WO1999064056A9 - Use of a cytokine regulatory agent to treat asthma - Google Patents

Abstract

The present invention relates to the use of a cytokine regulatory agent to reduce the severity of asthma.

Description

USE OF A CYTOKINE REGULATORY AGENT TO TREAT ASTHMA

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

The present invention relates generally to the fields of medicine and molecular pathology and, more specifically, to methods of using a cytokine regulatory agent to reduce the severity of asthma in an individual .

BACKGROUND INFORMATION

Cytokines are a class of proteins produced by macrophages and monocytes in response to viral or bacterial infection and in response to T cell stimulation during an immune response. Cytokines are normally present in very low concentrations in a tissue and mediate their effects through binding to high affinity receptors on specific cell types.

Various cytokines such as the interleukins (IL) , interferons (IF) and tumor necrosis factors (TNF) are produced during immune and inflammatory responses and control various aspects of these responses. Following induction of an immune or inflammatory response, the concentrations of the various cytokines increase at different times. For example, following exposure of a subject to bacterial endotoxin, TNF and interleukin-6 (IL-6) levels increase, followed a few hours later by increases in the levels of IL-1 and IL-8. The cytokines, including TNF, IL-1, IL-2, IL-6 and IL-8, mediate host defense responses, cell regulation and cell differentiation. These cytokines can induce fever in a subject, cause activation of T and B cells and affect the levels of other cytokines, which result in a cascade effect whereby other cytokines mediate the biological action of the first cytokine.

The activation of these and other cytokines is responsible for the tissue damage that occurs in various inflammatory conditions, including patho-immunogenic diseases such as asthma. Asthma is a disease of localized anaphylaxis, or atopy, and is characterized as an inflammatory disease. In some cases, asthma is triggered by exposure to allergens (allergic asthma) , while in other cases, asthma is triggered independent of allergen stimulation (intrinsic asthma) . Approximately 5% of the United States population has signs or symptoms of asthma.

Upon inhalation of an allergen, an immune response is initiated, resulting in the release of mediators of hypersensitivity including histamine, bradykinin, leukotrienes, prostaglandins, thromboxane A₂ and platelet activating factor. The initial phase of the asthmatic response also results in the release of chemotactic factors that recruit inflammatory cells such as eosinophils and neutrophils. The eosinophils and neutrophils are capable of releasing toxic enzymes, oxygen radicals and cytokines that can result in significant tissue damage. Clinical manifestations of these events include occlusion of the bronchial lumen with mucus, proteins and cellular debris; sloughing of the epithelium; thickening of the basement membrane; fluid buildup (edema) ; and hypertrophy of the bronchial smooth muscles . Cytokines have multiple biological activities and interact with more than one cell type. In addition, some cells interact with more than one type of cytokine. As a result, it has not been possible to prevent damage to healthy tissue by targeting one particular cytokine or cell type. For example, individual cytokine receptors or receptor antagonists that were designed to eliminate the biological effect due to one cytokine did not decrease mortality due to endotoxic shock, which is mediated by TNF, IL-1, IL-6 and IL-8.

A better approach for preventing tissue damage due to cytokines would be to regulate the expression of all or several of the cytokines involved in the response, without eliminating expression of any cytokine in its entirety. In this way, undesirable side effects such as complete immunosuppression can be prevented and homeostasis can be maintained.

Current methods for treating asthma are directed at airway obstruction and inflammation. For example, treatment with bronchodilators such as β-adrenergic agents results in smooth muscle relaxation of restricted airways and is often effective with mild forms of asthma. However, moderate to severe asthma often requires treatment with anti- inflammatory agents such as corticosteroids, which effectively modulate cytokine expression. Unfortunately, corticosteroids can cause complete immunosuppression and have other undesirable side effects such as inducing "wasting" syndrome, diabetes and osteoporosis. Even inhaled corticosteroids, which have less systemic effect, can cause adverse effects such as oral thrush.

In order to reduce the severity of asthma due to the expression of cytokines, it would be advantageous if the levels of cytokines that contribute to the deleterious effects associated with asthma could be regulated. Since the primary treatment for asthma that affects the inflammatory response induced by cytokines is administration of corticosteroids, it would be desirable to identify other anti-inflammatory agents that can effectively treat asthma, without producing the adverse side effects manifested by treatment with corticosteroids. Thus, a need exists for an effective method of treating an individual suffering from asthma, while minimizing the likelihood of undesirable side effects in the treated patient. The present invention satisfies this need and provides related advantages as well .

SUMMARY OF THE INVENTION

The present invention relates to the use of a cytokine regulatory agent (CRA) to reduce the severity of asthma. For example, the invention relates to the administration of a CRA having the structure X_ι-X₂- (D) Phe-Arg- (D) Trp-X₃, X₁-X₂-His- (D) Phe-Arg- (D) Trp-X₃ or X₄-X₅- (D) Phe-Arg- (D)Trp- X₃; where X_1# X₂, X₃, X₄ and X₅, when present, are amino acids or amino acid analogs, or modified forms of such structures; to an individual exhibiting or susceptible to asthma, wherein said CRA reduces the signs or symptoms of asthma in the individual.

BRIEF DESCRIPTION OF THE DRAWINGS

Figures 1A and IB show the effect of treatment with 300 μg/kg of HP 228 on airway mechanics in sheep challenged with Ascaris suum relative to historical controls. Figure 1A shows specific lung resistance (SR_L) , and Figure IB shows antigen-induced airway responsiveness (PC₄₀₀) . "BSL" indicates baseline values; "Post Antigen" indicates values obtained 24 hr after challenge with Ascaris suum antigen. Data are the mean ± standard error (SE) for three sheep, indicated by error bars.

Figures 2A and 2B show the effect of treatment with 100 μg/kg of HP 228 on airway mechanics in sheep challenged with Ascaris suum relative to historical controls. Figure 2A shows specific lung resistance (SR_L) , and Figure 2B shows antigen-induced airway responsiveness (PC₄₀₀) . Data are the mean ± SE for three sheep, indicated by error bars.

Figure 3 shows the effect of treatment with

300 μg/kg of HP 228 on specific lung resistance in sheep challenged with Ascaris suum relative to placebo controls. Values are mean + SE for six sheep. "*" indicates P<0.05 and "+" indicates P<0.10 vs HP 228 treated; repeated measures ANOVA followed by paired T-test .

Figure 4 shows the effect of treatment with 300 μg/kg of HP 228 on antigen-induced airway responsiveness (PC₄₀₀) in sheep relative to placebo controls. Values are mean ± SE for six sheep. " *" indicates P<0.05 vs Baseline (BSL) ; "+" indicates P<0.05 vs HP 228 treated; repeated measures ANOVA, followed by paired T-test.

Figure 5 shows the effect of 300 μg/kg of HP 228 on tissue kallikrein ("TK") activity in bronchoalveolar lavage fluids. The values are for six sheep and were evaluated using Friedman's non-parametric ANOVA, followed by Wilcoxon's paired test.

Figure 6 shows the effect of 300 μg/kg of HP 228 on albumin levels in bronchoalveolar lavage fluids. The values are mean + SE for six sheep. "*" indicates P<0.05 vs Baseline (BSL) and "+" indicates P<0.05 vs HP 228 treated; analyzed using Friedman's non- parametric ANOVA, followed by ilcoxon's paired test.

Figure 7 shows the effect of HP 228 on airway reactivity to methacholine in the ovalbumin-sensitized, challenged mouse. Enhanced pause ("Penh") indicates increased lung resistance. Circles represent challenged; squares represent sensitized-challenged; triangles represent treatment with 300 μg/kg HP 228 administered B.D., i.p.; inverted triangles represent treatment with 600 μg/kg HP 228 administered B.D., i.p.; diamonds represent 600 μg/kg HP 228 administered 4 times a day, i.p.; "x" represents 30 μg/animal administered 4 times a day, i.p.

Figure 8 shows the effect of HP 228 on bronchoalveolar lavage fluid cell number for macrophages ("MAC"), lymphocytes ("LYMPH"), neutrophils ("NEUT") and eosinophils ("EOS") in the ovalbumin-sensitized, challenged mouse. The bars represent (left to right) challenged (white) ; sensitized-challenged (black) ; treatment with 300 μg/kg HP 228 administered B.D., i.p. (right diagonal) ; treatment with 600 μg/kg HP 228 administered B.D., i.p. (left diagonal); treatment with 600 μg/kg HP 228 administered 4 times a day, i.p. (dark stipple) ; treatment with 30 μg/animal HP 228 administered 4 times a day, i.p. (light stipple).

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method of using a cytokine regulatory agent (CRA) to reduce the severity of asthma in an individual that has or is susceptible to this patho-immunogenic disease. CRAs are known in the art and described, for example, in U.S. Patent No. 5,420,109, issued May 30, 1995, and U.S. Patent No. 5,726,156, issued March 10, 1998, both of which are incorporated herein by reference (CRAs previously were known as "cytokine restraining agents") .

As disclosed herein, administration of a CRA can reduce the severity of asthma in an individual . As used herein, the term "reduce the severity, " when used in reference to the effect of a CRA on asthma, means that the onset and magnitude of signs or symptoms of the asthmatic response are reduced relative to an untreated individual. Such signs or symptoms include dyspnea, difficulty in or labored breathing, or shortness of breath, which can be accompanied by coughing, wheezing or anxiety.

Asthma is characterized by variable air flow obstruction and increased responsiveness of the airways to a variety of stimuli (see Rubin and Farber,

"Pathology" 2nd ed. , J.B. Lippincott Co., Philadelphia PA (1994); Kuby "Immunology" 3rd ed. , W.H. Freeman, New York NY (1997) ) . Asthma is clinically manifested as recurrent symptoms of wheezing, coughing and dyspnea. The bronchial hyper-responsiveness, or increased airway responsiveness, in asthma is believed to be an inflammatory reaction to diverse stimuli. Following exposure to a stimulus such as an allergen, drug, cold temperature or exercise, inflammatory mediators are released by activated macrophages, mast cells, eosinophils and basophils. These inflammatory mediators induce bronchoconstriction and increase vascular permeability and mucus secretion.

Extrinsic, or allergic, asthma is initiated in a sensitized person by inhalation of an allergen that interacts with IgE antibody bound to the surface of mast cells in the bronchial mucosa. The mast cells degranulate, releasing mediators of type I (immediate) hypersensitivity, including histamine, bradykinin, leukotrienes, prostaglandins, thromboxane A₂ and platelet activating factor (PAF) . Release of these molecules causes smooth muscle contraction, mucus secretion and increased vascular permeability and edema, each of which can cause airway obstruction. This early response phase occurs within minutes of allergen exposure.

During the late response phase of the asthmatic response, neutrophils, eosinophils and platelets are attracted to the bronchial wall by chemotactic factors, including leukotriene B₄ and chemotactic factors. Eosinophils release leukotriene B₄ and PAF, which stimulate bronchoconstriction and edema. The release of eosinophil granules further impairs mucociliary function and damages epithelial cells. The release of leukotriene B₄ and PAF by eosinophils also causes infiltration of more eosinophils, which prolongs and amplifies the asthmatic attack.

As exemplified herein, the effectiveness of administering a CRA such as that designated HP 228, having the sequence Ac-Nle-Gln-His- (D) Phe-Arg- (D) Trp-

Gly-NH₂, in the treatment of signs or symptoms of asthma was demonstrated using a sheep model of asthma (Abraham et al., . Pharmacol . Exp . Ther . 247:1004-1011 (1988), which is incorporated herein by reference) . Sheep are naturally sensitized to the Ascaris suum antigen and, when challenged with this allergen, have an increase in lung resistance, which is indicative of more difficulty breathing. Treatment with HP 228 resulted in a significant reduction of early and late phase specific lung resistance and airway responsiveness in sheep challenged with the allergen Ascaris suum (see Example

II) . In addition, treatment with HP 228 also caused reduction of albumin, which is a marker of inflammation, in bronchoalveolar lavage (BAL) fluids from allergen- challenged sheep (see Example III) . A mouse model of asthma also was used to demonstrate the effectiveness of HP 228 in treating signs or symptoms of asthma (Hamelmann et al . , Am. J. Respir. Crit. Care Med. 155:819-825 (1997), which is incorporated herein by reference) . Mice were sensitized by administration of ovalbumin, which causes mice to become sensitized to challenge with methacholine (Takeda et al . , J . Exp . Med . 186:449-454 (1997), which is incorporated herein by reference) . Treatment with HP 228 decreased airway hyper-responsiveness in ovalbumin-sensitized mice (see Example IV) . Treatment of ovalbumin-sensitized, challenged mouse with HP 228 also decreased infiltration of the lungs by neutrophils and eosinophils (see Example V) . Since eosinophils and neutrophils are responsible for releasing toxic enzymes, oxygen radicals and cytokines that lead to tissue damage and prolong the asthmatic attack, the decrease in eosinophil and neutrophil infiltration of lung, combined with the observed decrease in lung resistance in asthma models treated with HP 228, indicates that CRAs effectively reduce the severity of asthma in an individual .

The present invention is exemplified using HP 228. In general, however, a CRA useful for reducing the severity of asthma in an individual has the structure:

Xi-X₂- (D) Phe-Arg- (D)Trp-X₃, wherein

wherein Y¹ and Y² are independently a hydrogen atom, or are taken together to form a carbonyl or thiocarbonyl ; R is H, COCH₃, C₂H₅, CH₂Ph, COPh, COO-1-butyl, COOCH₂Ph,

CH₂CO- (polyethylene glycol) or A; R₂ is H, COCH₃, C₂H₅ or CH₂Ph; R₃ is a linear alkyl group having 1 to 6 carbon atoms or a branched or cyclic alkyl group having 3 to 6 carbon atoms; R₄ is (CH₂) _m-CONH₂, (CH₂) ,,,-CONHR_! or (CH₂)_m-CONHA; R₅ is OH, 0R₃, NH₂, SH, NHCH₃, NHCH₂Ph or A; and R₆ is H or R₃; and wherein "Ph" is C₆H₅; "m" is 1, 2 or 3; "n" is 0, 1, 2 or 3; and "A" is a carbohydrate having the general formula:

In addition, a CRA useful in the invention can have the structure : X₁ - X₂ -His - (D) Phe - Arg - (D) Trp - X₃ , wherein

wherein Y¹ and Y² are independently a hydrogen atom, or are taken together to form a carbonyl or thiocarbonyl ; R is H, C0CH₃, C₂H₅, CH₂Ph, COPh, COO-t-butyl, COOCH₂Ph, CH₂CO- (polyethylene glycol) or A; R₂ is H or COCH₃; R₃ is a linear alkyl group having 1 to 6 carbon atoms or a branched or cyclic alkyl group having 3 to 6 carbon atoms; R₄ is (CH₂) _m-CONH₂, (CH₂) -,-CO HRi or (CH₂) _m-CONHA; R₅ is OH, OR₃, NH₂, SH, NHCH₃, NHCH₂Ph or A; and R₆ is H or R₃; and wherein "Ph" is C₆H₅, "m" is 1, 2 or 3 , "n" is 0, 1, 2 or 3 , and "A" is a carbohydrate having the general formula:

(see U.S. Patent No. 5,420,109; supra, 1995)

A CRA useful in the invention also can have the structure :

X₄ X₅ - (D)Phe - Arg - (D) Trp - X₃, where

X₅ is His, H or COCH₃; and

wherein Y¹ and Y² are independently a hydrogen atom, or are taken together to form a carbonyl or thiocarbonyl; R_x is H, C0CH₃, C₂H₅, CH₂Ph, COPh, COO-t-butyl, COOCH₂Ph, CH₂CO- (polyethylene glycol) or A; R₂ is H or COCH₃; R₃ is a linear alkyl group having 1 to 6 carbon atoms or a branched or cyclic alkyl group having 3 to 6 carbon atoms; R₄ is (CH₂) _m-CONH₂, (CH₂) _m-CONHRi or (CH₂) _m-CONHA; R₅ is OH, OR₃, NH₂, SH, NHCH₃, NHCH₂Ph or A; and R₆ is H or R₃; and wherein "Ph" is C₆H₅, "m" is 1, 2 or 3, "n" is 0, 1, 2 or 3 , and "A" is a carbohydrate having the general formula

(see U.S. Patent No. 5,420,109, supra, 1995, which also discloses methods for making a CRA) .

Additionally, a CRA useful in the invention can have the structure :

X_x - X₂ -His - (D)Phe - Arg - (D)Trp - X₃, wherein

wherein Y¹ and Y² are independently a hydrogen atom, or are taken together to form a carbonyl or thiocarbonyl ; R₁ is H, COCH₃, C₂H₅, CH₂Ph, COPh, COO-t-butyl, COOCH₂Ph, CH₂CO- (polyethylene glycol) or A; R₂ is H or COCH₃; R₃ is a linear or branched alkyl group having 1 to 6 carbon atoms; R₄ is (CH₂) _ra-CONH₂, (CH₂) ,,,-CONHR_! or (CH₂) _m-CONHA; R₅ is OH, OR₃, NH₂, SH, NHCH₃ , NHCH₂Ph or A; and R₆ is H or R₃; and wherein "Ph" is C₆ 6H"5 ι ^■m¹ is 1, 2 or 3 , ^■n' is 0, 1, 2 or 3 , and "A" is a carbohydrate having the general formula :

For example, the invention provides peptides such as Nle-Gln-His- (D) Phe-Arg- (D) Trp-Gly-NH₂; Ac-Nle-Gln-His- (D) Phe-Arg- (D)Trp-Gly-NH₂ and

Ac- (cyclohexyl)Gly-Gln-His- (D) Phe-Arg- (D) Trp-Gly-NH₂, which can restrain cytokine activity.

Furthermore, a CRA useful in the invention can have the structure :

X₄ - His - (D)Phe - Arg - (D)Trp - X₃, wherein

wherein Y¹ and Y² are independently a hydrogen atom, or are taken together to form a carbonyl or thiocarbonyl; R_x is H, COCH₃, C₂H₅, CH₂Ph, COPh, COO-t-butyl, COOCH₂Ph, CH₂CO- (polyethylene glycol) or A; R₂ is H or COCH₃; R₃ is a linear or branched alkyl group having 1 to 6 carbon atoms or a branched or cyclic alkyl group having 3 to 6 carbon atoms; R₄ is (CH₂) _m-CONH₂, (CH₂) _m-CONHR₁ or (CH₂) _m-CONHA; R₅ is OH, OR₃, NH₂, SH, NHCH₃, NHCH₂Ph or A; and R₆ is H or R₃; and wherein "Ph" is C₆H₅, "m" is 1, 2 or 3, "n" is 0, 1, 2 or 3, and "A" is a carbohydrate having the general formula

For example, the invention provides His- (D) Phe-Arg- (D) Trp-Gly-NH₂; Ac-His- (D) Phe-Arg- (D)Trp-NH₂; His- (D) Phe-Arg- (D) Trp-OH ; and cyclo (His- (D) Phe-Arg- (D) Trp) , which can restrain cytokine activity.

In the above CRAs, X_x also can be selected from the group consisting of norleucine, norvaline, leucine or isoleucine. Additionally, in a CRA having a structure containing X_x and R₅, R₅ can be covalently bound to X_1# forming a cyclic peptide.

In general, a CRA is a peptide or a peptide- like structure such as a peptidomimetic or a peptoid (see Ecker and Crooke, Biotechnology 13:351-360 (1995), and Blondelle et al . , Trends Anal. Chem. 14:83-92 (1995), and the references cited therein, each of which is incorporated herein by reference) . Peptide cytokine regulatory agents as described herein are characterized, in part, by a core structure (D) Phe-Arg- (D) Trp . Amino acids are indicated herein by their commonly known three letter code, where " (D) " designates an amino acid having the "D" configuration, as compared to the naturally occurring (L) -amino acids. Where no specific configuration is indicated, one skilled in the art would understand the amino acid to be an (L) -amino acid. In the peptide and CRA structures exemplified herein, "Nle" is the three letter code for norleucine and "Ph" indicates a "phenyl" group (C₆H₅) . CRA peptides are written in the conventional manner, such that the amino-terminus (N-terminus) is shown to the left and the carboxyl-terminus (C-terminus) is shown to the right.

One skilled in the art would know that the choice of amino acids or amino acid analogs incorporated into the peptide will depend, in part, on the specific physical, chemical or biological characteristics required of the CRA. Such characteristics are determined, for example, by the route by which the CRA is administered. Selective modification of a reactive group in a peptide also can impart desirable characteristics to a CRA. For example, the N-terminus can be modified by acetylation or the C-terminus can be modified by amidation. Methods for modifying the N-terminus or

C-terminus of a peptide are well known in the art (see, for example, in U.S. Patent No. 5,420,109, supra , 1995).

The choice of modifications made to the reactive groups present on the peptide is determined by a desirable characteristic required in the CRA. A CRA having the structure Ac-Nle-Gln-His- (D) Phe-Arg- (D) Trp-Gly-NH₂ (HP 228) or the structure Ac-His- (D) Phe-Arg- (D) Trp-Gly-NH₂ is an example of a CRA that is modified both by acetylation at the N-terminus and by amidation at the C-terminus.

A cyclic peptide also can be an effective CRA. A cyclic peptide can be obtained by inducing the formation of a covalent bond between, for example, the amino group at the N-terminus of the peptide and the carboxyl group at the C-terminus. For example, the peptide, cyclo (His- (D) Phe-Arg- (D) Trp) , can be produced by inducing the formation of a covalent bond between His and (D)Trp. Alternatively, a cyclic peptide can be obtained by forming a covalent bond between a terminal reactive group and a reactive amino acid side chain or between two reactive amino acid side chains such as the sulfhydryl reactive groups present in cysteine residues. One skilled in the art would know that the choice of a particular cyclic peptide is determined by the reactive groups present on the peptide as well as the desired characteristic of the peptide. Cyclization of a CRA peptide can provide the CRA with increased stability in vivo .

In addition to the examples provided above, other representative cytokine regulatory agents include: 1 ) Ac -Nle - Gin - His _ (D) Phe - Arg _ (D) Trp -Gly-OH ;

2 ) Ac -Nle - Gin - His - (D) Phe - Arg - (D) Trp -Gly-OC₂H₅ ;

3 ) Ac-Nle - Gin - His - (D) Phe - Arg - (D) Trp -Gly-NH-NH₂ ;

4 ) Ac -Nle - Asn - His - (D) Phe - Arg - (D) Trp -Gly-NH₂ ; 5 ) Ac -Nle - Asn - His - (D) Phe - Arg - (D) Trp -Gly-OH ;

6 ) Ac -Nle - Gin - His - (D) Phe - Arg - (D) Trp - Gly-

NHCH₂CH₂Ph ;

7 ) Ac -Nle - Gin - His - (D) Phe - Arg - (D) Trp - Gly-

NHCH₂Ph ; 8 )

9) Gin - His - (D) Phe - Arg - (D) Trp - Gly-NH₂;

10) Ac-Gin - His - (D) Phe - Arg - (D)Trp - Gly-NH₂; 11) Ac-Nle - Gin - His - (D) Phe - Arg - (D)Trp-NH₂;

12) Ac-His - (D) Phe - Arg - (D)Trp(CH₂) - (NAc) Gly-NH₂ ; and

13) His - (D)Phe - Arg- (D)Trp(CH₂) - (NAc) Gly.

The invention also provides methods of reducing the severity of asthma in an individual by administering to the individual an effective dose of a CRA selected from the group consisting of:

(cyclohexyl) Gly-Gln-His- (D) Phe-Arg- (D) Trp-Gly; Ac- (cyclohexyl) Gly-Gln-His- (D) Phe-Arg- (D) Trp-Gly-NH₂; and cyclo (His- (D) Phe-Arg- (D) Trp) . The invention additionally provides methods of reducing the severity of asthma in an individual by administering to the individual an effective dose of the CRA (D) Phe-Arg- (D) Trp .

The invention further provides methods of reducing the severity of asthma in an individual by administering to the individual an effective dose of a CRA selected from the group consisting of: Nle-Gln-His- (D) Phe-Arg- (D) Trp-Gly-NH₂; His- (D) Phe-Arg- (D) Trp-Gly; His- (D) Phe-Arg- (D) Trp-Gly-NH₂; Ac-His- (D) Phe-Arg- (D)Trp-NH₂; His - (D) Phe-Arg- (D) Trp-OH ; His - (D) Phe-Arg- (D) Trp ; His - (D) Phe -Arg- (D) Trp-NH₂ ; Ac -His - (D) Phe-Arg- (D) Trp-OH ; and Ac -His - (D) Phe -Arg- (D) Trp-Gly-NH₂ .

Cytokine regulatory agents are synthesized using a modification of the solid phase peptide synthesis method of Merrifield (J. Am. Chem. Soc . 85:2149 (1964), which is incorporated herein by reference; see Example I) or can be synthesized using standard solution methods well known in the art (see, for example, Bodanszky, M. , Principles of Peptide Synthesis 2nd revised ed. (Springer-Verlag, 1988 and 1993) , which is incorporated herein by reference) . Peptides prepared by the method of Merrifield can be synthesized using an automated peptide synthesizer such as the Applied Biosystems 431A-01 Peptide Synthesizer (Mountain View, CA) or using the manual peptide synthesis technique described by Houghten, (Proc. Natl. Acad. ScΛ . . USA 82:5131 (1985), which is incorporated herein by reference) .

Peptides were synthesized using amino acids or amino acid analogs, the active groups of which were protected as required using, for example, a t-butyldicarbonate (t-BOC) group or a fluorenylmethoxy carbonyl (FMOC) group. Amino acids and amino acid analogs can be purchased commercially (Sigma Chemical Co.; St. Louis MO; Advanced ChemTec; Louisville KY) or synthesized using methods known in the art. Peptides synthesized using the solid phase method can be attached to resins including 4-methylbenzhydrylamine (MBHA) , 4- (oxymethyl) -phenyl acetamido methyl and 4- (hydroxymethyl) phenoxymethyl-copoly (styrene-1% divinylbenzene) (Wang resin) , all of which are commercially available, or to p-nitro benzophenone oxime polymer (oxime resin) , which can be synthesized as described by De Grado and Kaiser, J . Org . Chem . 47:3258 (1982), which is incorporated herein by reference (see Example I) .

One skilled in the art would know that the choice of amino acids or amino acid analogs incorporated into the peptide will depend, in part, on the specific physical, chemical or biological characteristics required of the cytokine regulatory peptide. Such characteristics are determined, in part, by the route by which the cytokine regulatory agent will be administered or the location in a subject to which the cytokine regulatory agent will be directed.

With regard to selective modification of the reactive groups in a peptide, the peptides can be manipulated while still attached to the resin to obtain N-terminal modified compounds such as an acetylated peptide or can be removed from the resin using hydrogen fluoride or an equivalent cleaving reagent and then modified. Compounds synthesized containing the C-terminal carboxyl group (Wang resin) can be modified after cleavage from the resin or, in some cases, prior to solution phase synthesis. Methods for modifying the N-terminus or C-terminus of a peptide are well known in the art and include, for example, methods for acetylation of the N-terminus or methods for amidation of the

C-terminus. Similarly, methods for modifying side chains of the amino acids or amino acid analogs are well known to those skilled in the art of peptide synthesis. The choice of modifications made to the reactive groups present on the peptide will be determined by the characteristics that the skilled artisan requires in the peptide.

A newly synthesized peptide can be purified using a method such as reverse phase high performance liquid chromatography (RP-HPLC; see Example I) or other methods of separation based on the size or charge of the peptide. Furthermore, the purified peptide can be characterized using these and other well known methods such as amino acid analysis and mass spectrometry (see Example I) .

It should be recognized that, while a CRA is referred to as a cytokine regulatory agent, no mechanism of action is proposed herein for the effectiveness of a CRA in reducing the severity of asthma. Thus, a CRA may reduce the severity of asthma by regulating cytokine activity or by some other mechanism that can be unrelated to cytokines.

A CRA generally is administered to an individual as a pharmaceutical composition comprising a cytokine regulatory agent and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are well known in the art and include aqueous solutions such as physiologically buffered saline or other solvents or vehicles such as glycols, glycerol, oils such as olive oil or injectable organic esters.

A pharmaceutically acceptable carrier can contain physiologically acceptable compounds that act, for example, to stabilize the cytokine regulatory agent or increase the absorption of the agent. Such physiologically acceptable compounds include, for example, carbohydrates, such as glucose, sucrose or dextrans, antioxidants, such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers or excipients . One skilled in the art would know that the choice of a pharmaceutically acceptable carrier, including a physiologically acceptable compound, depends, for example, on the route of administration of the cytokine regulatory agent and on the particular physico-chemical characteristics of the specific cytokine regulatory agent .

The present invention provides a method of reducing the severity of asthma in an individual by administering a CRA to the individual. One skilled in the art would know that a pharmaceutical composition comprising a cytokine regulatory agent can be administered to a subject having pathologically elevated cytokine activity by various routes including, for example, orally or parenterally, such as intravenously (i.v.), intramuscularly, subcutaneously, intraorbitally, intracapsularly, intraperitoneally (i.p.), intracisternally, intra-articularly or by passive or facilitated absorption through the skin using, for example, a skin patch or transdermal iontophoresis, respectively. Thus, a CRA can be administered by injection, intubation, orally or topically, the latter of which can be passive, for example, by direct application of an ointment or powder, or active, for example, using a nasal spray or inhalant. Administration of a CRA by inhalation is a particularly useful means of reducing the severity of asthma in an individual . Since cytokine levels in an individual with asthma are elevated locally in the bronchial tubes and lungs, one skilled in the art would recognize that a CRA can be suspended or dissolved in an appropriate pharmaceutically acceptable carrier and administered, for example, directly into the lungs using a nasal spray or inhalant. Alternatively, a CRA can be administered intravenously for systemic administration.

A CRA also can be administered as a topical spray, in which case one component of the composition is an appropriate propellant. The pharmaceutical composition also can be incorporated, if desired, into liposomes, microspheres or other polymer matrices (Gregoriadis, Liposome Technology. Vols. I to III, 2nd ed. (CRC Press, Boca Raton FL (1993) , which is incorporated herein by reference) . Liposomes, for example, which consist of phospholipids or other lipids, are nontoxic, physiologically acceptable and metabolizable carriers that are relatively simple to make and administer.

In order to reduce the severity of asthma in an individual, a CRA must be administered in an effective dose, which is about 0.01 to 100 mg/kg body weight per administration. The total treatment dose can be administered to a subject as a single dose, either as a bolus or by infusion over a relatively short period of time, or can be administered using a fractionated treatment protocol, in which the multiple doses are administered over a more prolonged period of time. One skilled in the art would know that the amount of a cytokine regulatory agent required to obtain an effective dose in a subject depends on many factors including the age and general health of the subject as well as the route of administration and the number of treatments to be administered. In view of these factors, the skilled artisan would adjust the particular dose so as to obtain an effective dose for regulating cytokine activity.

In an individual suffering from a more severe form of asthma, administration of a CRA can be particularly useful when administered in combination, for example, with a conventional agent such as a bronchodilator . The skilled artisan would administer a CRA, alone or in combination with a second agent, based on the clinical signs and symptoms exhibited by the individual and would monitor the effectiveness of such treatment using routine methods such as pulmonary function determination, radiologic, immunologic or, where indicated, histopathologic methods. The following examples are intended to illustrate but not limit the invention.

EXAMPLE I

Synthesis of CRAs

This example describes methods for the solid phase synthesis of peptide cytokine regulatory agents .

Cytokine regulatory agents having the amino acid sequences Ac-Nle-Gln-His- (D) Phe-Arg- (D) Trp-Gly-NH₂ (HP 228) and Ac-His- (D) Phe-Arg- (D) Trp-Gly-NH₂ were synthesized using a modification of the solid phase peptide synthesis method of Merrifield { supra, 1964) .

Essentially, MBHA resin containing a t-BOC glycine derivative (Advanced Chemtech; Louisville KY) was added to a reaction vessel suitable for solid phase peptide synthesis (see Houghten, supra, 1985) . The resin was washed three times with methylene chloride and the t-BOC protecting group was removed using trifluoroacetic acid (TFA) containing 1-2% anisole in methylene chloride. The resin then was washed with methylene chloride and treated with diisopropylethylamine.

The peptide was extended by the addition of 3.2 equivalents of N-formyl-BOC-protected D-tryptophan in dimethylformamide and 3.0 equivalents of dicyclohexylcarbodiimide . The reaction was monitored using ninhydrin and was allowed to proceed for 25 min, after which the resin was washed using methylene chloride. The procedure was repeated using di-tolulyl-BOC arginine, then with each of the desired protected amino acids until the appropriate pentapeptide or heptapeptide was synthesized.

The amino terminus of each peptide was acetylated by treating the sample with acetic anhydride, diisopropylethylamine and methylene chloride for 2 hr . Following synthesis of the peptides, the N-formyl protecting group on the tryptophan residue was removed using 20% piperidine in DMF and the resin was washed with methylene chloride. The peptide was cleaved from the resin using anhydrous hydrogen fluoride (HF) containing 10% anisole, the reaction mixture was concentrated and the residue was digested with aqueous acetic acid. The acetic acid fraction, which contained the digested sample, was removed and the residue was washed with water. The wash was added to the acetic acid fraction and the combined sample was concentrated.

Each of the resulting crude peptides was purified by RP-HPLC (Vydac, C-18 column, using a gradient of 1 to 60% solution B over 30 min (solution A is

0.1% TFA/water and solution B is 0.1% TFA/acetonitrile) . The acetylated heptapeptide was determined to be 98% pure by RP-HPLC (Vydac C-18 column, using isocratic 24% solution B; absorption determined at 215 nm) . The mass of each purified peptide was determined by plasma absorption mass spectrometry using a Biolon 20 Mass Analyzer time of flight detector. The mass was measured to be 985.2 daltons, which was the same as the expected molecular mass. The acetylated pentapeptide had a measured mass of 743 daltons and was greater than 97% pure.

EXAMPLE II

Reduction of Early and Late Phase Specific Lung Resistance and Airway Responsiveness in Allergen-Challenged Sheep

This example demonstrates that administration of HP 228 reduces early and late phase specific lung resistance and airway hyper-responsiveness in sheep challenged with allergen. Determination of specific lung resistance and airway hyper-responsiveness was performed essentially as described previously (Abraham et al . , supra 1988). All animals used in these experiments demonstrated both early and late airway responses to inhalation challenge with Ascaris suum antigen and were treated with procedures approved to assure humane care and use of experimental animals .

To measure airway mechanics, unsedated sheep were restrained in a cart in the prone position with their heads immobilized. After topical anesthesia of the nasal passages with a 2% lidocaine solution, a balloon catheter was advanced through one nostril into the lower esophagus. The animals were intubated with a cuffed endotracheal tube through the other nostril using a flexible fiberoptic bronchoscope as a guide. The cuff of the endotracheal tube was inflated only for the measurement of airway mechanics and during aerosol challenges to prevent discomfort . This procedure has no effect on airway mechanics. Pleural pressure was estimated with the esophageal balloon catheter, filled with one ml of air, which was positioned 5-10 cm from the gastroesophageal junction. In this position, the end expiratory pleural pressure ranges between -2 and -5 cm H₂0. Once the balloon was placed, it was secured so that it remained in position for the duration of the experiment .

Lateral pressure in the trachea was measured with a sidehole catheter (inner dimension 2.5 mm) advanced through and positioned distal to the tip of the endotracheal tube. Transpulmonary pressure, which is defined as the difference between tracheal and pleural pressure, was measured with a differential pressure transducer catheter system. For the measurement of pulmonary resistance (R_L) , the proximal end of the endotracheal tube was connected to a pneumotachograph (Fleisch, Dyna Sciences; Blue Bell PA) . The signals of flow and transpulmonary pressure were recorded on an oscilloscope recorder, which was linked to a DOS 386 computer for on-line calculation of R_L from transpulmonary pressure, respiratory volume (obtained by digital integration) and flow. Analysis of 5-10 breaths was used for the determination of R_L. Measurements of R_L were followed by measurements of thoracic gas volume (V_tg) . V_tg was measured in a constant volume body plethysmograph to obtain specific lung resistance (SR_L = R_L x V_tg) .

Aerosols of Ascaris suum extract (diluted 20:1 with phosphate buffered saline (PBS); 82,000 protein nitrogen units (PNU)/ml) were generated using a disposable medical nebulizer (RAINDROP, Puritan Bennett, Lenexa KS) , which produces an aerosol with a mass median aerodynamic diameter of 3.2 μm (geometric standard deviation, 1.9) as determined by a seven-stage Andersen cascade impactor. The output from the nebulizer was directed into a plastic t-piece, one end of which is connected to the inspiratory port of a Harvard respirator. To better control aerosol delivery, a dosimeter consisting of a solenoid valve and a source of compressed air (20 psi) was activated at the beginning of the inspiratory cycle of the Harvard respirator system for 1 sec. The aerosol was delivered at a tidal volume of 500 ml and a rate of 20 breaths per minute for 20 minutes. Each sheep was challenged with an equivalent dose of antigen (400 breaths) in the control and drug trial.

Carbachol aerosols were also generated with the nebulizer system described above. For the carbachol dose response curves, measurement of SR_L was repeated immediately after inhalation of buffer and after each administration of 10 breaths of increasing concentrations of carbachol solution (0.25%, 0.5%, 1.0%, 2.0% and 4.0% wt/vol) . To assess airway responsiveness, the cumulative carbachol dose in breath units (BU) that increased SR_L 400% over the post-buffer value (PC₄₀₀) was calculated from the dose response curve . One breath unit was defined as one breath of a 1% wt/vol carbachol solution.

For bronchoalveolar lavage (BAL) , the distal tip of a specially designed 80 cm fiberoptic bronchoscope was wedged into a randomly selected subsegmental bronchus. Lung lavage was performed by slow infusion and gentle aspiration of 3 x 30 ml aliquots of PBS, pH 7.4, at 39°C using 30 ml syringes attached to the working channel of the bronchoscope. For each lavage, a separate airway was used for each aliquot. The volume of the effluent collected from the BAL was measured to determine the return.

A preliminary study was performed to determine an optimized dose of HP 228 to be used. For these studies, baseline dose response curves to aerosol carbachol was obtained 1 to 3 days prior to antigen challenge. On the antigen challenge day, baseline values of SR_L were obtained, then the sheep were administered 300 μg/kg HP 228, i.v., 0.5 hr before antigen challenge. Post-drug measurements of SR_L were obtained, then the sheep were challenged with Ascaris suum antigen.

Measurements of SR_L were obtained immediately after challenge, hourly from 1 to 6 hrs after challenge, and on the half-hour from 6.5 to 8 hrs after challenge. Second and third doses of HP 228 were administered at 4 hr and 8 hr after antigen challenge. A fourth dose of HP 228 was administered 0.5 hr before the 24 hr post-challenge dose response curve. Measurements of SR_L were obtained 24 hr after challenge followed by the 24 hr post-challenge dose response curve. For these experiments, three sheep were used per dose and the results of the drug screen were compared to each sheep's historical control. Sheep were tested at doses of 300 μg/kg and 100 μg/kg.

Figures 1A and IB show the effect of 300 μg/kg of HP 228 on SR_L and airway hyper-responsiveness . The 300 μg/kg dose of HP 228 was administered i.v. at

-0.5 hr, 4 hr, 8 hr and 24 hr. Figure 1A, which shows SR_L, demonstrates that 300 μg/kg of HP 228 reduced the immediate bronchoconstriction and blocked the late response (4 to 8 hr period) . Figure IB, which shows the effect of HP 228 on post antigen- induced airway responsiveness, demonstrates that 300 μg/kg HP 228 blocked the 24 hr airway hyper-responsiveness, shown as a decrease in the PC₄₀₀. Control data are historical control. The historical control is a measurement of SR_L and airway hyper-responsiveness taken prior to the experiment in the same animal without treating with HP 228.

The effect of 100 μg/kg of HP 228 on SR_L and airway hyper-responsiveness is shown in Figures 2A and 2B. The 100 μg/kg dose of HP 228 was administered i.v. at -0.5 hr, 4 hr, 8 hr and 24 hr. The results in Figure 2A demonstrate that 100 μg/kg of HP 228 had no effect on immediate or late SR_L, and the results in Figure 2B demonstrate that this dose of HP 228 had no effect on 24 hr airway hyper-responsiveness . Control data are historical. Based on these results, 300 μg/kg of HP 228 was used for subsequent studies .

In subsequent studies, a randomized cross-over design in six sheep was used. Baseline dose response curves to aerosol carbachol were obtained 1 to 3 days prior to antigen challenge followed immediately by performance of BAL. Baseline values of SR_L were measured, then the sheep were treated with placebo or HP 228 (300 μg/kg, i.v.) 0.5 hr before antigen challenge on two separate occasions. Each treatment was separated by at least three weeks. Post-drug measurements of SR_L were obtained, then the sheep were challenged with Ascaris suum antigen. Measurements of SR_L were obtained immediately after challenge, hourly from 1 to 6 hr after challenge and on the half hour from 6.5 to 8 hr after challenge. Second and third doses of HP 228 were administered at 4 hr and 8 hr after antigen challenge. A fourth dose of HP 228 was administered 0.5 hr before the 24 hr post-challenge dose response curve. Following the last measurement (8 hr) , a second BAL was performed. Measurements of SR_L were obtained 24 hr after challenge followed by the 24 hr post challenge dose response curve and a third BAL.

The results of treatment with 300 μg/kg of

HP 228 are shown in Figure 3. HP 228 was administered i.v. at -0.5 hr, 4 hr, 8 hr and 24 hr. The control group was treated with placebo at the same time that the other group was treated with HP 228. In the placebo trial, antigen challenge resulted in characteristic early and late increases in SR_L. SR_L increased immediately after antigen challenge, then slowly returned toward baseline by 5 hr post challenge. SR_L then began to increase again during the late response period. Treatment with HP 228 reduced the peak early increase in SR_L (P<0.10) and significantly inhibited all other post challenge measurements of SR_L. Repeated measures of ANOVA analysis showed a highly significant effect of the drug on the changes in lung mechanics over time (P<0.0001) .

The effect of HP 228 on post -antigen induced airway hyper-responsiveness is shown in Figure 4. HP 228 was administered i.v. at -0.5 hr, 4 hr, 8 hr and 24 hr. In the control trial, all six sheep developed increased airway responsiveness to inhaled carbachol 24 hr after antigen challenge (P<0.01) as indicated by the fall in the PC_4oo- Treatment with HP 228 completely blocked this hyper-responsiveness . ANOVA analysis showed a highly significant effect of the drug on the changes in lung mechanics over time (P=0.03).

These results demonstrate that 300 μg/kg of

HP 228 reduced the immediate bronchoconstriction and blocked the late response in antigen-induced changes in specific lung resistance and blocked airway hyperresponsiveness .

EXAMPLE III

Reduction of Albumin Response in Bronchoalveolar Lavage

Fluids in Sheep

This example demonstrates that HP 228 reduces the albumin response measured in BAL fluids in sheep.

BAL was performed as described in Example II.

The BAL fluid was analyzed for cell content and for two markers of inflammation, albumin and kallikrein. For cellular analysis, the lavage return was centrifuged at 420 xg for 15 min, and the supernatant was decanted and saved frozen at -80°C for subsequent analysis. The cells were resuspended in buffered saline, and an aliquot was transferred to a hemocytometer chamber to estimate total cells. Total viable cells were estimated by trypan blue exclusion. A second aliquot of the cell suspension was spun in a cytometer (500 rpm for 10 min) and stained by "Diff-Quick" (VWR Scientific Products; West Chester PA) to identify cell populations. Five hundred cells per slide were identified to establish the differential cell count (100 X, oil objective) . Cell categories included epithelial cells, macrophages, lymphocytes, neutrophils, basophils, eosinophils and monocytes . Unidentifiable cells were grouped into a category termed "others." BAL fluids were analyzed for tissue kallikrein activity and albumin. Tissue kallikrein activity in unconcentrated BAL supernatants was measured by cleavage of (DL) Val-Leu-Arg pNA (p-Nitroanilide) . The Val in the peptide is a racemic mixture, and pNA is attached to Arg. 0.05 M Tris buffer, pH 8, was used to make a 0.2 mM stock solution of the tripeptide. For the assay, 200 μl of the tripeptide solution was placed in the well of a microtiter plate, followed by 100 μl aliquots of the recovered BAL supernatants, which were previously mixed with an inhibitory cocktail containing 25 μg/ml soybean trypsin inhibitor. Controls included wells with the tripeptide, alone, or the tripeptide plus 100 ng of human urinary kallikrein. Incubations were performed at room temperature for 24 hr and the kallikrein activity was determined by measuring the change in optical density (O.D.) between 0 and 24 hr. Albumin was measured with an ELISA method using a polyclonal rabbit antibody to bovine serum albumin (BSA) and a mouse anti-BSA antibody (Mariassy et al . , J. Clin. Immunol. 93:585-593 (1994), which is incorporated herein by reference) .

No significant increase in the total number of cells recovered in BAL was observed for either the control or HP 228 treated group. Similarly, no significant difference in the cell differentials was observed for either group.

The effect of HP 228 on tissue kallikrein activity in BAL is shown in Figure 5. A 300 μg/kg dose of HP 228 was administered, i.v., at -0.5 hr, 4 hr, 8 hr and 24 hr. Analysis of BAL fluid for tissue kallikrein showed no significant differences either within or between trials.

The effect of HP 228 on albumin levels in BAL is shown in Figure 6. A 300 μg/kg dose of HP 228 was administered i.v. at -0.5 hr, 4 hr, 8 hr and 24 hr . As shown in Figure 6, there was a significant suppression of the albumin response in the HP 228 treated animals at both 8 and 24 hr post challenge. These results are consistent with an anti-inflammatory effect.

These results demonstrate that HP 228 reduced the albumin levels in BAL fluids, indicating that HP 228 inhibits the inflammatory response to "antigen" in sheep lung.

EXAMPLE IV

Decreased Airway Reactivity to Methacholine in Ovalbumin-sensitized. Challenged Mouse

This example demonstrates that HP 228 decreases airway reactivity in response to methacholine in ovalbumin-sensitized, challenged mouse.

Balb/C mice were sensitized to ovalbumin by administering 20 μg of ovalbumin i.p. on days 1 and 14. On days 26, 27 and 28, mice were challenged with aerosol administration of 1% ovalbumin. On days 30 and 31, assays were performed to determine serum antibody and BAL fluid cytokine levels, to analyze cellular infiltration in lungs, to determine airway hyper-responsiveness to inhaled methacholine and to perform immunohistochemistry of lung sections essentially as described previously (Takeda et al . , supra) .

Briefly, airway responsiveness was assessed as a change in airway function after challenge with aerosolized methacholine (MCh) via the airways. Anesthetized, tracheostomized mice were mechanically ventilated and lung function was assessed using methods similar to those described by Martin et al . (J. Appl . Physiol. 64:2318-2323 (1988), which is incorporated herein by reference) . A four-way connector was attached to the tracheostomy tube, with two ports connected to the inspiratory and expiratory sides of a ventilator (model 683; Harvard Apparatus; South Natick MA) . Ventilation was achieved at 160 breaths/min and a tidal volume of

0.15 ml with a positive end-expiratory pressure of 2-4 cm H₂0.

The Plexiglas chamber containing the mouse was continuous with a 1.0 liter glass bottle filled with copper gauze to stabilize the volume signal for thermal drift . Transpulmonary pressure was detected by a pressure transducer with one side connected to the fourth port of the four-way connector and the other side connected to a second port on the plethysmograph. Changes in lung volume were measured by detecting pressure changes in the plethysmographic chamber through a port in the connecting tube with a pressure transducer and then referenced to a second copper gauze- filled 1.0 liter glass bottle. Flow was measured by digital differentiations of the volume signal. Lung resistance (R_L) and dynamic compliance (Cdyn) were continuously computed (Labview; National Instruments; Austin, TX) by fitting flow, volume, and pressure to an equation of motion.

Aerosolized agents were administered for 10 sec with a tidal volume of 0.5 ml (DiCosmo et al . , J. Clin. Invest . 94:2028-2035 (1994), which is incorporated herein by reference) . From 20 sec up to 3 min after each aerosol challenge, the data of R_L and Cdyn were continuously collected. Maximum values of R_L and minimum values of Cdyn were taken to express changes in murine airway function.

For bronchoalveolar lavage and lung cell isolation, lungs were lavaged via the tracheal tube with Hanks' Balanced Salt Solution (HBSS) (1 x 1 ml at 37°C) immediately after assessment of airway hyper-responsiveness . The volume of collected bronchoalveolar lavage (BAL) fluid was measured in each sample and numbers of leukocytes were counted (Coulter Counter; Coulter Corporation; Hialeah, FL) . Cells in lung tissue were isolated and counted as previously described (Oshiba et al . , J. Clin. Invest. 97:1398-1408 (1996), which is incorporated herein by reference).

For histologic and immunochemistry studies, lungs were inflated, after perfusion via the right ventricle, through the trachea with 2 ml air and then fixed in 10% formalin by immersion. Blocks of the left lung tissue were cut from around the main bronchus and embedded in paraffin blocks. 5 μm tissue sections were affixed to microscope slides and deparaffinized. The slides were then stained with Astra Blue/Vital New Red and mast cells and eosinophils were examined under light microscopy (Duffy et al . , J. Histotechnol . 16:143-144 (1993) , which is incorporated herein by reference) .

Cells containing eosinophilic major basic protein (MBP) were identified by immunohistochemical staining as previously described using a rabbit anti-mouse MBP (Hamelmann et al . , supra) . Numbers of eosinophils in the submucosal tissue around central airways were analyzed using the IPLab2 software (Signal Analytics; Vienna VA) counting four different sections per animal (Hamelmann et al . , supra) .

As shown in Figure 7, mice sensitized to ovalbumin and then challenged with methacholine (MCh) had increased lung resistance with increasing amounts of methacholine as measured by enhanced pause ("Penh"), which is a measurement of the pause in normal breathing and is used as a measure of airway resistance. Mice (groups of 4 mice per dose group) were treated i.p. with either 300 μg/kg, 2 times a day (B.D.); 600 μg/kg, B.D.; 600 μg/kg, 4 times a day; or 30 μg/animal, 4 times a day. HP228 dosing started on day 28, just before aerosol challenge with ovalbumin solution. Treatment with various concentrations and various regimens of HP 228 caused decreased lung resistance compared to sensitized- challenged mice. The most effective dosage was 600 μg/kg administered 4 times a day.

These results demonstrate that treatment with

HP 228 causes decreased lung resistance in mice sensitized with ovalbumin and subsequently challenged with methacholine and confirm the general effectiveness of a CRA for reducing the signs and symptoms associated with asthma.

EXAMPLE V

Reduction in Neutrophils and Eosinophils in Bronchoalveolar Lavage Fluid from Ovalbumin-sensitized- challenged Mouse

This example demonstrates that treatment with

HP 228 causes a decrease in the number of neutrophils and eosinophils in BAL fluids of ovalbumin-sensitized, challenged mice.

Mice were sensitized to ovalbumin as described in Example IV. HP 228 was administered i.p. to mice with the following regimen: 300 μg/kg, B.D.; 600 μg/kg, B.D.; 600 μg/kg, 4 times a day; or 30 μg/animal, 4 times a day. BAL fluid was obtained from mice as described in Example IV. The number of macrophages, lymphocytes, neutrophils and eosinophils were determined in BAL fluid essentially as described previously (Hamelmann et al . , supra) .

Briefly, lung cells were isolated as previously described (Lavnikova et al . , Am. J. Respir. Cell Mol . Biol . 8:384- 392 (1993) , which is incorporated herein by reference) . Lungs were perfused with warmed (37°C) calcium- and magnesium-free HBSS containing 10% fetal calf serum (FCS), 0.6 mM ethylenediaminetetraacetic acid (EDTA) , 100 U/ml penicillin and 100 μg/ml streptomycin via the right ventricle at a rate of 4 ml/min for 4 min. Lungs were removed and cut into 300 μm pieces. Four ml of HBSS containing 175 U/ml collagenase (type IA; Sigma) , 10% FCS, 100 U/ml penicillin and 100 μg/ml streptomycin were added to the minced lungs and incubated for 60 min in an orbital shaker at 37°C. The digested lungs were sheared with a sterile 20-gauge needle and filtered through 45- and 15-μm filters. Filters were washed with HBSS/2% FCS (45 μm: 1 x 10 ml; 15 μm: 2 x 10 ml) . Cells were resuspended in HBSS and counted with a hemocytometer, and cytospin slides were prepared. Slides were stained with Leukostat (Fisher Diagnostics; Fair Lawn, NJ) , and cell differentiation percentages were determined by counting at least 300 cells using light microscopy.

The effect of HP 228 on cell number in BAL fluid of ovalbumin-sensitized, challenged mouse is shown in Figure 8. Compared to the ovalbumin-sensitized, challenged mouse, there was little difference in the number of macrophages (MAC) or lymphocytes (LYMPH) in mice treated with any regimen of HP 228. In contrast, there was a reduced number of neutrophils (NEUT) and eosinophils (EOS) with certain treatment regimens of HP 228. In particular, the number of neutrophils in BAL fluid was significantly reduced by treatment with HP 228 at 300 μg/kg, B.D., 600 μg/kg, B.D. or 600 μg/kg, 4 times a day. Eosinophils were significantly reduced relative to ovalbumin-sensitized, challenged mouse by treatment with 600 μg/kg, 4 times a day. These results demonstrate that treatment with HP 228 reduced the infiltration of neutrophils and eosinophils in mouse lung in ovalbumin-sensitized, challenged mouse. Since eosinophils and neutrophils are associated with the pathogenesis of asthma, these results demonstrate that HP 228 can be an effective treatment for reducing the severity of asthma.

Although the invention has been described with reference to the examples provided above, it should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the claims.

Claims

We claim :

1. A method of reducing the severity of asthma in an individual, comprising administering to the individual an effective dose of a cytokine regulatory agent (CRA) having the structure:

X₁-X₂- (D) Phe-Arg- (D) Trp-X₃, wherein

, H or COCH₃ or absent ;

wherein Y¹ and Y² are independently a hydrogen atom, or are taken together to form a carbonyl or thiocarbonyl ; R is H, COCH₃, C₂H₅, CH₂Ph, COPh, COO-t-butyl, COOCH₂Ph, CH₂C0- (polyethylene glycol) or A; R₂ is H, COCH₃, C₂H₅ or CH₂Ph; R₃ is a linear alkyl group having 1 to 6 carbon atoms or a branched or cyclic alkyl group having 3 to 6 carbon atoms; R₄ is (CH₂) _m-CONH₂, (CH₂)_m-CONHR₁ or (CH₂)_m-CONHA; R₅ is OH, 0R₃, NH₂ , SH, NHCH₃, NHCH₂Ph or A; and R₆ is H or R₃; and wherein "Ph" is C₆H₅; "m" is 1, 2 or 3; "n" is 0, 1, 2 or 3 ; and "A" is a carbohydrate having the general formula :

2. The method of claim 1, wherein the amino terminus of said CRA is modified.

3. The method of claim 2, wherein the amino terminus of said CRA is modified by acetylation.

4. The method of claim 1, wherein the carboxyl terminus of said CRA is modified.

5. The method of claim 4, wherein the carboxyl terminus of said CRA is modified by amidation.

6. The method of claim 1, wherein R is selected from the group consisting of H, C₂H₅ and CH₂Ph.

7. The method of claim 1, wherein R_x and R₂ are each H.

8. The method of claim 1, wherein X is selected from the group consisting of norleucine, norvaline, leucine or isoleucine.

9. The method of claim 1, wherein R₅ is covalently bound to X_lf said covalent bond forming a cyclic peptide.

10. The method of claim 1, wherein said CRA has the structure:

Ac-Nle-Gln-His- (D) Phe-Arg- (D) Trp-Gly-NH₂.

11. A method of reducing the severity of asthma in an individual, comprising administering to the individual an effective dose of a CRA selected from the group consisting of:

(cyclohexyl) Gly-Gln-His- (D) Phe-Arg- (D) Trp-Gly; Ac- (cyclohexyl) Gly-Gln-His- (D) Phe-Arg- (D) Trp- Gly-NH₂; and cyclo (His- (D) Phe-Arg- (D) Trp) .

12. A method of reducing the severity of asthma in an individual, comprising administering to the individual an effective dose of the CRA (D) Phe-Arg- (D) Trp.

13. A method of reducing the severity of asthma in an individual, comprising administering to the individual an effective dose of a CRA selected from the group consisting of: Nle-Gln-His - (D) Phe-Arg- (D) Trp-Gly-NH₂ ;

His- (D) Phe-Arg- (D) Trp-Gly;

His - (D) Phe-Arg- (D) Trp-Gly-NH₂ ;

Ac-His- (D) Phe-Arg- (D) Trp-NH₂ ;

His- (D) Phe-Arg- (D) Trp-OH ; His- (D) Phe-Arg- (D) Trp;

His- (D) Phe-Arg- (D) Trp-NH₂ ;

Ac-His - (D) Phe-Arg- (D) Trp-OH ; and

Ac-His - (D) Phe-Arg- (D) Trp-Gly-NH₂ .

14. A method of reducing the severity of asthma in an individual, comprising administering to the individual an effective dose of a CRA selected from the group consisting of: Ac-Nle-Gln-His- (D) Phe-Arg- (D) Trp-Gly-OH;

Ac-Nle-Gln-His- (D) Phe-Arg- (D) Trp-Gly-OC₂H₅;

Ac-Nle-Gln-His- (D) Phe-Arg- (D) Trp-Gly-NH-NH₂;

Ac-Nle-Asn-His- (D) Phe-Arg- (D) Trp-Gly-NH₂;

Ac-Nle-Asn-His- (D) Phe-Arg- (D) Trp-Gly-OH ; Ac-Nle-Gln-His - (D) Phe-Arg- (D) Trp-Gly-NHCH₂CH₂Ph ;

Ac-Nle-Gln-His- (D) Phe-Arg- (D) Trp-Gly-NHCH₂Ph;

Gln-His- (D) Phe-Arg- (D) Trp-Gly-NH₂ ; Ac-Gln-His - (D) Phe-Arg- (D) Trp-Gly-NH₂ ; Ac -Nle-Gln-His- (D) Phe-Arg- (D) Trp-NH₂ ; Ac-His- (D) Phe-Arg- (D) Trp (CH₂) - (NAc) Gly-NH₂ ; and His- (D) Phe-Arg- (D) Trp ( CH₂) - (NAc) Gly .