and Neutrophil Accumulation and Edema or Tissue Damage Perform a histological examination. Count other skin biopsy specimens with a gamma counter And evaluate the albumin flux into the infused skin. Next, the skin after administration of the inhibitor Skin irritation can be compared to controls of the same time. Ideally, the same animal should be used. Small It is recommended to make at least 4 measurements for each experimental group. B.ARDS IL-8 inhibitor treatment   The best model of adult respiratory distress syndrome (ARDS) in humans is probably the minipig circulation It may have introduced Gram-negative bacteria into the blood. Similar to human ARDS, this ARDS To determine whether IL-8 is the major neutrophil activator in the model To this end, this model is used with a test method similar to that performed in humans. mini To assess the effect of IL-8 administration by the intravenous and intrapulmonary routes in pigs.   In the first step, neutrophil influx into the lung and enzyme release were assessed, as assessed above. IL-8 is administered to minipigs by the route that is most suitable for causing it. These neutrophil actions Inhibit enzyme release, especially to determine the appropriate dose to use to prevent However, in order to determine the dose of peptide that does not suppress neutrophil influx, the peptide Administer   In the next stage, acute minipig caused by circulating Gram-negative bacteria Use the lung injury model. The peptide of interest is administered to an animal to suppress the lung injury of the peptide. Evaluate the control effect. Number of neutrophils in bronchoalveolar fluid, enzyme release into lung parenchyma, circulation The extent of protein leakage from the blood to the lungs is used as an indicator for this test.   IL-8 influx neutrophils into the lungs, enzyme release and / or ARDS-like tissue damage to the lungs If desired, administer the peptide of interest to suppress neutrophil function Determine the appropriate dose to use. In these studies, various doses of radioactivity The peptide is administered first. Next, the plasma concentration and the form of radioactivity are determined. these The data for plasma clearance, half-life, and steady-state distribution were It can be used to determine an effective dose range. C.Treatment protocol   Due to the precautions that are always associated with all new drugs, the combination with the IL-8 peptide inhibitors of the present invention The product has not been clinically tested in humans. But admitted Their apparent in vitro activity in the model makes the present invention useful as an anti-inflammatory agent. It is considered to show usability. Clinical trials go through a formal process and, of course, follow FDA procedures. Therefore, it will be implemented. The following specific methods will carry out the present invention in various clinical situations. It is the best method currently considered by the present inventors to apply.   A pharmaceutical composition containing a peptide inhibitor of IL-8 was used to treat bronchial inflammation, cystic fibrosis, chest Lung diseases such as intracavitary extension, asthma, bronchitis and ARDS, as well as skin such as psoriasis and dermatitis Diseases, and joint diseases such as rheumatoid arthritis, pseudogout, inflammatory bowel disease, For the treatment of myocardial damage after reperfusion or other diseases associated with cancer and increased cell proliferation Is also believed to prove useful.   These peptides are responsible for lung inflammation such as ARDS, chronic bronchitis and cystic fibrosis. Appropriate therapies for these diseases are described as they appear to be particularly suitable for suppression. For treatment of ARDS or cystic fibrosis, preferably non-injection such as intravenous injection, intramuscular injection, subcutaneous injection, etc. Oral administration is used. However, aerosols or inhalants can also be used. Parenteral injection A method for preparing a customary peptide formulation, particularly an injectable formulation, is described in the preferred embodiment herein. Are described in detail in. If you want to use the method related to the present invention, there are several Inhalants are described below.   An inhalant is a medicinal product designed to reach a patient's respiratory tree with a drug or compound It is an agent. The administered inhalant or mist reaches the affected area and fills the bronchi and nasal passages. Relieves blood symptoms. Inhalants can be administered intranasally or orally by the respiratory route. Sucking Immersion should be administered only if the droplets are sufficiently fine and homogeneous so that the fog can reach the bronchi. As long as it is valid.   Another group of products, also known as inhalants, often known as breathing agents, A solution of drug in a liquefied gas propellant consisting of powdered drug or liquid drug Or by using a special delivery system such as a drug aerosol containing suspension Then, it is administered to the respiratory tract. Emitted from the appropriate valve and oral adapter Then, a metered dose of inhalant is delivered to the patient's respiratory tract.   Particle size is very important for the administration of this type of formulation. Immersed in the alveolar space Optimum particle size for transmission is reported to be on the order of 0.5 to 7 μm. You. Pressurized aerosols produce very fine mist, which makes them extremely useful. It is profitable.   The anti-IL-8 peptides described herein are intravenously administered as a single agent or combination. Those that can reduce the inflammation of ARDS and cystic fibrosis by administration it is conceivable that. The dose range administered is about 500 to about 1000 mg / day, or about 0.8. It is estimated to be approximately 16.56 mg / kg / hr from 3 mg / kg body weight / hour (mg / kg / hr).   Of course, to treat many other diseases associated with neutrophil activation and inflammation, IL It must be remembered that a wide variety of other pharmaceutical formulations of -8 inhibitors can be prepared and used Yes.   All of the compositions and methods disclosed and claimed herein are within the scope of this disclosure. It can be prepared and carried out without undue burden of experimentation. is there. The compositions and methods of the present invention are reported in terms of the preferred embodiments. Without departing from the concept, spirit and scope of the present invention. Changes may be made in the method steps or series of steps described. Will be apparent to those familiar with the technology. More specifically, chemical and raw Replace certain substances with physical relevance with the substances described here to It is clear that similar or similar results are achieved. Person familiar with this technology Such similar substitutes and modifications apparent to those skilled in the art are subject to the scope of the appended claims. Within the spirit, scope and concept of the invention as defined by the box it is conceivable that.

─────────────────────────────────────────────────── ─── Continuation of front page (51) Int.Cl. ⁶ Identification number Internal reference number FI A61K 38/00 ACD A61K 37/02 ADS ACL ACL ACL ABG ADS ADA (81) Designated country EP (AT, BE, CH) , DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ, CF, CG, CI, CM, GA, GN, ML, MR) , NE, SN, TD, TG), AT, AU, BB, BG, BR, BY, CA, CH, CZ, DE, DK, ES, FI, GB, HU, JP, KP, KR, KZ, LK , LU, LV, MG, MN, MW, NL, NO, NZ, PL, PT, RO, RU, SD, SE, SK, UA, US UZ, VN (72) Inventor Shinichiro Hayashi United States, 75703 Texas, Tyler, Sigpen Drive 202, Aperture 1220 (72) Inventor Kurduska, Anna Kay United States, 75703 Texas, Tyler, Shiloh Road 710, Aper Torment 1228 (72) Inventor Tatur, Ronald Earl United States, 92025 California, Escondido, Vista del Sembrado 2704

Claims (1)

[Claims] 1. A CXC intercrine molecule or intercrine target cell was prepared from a peptide having an amino acid sequence Arg Arg Trp Trp Cys Xaa ₁ (SEQ ID NO: 23) (Xaa ₁ may be any amino acid residue) with a length of 6 to about 14 residues. A method of inhibiting a CXC intercrine family molecule, comprising contacting with a biologically effective amount of the composition. 2. The method according to claim 1, wherein the CXC intercrine group molecule is IL-8. 3. The method of claim 1, wherein the CXC intercrine group molecule is GRO. 4. The method according to claim 1, wherein the CXC intercrine group molecule is MIP2β. 5. The method of claim 1, wherein the composition comprises an acylated peptide. 6. 6. The method of claim 5, wherein the composition comprises an N-terminally acylated peptide. 7. The method of claim 1, wherein the composition comprises a C-terminal amidated peptide. 8. The method of claim 1, wherein the composition comprises an N-terminally acylated, C-terminally amidated peptide. 9. The method of claim 1, wherein the composition comprises a D-amino acid peptide. 10. The method of claim 1, wherein the composition comprises an L-amino acid peptide. 11. The method of claim 1, wherein the composition comprises an L-amino acid peptide and a D-amino acid peptide. 12. The method of claim 1, wherein the composition comprises a peptide containing both L-amino acids and D-amino acids. 13. The method of claim 1, wherein the composition comprises a peptide comprising 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 comprising 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 comprising 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 comprising 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 comprising 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 comprising 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 comprising 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 comprising 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 comprising 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 comprising 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 comprising 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 comprising 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 comprising 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 comprising 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 comprising 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 comprising 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 comprising 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 comprising 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 comprising 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 comprising 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 comprising the amino acid sequence Arg Arg Trp Trp Cys Arg Xaa ₂ (SEQ ID NO: 2) (Xaa ₂ can be any amino acid residue). 34. 34. The method of claim 33, wherein the composition comprises a peptide comprising the amino acid sequence Arg Arg Trp Trp Cys Arg Ala (SEQ ID NO: 3). 35. 34. The method of claim 33, wherein the composition comprises a peptide comprising the amino acid sequence Arg Arg Trp Trp Cys Arg Cys (SEQ ID NO: 4). 36. 34. The method of claim 33, wherein the composition comprises a peptide comprising the amino acid sequence Arg Arg Trp Trp Cys Arg Asp (SEQ ID NO: 5). 37. 34. The method of claim 33, wherein the composition comprises a peptide comprising the amino acid sequence Arg Arg Trp Trp Cys Arg Glu (SEQ ID NO: 6). 38. 34. The method of claim 33, wherein the composition comprises a peptide comprising the amino acid sequence Arg Arg Trp Trp Cys Arg Phe (SEQ ID NO: 7). 39. 34. The method of claim 33, wherein the composition comprises a peptide comprising the amino acid sequence Arg Arg Trp Trp Cys Arg Gly (SEQ ID NO: 8). 40. 34. The method of claim 33, wherein the composition comprises a peptide comprising the amino acid sequence Arg Arg Trp Trp Cys Arg His (SEQ ID NO: 9). 41. 34. The method of claim 33, wherein the composition comprises a peptide comprising the amino acid sequence Arg Arg Trp Trp Cys Arg Ile (SEQ ID NO: 10). 42. 34. The method of claim 33, wherein the composition comprises a peptide comprising the amino acid sequence Arg Arg Trp Trp Cys Arg Lys (SEQ ID NO: 11). 43. 34. The method of claim 33, wherein the composition comprises a peptide comprising the amino acid sequence Arg Arg Trp Trp Cys Arg Leu (SEQ ID NO: 12). 44. 34. The method of claim 33, wherein the composition comprises a peptide comprising the amino acid sequence Arg Arg Trp Trp Cys Arg Met (SEQ ID NO: 13). 45. 34. The method of claim 33, wherein the composition comprises a peptide comprising the amino acid sequence Arg Arg Trp Trp Cys Arg Asn (SEQ ID NO: 14). 46. 34. The method of claim 33, wherein the composition comprises a peptide comprising the amino acid sequence Arg Arg Trp Trp Cys Arg Pro (SEQ ID NO: 15). 47. 34. The method of claim 33, wherein the composition comprises a peptide comprising the amino acid sequence Arg Arg Trp Trp Cys Arg Gln (SEQ ID NO: 16). 48. 34. The method of claim 33, wherein the composition comprises a peptide comprising the amino acid sequence Arg Arg Trp Trp Cys Arg Arg (SEQ ID NO: 17). 49. 34. The method of claim 33, wherein the composition comprises a peptide comprising the amino acid sequence Arg Arg Trp Trp Cys Arg Ser (SEQ ID NO: 18). 50. 34. The method of claim 33, wherein the composition comprises a peptide comprising the amino acid sequence Arg Arg Trp Trp Cys Arg Thr (SEQ ID NO: 19). 51. 34. The method of claim 33, wherein the composition comprises a peptide comprising the amino acid sequence Arg Arg Trp Trp Cys Arg Val (SEQ ID NO: 20). 52. 34. The method of claim 33, wherein the composition comprises a peptide comprising the amino acid sequence Arg Arg Trp Trp Cys Arg Trp (SEQ ID NO: 21). 53. 34. The method of claim 33, wherein the composition comprises a peptide comprising 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 comprising the amino acid sequence Gln Ile Pro Arg Arg Ser Trp Cys Arg Phe Leu Phe (SEQ ID NO: 52). 55. The composition has 2 to 41 different peptides having an amino acid sequence of any one of the amino acid sequences presented in SEQ ID NO: 1, SEQ ID NO: 3 to SEQ ID NO: 22, SEQ ID NO: 24 to SEQ ID NO: 42 or SEQ ID NO: 52. The method of claim 1, comprising: 56. 56. The method of claim 55, wherein the composition comprises two different peptides. 57. 56. The method of claim 55, wherein the composition comprises 5 different peptides. 58. 56. The method of claim 55, wherein the composition comprises 10 different peptides. 59. 56. The method of claim 55, wherein the composition comprises 20 different peptides. 60. 56. The method of claim 55, wherein the composition comprises 30 different peptides. 61. 56. The method of claim 55, wherein the composition comprises 41 different peptides. 62. 56. The method of claim 55, wherein the composition comprises a peptide comprising Arg Arg Trp Trp Cys Arg (SEQ ID NO: 1) and a peptide comprising 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 pharmaceutically acceptable form. 64. 64. The method of claim 63, wherein the pharmaceutical composition is formulated as an injectable, nasal spray, inhalant, aerosol, cream, gel, micelle or liposome capsule form or incorporated into a biocompatible sustained release capsule. . 65. 66. The method of claim 64, wherein the pharmaceutical composition is formulated as an intravenous, intramuscular, or subcutaneous injection. 66. 66. The method of claim 64, wherein the pharmaceutical composition is formulated as a nasal spray, inhalant, and aerosol. 67. 64. The method of claim 63, wherein the CXC intercrine group molecule or intercrine target cell is present in the body of an animal and an effective amount of the pharmaceutical composition is administered to the animal. 68. A composition comprising neutrophils and IL-8 is provided with an amino acid sequence Arg Arg Trp Trp Cys Xaa ₁ (SEQ ID NO: 23) in an amount effective to reduce neutrophil enzyme release rather than chemotaxis. Enzyme of neutrophils, rather than chemotaxis of neutrophils, comprising contacting with a composition comprising a peptide of 6 to about 14 residues in length, wherein (Xaa ₁ can be any amino acid residue) Methods for preferentially reducing emissions. 69. 69. The method of claim 68, wherein neutrophils and IL-8 are present in the body of the animal and an effective amount of the pharmaceutical composition is administered to the animal in a pharmaceutically acceptable form. 70. A biologically effective peptide containing 6 to about 14 residues in length, which contains the amino acid sequence Arg Arg Trp Trp Cys Xaa ₁ (SEQ ID NO: 23) (Xaa ₁ may be any amino acid residue) in inflamed animals. A method of reducing inflammation comprising administering an amount of a pharmaceutical composition. 71. 71. The method of claim 70, wherein the inflammation is associated with adult respiratory distress syndrome (ARDS) or cystic fibrosis. 72. CXC intercrine group molecules can be inhibited, and the amino acid sequence Arg Arg Trp Trp Cys Xaa ₁ Xaa ₂ (SEQ ID NO: 57) (Xaa ₁ and Xaa ₂ may be any amino acid residue) is contained in about 7 to about 14 residues. A pharmaceutical composition in a pharmaceutically acceptable form, which comprises a base-length peptide. 73. 73. The pharmaceutical composition of claim 72, wherein the composition comprises a peptide capable of inhibiting IL-8. 74. 73. The pharmaceutical composition of claim 72, wherein the composition comprises a peptide capable of inhibiting GRO. 75. 73. The pharmaceutical composition of claim 72, wherein the composition comprises a peptide capable of inhibiting MIP2β. 76. 73. The pharmaceutical composition of claim 72, wherein the composition comprises an N-terminally acylated peptide or a C-terminally amidated peptide. 77. 73. The pharmaceutical composition of claim 72, wherein the composition comprises an N-terminally acylated, C-terminally amidated peptide. 78. 73. The pharmaceutical composition according to claim 72, wherein the composition comprises a peptide comprising the amino acid sequence Arg Arg Trp Trp Cys Arg Xaa ₂ (SEQ ID NO: 2) (Xaa ₂ can be any amino acid residue). 79. 79. The pharmaceutical composition of claim 78, wherein the composition comprises a peptide comprising an amino acid sequence according to any one of the amino acid sequences set forth in SEQ ID NOs: 3-22. 80. 80. The pharmaceutical composition according to claim 79, wherein the composition comprises 2 to 20 different peptides comprising an amino acid sequence according to any one of the amino acid sequences set forth in SEQ ID NO: 3 to SEQ ID NO: 22. 81. 73. The medicament according to claim 72, wherein the composition is formulated as an injectable, nasal spray, inhalant, aerosol, cream, gel, micelle or liposome capsule form or incorporated into a biocompatible sustained release capsule. Composition. 82. A peptide which is capable of inhibiting IL-8, GRO or MIP2β, and which has an amino acid sequence according to any one of the amino acid sequences shown in SEQ ID NOs: 3 to 22 and has a length of 7 to about 14 residues.