HRP980323A2 - Peptides and compounds that bind to the il-5 receptor - Google Patents

Description

Basics of invention

The described invention relates to peptides and compounds that bind to interleukin 5 (IL-5R) receptors, methods for testing interleukin 5 (IL-5), as well as methods for suppressing the binding of IL-5 to IL-5R. The invention has application in the field of biochemistry and medicinal chemistry, and in particular provides IL-5 antagonists for use in the treatment of human diseases.

Interleukin-5 (IL-5 or IL5) is a lymphokine secreted by T cells and mammary gland cells, which has a biological effect on B cells and eosinophils. In hematopoiesis and blood cell formation in mice, IL-5 is a selective signal for proliferation and for differentiation of eosinophilic origin. See Yamaguchi et al.: "J. Exp. Med.", 167, p. 43-56, (1988). In this regard, IL-5 function shows analogies with colony stimulating factors for other myeloid species. Human (h) IL-5 is very potent in activating human eosinophils. See Lopez et al.: "J. Exp. Med.", 167, p. 219-224, (1988); and Saito et al.: "Proc. Natl. Acad. Sci. USA", 85, p. 2288-2292, (1988).

IL-5 directs its action through the cell membrane receptor complex. This complex was analyzed physicochemically on mouse and human systems. Mouse lines of pre-B cells, whose growth depends on IL-5, were developed from bone marrow and were used for IL-5 receptor analysis. See Rolink et al.: "J. Exp. Med.", 169, p. 1693-1701, (1989). The human IL-5 receptor can be studied on a subclone of the promyelocytic cell line HL60 induced towards eosinophilic differentiation. See Plaetinck et al.: "J. Exp. Med.", 172, p. 683-691, (1990).

Eosinophilic differentiation is initiated by sodium butyrate. Only high-affinity IL-5 binding sites (Kd = 30 pM) can be found on these cells. However, cross-linking studies revealed the existence of two polypeptide sequences involved in IL-5 binding, ie, the IL-5R-� and IL-5R-� sequences. Canadian patent no. 2,058,003 (Devos et al.), describe a recombinant human IL-5R sequence or portions thereof, the DNA sequence coding for such receptor or portions thereof, and host cells transformed with such vectors. European patent no. 475,748 (Takatsu et al.), show an isolated cDNA sequence coding for the murine and human IL-5 receptor.

The soluble human IL-5R-� array can be used as an IL-5 antagonist in chronic asthma or other disease states with pronounced eosinophilia. Eosinophils are white blood cells of granulocyte origin. Their normal function, it seems, is to fight parasitic infections, especially infections with intestinal worms. However, their accumulation in tissues (a condition called eosinophilia) is also associated with several disease states, especially asthma. Damage to the epithelial lining of the bronchial ducts in severe asthma attacks is believed to be largely caused by compounds released by degranulated eosinophils.

In US patent no. 5,096,704, the application of compounds that block the stimulatory effects of IL-5, in order to suppress the production and accumulation of eosinophils, is specifically shown. The stimulatory effects of IL-5 are blocked by administration of an effective amount of a human interleukin-5 antagonist, preferably using monoclonal antibodies or binding compositions derived therefrom using standard techniques. Monoclonal antibodies were selected for their ability to suppress IL-5-induced effects in standard IL-5 bioassays, such as their ability to stimulate the growth and development of eosinophils in in vitro colony formation assays, and their ability to increase in vitro proliferation of in vivo treated BCL1 lymphoma cells . The use of antibody fragments, for example Fab fragments, has also been disclosed.

Currently, glucocorticoid steroids are the most effective drugs for the treatment of acute conditions of allergic diseases, such as asthma. However, long-term use of steroids is associated with certain side effects. Moreover, it appears that steroids do not interfere with the production or accumulation of granulocytic cells, such as eosinophils, in the affected tissues. The availability of alternative or complementary approaches to the treatment of eosinophilia-related disorders would have important clinical applications.

The availability of cloned IL-5R genes, including a soluble IL-5R derivative, facilitates the search for agonists and antagonists of these important receptors. The availability of recombinant receptor proteins enables the study of the interaction between receptors and ligands in a number of systems for the generation of arbitrary and semi-arbitrary peptide diversity. These systems include the "peptides on plasmids" system described in US Pat. 5,270,170; the "peptides on phage" system described in US patent no. 5,432,018, and in Cwirl et al.: "Proc. Natl. Acad. Sci. USA", 87, p. 6378-6382, (1990); and the "very large scale synthesis of immobilized polymers" system described in US Pat. No. 5,143,854, PCT patent file no. 90/15070, published on December 13, 1990; Fodor et al.: "Science", 251, p. 767-773, February 15, 1991, Dower and Fodor: "Ann. Rep. Med. Chem.", 26, p. 271-280, (1991), each of the cited patent applications and publications being incorporated herein by reference.

Asthma has become the most common chronic disease in industrialized countries.

Conventional procedures and therapeutic agents may not be completely effective in the treatment of asthma or other immune-induced inflammatory diseases in the entire patient population. In addition, there remains a need for compounds that bind or otherwise interact with the IL-5R, both for the study of important biological actions induced by this receptor, and for the treatment of diseases. The described invention provides such compounds.

Brief overview of the invention

The invention is directed in part to the novel and unexpected discovery that defined peptides and low molecular weight peptide mimics have strong IL-5R binding properties. Accordingly, such peptides and peptide mimetics are useful for therapeutic purposes in the treatment of conditions driven by IL-5, or associated with dysregulated IL-5 production or dysresponsiveness to IL-5, and can be used to suppress production and accumulation. eosinophils. These compounds will find special use in the treatment of asthma. The invention thus described also describes a method for treating a patient who has a disorder amenable to treatment with an IL-5 inhibitor, whereby the patient receives, or is given, a therapeutically effective dose or amount of a compound according to the invention described.

Peptides and peptide mimetics suitable for therapeutic and/or diagnostic purposes have an IC50 of about 2 mM or less, as determined by the binding affinity assay set forth in Example 2 below, where a lower IC50 refers to a stronger binding affinity for the IL-5R. For pharmaceutical purposes, peptides and peptide mimics preferably have an IC50 of no greater than about 100 µm. In one preferred embodiment, the molecular weight of the peptide or peptide mimic is from about 250 to about 5,000 daltons.

When used for diagnostic purposes, peptides and peptide mimics are preferably labeled with some detectable label, and thus unlabeled peptides and peptide mimics serve as intermediates in the preparation of labeled peptides and peptide mimics.

Peptides that meet the set criteria for molecular mass and affinity for binding to IL-5R contain 12 or more amino acids, whereby the amino acids can be natural or synthetic (not occurring in nature). Peptide mimics include peptides that include one or more of the following modifications:

- peptides in which one or more peptidyl [-C(O)NR-] bonds are replaced by some non-peptidyl bond such as -CH2-carbamate bond [-CH2-OC(O)NR-], phosphonate bond, -CH- sulfonamide [-CH2-S(O)2NR-] bond, urea [-NHC(O)NH-] bond, -CH2-secondary amine bond or some alkylated peptidyl bond [-C(O)NR6, where R6 is lower alkyl ];

- peptides where the N-terminus is derivatized to one -NRR1 group, to one -NRC(O)R group, to one -NRC(O)OR group, to one -NRS(O)2R group, to one -NHC( O)NHR group, where R and R1 are hydrogen or lower alkyl, provided that R and R1 are not both hydrogen, to one succinimide group, to one benzyloxycarbonyl-NH-(CBZ-NH-) group, or to one benzyloxycarbonyl-NH - a group having 1 to 3 substituents on the phenyl ring selected from the group comprising lower alkyl, lower alkoxy, chloro and bromo; or

- peptides in which the C-terminus is derivatized to -C(O)R2, where R2 is selected from the group consisting of lower alkoxy, and -NR3R4 where R3 and R4 are independently selected from the group consisting of hydrogen and lower alkyl .

Accordingly, recommended peptides and peptide mimetics include compounds having:

(1) molecular weight less than about 5,000 daltons, and

(2) affinity for binding to IL-5R expressed through an IC50 not greater than about 100 µm,

wherein, starting from zero to all, -C(O)NH- bonds of the peptide are replaced by one bond selected from the group consisting of one -CH2OC(O)NR- bond, one phosphonate bond, one -CH2S(O )2NR- bond, one CH2NR- bond, and one -C(O)NR6 bond, and one -NHC(O)NH- bond, where R is hydrogen or lower alkyl, and R6 is lower alkyl,

wherein the N-terminus of said peptide or peptide mimic is selected from the group consisting of one -NRR1 group, one -NRC(O)R group, one -NRC(O)OR group, one -NRS(O)2R group, one - an NHC(O)NHR group, one succinimide group, one benzyloxycarbonyl-NH-(CBZ-NH-) group, and one benzyloxycarbonyl-NH- group having 1 to 3 substituents on the phenyl ring selected from the group consisting of lower alkyl, lower alkoxy , chloro and bromo, where R and R1 are independently selected from the group consisting of hydrogen or lower alkyl, and

wherein the C-terminus of said peptide or peptide mimic has the formula -C(O)R2, where R2 is selected from the group consisting of lower hydroxy, lower alkoxy, and -NR3R4, where R3 and R4 are independently selected from the group consisting of consists of hydrogen and lower alkyl, and where the nitrogen atom of the -NR3R4 group can possibly be the amino group of the N-terminus of the peptide thus forming a cyclic peptide,

and their physiologically acceptable salts.

In a related embodiment, the invention is directed to a labeled peptide or peptide mimic, comprising the above-described peptide or peptide mimic to which a detectable label is covalently attached.

In a particularly preferred embodiment, the peptides are 20 to 40 or more amino acid residues in length, preferably 12 to 25 amino acid residues in length, and contain a basic amino acid sequence selected from the following:

C W R S V A T H T W F C G (SEQ ID NO:1);

C W R S V A T H T W F C G E (SEQ ID NO:2);

C W R S V A T H T W F C G E E (SEQ ID NO:3);

E G D C W R S V A T H T W M C G V E (SEQ ID NO:4);

E V E C W R S V A T H T W F C G E D (SEQ ID NO:5);

G G G V E V C T R S V A T H S V C G I D (SEQ ID NO:6);

L R R A S L G G C W R S V A T H T W F C G E E (SEQ ID NO:7);

V D E C W R L V A T H T W F C G D D (SEQ ID NO:8);

V D E C W R S V A T H T W F C G E E (SEQ ID NO:9);

V E D C W R S V A T H T W F C G E D (SEQ ID NO:10);

V L D C W R S V A T H S W F C G E D (SEQ ID NO:11);

V V D C W R S V A T H S W F C G E E (SEQ ID NO:12);

V V D C W R S V A T H T W F C G (SEQ ID NO:13);

V V D C W R S V A T H T W F C G E (SEQ ID NO:14);

V V D C W R S V A T H T W F C G E D (SEQ ID NO:15);

C W R S V A T H T W F C G E E-(NH2) (SEQ ID NO:16);

C W R S V A T H T W F C G E-(NH2) (SEQ ID NO:17);

C W R S V A T H T W F C G-(NH2) (SEQ ID NO:18);

L R R A S L G G C W R S V A T H T W F C G E E-(NH2) (SEQ ID NO:19);

V V D C W R S V A T H T W F C G E-(NH2) (SEQ ID NO:20); and

V V D C W R S V A T H T W F C G-(NH2) (SEQ ID NO:21).

The invention also relates to pharmaceutical compositions containing one or more of the compounds described here and a physiologically acceptable carrier. These pharmaceutical compositions can take a variety of forms, including oral dosage forms, as well as powders and solutions for inhalation and solutions for injections and infusions.

DESCRIPTION OF SPECIFIC PERFORMANCES

I. Definitions and general parameters

The following definitions are set forth to illustrate the meaning and scope of various terms used in the invention.

As used herein, the term "lower" refers to a group having between one and six carbon atoms.

As used herein, the term "alkyl" refers to a straight or branched chain hydrocarbon having from 1 to 10 carbon atoms.

As used herein, the term "alkoxy" refers to the group RaO-, where Ra is alkyl.

The term "pharmaceutically acceptable salts" refers to non-toxic alkali metal, alkaline earth metal and ammonium salts commonly used in the pharmaceutical industry, including sodium, potassium, lithium, calcium, magnesium, barium, ammonium and protamine zinc salts, which are prepared by known procedures. The term also includes non-toxic acid addition salts, which are usually prepared by reacting the components of this invention with a suitable organic or inorganic acid.

The term "pharmaceutically acceptable acid addition salts" refers to those salts which retain biological activity and free base properties, and which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid acid, phosphoric acid and the like, and with organic acids such as acetic acid, propionic acid. glycolic acid, pyruvic acid, oxalic acid, malic acid, malonic acid, succinic acid, maleic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. For a description of pharmaceutically acceptable acid addition salts as prodrugs, see Bundgaard H.: "Supra".

The term "pharmaceutical acceptable ester" refers to those esters which, after hydrolysis, retain the ester bonds, biological effectiveness and properties of the carboxylic acid or alcohol, and are not biologically undesirable in any other way. For a description of pharmaceutically acceptable esters as prodrugs, see Bundgaard H.: "Design of Prodrugs", Elsevier Science Publishers, Amsterdam, (1985). These esters are usually formed from the corresponding carboxylic acid and some alcohol. In general, the formation of esters can be accomplished by conventional synthesis techniques. See for example March: "Advanced Organic Chemistry", 3rd edition, John Wiley & Sons, New York, p. 1157, (1985) and references therein, and Mark et al.: "Encyclopedia of Chemical Technology", John Wiley & Sons, New York, (1980). The alcohol component of the ester will typically contain: (1) a C 2 -C 12 aliphatic alcohol which may or may not contain one or more double bonds, and may or may not contain branched carbon chains; or (2) a C7-C12 aromatic or heteroaromatic alcohol. The invention also encompasses the use of those compositions which are both esters as described herein, and at the same time are their pharmaceutically acceptable acid addition salts.

The term "pharmaceutically acceptable amide" refers to those amides which, upon hydrolysis of the amide bond, retain the biological effectiveness and properties of the carboxylic acid or amine, and are not biologically undesirable in any other way. For a description of pharmaceutically acceptable amides as prodrugs, see Bundgaard H.: "Design of Prodrugs", Elsevier Science Publishers, Amsterdam, (1985). These amides are usually formed from the corresponding carboxylic acid and an amine. In general, the formation of amides can be carried out via conventional synthesis techniques. See for example March: "Advanced Organic Chemistry", 3rd edition, John Wiley & Sons, New York, p. 1152, (1985), and Mark et al.: "Encyclopedia of Chemical Technology", John Wiley & Sons, New York, (1980). The invention also encompasses the use of those compositions which are both amides as described herein, and at the same time are their pharmaceutically acceptable acid addition salts.

The term "pharmaceutically or therapeutically acceptable carrier" refers to a carrier medium that does not affect the effectiveness of the biological action of the active ingredients, and that is not toxic to the host or patient.

The term "stereoisomer" refers to a chemical compound having the same molecular weight, chemical composition, and constitution as one another, but with atoms grouped differently. In fact, certain identical chemical groups have different orientations in space, and because of this, when they are pure, they have the ability to rotate the plane of polarized light. However, some pure stereoisomers may have an optical rotation that is so small that it cannot be detected with existing instruments. The compounds according to this invention may have one or more asymmetric carbon atoms, and therefore include various stereoisomers. All stereoisomers are included within the scope of the invention.

The term "therapeutically or pharmaceutically effective amount" applied to compositions according to the described invention, refers to an amount of the composition sufficient to cause the desired biological result. This result can be the alleviation of the signs, symptoms or causes of a disease, or some other desired change of a biological system. With the described invention, the result will usually be associated with a reduction in the immune and/or inflammatory response to infection or tissue injury.

Amino acid residues in peptides have the following abbreviations: phenylalanine is Phe or F; leucine is Leu or L; isoleucine is Ile or I; methionine is Met or M; valine is Val or V; serine is Ser or S; proline is Pro or P; threonine is Thr or T; alanine is Ala or A; tyrosine is Tyr or Y; histidine is His or H; glutamine is Gln or Q; asparagine is Asn or N; lysine is Lys or K; aspartic acid is Asp or D; glutamic acid is Glu or E; cysteine is Cys or C; tryptophan is Trp or W; arginine is Arg or R; and glycine is Gly or G.

In addition to peptides consisting only of natural amino acids, peptide simulants or peptide analogs are also provided. Peptide analogs are commonly used in the pharmaceutical industry as non-peptide drugs with properties analogous to those of the parent peptides. These types of non-peptide compounds are called "peptide stimulants" or "peptidostimulants" (Fauchere J.: "Adv. Drug Res.", 15, p. 19, (1986); Veber and Freidinger: "TINS" p. 392, (1085); and Evans et al.: "J. Med. Chem.", 30, p. 1229, (1987), which are incorporated herein by reference). Peptide mimics that are structurally similar to therapeutically useful peptides can be used to produce equivalent or enhanced therapeutic or prophylactic activity. In general, peptidostimulants are structurally similar to a parent polypeptide (ie, a polypeptide that has biological or pharmacological activity), such as a native polypeptide that binds to a receptor, but have one or more peptide bonds optionally replaced with one bond selected from which consists of: -CH2NH-, -CH2S, -CH2-CH2-, -CH=CH- (cis and trans), -COCH2-, CH(O) CH2- and -CH2SO-, methods known in the art and further described in the following literature: Spatola A.F.: "Chemistry and Biochemistry of Amino Acids"; B. Winstein: "Peptides and Proteins", published by Marcel Dekker, New York, p. 267, (1983); Spatola A.F.: "Vega Data, Peptide Backbone Modifications" (General Review), Volume 1, Issue 3, (March 1983); Morley: "Trends Pharm Sci" (overview), p. 463-468, (1980); Hudson D. et al.: "Int J Pept Prot Res", 14, p. 177-185, (1979), (-CH2NH-, CH2CH2-); Spatola et al.: "Life Sci" 38, p. 1243-1249, (1986), (-CH 2 S); Hann: "J. Chem. Soc. Perkin Trans. I", p. 307-314, (1982), (-CH-CH-, cis and trans); Almquist et al.: "J Med Chem.", 23, p. 1391-1398, (1980), (-COCH 2 ); Jennings-White et al.: "Tetrahedron Lett.", 23, 2533, (1982), (-COCH2-); Szelke et al.: European Patent Application EP 45665 CA, 97, 39405 (1982), (-CH(OH)CH2-); Holladay et al.: "Tetrahedron Lett.", 24, p. 4401-4404, (1983), (-C(OH)CH2-); and Hruby: "Life Sci.", 31, p. 189-198, (1982), (-CH2-S-); all included here as reference literature. A particularly recommended non-peptide bond is -CH2NH-. These peptide mimics can have significant advantages over polypeptide versions, including, for example, more economical production, greater chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (for example, a broad spectrum of biological actions), reduced antigenicity, and others. Labeling of peptide stimuli is usually associated with the covalent attachment of one or more labels, directly or via an intermediary (for example an amide group), to a non-interfering position or positions on the peptide stimuli predicted by quantitative structure-activity data and/or molecular modeling. These non-interfering sites are typically sites that do not form direct contact with the macromolecule or macromolecules (for example, molecules of the immunoglobulin superfamily) to which the peptide inducer binds to produce a therapeutic effect. Derivatization (eg labeling) of peptide stimulants should not significantly interfere with the desired biological or pharmacological activity of the peptide stimulant. In general, receptor-binding peptide agonists bind to the receptor with high affinity and have appreciable biological activity (for example, they are agonists or antagonists of one or more receptor-induced phenotypic changes).

Systematic substitution of one or more amino acids of one of the consensus sequences with one D-amino acid of the same type (for example D-lysine instead of L-lysine) can be used to generate more stable peptides. In addition, restricted peptides having a single consensus sequence or a substantially identical variant of the consensus sequence can be generated by known procedures (Rizo and Gierasch: "Ann. Rev. Biochem.", 61, p. 387, (1992), herein included as reference literature), for example by adding internal cysteine residues capable of forming intramolecular disulfide bridges that cyclize peptides.

"Acm" refers to an acetamidomethyl protecting group.

II. Review

The disclosed invention provides compounds that bind to the IL-5R. These compounds represent "parent" peptide compounds and "derivative" compounds, made to have the same or similar molecular structure or shape as the parent compounds, but to differ from the parent compounds in their susceptibility to hydrolysis or proteolysis and/or in their other biological properties, such as increased affinity for the receptor. The described invention also provides compositions containing a compound that effectively binds IL-5R, blocks IL-5, and in particular, a compound useful for treating disorders associated with overexpression of IL-5 or with the production and accumulation of eosinophils.

III. Identification of peptides that bind to IL-5R

A. Receptor

Immobilized � array, � array and heterodimer as well as extracellular regions of individual IL-5 receptor arrays were produced in recombinant host cells. DNA encoding IL-SR obtained by PCR processing of cDNA from TF-1 cells using examples obtained from published receptor sequences. See Murat: "J. Exp. Med.", 175, p. 341-351, (1992); and Hayashida: "Proc. Natl. Acad. Sci. USA", 87, p. 9655-9659, (1990), which are incorporated herein by reference. One useful form of IL-5R is made by expressing the protein as a soluble protein in baculovirus-transformed host cells using standard procedures, while another useful form is made with a signal peptide for protein secretion and glycophospholipid membrane anchoring. This form of fastening is called "PIG-bonding". See Caras and Wendel: "Science", 243, p. 1196-1198, (1989); and Lin et al.: "Science", 249, p. 677-679, (1990).

Using the PIG-binding system, one can cleave the receptor from the cell surface expressing the receptor (for example, transformed CHO cells selected for high levels of receptor expression by a single cell selector) using phospholipase C. The cleaved receptor still contains a single carboxy-terminated amino acid sequence, called "HPAP terminus", from a membrane binding signal protein and can be immobilized without further purification. Recombinant receptor protein can be immobilized by coating wells of microtiter plates with an antibody (Ab 179) against HPAP termination, blocking non-specific binding with bovine serum albumin (BSA) in PBS, and then binding the separated recombinant receptor to the antibody. Using this procedure, the immobilization reaction should be performed with different concentrations of receptor to determine the optimal amount for a given preparation, since different recombinant protein preparations often contain different amounts of the desired protein. In addition, during the affinity enhancement process, it should be checked that the immobilizing antibody is completely blocked (with IL-5R or some other blocking compound). An otherwise unblocked antibody may bind unwanted phages during the affinity enhancement procedure. Peptides that bind to the immobilizing antibody can be used to block the unbound sites that remain after immobilizing the receptor to avoid this problem, or the receptor can be immobilized directly to the microtiter plate wells without the help of an immobilizing antibody. See Koller et al.: "Anal. Biochem.", 250, p. 51-60, (1997).

To distinguish higher affinity peptides, a monovalent receptor probe is often used. This probe can be made using protein kinase A, to phosphorylate a single kemptid sequence fused to the C-terminus of the soluble receptor. "Engineered" forms of the IL-5 receptor � and � sequences are then expressed in host cells, usually CHO cells. After Pl-PLC collection of the receptor, the receptor is labeled for highly specific activity with 33P or 32P for use as a monovalent probe for identifying high affinity ligands using colony lifters.

B. Peptides

Recommended search procedures for easier identification of peptides that bind to the IL-5R include first identifying the basic peptides that bind to the receptor, and then finding other peptides that are similar to the basic peptides. Specifically, using peptides based on pIII or pVIII on a phage system, a freely accessible library can be searched to find a phage representing a peptide that binds to the IL-5R. The DNAs were sequenced to determine the sequences of the peptides distributed on the surface of the phages. The "peptide on plasmid" technique was used to search for peptides and to study mutagenesis, which is described in detail in US Pat. 5,338,665, which is incorporated herein by reference. In addition, peptide-free screening and mutagenesis studies were performed using a modified C-terminal Lac-I display system in which the display valence was reduced ("head dimer" display system).

Clones specific for the �-string and clones capable of specific binding to the �-string of IL-5R were identified. The sequences of these peptides serve as the basis for the creation of other libraries, designed to contain a high frequency of derivatives of the initially identified peptides.

A representative list of recommended compounds according to the invention is shown in the following tables 1 and 2.

Table 1

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In addition to the aforementioned compounds, the recommended compounds according to the invention also include various derivatives of the aforementioned compounds, which will be discussed in more detail later. Thus, for example, additional recommended compounds include, but are not limited to:

Table 2

[image]

where -(NH2) indicates that the carboxy terminus of the compound is amidated.

C. Affinity

A number of methods can be used to determine IC50 values. Thus, for example, a [125-I] IL-5 binding assay was used to determine whether the peptides inhibit IL-5 binding to the extracellular domain of the IL-5 receptor array. Alternatively, a microphysiometric assay was used for some peptides to determine whether the peptide blocked the response of TF-1 cells to IL-5 (5 ng/ml).

To determine whether these peptides had any effect on IL-5-induced signal transduction, they were tested for IL-5R agonistic and antagonistic activity in a microphysiometric assay using the IL-responsive human leukemia cell line TF-1. -5. After stopping the administration of IL-5 overnight, these cells showed a sudden and large increase in metabolic activity after the addition of IL-5 to the cell culture medium. One recommended compound was tested using a TF-1 cell microphysiometric assay, and was found to almost completely block the response of the cells to 400 pM IL-5 when tested at a concentration of 10 µM. The peptide had a high enough affinity to allow obtaining a precise IC50 value by microphysiometric testing.

Typically, IC50 values were determined using the free peptide. The IC50 value can be determined using the free peptide, which can optionally be amidated at the C-terminus, or can be prepared as an ester or other carboxy amide. For peptides identified from plasmid-based peptide libraries, IC50 values were typically evaluated on both the parent MBP-fusion and the corresponding synthetic peptide. To restore the exact sequence expressed by the phage, the N-terminal and C-terminal amino acids of synthetic peptides are often preceded by one or two glycine residues. These glycines are not thought to be necessary for binding or for action.

Peptides and peptide mimics that have an IC50 greater than about 100 mM do not have sufficient binding to enable them to be used either in the diagnostic or therapeutic aspects of the invention. Preferably, for diagnostic purposes, the peptides and peptide mimics have an IC50 of about 2.5 mM or less, and for pharmaceutical purposes, the peptides and peptide mimics have an IC50 of about 2 mM or less.

The binding peptide sequence also provides a way to determine the minimum size of an IL-5R binding compound of the invention. Using the "encoded synthetic library" (ESL) system described in US patent application no. 946,239, filed October 16, 1992, which is a continuation of US patent application no. 762,522, filed Oct. 18, 1991, the "synthesis of very highly immobilized polymers" system described in US patent applications no. 492,462 filed March 7, 1990; no. 624,120 filed on December 6, 1990; and no. 805,727 filed Dec. 6, 1991, not only can the minimum size of a peptide with such activity be determined, but all peptides that form a group of peptides that differ from the recommended scheme (or from the minimum size of that scheme) by one, two or more remains. This collection of peptides can then be screened for their ability to bind to the IL-5 receptor. These immobilized polymer synthesis systems or other peptide synthesis procedures can also be used to synthesize truncated analogs, deleted analogs, substituted analogs, and combinations thereof for all peptide compounds of the invention.

The peptides of the invention can be prepared by conventional procedures known in the art, for example using standard solid phase techniques. Standard procedures include exclusive solid phase synthesis, partial solid phase synthesis procedures, condensation of fractions, classical solution synthesis, and even recombinant DNA technology. See, for example Merrifield J.: "Am. Chem. Soc.", 85, p. 2149, (1963), which is incorporated herein by reference. Solid-phase synthesis usually starts from the C-terminus of the peptide using a single alpha-amino protected resin.

A suitable starting material can be prepared, for example by binding the required alpha-amino acid to a chloromethylated resin, a hydroxymethylated resin or to a benzhydrylamine resin. One such chloromethylated resin is sold under the trade name BIO-BEADS SX-1 by Bio Rad Laboratories, Richmond, CA, and the preparation of the hydroxymethyl resin is described by Bodonszky et al.: "Chem. Ind.", 38, p. 1597, London, (1966). Benzhydrylamine (BHA) resin is described by Pietta and Marshall: "Chem. Commn.", p. 650, (1970), and is commercially available from Beckman Instruments, Inc., Palo Alto, CA, in the hydrochloride form.

Thus, the compounds according to the invention can be prepared by coupling one alpha-amino protected amino acid with a chloromethylated resin using, for example, a cesium bicarbonate catalyst, according to the procedure described by Gisin: "Helv. Chim. Acta", 56, p. 1467, (1973). After initial attachment, the alpha-amino protecting group is removed by a choice of reagents, including solutions of trifluoroacetic acid (TFA) or hydrochloric acid (HCl), in organic solvents at room temperature.

Alpha-amino protecting groups are known to be useful in stepwise peptide synthesis. Included are acyl protecting groups (for example formyl, trifluoroacetyl, acetyl), aromatic urethane protecting groups (for example benzyloxycarbyl Cbz and substituted Cbz), aliphatic urethane protecting groups (for example t-butyloxycarbonyl Boc, isopropyloxycarbonyl, cyclohexyloxycarbonyl) and alkyl protecting groups ( for example benzyl, triphenylmethyl). Recommended protecting groups are Boc and Fmoc. Side chain protecting groups remain intact during ligation and are not removed during deprotection of the amino terminus or during ligation. Side chain protecting groups must be removable upon completion of synthesis of the final peptide and under reaction conditions that will not alter the target peptide.

Side chain protecting groups for Tyr include tetrahydropyranyl, tert-butyl, trityl, benzyl, Cbz, Z-Br-Cbz, and 2,5-dichlorobenzyl. Side chain protecting groups for Asp include benzyl, 2,6-dichlorobenzyl, methyl, ethyl and cyclohexyl. Side chain protecting groups for Thr and Ser include acetyl, benzoyl, trityl, tetrahydropyranyl, benzyl, 2,6-dichlorobenzyl and Cbz. The side chain protecting group for Thr and Ser is benzyl. Side chain protecting groups for Arg include nitro, tosyl (Tos), Cbz, adamantyloxycarbonyl mesitoylsulfonyl (Mts) or Boc. Side chain protecting groups for Lys include Cbz, 2-chlorobenzyloxycarbonyl (2-Cl-Cbz), 2-bromobenzyloxycarbonyl (2-BrCbz), Tos or Boc.

After removal of the alpha-amino protecting group, the remaining protected amino acids are attached stepwise in the desired order. An excess of each of the protected amino acids with a suitable carboxyl group activator such as dicyclohexylcarbodiimide (DCC) in solution, for example a mixture of methylene chloride (CH 2 Cl 2 ) and dimethyl formamide (DMF), is usually used.

After the desired amino acid sequence is completed, the desired peptide is cleaved from the resin substrate by treatment with a reagent such as trifluoroacetic acid or hydrofluoric acid (HF), which not only cleaves the peptide from the resin, but also cleaves the remaining side protecting groups. of strings. When a chloromethylated resin is used, treatment with hydrofluoric acid leads to the formation of free peptide acids. When benzhydrylamino acid is used, treatment with hydrofluoric acid immediately gives the free peptide amides. Alternatively, when using a chloromethylated resin, the side-chain-protected peptide can be cleaved by treating the peptide resin with ammonia to give the desired side-chain-protected amide, or with an alkylamine to give the side-chain-protected alkylamide or dialkylamide. The side chains are then deprotected in the usual way by treatment with hydrofluoric acid to give free amides, alkylamides or dialkylamides.

When preparing the compounds according to the invention, resins are used that were used in the preparation of peptide acids, and the peptide with protected side chains is separated with a base and a suitable alcohol, for example methanol. The side chains are then deprotected in the usual manner by treatment with hydrofluoric acid to give the desired ester. These procedures for synthesizing solid phase peptides are known and are described in detail by Stewart: "Solid Phase Peptide Syntheses", Freeman and Co., San Francisco, (1969).

These procedures can be used to synthesize peptides in which the amino acids are different from the 20 naturally occurring genetically encoded amino acids, which are substituted in one, two or more positions of any of the compounds of the invention. Thus, for example, naphthylalanine can replace tryptophan, thus facilitating synthesis. Other synthetic amino acids that can be substituted into peptides according to the described invention include L-hydroxypropyl, L-3,4-dihydroxyphenylalanyl, � amino acids such as L-�-hydroxylisyl and D-�-methylalanyl, L-�-methylalanyl, � amino acids and isoquinolyl. D amino acids and unnatural synthetic amino acids can also be incorporated into peptides according to the described invention.

The natural side chains of the 20 genetically encoded amino acids (or D amino acids) can be replaced with other side chains, for example groups such as alkyl, lower alkyl, cyclic 4-, 5-, 6- to 7-membered alkyl, amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy, carboxy and their lower ester derivatives, and with 4-, 5-, 6- to 7-membered hetrocycles. In particular, a proline analog can be used where the ring size of the proline residue has been changed from 5 members to 4, 6 or 7 members. Cyclic groups can be saturated or unsaturated, and if they are unsaturated they can be aromatic or non-aromatic.

Peptides are usually synthesized as the free acid, but as mentioned above, they can also be easily prepared as an amide (ie indicated with one -(NH2) at the carboxy terminus), or an ester. It is also possible to modify the amino and/or carboxy terminus of the peptide compounds of the invention to produce other compounds of the invention. Modifications to the amino terminus include methylation (ie -NHCH3 or -NH(CH3)2), acetylation, addition of a single carbobenzoyl group, or blocking of the amino terminus with any blocking group containing a single carboxylate functional group defined by RCOO-, where R is selected from the group consisting of naphthyl, acridinyl, steroidyl, and the like. Modifications to the carboxy terminus include replacement of the free acid with a single carboxyamide group, or formation of a cyclic lactam at the carboxy terminus to introduce structural constraints.

Modifications to the amino terminus are as outlined above, including alkylation, acetylation, addition of one carbobenzoyl group, formation of one succinimide group, etc. Specifically, the N-terminal amino group can then be reacted as follows:

(a) to form an amide group of the formula RC(O)NH- where R is as defined above, by reacting with a halide-anhydride (for example RC(O)Cl) or an acid anhydride. Typically, the reaction can be carried out by contacting an approximately equimolar or excess amount (for example about 4 equivalents) of the halide-anhydride with the peptide in some inert solvent (for example dichloromethane) which preferably has an excess (for example about 10 equivalents) of a tertiary amine, as which is diisopropylethylamine, to remove the acid generated during the reaction. The reaction conditions are otherwise usual (eg room temperature, duration 30 minutes). Alkylation of the terminal amino to allow N-substitution with a lower alkyl, followed by reaction with one halide-anhydride, as described above, will give an N-alkyl amide group of the formula RC(O)NR-;

(b) to form a succinimide group by reacting with succinic anhydride. As before, an approximately equimolar amount or excess of succinic anhydride (for example about 5 equivalents) can be used and the amino group is converted to succinimide by well-known procedures, including the use of an excess (for example 10 equivalents) of a tertiary amine such as diisopropylethylamine in a suitable inert solvent (for example dichloromethane). See, for example, Wollenberg et al.: US Patent No. 4,612,132, incorporated herein in its entirety by reference. It is understood that the succinic group can be substituted with, for example (C2-C6) alkyl or -SR substituents, which are prepared in a conventional manner to provide a substituted succinimide at the N-terminus of the peptide. Such alkyl substituents are prepared by reacting a lower olefin (C2-C6) with maleic anhydride in the manner described by Wollenberg et al. (see above), and -SR substituents are prepared by reacting RSH with maleic anhydride, where R is as defined forward;

(c) to form one benzyloxycarbonyl-NH- or substituted benzyloxycarbonyl-NH- group by reacting with an approximately equivalent amount or excess of Cbz-Cl (ie benzyloxycarbonyl chloride) or substituted Cbz-Cl, in a suitable inert solvent (for example dichloromethane) which preferably contains one tertiary amine to remove the acid generated during the reaction;

(d) to form one sulfonamide group by reacting with an equivalent amount or excess (eg 5 equivalents) of R-S(O)2Cl in a suitable solvent (dichloromethane) to convert the terminal amine to a sulfonamide, where R is as defined above. Preferably, the inert solvent contains an excess of a tertiary amine (eg 10 equivalents) such as diisopropylethylamine, to scavenge the acid generated during the reaction. The reaction conditions are usual (eg room temperature, duration 30 minutes);

(e) to form one carbamate group by reacting with one equivalent amount or excess (for example 5 equivalents) of R-OC(O)Cl or R-OC(O)OC6H4-p-NO2, in a suitable inert solvent (for example dichloromethane) , to convert the terminal amine to a carbamate where R is as defined above. Preferably, the inert solvent contains an excess of a tertiary amine (eg 10 equivalents) such as diisopropylethylamine, to scavenge the acid generated during the reaction. The reaction conditions are usual (eg room temperature, duration 30 minutes); and

(f) to form one urea group by reacting with one equivalent amount or excess (eg 5 equivalents) of R-N=C=O, in a suitable inert solvent (eg dichloromethane) to convert the terminal amine to urea (ie RNHC(O)NH -) group, where R is as defined above. Preferably, the inert solvent contains an excess of a tertiary amine (eg 10 equivalents) such as diisopropylethylamine, to scavenge the acid generated during the reaction. The reaction conditions are usual (eg room temperature, duration about 30 minutes).

In the preparation of peptide stimulants, in which the C-terminal carboxyl group is replaced with an ester (ie -C(O)OR, where R is as defined above), the resins used in the preparation of peptide acids are used, and the peptide with protected side chains is separated with a base and a suitable alcohol, for example methanol. The side chains are then deprotected, in the usual way by treatment with hydrofluoric acid, to give the desired ester.

When preparing peptide stimulants where the C-terminal carboxyl group is replaced with amide -C(O)NR3R4, one benzhydrylamine resin is used as a solid substrate for peptide synthesis. Upon completion of the synthesis, treatment with hydrofluoric acid to release the peptide from the substrate directly yields the free peptide amide (ie, the C-terminus is -C(O)NH2). Alternatively, the use of a chloromethylated resin during peptide synthesis, coupled with reaction with ammonia, to separate the peptide with protected side chains from the substrate, gives the free peptide amide, and reaction with a single alkylamine or dialkylamine gives the alkylamide or dialkylamide with protected side chains (i.e., C-terminus is -C(O)NRR1, where R and R1 are as defined above). The side chains are then deprotected in the usual way by treatment with hydrofluoric acid to give free amides, alkyl amides or dialkyl amides.

In an alternative embodiment, the C-terminal carboxyl group or some C-terminal ester can be cyclized with an internal -OH or ester (-OR) carboxyl group or ester with an N-terminal amino group to form a cyclic peptide. Thus, for example after synthesis and separation to obtain the peptide acid, the free acid is converted into an activated ester with a suitable carboxyl group activator such as dicyclohexylcarbodiimide (DCC) in solution, for example in mixtures of methylene chloride (CH2Cl2) and dimethylformamide (DMF) . A cyclic peptide is then formed by internal replacement of the activated ester site with an N-terminal amine. Internal cyclization, unlike polymerization, can be enhanced by using very dilute solutions. These procedures are otherwise well known.

One can also cyclize the peptides of the invention, or one can incorporate a deamino or decarboxy residue at the ends of the peptide, so that there is no terminal amino or carboxyl group that would reduce sensitivity to proteases or limit the conformation of the peptide. The C-terminal groups of the compounds according to this invention include amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy and carboxy, as well as their lower ester derivatives and their pharmaceutically acceptable salts.

Peptides can also be easily modified with phosphorylation, and other procedures for making derivatives of peptide compounds according to the described invention are described by Hruby et al.: "Biochem J.", 268 (2), p. 249-262, (1990), incorporated herein by reference. In this way, the peptides according to the invention also serve as structural models for non-peptide compounds with similar biological activity. Those skilled in the art will know that they have at their disposal a number of techniques for making compounds with the same or similar biological activity as the parent peptide compound, but with more favorable activity than the parent compound in terms of solubility, stability, and susceptibility to hydrolysis and proteolysis. See Morgan and Gainor: "Ann. Rep. Med. Chem.", 24, p. 243-252, (1989), incorporated herein by reference. These techniques involve replacing the peptide core with a core composed of phosphonates, amidates, carbamates, sulfonamides, secondary amines, or N-methylamino acids.

Peptide mimics in which one or more peptidyl bonds [-C(O)NH-] are replaced by such bonds as one -CH2-carbamate bond, one phosphonate bond, one -CH2-sulfonamide bond, one urea bond, one secondary amine linkage (-CH2NH-), and alkylated peptidyl linkage [-C(O)NR6- where R6 is lower alkyl) are prepared during conventional peptide synthesis by simply substituting an appropriately protected amino acid analog for the amino acid reagent at the appropriate point during the synthesis.

Suitable reagents include, for example, amino acid analogues in which the carboxyl group of the amino acid has been replaced by a group suitable for forming one of the aforementioned linkages. Thus, for example, if one wishes to replace one -C(O)NR- bond in a peptide with one -CH2-carbamate bond (-CH2OC(O)NR-), then the carboxyl (-COOH) group of a suitably protected amino acid converts with usual procedures into one -OC(O)Cl functional group or into a para-nitrocarbonate-OC(O)O-C6H4-p-NO2 functional group. The reaction of one or the other of these functional groups with a free amine or an alkylated amine at the N-terminus of a partially formed peptide located on a solid support leads to the formation of a -CH2OC(O)NR- bond. For a more detailed description of the formation of such -CH2-carbamate bonds, see Cho et al.: "Science", 26, p. 1303-1305, (1993).

Similarly, replacement of one amido bond in a peptide with one phosphonate bond can be accomplished in the manner disclosed in US Pat. Nos. 5,359,115 and 5,420,328, the disclosures of which are incorporated herein by reference in their entirety.

Replacement of an amido bond in a peptide with a -CH2-sulfonamide bond can be achieved by reducing the carboxyl (-COOH) group of a suitably protected amino acid to a -CH2OH group and then converting the hydroxyl group to a suitable leaving group such as a tosyl group with usual procedures. Reaction of the tosylated derivative with, for example, thioacetic acid, followed by hydrolysis and oxidative chlorination, will yield a -CH2-S(O)2Cl functional group that replaces the carboxyl group of an otherwise suitably protected amino acid. The use of this suitably protected amino acid analogue in peptide synthesis ensures the inclusion of one -CH2S(O)2NR-bond, which replaces the amido bond in the peptide, which enables obtaining a peptide stimulant. For a more complete description of the conversion of the carboxyl group of an amino acid to a single -CH2S(O)2Cl group, see for example Boris Weinstein: "Chemistry & Biochemistry of Amino Acids, Peptides and Proteins", vol. 7, p. 267-357, published by Marcel Dekker In., New York, (1983), incorporated herein by reference.

Replacement of an amido bond in a peptide with a single urea bond can be performed in the manner disclosed in US patent application no. 08/147,805 which is incorporated herein in its entirety as a reference.

Secondary amine bonds where one -CH2NH- bond replaces the amido bond in the peptide can be prepared using, for example, a suitably protected dipeptide analogue where the carbonyl bond of the amido bond has been reduced to one CH2 group by conventional methods. Thus, for example, in the case of diglycine, the reduction of the amide to the amine will give, after deprotection, H2NCH2CH2NHCH2COOH which is then used in the N-protected form in the next coupling reaction. The preparation of such analogs by reducing the carbonyl group of the amido bond in dipeptides is known in the art.

A suitably protected amino acid analogue is used in conventional peptide synthesis in the same way as the corresponding amino acid would be used. So, for example, typically about 3 equivalents of the protected amino acid analog are used in this reaction. An inert organic solvent such as methylene chloride or DMF is used, and when an acid is generated as a by-product of the reaction, the reaction solvent will usually contain an excess of a tertiary amine to scavenge the acid generated during the reaction. A particularly recommended tertiary amine is diisopropylethylamine, which is usually used in a tenfold increased amount. The reaction leads to the incorporation into the peptide mimic of an amino acid analogue that has a non-peptide bond. Such substitution can be repeated as desired so that the amido bonds in the peptide, from zero to all, are replaced with non-amido bonds.

One can also cyclize the peptides of the invention, or one can incorporate a deamino or decarboxy residue at the ends of the peptide, so that there is no terminal amino or carboxyl group that would reduce sensitivity to proteases or limit the conformation of the peptide. The C-terminal groups of the compounds according to this invention include amide, amide lower alkyl, amide di(lower alkyl), lower alkoxy, hydroxy and carboxy, as well as their lower ester derivatives and their pharmaceutically acceptable salts.

The compounds according to the described invention can exist in a cyclized form with one intramolecular disulfide bond between thiol groups of cysteine. Alternatively, a single intermolecular disulfide bond can be produced between the thiol groups of cysteine, so that a dimeric (or more oligomeric) compound is obtained. One or more cysteine residues can also be substituted with one homocysteine. These intramolecular or intermolecular disulfide derivatives can be represented schematically as follows:

[image]

where m and n are independently 1 or 2.

Other embodiments of the invention provide analogs of these disulfide derivatives in which one of the sulfur atoms is replaced by a CH 2 group or another sulfur isostere. These analogs can be made via intramolecular or intermolecular displacement, using known procedures as shown below:

[image]

where p is 1 or 2. A person skilled in the art will readily appreciate that this shift can also be accomplished using other homologues of the �-amino-�-butyric acid and homocysteine derivative shown above.

Alternatively, the amino terminus of the peptide may be capped with one alpha-substituted acetic acid, wherein the alpha substituent is a leaving group, such as one �-halo acetic acid, for example �-chloro acetic acid, �-bromo acetic acid or � -iodoacetic acid. The compounds according to the described invention can be cyclized or dimerized by displacement of the leaving group with sulfur from a cysteine or homocysteine residue. See, for example, Barker et al.: "J. Med. Chem.", 35, p. 2040-2048, (1992), and Or et al.: "J. Org. Chem.", 56, p. 3146-3149, (1991), both incorporated herein by reference.

In addition to the previously described modifications of the N-terminus and C-terminus, compounds according to the invention, including peptide mimics, can be modified with one or more numerous hydrophilic polymers, or covalently linked with them. A suitable derivative may have increased solubility and circulation half-life and masked immunogenicity. Non-protein polymers suitable for use in accordance with the described invention include, but are not limited to, polyalkylethers such as, for example, polyethylene glycol and polypropylene glycol, polylactic acid, polyglycolic acid, polyoxyalkenes, polyvinyl alcohol, polyvinylpyrrolidone, cellulose and cellulose derivatives, dextran and dextran derivatives, etc. Generally speaking, these hydrophilic polymers have an average molecular weight in the range of about 500 to about 100,000 daltons, preferably from about 200 to about 40,000 daltons, and even more preferably from about 5,000 to about 20,000 daltons. In preferred embodiments, these hydrophilic polymers have an average molecular weight of about 5,000 daltons, 10,000 daltons, or 20,000 daltons.

The compounds according to the invention can be derivatized with the mentioned polymers or combined with them, using any of the procedures presented in the following documents: Zallipsky: "Bioconjugate Chem.", 6, p. 150-165, (1995); Monfardini et al.: "Bioconjugate Chem.", 6, p. 62-69, (1995); US patent no. 4,640,835; US patent no. 4,496,689; US patent no. 4,670,417; US patent no. 4,791,192; US patent no. 4,179,337; or WO 95/34326, all incorporated herein by reference in their entirety.

In one currently recommended embodiment, the compounds according to the described invention are derivatized with polyethylene glycol (PEG). PEG is a linear, water-soluble polymer of repeated units of ethylene oxide, with two terminal hydroxyl groups. PEGs are classified according to their molecular weight, which is usually in the range of 5,000 daltons to about 20,000 daltons. The PEGs attached to the compounds of the described invention can be branched or unbranched. See, for example, Monfardini et al.: "Bioconjugate Chem.", 6, pp. 62-69, (1995). PEGs are commercially available from Shearwater Polymers Inc. (Huntsville, Alabama), Sigma Chemicals, and other manufacturers. These PEGs include, but are not limited to, monomethoxypolyethylene glycol (MePEG-OH), monomethoxypolyethylene glycol succinate (MePEG-S-NHS), monomethoxy polyethylene glycol amine (MePEG-NH2), monomethoxypolyethylene glycol tresylate (MePEG-TRES ) and monomethoxypolyethylene glycol-imidazole-carbonyl (MePEG-IM).

Briefly, in one exemplary embodiment, the hydrophilic polymer used, for example PEG, is preferably capped at one end with a non-reactive protecting group such as methoxy or ethoxy. The polymer is then activated at the other end by reacting with a suitable activating agent, such as a cyanuric acid halide (for example, cyanuric acid chloride, bromide or fluoride), a diimidazole, an anhydride reagent (for example, dihalo succinic anhydride, such as dibromo succinic anhydride), acyl azide, p-diazonium benzyl ether, 3-(p-diazoniumphenoxy)-2-hydroxypropyl ether and the like. The activated polyvinyl is then reacted with a compound according to the described invention to produce a compound derivatized with a polymer. Alternatively, one functional group in the compounds of the invention may be activated to react with the polymer, or the two groups may be joined in a directed coupling reaction using known coupling procedures. It will be readily appreciated that the compounds of the invention may be derivatized with PEG using countless other reaction schemes known and used by those skilled in the art.

In addition, in some preferred embodiments of the invention that have one or more cysteine residues in the compound, one or more cysteines may have a suitable protecting group, preferably an Acm group, to prevent or hinder unwanted disulfide formation.

V. Testing in vivo and in vitro

The action of the compounds according to the described invention can be evaluated in vivo in one of the numerous animal models of asthma. See Larson: "Experimental Models of Reversible Airway Obstruction" in The Lung, Scientific Foundations; West et al.: "Crystal", published by Raven Press, New York, (1991); and Warner et al.: "Am. Rev. Respir. Dis.", 141, p. 253-257, (1990). An ideal animal model will replicate the main clinical and physiological features of human asthma, including: airway hypersensitivity to chemical triggers and physical stimulation; removal of obstructions in the airways using drugs useful in human asthma (�-adrenergics, methylxanthines, corticosteroids and the like); airway inflammation with infiltration of activated leukocytes; and chronic inflammatory degenerative changes, such as basement membrane thickening, smooth muscle hypertrophy, and epithelial damage. Animal species used as animal models include mice, rats, guinea pigs, rabbits, dogs and sheep. All of them have some limitations, and the appropriate choice of animal model depends on the question to be asked.

The initial asthmatic reaction can be evaluated in guinea pigs and dogs, and especially with mixed rabbits and greyhounds, which develop a non-specific increased responsiveness of the airways to numerous non-allergic substances such as methacholine and citric acid. Certain selected sheep show a double reaction after antigenic infection with Ascaris proteins. In double responder animals, the initial asthmatic response (IAR) was followed by a late asthmatic response (LAR) at 6-8 hours post challenge. Hypersensitivity to the cholinergic agonist carbachol increases 24 hours after antigen challenge in those animals in which LAR occurs.

The allergic sheep model can be used to evaluate the potential anti-asthmatic effect of the compounds according to the described invention. Administration of compositions comprising aerosol solutions of the compounds of this invention to allergic sheep before or after exposure to specific allergens will show that such compositions reduce or eliminate subsequent asthmatic response and subsequent hypersensitivity.

The compounds of this invention are also useful for the treatment of other immune-mediated inflammatory disorders in which tryptase activity contributes to the pathological condition. These diseases include inflammatory diseases associated with mammary gland cells, then rheumatoid arthritis, rheumatoid spondylitis, osteoarthritis, gouty arthritis and other arthritic conditions, inflammatory bowel disease, stomach ulcers and various skin conditions.

The efficacy of the compounds according to the present invention in the treatment of the majority of immune-induced inflammatory disorders can be verified either by in vivo or in vitro procedures. Thus, the anti-inflammatory efficiency of the compounds according to this invention can be demonstrated using known tests, such as the reverse passive Arthus reaction (RPAR)-PAW technique (see, for example, Ganguly et al.: US patent no. 5,126,352, (1992 )). Tests to determine the therapeutic value of compounds in the treatment of various skin diseases, such as hyperproliferative skin disease, are well known, such as the mouse ear test with arachidonic acid. The compounds according to the described invention can be evaluated for their antiulcer activity using the procedures described by Chiu et al.: "Archives Internationales de Pharmacodynamie et de Therapie", 270, p. 128-140, (1984).

YOU. Applications in vitro

The compounds of the invention are useful in vitro as unique tools for understanding the biological role of IL-5, including the evaluation of a number of factors that are thought to influence, or be influenced by, IL-5 production and the receptor binding process. The disclosed compounds are also useful in the development of other compounds that bind to the IL-5R, as these compounds provide important structure-activity relationship information that should facilitate such development.

The compounds are also useful as comparative inhibitors or indicators for the search for new blockers of the IL-5 receptor. In such assays, the compounds of the invention may be used without modification, or may be modified in various ways, for example by labeling, such as covalent or non-covalent binding to a nucleus, which indirectly or directly provides a detectable signal.

Possibilities for indirect labeling include biotinylation of one of the components followed by binding to avidin attached to one of the previously mentioned labeling groups. The compounds may also include mediators or linkers in cases where the compounds need to be attached to a solid substrate.

The compositions and methods of the described invention can also be used in vitro to test a patient's sensitivity to variable treatment regimens for disorders associated with overproduction of IL-5 or improper response to IL5, using an in vitro diagnostic procedure in which a sample is obtained from the patient and is treated with a compound according to the described invention that binds to IL-5R and blocks IL-5, in order to determine the efficiency and amount of the compound required to obtain the desired effect. Blocking and dosing compounds can be changed. Once the blocking compounds are searched, the doctor will be able to choose the appropriate treatment and dosage, and give it to the patient based on the results. Therefore, this invention encompasses the use of the blocking compounds of this invention in numerous diagnostic kits and test procedures.

V. Applications in vivo

The compounds of the invention can also be administered to warm-blooded animals, including humans, to block the binding of IL-5 to the IL-5R in vivo. Accordingly, the described invention encompasses methods for the treatment of IL-5-related disorders comprising administering a compound of the invention in amounts sufficient to block or suppress the binding of IL-5 to the IL-5R in vivo. Thus, for example, peptides and compounds according to the invention can be administered for the treatment of symptoms related to excessive production of IL-5 or for improper response to IL-5. The compositions and methods described herein will find use in the treatment and/or prevention of a number of disorders associated with IL-5.

According to one embodiment, the compositions according to the described invention are useful for preventing or alleviating asthma. When using the compositions according to the described invention to treat asthma, the compounds will usually be administered prophylactically, before exposure to an allergen or other precipitating factor, or after such exposure. The compounds according to the described invention are particularly useful in mitigating tissue destruction in the later phase that occurs in seasonal and permanent rhinitis. Another aspect of the described invention is directed to the prevention and treatment of other immune-induced inflammatory disorders associated with mammary gland cells, such as urticaria and angioedema, eczematous dermatitis (hereditary allergic dermatitis) and increased sensitivity, as well as hyperproliferative skin disease, gastric ulcer and similar to.

Accordingly, the described invention also provides pharmaceutical compositions containing, as an active ingredient, at least one of the peptides or peptide simulants according to the invention together with a pharmaceutical carrier or solvent. The compounds of this invention can be administered orally, via the lung, parenterally (by intramuscular, intraperitoneal, intravenous (i.v.) injection), by inhalation (via a fine powder formulation), transdermally, nasally, vaginally and rectally, or by sublingually. they can be formulated in dosage forms suitable for each route of drug administration.

The compounds of the invention when used to treat asthma will typically be formulated as aerosols. The term "aerosol" includes any gas-borne suspended phase of the compounds of this invention that can be inhaled into the bronchioles or into the nasal passages. More specifically, an aerosol comprises a gas-borne suspension of droplets of the compounds of this invention, which may be produced in a metered dose inhaler or in a nebulizer, or in a fine atomizer. Aerosol also includes a composition in the form of a dry powder of a compound according to the described invention suspended in air or another carrier gas, which can be administered by forced inhalation, for example from an inhaler.

For the solutions used to make aerosols according to the described invention, the recommended concentration range of the compounds according to the described invention is 0.1 - 100 mg/ml, more preferably 0.1 - 30 mg/ml, and most preferably 1 - 10 mg/ml. The solutions are usually buffered with some physiologically compatible buffer, such as phosphate or bicarbonate. The usual pH range is 5 to 9, preferably 6.5 to 7.8, and most preferably 7.0 to 7.6. Sodium chloride is usually added to adjust the osmotic permeability in the physiological range, preferably within 10% of isotonic.

Suspensions of compounds according to the described invention in hydrofluoroalkane propellant gases, especially 1,1,1,2-tetrafluoro ethane 1,1,1,2,3,3,3-heptafluoro propane, possibly in the presence of some surfactant and/or common solvent (for example ethanol) in a pressurized box, can be provided together with a suitable discharge device, for the purpose of treating the aforementioned respiratory disorders, especially asthma and allergic rhinitis.

Formulations of these solutions to form inhalation aerosols are described in Remington: "Pharmaceutical Sciences" and also in Ganderton and Jones: "Drug Delivery to the Respiratory Tract", ed. Ellis Horwood, (1987); Gonda: "Critical Reviews in Therapeutic Drug Carrier Systems", 6, p. 273-313, (1990); and Raeburn et al.: "J. Pharmacol. Toxicol. Methods", 27, p. 143-159, (1992).

Solutions of the compounds according to the described invention can be converted into aerosols by any method routinely used to make aerosolized pharmaceutical inhalation agents. Generally speaking, these procedures involve pressurizing or providing means to pressurize a solution box, usually by means of some inert carrier gas, and passing the pressurized gas through a small opening, thereby drawing droplets of the solution into the mouth and trachea of the subject being administered the drug. . A mouthpiece is usually placed at the exit of the opening to facilitate introduction into the mouth and trachea.

In one embodiment, the devices contain solutions of compounds according to the described invention, associated with or contained in any of the conventional means of forming aerosols for the treatment of asthma, such as metered dose inhalers, jet nebulizers or ultrasonic nebulizers. If necessary, these devices can have a mouthpiece placed around the opening.

In one embodiment for the treatment of allergic rhinitis, the device may contain a compound according to the described invention in one nasal spray.

A dry powder containing a compound according to the described invention, if necessary with some additive, represents another embodiment of the described invention. It can be administered using an inhaler for powder medicines containing the powder described above.

The compounds of the invention can also be used in the treatment of immune-induced inflammatory skin conditions, such as urticaria, angioedema, eczematous dermatitis, and hyperproliferative skin disease, for example psoriasis, in mammals. As a result of local administration of a compound according to the invention, relief of symptoms can be expected. In this way, a subject suffering from an immune-induced inflammatory skin condition can expect a reduction in skin peeling, skin redness, spot size, itching and other symptoms associated with the skin condition. The dosage of medication and the length of time required for successful treatment may vary for each individual patient, but experts will be able to recognize these variations and adjust the course of treatment.

The invention also includes preparations for external topical application to the skin, comprising a compound of the described invention, usually in concentrations in the range of about 0.001% to 10%, together with some non-toxic pharmaceutically acceptable carrier. These topical preparations for external application may be prepared by combining the active ingredient of this invention with conventional pharmaceutical solvents and carriers used in topical external dry, liquid, cream and aerosol formulations. Ointments and creams may, for example, be formulated with a water or oil base with the addition of suitable thickening and/or gelling agents. These bases may contain water and/or an oil such as liquid paraffin or a vegetable oil such as peanut oil or castor oil. Thickeners that can be used in accordance with the nature of the base include soft paraffin, aluminum stearate, cetostearyl alcohol, propylene glycol, polyethylene glycols, lanolin, hydrogenated lanolin, beeswax and the like.

Lotions can be formulated with a water or oil base, and will generally contain one or more stabilizers, emulsifiers, dispersing agents, suspending agents, thickeners, coloring agents, perfumes and the like.

Powders can be formed using any suitable powder base, for example talc, lactose, starch and the like. Droplets may be formulated with an aqueous base or a non-aqueous base, which may also include one or more dispersing agents, suspending agents, solubilizing agents, and the like.

External topical pharmaceutical compositions according to this invention may also comprise one or more preservatives or bacteriostatic agents, for example methyl hydroxybenzoate, propyl hydroxybenzoate, chlorocresol, benzalkonium chloride, and the like. External topical compositions may also contain other active ingredients such as agents against microscopic organisms, especially antibiotics, anesthetics, analgesics and antipruritic agents.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one inert pharmaceutically acceptable carrier, such as sucrose, lactose or starch. It is normal practice that these dosage forms, in addition to inert solvents, may also contain additional substances, for example lubricants such as magnesium stearate. In the case of capsules, tablets and pills, the dosage forms may also contain buffering agents. Tablets and pills can additionally be prepared with an enteric coating.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, wherein elixirs include commonly used inert solvents such as water. In addition to these inert solvents, the compositions also include auxiliaries such as wetting agents, emulsifying and suspending agents, and sweeteners and agents for imparting taste and smell.

Means according to this invention for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions or emulsions. Examples of non-aqueous solvents or carriers are propylene glycol, polyethylene glycol, vegetable oils, such as olive and corn oil, gelatin, and injectable organic esters, such as ethyl oleate. These dosage forms may also contain additives such as preservatives, wetting agents, emulsifiers and dispersing agents. They can be sterilized, for example, by filtering through a bacteria-retaining filter, including a sterilizing agent in the composition, irradiating the composition, or heating the composition. They may also be produced using sterile water, or some other sterile injection medium, immediately prior to use.

Compositions for rectal or vaginal administration are preferably suppositories which may contain, in addition to the active substance, additives such as coconut oil or wax for suppositories. Compositions for nasal or sublingual administration may also be prepared with known standard additives.

It is certainly understood that the compositions and compounds according to this invention can be used in combination with other agents that have the ability to modulate the synthesis, release and/or binding of IL-5, and with other agents for the treatment of immune-induced inflammatory disorders, especially asthma. The β-adrenergic agonists are particularly useful in these combinations, since they provide symptomatic relief of the initial asthmatic response, while the compounds of the present invention provide relief of the later asthmatic response. Recommended α-adrenergic agonists in these solutions include any of the common α-agonists used to relieve asthma, such as albuterol, terbutaline, formoterol, fanoterol or prenaline.

Other agents useful in combination with the compounds of the present invention include anticholinergics such as ipratopium bromide, and anti-inflammatory corticosteroids (adrenocortical steroids) such as beclomethasone, triamcinolone, flurizolid or dexamethasone.

Compositions containing the compounds may be administered for prophylactic and/or therapeutic treatment. In therapeutic applications, the compositions are administered to a patient already suffering from a disease, as described above, in an amount sufficient to cure or at least partially stop the symptoms of the disease and its complications. The amount sufficient to achieve this is defined as a "therapeutically effective dose". The amounts effective for such use will depend on the severity of the disease, and on the weight and general condition of the patient.

For prophylactic use, compositions containing the compounds according to the invention are administered to a patient susceptible to a particular disease, or otherwise at risk for him. Such an amount is defined as a "prophylactically effective dose". With this application, the exact amounts again depend on the patient's condition, health and weight.

The amounts of IL-5 blocking compounds required for effective therapy will depend on many different factors, including the route of administration, the site to which it is directed, the physiological state of the patient, and other medications administered to the patient. Therefore, treatment doses should be adjusted to optimize safety and efficacy. Doses used in vitro can usually provide useful guidance as to the amounts useful for in situ administration of these reagents. Animal studies of effective doses for the treatment of certain disorders will provide predictable indications for human dosing. Various considerations are described by, for example, Goodman and Gilman: "The Pharmacological Basis of Therapeutics", 8th ed., Pergamon Press, (1990); and Remington: "Pharmaceutical Sciences", 7th ed., Mack Publishing Co., Easton, Penn, (1985), all incorporated by reference.

The peptides and peptide mimics of the present invention are effective in the treatment of IL-5 induced conditions when administered at a dose of from about 0.001 mg to about 10 mg/kg of body weight per day. The specific dose to be used is regulated by the specific condition being treated, through which the drug is administered, as well as the assessment of the treating clinician, which depends on factors such as the severity of the condition, age and general condition of the patient, and the like.

Although only recommended embodiments of the invention are specifically described above, it is understood that modifications and variants of the invention are possible without departing from the spirit and desired scope of the invention.

ABBREVIATIONS

DMEM Dulbecco's minimal essential medium

DMEM/F12 Dulbecco's minimal essential medium / Hamm's F12 medium

ng/ml nanogram/milliliter

min minutes

µl microliter

µl/mg microliter/milligram

BSA protein from bovine serum

PBS phosphate buffered saline

Example 1

Peptide synthesis in solid phase

The various peptides of the invention were synthesized using Merrifield's solid phase peptide synthesis technique (see Steward and Young: "Solid Phase Peptide Synthesis", 2nd ed., Pierce Chemical, Rockford, IL, (1984); and Merrifield: " J. Am. Chem. Soc.", 85, p. 2149, (1963)) on a Milligen/Biosearch 9600 automated instrument or on an Applied Biosystems Inc. Model 431A peptide synthesizer. Peptides were generated using standard protocols from Applied Biosystems Inc., System Software version 1.01. Each binding was performed in 1 to 2 hours with BOP (benzothiazolyl N-octrisdimethylaminophosphonium hexafluorophosphate) and HOBt (1-hydroxybenzothiazole).

The resin used was HMP resin or PAL (Milligen/Biosearch), which is a cross-linked polystyrene resin with 5-(4'-Fmoc-aminomethyl-3,4'-dimethoxyphenoxy)valeric acid as a linker. The use of PAL resin provides a single amide functional group at the carboxyl terminus after separation of the peptide from the resin. After cleavage, the HMP resin produces a single carboxylic acid nucleus at the C-terminus of the final product. Most reagents, resins, and protected amino acids (free or on resin) were purchased from Millipore or Applied Biosystems Inc.

The Fmoc group was used for amino protection during the binding procedure. Primary amino protection on amino acids was achieved with Fmoc, and the side chain protecting groups were t-butyl for serine, tyrosine, asparagine, glutamic acid and threonine, trityl for glutamine, Pmc (2,2,5,7,8-pentamethylchromic sulfonate) for arginine, N-t-butyloxycarbonyl for tryptophan, N-trityl for histidine and glutamine, and S-trityl for cysteine.

Removal of the peptide from the resin and simultaneous removal of the protection from the functional groups of the side chains was achieved by treatment with regans K or one of its slight modifications. Alternatively, in the synthesis of these peptides, with one amidated carboxyl terminus, the fully completed peptide was separated with a mixture of 90% trifluoroacetic acid, 5% ethanedithiol, and 5% water, initially at 4 °C, with a gradual rise to room temperature. Deprotected peptides were precipitated with diethyl ether. In all cases, purification was performed by preparative, opposite-phase, high-performance liquid chromatography on a C18 bonded silica gel column with an acetonitrile/water gradient in 0.1% trifluoroacetic acid. Homogeneous peptides were analyzed by mass spectrometry, fast atom bombardment, or electrospray mass spectrometry and amido acid analysis when necessary.

Example 2

Biological tests

The biological activity of synthetic peptides and MBP-peptide fusions were measured using a Cytosensor microphysiometer (Molecular Devices) to record the metabolic response of TF-1 cells (a human leukemia cell line) to IL-5, in the presence and absence of peptide. After overnight incubation without IL-5, these cells showed a strong increase in metabolic activity when IL-5 was added to the medium. This increase was measured with a microphysiometer as an increase in the acidification rate of a weakly buffered tissue culture medium.

TF-1 cells were seeded into microphysiometer chambers at a density of 1.5 × 105 cells per chamber, and grown overnight in DMEM tissue culture medium containing 10% bovine serum but lacking 1 ng/ml IL-5. (R&D Systems) which is required for the long-term maintenance of these cells in culture. Chambers were then placed in a microphysiometer and incubated with lightly buffered DMEM/F12 medium containing 1% human serum protein until baseline acidification rate was established. IL-5 was administered in the amount of 10 ng/ml for 25 minutes in the constant presence of the tested peptide. Then the chambers were washed with fresh medium.

Typically, maximal responsiveness to IL-5 occurred within 20 minutes after the initiation of IL-5 addition to the medium. In the absence of peptide, this reaction was usually accompanied by a 1.5- to 2-fold increase in the acidification rate of the medium. All peptides were able to reduce or completely block the responsiveness of TF-1 cells to IL-5. Other, arbitrarily chosen control peptides, of the same or higher concentrations, had no effect. The tested peptides also had no effect on the strong microphysiometric response of TF-1 cells to TNF�, which indicates that the tested peptides exerted their effect by specifically antagonizing the action of IL-5. The IC50 for test peptides is defined as the concentration of peptide that produces a 50% reduction in maximal response to IL-5, compared to response to IL-5 alone.

Example 3

Binding affinity

The binding affinities of the synthetic peptides for IL-5R� were measured in a comparative binding assay, using radioiodinated IL-5. Immulon 4 (Dynatech) microtiter plates were coated with streptavidin (Sigma) by incubating 100 µl of a 50 µg/ml solution in PBS for 30 min at 37 °C. The wells were blocked with 200 µl of 1% BSA in PBS for 15 minutes, at 37 °C, then with 100 µl of biotinylated monoclonal antibody, labeled mAb 179, at 5 µg/ml in PBS. Solubilized IL-5Rα was then immobilized in cups by incubating 100 µl of the collected receptor solution diluted 1:5000 in PBS/0.1% BSA for 1 hour at 4 °C. After washing off the unbound receptors, 40 µl of various concentrations of the test peptide diluted in PBS/0.1% BSA were added to the cups, followed by 50 µl of a constant concentration of [125 I]IL-5 (Amersham). The binding reaction mixtures were incubated at 4 °C for 2 hours, and then washed with PBS to remove unbound [125 I]IL-5. Bound [125 I]IL-5 was determined by counting. Total binding was defined by the amount of [125 I]IL-5 bound in the presence of 30 nM IL-5. Peptide binding data were analyzed to determine the concentration of peptide required to reduce specific [125 I]IL-5 binding by 50% (IC50). Under the described conditions, the obtained IC50 values should be similar to the displacement constant (Kd) of the peptide for IL-5R�.

All articles and references in this application, including patent documents, are included as references.

LIST OF SEQUENCES

(1) GENERAL INFORMATION

(i) APPLICANT: Glaxo Group Limited

(ii) TITLE OF THE INVENTION: Peptides and compounds that bind to the IL-5 receptor

(iii) NUMBER OF SEQUENCES: 21

(iv) ADDRESS FOR CORRESPONDENCE:

(A) ADDRESSEE: Glaxo Group Limited

(B) STREET: Glaxo Wellcome House, Berkeley Avenue

(C) CITY: Greenford

(D) STATE:

(E) COUNTRY: GREAT BRITAIN

(F) ZIP: UB6 ONN

(v) COMPUTER READABLE FORM:

(A) MEDIA TYPE: Diskette

(B) COMPUTER: IBM compatible

(C) OPERATING SYSTEM: DOS

(D) SOFTWARE: FastSEQ for Windows Version 2.0

(vi) INFORMATION ON THE CURRENT APPLICATION:

(A) APPLICATION NUMBER:

(B) DATE OF SUBMISSION:

(C) CLASSIFICATION:

(vii) DATA ON PREVIOUS APPLICATION:

(A) APPLICATION NUMBER: 9712410.1

(B) FILED DATE: June 14, 1987.

(viii) INFORMATION ABOUT THE ATTORNEY, REPRESENTATIVE:

(A) NAME: Quillin Helen K

(B) REGISTRATION NUMBER:

(C) REFERENCE NUMBER/CASE NUMBER: PK3293

(ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 01714934060

(B) TELEFAX: 01819668838

(C) TELEX:

(2) INFORMATION FOR SEQ ID NO:1:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid

(C) CHAINING: single

(D) TOPOLOGY: linear

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:

Cys Trp Arg Ser Val Ala Thr His Thr Trp Phe Cys Gly

(2) INFORMATION FOR SEQ ID NO:2:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) CHAINING: single

(D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Cys Trp Arg Ser Val Ala Thr His Thr Trp Phe Cys Gly Glu

(2) INFORMATION FOR SEQ ID NO:3:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid

(C) CHAINING: single

(D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:

Cys Trp Arg Ser Val Ala Thr His Thr Trp Phe Cys Gly Glu Glu

(2) INFORMATION FOR SEQ ID NO:4:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 19 amino acids

(B) TYPE: amino acid

(C) CHAINING: single

(D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:

Glu Gly Asp Cys Trp Arg Ser Val Ala Thr His Thr Trp Met Cys Gly Val Glu

(2) INFORMATION FOR SEQ ID NO:5:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 19 amino acids

(B) TYPE: amino acid

(C) CHAINING: single

(D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:

Glu Cal Glu Cys Trp Arg Ser Val Ala Thr His Thr Trp Phe Cys Gly Glu Asp

(2) INFORMATION FOR SEQ ID NO:6:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 21 amino acids

(B) TYPE: amino acid

(C) CHAINING: single

(D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:

Gly Gly Gly Val Glu Cys Thr Arg Ser Val Ala Thr His Ser Trp Val Cys Gly Ile Asp

(2) INFORMATION FOR SEQ ID NO:7:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 24 amino acids

(B) TYPE: amino acid

(C) CHAINING: single

(D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:

Leu Arg Arg Ala Ser Leu Gly Gly Cys Trp Arg Ser Val Ala Thr His Thr Trp Phe Cys Gly Glu glu 20

(2) INFORMATION FOR SEQ ID NO:8:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 18 amino acids

(B) TYPE: amino acid

(C) CHAINING: single

(D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:

Val Asp Glu Cys Trp Arg Leu Val Ala Thr His Thr Trp Phe Cys Gly Asp Asp

(2) INFORMATION FOR SEQ ID NO:9:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 18 amino acids

(B) TYPE: amino acid

(C) CHAINING: single

(D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:

Val Asp Glu Cys Trp Arg Ser Val Ala Thr His Thr Trp Phe Cys Gly Glu Glu

(2) INFORMATION FOR SEQ ID NO:10:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 19 amino acids

(B) TYPE: amino acid

(C) CHAINING: single

(D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:

Val Glu Aso Cys Trp Arg Ser Val Ala Thr His Thr Trp Phe Cys Gly Glu Asp

(2) INFORMATION FOR SEQ ID NO:11:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 18 amino acids

(B) TYPE: amino acid

(C) CHAINING: single

(D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:

Va, Leu Asp Cys Trp Arg Ser Val Ala Thr His Ser Trp Phe Cys Gly Glu Asp

(2) INFORMATION FOR SEQ ID NO:12:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 18 amino acids

(B) TYPE: amino acid

(C) CHAINING: single

(D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:

Val Val Asp Cys Trp Arg Ser Val Ala Thr His Ser Trp Phe Cys Gly Glu Glu

(2) INFORMATION FOR SEQ ID NO:13:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid

(C) CHAINING: single

(D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:

Val Val Asp Cys Trp Arg Ser Val Ala Thr His Thr Trp Phe Cys Gly

(2) INFORMATION FOR SEQ ID NO:14:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 17 amino acids

(B) TYPE: amino acid

(C) CHAINING: single

(D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:

Val Val Asp Cys Trp Arg Ser Val Ala Thr His Thr Trp Phe Cys Gly Glu

(2) INFORMATION FOR SEQ ID NO:15:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 18 amino acids

(B) TYPE: amino acid

(C) CHAINING: single

(D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:

Val Val Asp Cys Trp Arg Ser Val Ala Thr His Thr Trp Phe Cys Gly Glu Asp

(2) INFORMATION FOR SEQ ID NO:16:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 15 amino acids

(B) TYPE: amino acid

(C) CHAINING: single

(D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:

Cys Trp Arg Ser Val Ala Thr His Thr Trp Phe Cys Gly Glu Glu

(2) INFORMATION FOR SEQ ID NO:17:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 14 amino acids

(B) TYPE: amino acid

(C) CHAINING: single

(D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:

Cys Trp Arg Ser Val Ala Thr His Thr Trp Phe Cys Gly Glu

(2) INFORMATION FOR SEQ ID NO:18:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 13 amino acids

(B) TYPE: amino acid

(C) CHAINING: single

(D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:

Cys Trp Arg Ser Val Ala Thr His Thr Trp Phe Cys Gly

(2) INFORMATION FOR SEQ ID NO:19:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 23 amino acids

(B) TYPE: amino acid

(C) CHAINING: single

(D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:

Leu Arg Arg Ala Ser Leu Gly Gly Cys Trp Arg Ser Val Ala Thr His Thr Trp Phe Cys Gly Glu Glu

(2) INFORMATION FOR SEQ ID NO:20:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 17 amino acids

(B) TYPE: amino acid

(C) CHAINING: single

(D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:

Val Val Asp Cys Trp Arg Ser Val Ala Thr His Thr Trp Phe Cys Gly Glu

(2) INFORMATION FOR SEQ ID NO:21:

(i) CHARACTERISTICS OF THE SEQUENCE:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid

(C) CHAINING: single

(D) TOPOLOGY: linear

(ii) TYPE OF MOLECULE: Peptide

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:

Val Val Asp Cys Trp Arg Ser Val Ala Thr His Thr Trp Phe Cys Gly

Claims (19)

1. A compound that binds to the IL-5 receptor, characterized by the fact that this compound comprises one basic sequence of amino acids: C W R S V A T H T W F C G (SEQ ID NO:1); C W R S V A T H T W F C G E (SEQ ID NO:2); C W R S V A T H T W F C G E E (SEQ ID NO:3); E G D C W R S V A T H T W M C G V E (SEQ ID NO:4); E V E C W R S V A T H T W F C G E D (SEQ ID NO:5); G G G V E V C T R S V A T H S V C G I D (SEQ ID NO:6); L R R A S L G G C W R S V A T H T W F C G E E (SEQ ID NO:7); V D E C W R L V A T H T W F C G D D (SEQ ID NO:8); V D E C W R S V A T H T W F C G E E (SEQ ID NO:9); V E D C W R S V A T H T W F C G E D (SEQ ID NO:10); V L D C W R S V A T H S W F C G E D (SEQ ID NO:11); V V D C W R S V A T H S W F C G E E (SEQ ID NO:12); V V D C W R S V A T H T W F C G (SEQ ID NO:13); V V D C W R S V A T H T W F C G E (SEQ ID NO:14); V V D C W R S V A T H T W F C G E D (SEQ ID NO:15); C W R S V A T H T W F C G E E-(NH2) (SEQ ID NO:16); C W R S V A T H T W F C G E-(NH2) (SEQ ID NO:17); C W R S V A T H T W F C G-(NH2) (SEQ ID NO:18); L R R A S L G G C W R S V A T H T W F C G E E-(NH2) (SEQ ID NO:19); V V D C W R S V A T H T W F C G E-(NH2) (SEQ ID NO:20); or V V D C W R S V A T H T W F C G-(NH2) (SEQ ID NO:21) and their dimers and oligomers, which have (a) a molecular weight of less than about 5,000 daltons, and (b) IL-5R binding affinity expressed as an IC50 not greater than about 100 µm, wherein, starting from zero to all, the -C(O)NH- bonds of the peptide are replaced by one bond selected from the group consisting of one -CH2OC(O)NR- bond, one phosphonate bond, one -CH2S(O )2NR- bond, one CH2NR- bond, and one -C(O)NR6 bond, and one -NHC(O)NH- bond where R is hydrogen or lower alkyl and R6 is lower alkyl, wherein the N-terminus of said peptide or peptide mimic is selected from the group consisting of one -NRR1 group, one -NRC(O)R group, one -NRC(O)OR group, one -NRS(O)2R group, one - NHC(O)NHR group, one succinimide group, one benzyloxycarbonyl-NH-(CBZ-NH-) group, and one benzyloxycarbonyl-NH- group, having 1 to 3 substituents on the phenyl ring selected from the group consisting of lower alkyl, lower alkoxy, chloro and bromo, wherein R and R1 are independently selected from the group consisting of hydrogen or lower alkyl, and wherein the C-terminus of said peptide or peptide mimic has the formula -C(O)R2, where R2 is selected from the group consisting of lower hydroxy, lower alkoxy, and -NR3R4 where R3 and R4 are independently selected from the group consisting of hydrogen and lower alkyl, and where the nitrogen atom of the -NR3R4 group may optionally be the amino group of the N-terminus of the peptide so that a cyclic peptide is formed, and their physiologically acceptable salts. 2. The compound according to claim 1, characterized in that the cysteines are cyclized via one intramolecular disulfide bond. 3. Compound according to claim 1 or 2, characterized in that the carboxy terminus is amidated. 4. A compound according to any of the preceding claims, characterized in that it is used in medicine. 5. Pharmaceutical composition, characterized in that it contains one compound according to claims 1 to 3, in combination with one pharmaceutically acceptable carrier. 6. Composition according to claim 6, characterized in that it is used for the treatment of immune-induced inflammatory disorders. 7. An aerosol composition, characterized in that it contains one compound according to any one of claims 1 to 3, in a pharmaceutically acceptable carrier solution or in the form of a dry powder suitable for use in an aerosol. 8. Composition according to any one of claims 5 to 7, characterized in that the compound according to any one of claims 1 to 3 is present in said carrier in a concentration of 0.1 to 100 mg/ml. 9. The composition according to any one of claims 5 to 8, characterized in that it further contains one α-adrenergic agonist compound. 10. Composition according to any one of claims 5 to 9, characterized in that it contains an anti-inflammatory corticosteroid. 11. The composition according to any one of claims 5 to 10, characterized in that it contains ipratropium bromide. 12. The composition according to any one of claims 5 to 11, characterized in that it is used for the treatment of asthma. 13. An aerosol device, characterized in that it contains a composition according to any one of claims 7 to 12, and means for converting this solution or dry powder into an aerosol form suitable for inhalation. 14. A method for treating a patient suffering from an immune-induced inflammatory disorder, characterized in that it comprises administering to the patient a therapeutically effective dose of a compound according to any one of claims 1 to 3. 15. A method for treating a patient suffering from a disorder associated with improper production of IL-5 or improper response to IL-5, characterized in that it comprises administering to the patient a therapeutically effective dose of a compound according to any one of claims 1 to 3. 16. A method for treating a patient suffering from a disorder associated with improper production and accumulation of eosinophils, comprising administering to the patient a therapeutically effective dose of a compound according to any one of claims 1 to 3. 17. The method according to one of claims 14 to 16, characterized in that the disorder is asthma. 18. A compound according to any one of claims 1 to 3, characterized in that it is used for the production of a medicament for the treatment of disorders associated with improper production of IL-5 or improper response to IL-5. 19. The compound according to any one of claims 1 to 3, characterized in that it is used for the production of medicaments for the treatment of asthma.