WO2011086557A1 - Peptides for the treatment of respiratory diseases - Google Patents

Abstract

The present invention relates to uses of peptides derived from human plasminogen activator inhibitor-1 (PAI-1) for inhibiting airway smooth muscle contractility. Particularly, the present invention relates to peptides of 7 to 30 amino acid residues corresponding to the amino acids 363-392 of human PAI-1 for the treatment of asthma.

Description

PEPTIDES FOR THE TREATMENT OF RESPIRATORY DISEASES

FIELD OF THE INVENTION

The present invention relates to uses of peptides derived from human plasminogen activator inhibitor- 1 (PAI-1) for inhibiting airway smooth muscle contractility. Particularly, the present invention relates to peptides of 7 to 30 amino acid residues corresponding to the amino acids 363-392 of human PAI-1 for the treatment of asthma.

BACKGROUND OF THE INVENTION

In the second half of the 20th century, asthma has become a worldwide health concern and its prevalence continues to rise. The pathogenesis of asthma is complex and includes airway smooth muscle cell (SMC) contraction, inflammation, edema, altered mucus production and airway remodeling, among which contractility of airway SMC is of central importance. The cellular mechanisms that lead to excessive SMC contraction are currently only partially understood. N-methyl-D-aspartate receptors (NMDA-Rs) have been identified in the large airways where they have been implicated in the regulation of vascular tone and airway contractility in experimental systems. However, the involvement of NMDA-Rs in the physiological regulation of airway contractility and the endogenous mediators that might engage these receptors in patients with asthma have not been fully elucidated. Clues to the function of NMDA-Rs within the lung may come from their activity in other tissues. For example, NMDA-Rs play important physiological as well as pathological roles within the central nervous system (CNS). Several groups have shown that plasminogen activators (PAs) initiate signal transduction through NMDA-Rs (Armstead et al., 2005, Develop. Brain Res., 156: 139- 146; Nicole et al., 2001 , Nat Med. 7:59-64). Overstimulation of NMDA-Rs by glutamate released as a consequence of head trauma or stroke has deleterious effects by causing neuronal apoptosis and cerebral vasodilation. Moreover, tissue type plasminogen activators (tPA) and urokinase-type plasminogen activator (uPA) act through NMDA-Rs to impair cerebral vasodilation in response to hypercapnea in the setting of hypoxia/ischemia.

The molecular basis of the interaction between PAs and NMDA-Rs is incompletely understood. Tissue type PA forms a complex with the NMDA-R1 expressed by cortical neuronal cells in culture. This precedes the cleavage of its NR1 subunit, which is followed by enhancement of intracellular Ca⁺⁺ concentrations and cell death. It is uncertain whether cell death results from occupancy or cleavage of the NR1 subunit or from events downstream of the binding. It has recently been shown that tPA also causes vasodilatation in the mouse whisker barrel cortex due to phosphorylation of neuronal nitric oxide synthase leading to release of NO. Even less is known about the interaction between uPA and NMDA-R1.

PAs have been implicated in the development of asthma. The level of uPA and PAI-1 is elevated in the sputum of asthmatic patients and associations between asthma and polymorphisms in the uPA and PAI-1 genes have been identified (Kowal et al., 2007, Int. Arch. Allergy Immunol., 144:240-246; Kowal et al., 2008, Folia Histochem Cytobiol 46: 193-198; Chu et al., 2006, Amer. J. Respir. Cell Mol. Biol. 35:628-638; Cho et al., 2004, Exp. Biol. Med. 229:138-146). uPA is induced in human bronchial epithelial cells by mechanical activation in vitro and in vivo (Chu et al., 2006, Amer. J. Respir Cell. Mol. Biol. 35:628-638). uPA is activated by mast cell tryptase, promotes the adhesion of airway eosinophils to ICAM-1 and VCAM-1 after allergen challenge, and potentiates PDGF-induced chemotaxis of human airway SMC. uPA and PAI-1 have also been implicated in the deposition of extracellular matrix and in other aspects of airway remodeling. Each of these outcomes suggests that chronic activation of the PA system may contribute to the development of asthma. On the other hand, subepithelial fibrin, which is characteristic of chronic asthma, enhances airway hyperresponsiveness, and aerosolized tPA and uPA have been reported to reduce airway remodeling through their fibrinolytic activity.

The explanation for this dichotomy might lie in the fact that PAs are multifunctional proteins with beneficial and deleterious effects as clearly exemplified by their activities in the CNS. For example, in the case of stroke, the beneficial effects of fibrinolysis may be offset by neuronal apoptosis and vasodilation. It was shown that the beneficial and deleterious effects of PAs in the CNS are mediated through different pathways and involve different domains within the molecules. Furthermore, it was demonstrated that it is possible to dissociate the salutary and deleterious effects and to utilize these differences to improve outcome (Armstead et al., 2006, Nat. Neurosci. 9: 1 150 - 1 155).

The expression of NMDA-Rs in airways suggests a mechanism by which airway contractility may be regulated physiologically or may be disordered in reactive airway disease in response to endogenous PAs. However, a direct role for either uPA or tPA in regulating bronchial contractility and their role, if any, in the activation of NMDA-Rs in this process have not been demonstrated.

International Patent Application Publication No. WO 01/51085 discloses methods for treating asthma or chronic obstructive pulmonary disease comprising administering a plasminogen activator inhibitor- 1 (PAI-1) antagonist to reduce PAI-1 activity in the lung tissue.

International Patent Application Publication No. WO 01/97752 to Cines and Higazi discloses compositions comprising one or more domains of urokinase-type plasminogen activator (uPA) to modulate the contractility and or angiogenic activity of a mammalian muscle or endothelial cell or tissue for use in the treatment of a disease having as a symptom of abnormal muscle cell or tissue contractility and/or abnormal angiogenic activity.

International Patent Application Publication No. WO 03/095476 to the inventor of the present invention teaches administration of the peptide EEIIMD or the peptide acetyl-RMAPEEIIMDRPFLYVVR-amide in combination with one or more fibrinolytic agents for enhancing the fibrinolytic activity, reducing the side effects due to vasoactivity caused by the fibrinolytic agents, and/or for prolonging the half lives of the fibrinolytic agents. Particularly, WO 03/095476 relates to combination compositions comprising the polypeptide EEIIMD and/or the Ac-RMAPEEIIMDRPFLYVVR-amide peptide and one or more currently used plasminogen activators.

International Patent Application Publication No. WO 2008/018084 to Higazi and Cines discloses the use of the peptide EEIIMD or 6-mer peptide analogs thereof in preventing neuronal damage and in treating brain injury.

International Patent Application Publication No. WO 2009/013753 to the inventor of the present invention discloses 18-mer peptides derived from human plasminogen activator inhibitor- 1 and uses thereof for treating thromboembolic diseases and pathological conditions associated with neurological damage. Nassar et al. (Amer. J. Resp. Cell Mol. Biol., 43: 703-71 1, January 22, 2010) disclose the involvement of plasminogen activators and type 1 N-methyl-D-aspartate receptors (NMDA-Rls) in airway contractility and show that plasminogen activator inhibitor- 1 (PAI-1) as well as a PAI-1 derived hexapeptide that recognizes the tPA and uPA docking domains inhibited PAs binding to NMDA-R1.

There is still an unmet need for improved and highly effective means for inhibiting airway smooth muscle contractility, and particularly for treating asthma.

SUMMARY OF THE INVENTION

The present invention provides methods for inhibiting airway smooth muscle contractility comprising administering to a subject in need of such treatment an isolated peptide consisting of 7 to 30 amino acid residues corresponding to the amino acid segment 363-392 of human plasminogen activator inhibitor-1 (PAI-1).

It is now disclosed for the first time that tissue-type plasminogen activator

(tPA) or urokinase (uPA) increased tracheal contractility induced by acetylcholine. The increase in tracheal contractility by tPA or uPA was found to be mediated by type 1 N- methyl-D aspartate receptors (NMDA-Rls). Binding of tPA or uPA to NMDA-Rls reversed the relaxation of tracheal smooth muscle cells (SMCs) mediated by NMDA- Rl s and thereby induced tracheal contractility.

It is further disclosed that the procontractile activity of plasminogen activators on airway smooth muscle is dependent of their catalytic activity. Catalytically active plasminogen activators were shown to exert contractile activity while catalytically inactive plasminogen activators were essentially inactive.

Unexpectedly, it is now disclosed that 18-mer peptides corresponding to the amino acid sequence at positions 369-386 of human PAI-1, and particularly the 18-mer peptide of the amino acid sequence acetyl-RMAPEEIIMDRPFLYVVR-amide (SEQ ID NO: l), prevented tracheal contractility induced by tPA or uPAs. The peptide of SEQ ID NO: 1 was shown to be highly stable.

According to one aspect, the present invention provides a method for treating asthma comprising administering to a subject having or at risk of having asthma a pharmaceutical composition comprising a therapeutically effective amount of an isolated peptide of the amino acid sequence as set forth in SEQ ID NO:2: R i - Arg-Met- Ala-Pro-X i -X₂-Ile-Ile-Met-X₃-Arg-Pro-Phe-Leu-X4-Val-Val- Arg-R₂ wherein Rj is selected from the group consisting of a hydrogen, acyl, alkyl, an amino blocking group, and 1 to 6 amino acid residues; X_\ is selected from the group consisting of Asp, Glu, and Arg; X₂ is selected from the group consisting of Asp and Glu; X₃ is selected from the group consisting of Asp and Glu; X4 is selected from the group consisting of Phe and Tyr; and R₂ is selected from the group consisting of a carboxyl, amide, alcohol, ester, a carboxyl blocking group, and 1 to 6 amino acid residues; or a fragment thereof, wherein the fragment consists of 7 to 17 amino acid residues comprising the amino acid sequence Xi-X₂-Ile-Ile-Met-X₃; the pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

According to some embodiments, Xi is Glu, X₂ is Glu, and X₃ is Asp. According to additional embodiments, Ri is acetyl and R₂ is amide.

According to further embodiments, the peptide to be used for treating asthma is selected from the group consisting of SEQ ID NOs: l, 3 to 9. According to a certain embodiment, the peptide to be used has the amino acid sequence as set forth in SEQ ID NO: l . According to another embodiment, the peptide to be used has the amino acid sequence as set forth in SEQ ID NO:6.

According to some embodiments, asthma is selected from the group consisting of allergic or extrinsic asthma and idiosyncratic or intrinsic asthma.

According to another aspect, the present invention discloses a method for inhibiting airway smooth muscle contractility comprising administering to a subject in need of such treatment a pharmaceutical composition comprising a therapeutically effective amount of an isolated peptide of the amino acid sequence as set forth in SEQ ID NO:2:

R, -Arg-Met- Ala-Pro-X, -X₂-Ile-Ile-Met-X₃-Arg-Pro-Phe-Leu-X₄-Val-Val-Arg-R₂ wherein R_\ is selected from the group consisting of a hydrogen, acyl, alkyl, an amino blocking group, and 1 to 6 amino acid residues; X] is selected from the group consisting of Asp, Glu, and Arg; X₂ is selected from the group consisting of Asp and Glu; X₃ is selected from the group consisting of Asp and Glu; X4 is selected from the group consisting of Phe and Tyr; and R₂ is selected from the group consisting of a carboxyl, amide, alcohol, ester, a carboxyl blocking group, and 1 to 6 amino acid residues; or a fragment thereof, wherein the fragment consists of 7 to 17 amino acid residues comprising the amino acid sequence Xi-X₂-Ile-Ile-Met-X₃; the pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

According to some embodiments, Xj is Glu, X₂ is Glu, and X₃ is Asp. According to additional embodiments, Ri is acetyl and R₂ is amide.

According to further embodiments, the peptide to be used for inhibiting airway smooth muscle contractility is selected from the group consisting of SEQ ID NOs: l , 3 to 9. According to a certain embodiment, the peptide to be used has the amino acid sequence as set forth in SEQ ID NO: l . According to another embodiment, the peptide to be used has the amino acid sequence as set forth in SEQ ID NO:6.

According to some embodiments, airway smooth muscle contractility is bronchial smooth muscle contractility or bronchospasm. According to certain embodiments, bronchospasm is associated with bronchitis. According to further embodiments, airway smooth muscle contractility is tracheal smooth muscle contractility.

According to further embodiments, the pharmaceutical composition is administered by intranasal, intravenous, subcutaneous, intramuscular, intraperitoneal, oral, topical, intradermal, transdermal, epidural, ophthalmic, vaginal or rectal administration route. According to a certain embodiment, the pharmaceutical composition is administered by intranasal administration route such as by inhalation.

According to yet further embodiments, the pharmaceutical composition is formulated in a form selected from the group consisting of a solution, spray, suspension, emulsion, tablet, capsule, gel, powder, cream, depot, and a sustained- release formulation.

According to another aspect, the present invention provides a pharmaceutical composition comprising an isolated peptide of the amino acid sequence as set forth in SEQ ID NO:2:

Ri-Arg-Met-Ala-Pro-Xi-X₂-Ile-Ile-Met-X₃-Arg-Pro-Phe-Leu-X4-Val-Val-Arg-R₂ wherein Ri is selected from the group consisting of a hydrogen, acyl, alkyl, an amino blocking group, and 1 to 6 amino acid residues; X] is selected from the group consisting of Asp, Glu, and Arg; X₂ is selected from the group consisting of Asp and Glu; X₃ is selected from the group consisting of Asp and Glu; X4 is selected from the group consisting of Phe and Tyr; and R₂ is selected from the group consisting of a carboxyl, amide, alcohol, ester, a carboxyl blocking group, and 1 to 6 amino acid residues; or a fragment thereof, wherein the fragment consists of 7 to 17 amino acid residues comprising the amino acid sequence Xi-X₂-Ile-Ile-Met-X₃, for treating asthma.

According to further aspect, the present invention provides a pharmaceutical composition comprising an isolated peptide of the amino acid sequence as set forth in SEQ ID NO:2:

R₁-Arg-Met-Ala-Pro-Xi-X₂-Ile-Ile-Met-X₃-Arg-Pro-Phe-Leu-X₄-Val-Val-Arg-R₂ wherein R_\ is selected from the group consisting of a hydrogen, acyl, alkyl, an amino blocking group, and 1 to 6 amino acid residues; Xi is selected from the group consisting of Asp, Glu, and Arg; X₂ is selected from the group consisting of Asp and Glu; X₃ is selected from the group consisting of Asp and Glu; X4 is selected from the group consisting of Phe and Tyr; and R₂ is selected from the group consisting of a carboxyl, amide, alcohol, ester, a carboxyl blocking group, and 1 to 6 amino acid residues; or a fragment thereof, wherein the fragment consists of 7 to 17 amino acid residues comprising the amino acid sequence Xi-X₂-Ile-Ile-Met-X₃, for inhibiting airway smooth muscle contractility.

These and further embodiments will be apparent from the figures, detailed description and examples that follow. BRIEF DESCRIPTION OF THE FIGURES

FIGs. 1A-B show the effect of uPA on the contraction of tracheal rings. FIG. 1 A shows the effect of increasing concentrations of acetylcholine (Ach). Contraction of tracheal rings was induced by Ach at the indicted concentrations in the absence (□) or presence of 20 nM uPA (■). The molar concentration of Ach is displayed in logarithmic units and the response expressed as a percent of the maximum contraction observed using KCl. FIG. IB shows the effect of increasing concentrations of uPA on Ach EC₅₀ of tracheal ring contraction. The mean ± SD of 6 experiments is shown.

FIG. 2 shows the effect of tPA on the contraction of murine tracheal rings. Contraction of tracheal rings was induced by Ach at the indicted concentrations in the absence (■) or presence of 1 nM (□) or 20 nM (i.) tPA. The mean ± SD of 6 experiments is shown. FIGs. 3A-B show the involvement of PA catalytic activity in the contraction of tracheal and aortic rings. FIG. 3A shows that catalytically inactive PAs variants fail to stimulate contractility of tracheal rings. Contraction of tracheal rings was induced by Ach in the absence (Cont) or presence of 20 nM wild-type tPA or catalytically inactive tPA-ser⁴⁸¹ala (mut tPA) or wild-type uPA or catalytically inactive uPA-ser³⁵⁶ala (mut tPA). The mean ± SD of 3 experiments is shown. FIG. 3B shows that catalytically inactive PAs variants stimulate contractility of aortic rings. Contraction of aortic rings was induced by phenylephrine in the absence (Cont) or presence of 20 nM catalytically inactive mut tPA or mut uPA. The mean ± SD of 3 experiments is shown.

FIGs. 4A-C show the involvement of NMDA-Rs in the contraction of tracheal rings. FIG. 4A. NMDA-Rs mediate the relaxation of tracheal SMC induced by glutamate. Contraction of tracheal rings was induced by Ach in the absence (Control) or presence of tPA (20 nM), uPA (20 nM), the NMDA-R antagonist MK801 (MK801) (100 nM) or the NMDA-R agonist glutamate (150 μΜ). The mean ± SD of 3 experiments is shown. FIG. 4B. Involvement of NOS in NMDA-R mediated relaxation of tracheal SMC induced by glutamate. Contraction of tracheal rings was induced by Ach in the absence (Control) or presence of N (G)-nitro-L-arginine methyl ester (L- NAME; 50 μΜ) alone or together with tPA (20 nM), uPA (20 nM), MK801 (100 nM) or glutamate (150 μΜ). The mean ± SD of 3 experiments is shown. FIG. 4C. Involvement of the epithelium in NMDA-R mediated relaxation of tracheal SMC induced by glutamate. Contraction of denuded tracheal rings was induced by Ach in the absence (Control) or presence of tPA (20 nM), uPA (20 nM), MK801 (100 nM) or glutamate (150 μΜ). The mean ± SD of 3 experiments is shown.

FIG. 5 shows that the 18-mer peptide derived from PAI-1 inhibits the effect of PAs on tracheal contractility. The PAI-1 derived peptide inhibits the pro-contractile activity of tPA and uPA. Contraction of tracheal rings was induced by Ach in the absence or presence of tPA (20 nM) or uPA (20 nM) in the absence or presence of the PAI-1 derived peptide of SEQ ID NO: l (1 μΜ). The mean ± SD of 3 experiments is shown. DETAILED DESCRIPTION OF THE INVENTION

The present invention is based in part on the unexpected findings that 18-mer peptides corresponding to amino acids 369-386 of human PAI-1 were capable of inhibiting airway contractility induced by tPA or uPA in animal models.

While previous studies disclosed PAI-1 antagonists for treating asthma and chronic obstructive pulmonary diseases by virtue of their effect to inhibit PAI-1 activity and hence to maintain plasminogen activator activity, the present invention discloses peptides derived from the docking site of PAI-1 which bind to plasminogen activators and hence inhibit their contractility activity on tracheal smooth muscle. The present invention discloses that inhibition of the pro-contractile activity of tPA or uPA on airway smooth muscle by peptides corresponding to the amino acid sequence 363-392 of human PAI-1 is beneficial for the treatment of respiratory diseases associated with smooth muscle contractility, such as asthma and bronchitis. In addition, the present invention discloses that the mechanism of action of plasminogen activators on airway smooth muscle contraction is different from that of vascular smooth muscle contraction. While catalytically inactive plasminogen activators were shown to enhance the contractility of isolated aortic rings, such catalytically inactive plasminogen activators did not exert procontractile activity on tracheal rings.

The present invention provides isolated peptides derived from PAI-1 for inhibiting airway smooth muscle contractility, and particularly for the treatment of asthma and bronchitis. Particularly, the present invention provides uses of a peptide having the amino acid sequence as set forth in SEQ ID NO:2:

R Arg-Met-Ala-Pro-Xi^-Ile-Ile-Met-X^Arg-Pro-Phe-Leu-^-Val-Val-Arg-^ wherein Rj is selected from the group consisting of a hydrogen, acyl, alkyl, an amino blocking group, and 1 to 6 amino acid residues; Xj is selected from the group consisting of Asp, Glu, and Arg; X₂ is selected from the group consisting of Asp and Glu; X₃ is selected from the group consisting of Asp and Glu; X4 is selected from the group consisting of Phe and Tyr; and R₂ is selected from the group consisting of a carboxyl, amide, alcohol, ester, a carboxyl blocking group, and 1 to 6 amino acid residues; or a fragment thereof, wherein the fragment consists of 7 to 17 amino acid residues comprising the amino acid sequence Xi-X₂-Ile-Ile-Met-X₃, for inhibiting airway smooth muscle contractility.

Thus, the present invention provides uses of 7 to 30-mer peptides comprising preferably the sequence EEIIMD as set forth in SEQ ID NO: 10 derived from human PAI-1 (SEQ ID NO: 1 1) or analogs or derivatives thereof for inhibiting airway smooth muscle contractility, and particularly, for the treatment of respiratory diseases associated with bronchospasm, such as asthma and bronchitis.

According to some embodiments, Ri consists of 1 to 6 amino acid residues corresponding to the amino acid sequence AVIVSA (SEQ ID NO: 12) at positions 363- 368 of human PAI-1. According to further embodiments, R₂ consists of 1 to 6 amino acid residues corresponding to the amino acid sequence HNPTGT (SEQ ID NO: 13) at positions 387-393 of human PAI-1. Thus, the present invention encompasses peptides of 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acid residues which comprise the sequence X]-X₂-Ile-Ile-Met-X₃.

Specifically, the present invention relates to the following peptides:

1) Ac-RMAPEEIIMDRPFLYVVR-amide (SEQ ID NO: 1 )

2) RMAPEEIIMDRPFLYVVR (SEQ ID NO:3)

3) Ac-RMAPEEIIMDRPFLYVVR (SEQ ID NO:4)

4) RMAPEEIIMDRPFLYVVR-amide (SEQ ID NO:5)

5) Ac-RMAPEEIIMDRPFLFVVR-amide (SEQ ID NO:6)

6) RMAPEEIIMDRPFLFVVR (SEQ ID NO:7)

7) Ac-RMAPEEIIMDRPFLFVVR (SEQ ID NO:8)

8) RMAPEEIIMDRPFLFVVR-amide (SEQ ID NO:9)

The term "peptide" as used throughout the specification and claims designates a linear series of amino acid residues connected one to the other by peptide bonds. The amino acid residues are represented throughout the specification and claims by one- letter or three-letter codes according to IUPAC conventions.

The term "amino acid" or "amino acid residue" is understood to include the 20 naturally occurring amino acids.

The peptides of the present invention can be isolated by any protein purification method known in the art. For example, PAI-1 can be subjected to one or more proteolytic enzymes to yield a mixture of peptides, which can further be purified by any protein purification method known in the art to obtain the isolated peptides. Alternatively or additionally, PAI-1 can be cleaved by chemical agents such as, for example, CNBr to yield a mixture of peptides that can be further purified to obtain isolated peptides.

The peptides of the present invention can also be prepared by methods well known in the art including chemical synthesis or recombinant DNA technology.

A preferred method of synthesizing the peptides of the present invention involves solid-phase peptide synthesis utilizing a solid support as described by Merrifield (see J. Am. Chem. Soc, 85:2149, 1964). Large-scale peptide synthesis is described, for example, by Andersson et al. (Biopolymers 55(3): 227-50, 2000). Examples of solid phase peptide synthesis methods include the BOC method, which utilizes tert-butyloxycarbonyl as the a-amino protecting group, and the FMOC method, which utilizes 9-fluorenylmethyloxcarbonyl to protect the a-amino of the amino acid residues, both methods are well-known by those of skill in the art. Alternatively, the peptides of the present invention can be synthesized by standard solution synthesis methods (see, for example, Bodanszky, M., Principles of Peptide Synthesis, Springer- Verlag, 1984).

The peptides useful for practicing the present invention need not be identical to the amino acid sequence RMAPEEIIMDRPFLFVVR (SEQ ID NO:7) derived from amino acids 369-386 of human PAI-1 so long as each of these peptides is capable of reducing, preventing and/or abolishing airway contraction or alternatively capable of treating a respiratory disease such as asthma or bronchitis.

The term "analog" includes any peptide comprising altered sequence by amino acid substitutions, deletions, or chemical modifications of the peptides listed herein above and which displays inhibitory activity on airway smooth muscle contractility. By using "amino acid substitutions", it is meant that functionally equivalent amino acid residues are substituted for residues within the sequence resulting in a silent change. For example, one or more amino acid residues within the sequence can be substituted by another amino acid of a similar polarity, which acts as a functional equivalent, resulting in a silent alteration. Substitutes for an amino acid within the sequence may be selected from other members of the class to which the amino acid belongs. For example, the non-polar (hydrophobic) amino acids include alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan and methionine. The polar neutral amino acids include glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine. The positively charged (basic) amino acids include arginine, lysine and histidine. The negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Such substitutions are known as conservative substitutions. Additionally, a non-conservative substitution may be made so long as the inhibitory effect on airway smooth muscle contraction of the peptide analog is maintained if compared to that of the peptide of SEQ ID NO: l . It will be appreciated that the present invention encompasses peptide analogs, wherein at least one amino acid is substituted by another amino acid to produce a peptide analog having increased stability or longer half-life as compared to the peptides listed herein above.

While the amino acid residues of the peptide sequences set forth herein above are all in the "L" isomeric form, residues in the "D" isomeric form can substitute any L- amino acid residue so long as the peptide analog retains the relaxation activity on airway smooth muscle exhibited by the peptide of SEQ ID NO: 1. Production of a retro- inverso D-amino acid peptide analog where the peptide is made with the same amino acids as disclosed, but at least one amino acid, and perhaps all amino acids are D-amino acids is well known in the art. When all of the amino acids in the peptide analog are D- amino acids, and the N- and C-terminals of the peptide analog are reversed, the result is an analog having the same structural groups being at the same positions as in the L- amino acid form of the peptide. However, the peptide analog is more stable to proteolytic degradation and is therefore useful in many of the applications recited herein.

The present invention further encompasses peptide derivatives of the peptides listed herein above. The term "derivative" refers to a peptide having an amino acid sequence that comprises the amino acid sequence of the peptide of the invention, in which one or more of the amino acid residues is subjected to chemical derivatizations by a reaction of side chains or functional groups, where such derivatizations do not destroy the anti-contractive activity of the peptide derivative. Chemical derivatization of amino acid residues include, but are not limited to, acetylation, amidation, glycosylation, oxidation, reduction, myristylation, sulfation, acylation, ADP- ribosylation, cyclization, disulfide bond formation, hydroxylation, iodination, and methylation.

The peptide derivatives according to the principles of the present invention also include bond modifications, including but not limited to CH₂-NH, CH₂-S, CH₂-S=0, 0=C-NH, CH₂-0, CH₂-CH₂, S=C-NH, CH=CH, and CF=CH and backbone modifications. Peptide bonds (-CO-NH-) within the peptide may be substituted, for example, by N-methylated bonds (-N(CH3)-CO); ester bonds (-C(R)H-C-O-O-C(R)- N); ketomethylene bonds (-CO-CH2-); ct-aza bonds (-NH-N(R)-CO-), wherein R is any alkyl group, e.g., methyl; carba bonds (-CH2-NH-); hydroxyethylene bonds (-CH(OH)- CH2-); thioamide bonds (-CS-NH-); olefinic double bonds (-CH=CH-); and peptide derivatives (-N(R)-CH2-CO), wherein R is the "normal" side chain, naturally presented on the carbon atom. These modifications can occur at any of the bonds along the peptide chain and even at several (2-3) at the same time.

Blocking groups are well known to those of skill in the art as are methods of coupling such groups to appropriate residue(s) of the peptides of the present invention (see, e.g., Greene et al., (1991) Protective Groups in Organic Synthesis, 2nd ed., John Wiley & Sons, Inc. Somerset, N.J.).

In certain embodiments, the terminal amino acids of the peptides of the invention are blocked with a protecting or blocking group. A wide number of blocking groups are suitable for this purpose. Such groups include, but are not limited to, acetyl and alkyl groups for N-terminal protection. In certain embodiments, alkyl chains as in fatty acids, e.g., propeonyl, formyl, and others, can be used. Carboxyl blocking groups include, but are not limited to, amides, esters, and ether-forming blocking groups. Other blocking groups include, but are not limited to Fmoc, t-butoxycarbonyl (t-BOC), 9- fluoreneacetyl group, 1-fluorenecarboxylic group, 9-florenecarboxylic group, 9- fluorenone-l-carboxylic group, benzyloxycarbonyl, Xanthyl (Xan), Trityl (Trt), 4- methyltrityl (Mtt), 4-methoxytrityl (Mmt), 4-methoxy-2,3,6-trimethylbenzenesulphonyl (Mtr), Mesitylene-2-sulphonyl (Mts), 4,4-dimethoxybenzhydryl (Mbh), Tosyl (Tos), 2,2,5, 7,8-pentamethyl chroman-6-sulphonyl (Pmc), 4-methylbenzyl (MeBzl), 4- methoxybenzyl (MeOBzl), Benzyloxy (BzlO), Benzyl (Bzl), Benzoyl (Bz), 3-nitro-2- pyridinesulphenyl (Npys), l-(4,4-dimentyl-2,6-diaxocyclohexylid-ene)ethyl (Dde), 2,6- dichlorobenzyl (2,6-DiCl-Bzl), 2-chlorobenzyloxycarbonyl (2-C1--Z), 2- bromobenzyloxycarbonyl (2— Br-Z), Benzyloxymethyl (Bom), cyclohexyloxy (cHxO), t-butoxymethyl (Bum), t-butoxy (tBuO), t-Butyl (tBu), Acetyl (Ac), and Trifluoroacetyl (TFA).

Also included as chemical derivatives are those peptides which contain one or more naturally occurring amino acid derivatives of the twenty standard amino acid residues. For example: 4-hydroxyproline may be substituted for proline; 5- hydroxylysine may be substituted for lysine; 3-methylhistidine may be substituted for histidine; homoserine may be substituted or serine; and ornithine may be substituted for lysine. The peptides may also contain non-natural amino acids. Examples of non- natural amino acids are norleucine, ornithine, citrulline, diaminobutyric acid, homoserine, isopropyl Lys, 3-(2'-naphtyl)-Ala, nicotinyl Lys, amino isobutyric acid, and 3-(3'-pyridyl-Ala). The peptides may also contain non-protein side chains. In addition to the above, the peptides of the present invention may also include one or more non-amino acid monomers (e.g., fatty acids, complex carbohydrates, and the like).

The present invention includes conjugates of the peptides of the invention. The term "conjugate" is meant to define a peptide of the present invention coupled to or conjugated with another protein or polypeptide. Such conjugates may have advantages over the peptides themselves. Such conjugates can be made by protein synthesis, e. g., by use of a peptide synthesizer, or by ligating the appropriate nucleic acid sequences encoding the desired amino acid sequences to each other by methods known in the art, in the proper coding frame, and expressing the chimeric protein by methods commonly known in the art.

A peptide of the present invention may also be conjugated to itself or aggregated in such a way as to produce a large complex containing the peptide. Such large complexes may be advantageous because they may have new biological properties such as longer half-life in circulation or greater activity.

Pharmaceutical compositions and administration routes

The present invention provides methods for inhibiting smooth muscle contractility, and particularly for treating a respiratory disease associated with bronchospasm such as asthma or bronchitis comprising administering to a subject in need of such treatment a pharmaceutical composition comprising a therapeutically effective amount of a peptide of SEQ ID NO:2 and a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutical composition" refers to a preparation of one or more of the peptides described herein with other chemical components such as pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of an active ingredient to an organism.

The term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

The pharmaceutical compositions of the present invention can be formulated as pharmaceutically acceptable salts of the peptides of the present invention. The term "pharmaceutically acceptable salts" refers to salts prepared from pharmaceutically acceptable non-toxic bases or acids including inorganic or organic bases and inorganic or organic acids. Salts derived from inorganic bases include aluminum, ammonium, calcium, copper, ferric, ferrous, lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc, and the like. Particularly preferred are the ammonium, calcium, magnesium, potassium, and sodium salts. Salts derived from pharmaceutically acceptable organic non-toxic bases include salts of primary, secondary, and tertiary amines, substituted amines including naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as arginine, betaine, caffeine, choline, Ν,Ν'-dibenzylethylenediamine, diethylamine, 2-diethylaminoethanol, 2- dimethylaminoethanol, ethanolamine, ethylenediamine, N-ethyl-morpholine, N- ethylpiperidine, glucamine, glucosamine, histidine, hydrabamine, isopropylamine, lysine, methylglucamine, morpholine, piperazine, piperidine, polyamine resins, procaine, purines, theobromine, triethylamine, trimethylamine, tripropylamine, tromethamine, and the like.

Salts may be prepared from pharmaceutically acceptable non-toxic acids, including inorganic and organic acids. Such acids include acetic, benzenesulfonic, benzoic, camphorsulfonic, citric, ethanesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic acid, and the like. Particularly preferred are citric, hydrobromic, hydrochloric, maleic, phosphoric, sulfuric, and tartaric acids.

The term "carrier" refers to a diluent or vehicle that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under this term. Pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions.

The pharmaceutical compositions of the invention can further comprise an excipient. The term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, trehalose, gelatin, sodium stearate, glycerol monostearate, talc, sodium chloride, glycerol, propylene glycol, and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates or phosphates. Antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose are also envisioned.

The pharmaceutical compositions of the present invention can be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.

Typically, pharmaceutical compositions, which contain peptides as active ingredients are prepared as injectable, either as liquid solutions or suspensions. However, solid forms, which can be suspended or solubilized prior to injection, can also be prepared. The compositions can also take the form of emulsions, tablets, capsules, gels, syrups, slurries, powders, creams, depots, sustained-release formulations and the like.

Methods of introduction of a pharmaceutical composition comprising a peptide of the invention include, but are not limited to, intravenous, subcutaneous, intramuscular, intraperitoneal, oral, topical, intradermal, transdermal, intranasal, epidural, ophthalmic, vaginal and rectal routes. The pharmaceutical compositions can be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), and may be administered together with other therapeutically active agents. The administration may be localized, or may be systemic. Pulmonary administration can also be employed, e.g., by use of an inhaler or neubilizer.

Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers and optionally comprising excipients which facilitate processing of the active ingredients into preparations that can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible solutions such as Hank's solution, Ringer's solution, or physiological salt buffer.

For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

For oral administration, the pharmaceutical composition can be formulated readily by combining the active ingredients with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmaceutical preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable excipients as desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, and sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof, such as sodium alginate, may be added.

Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

Pharmaceutical compositions that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate, and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.

For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

For administration by nasal inhalation, the active ingredients for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a neubilizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro- tetrafluoroethane, or carbon dioxide. In the case of a pressurized aerosol, the dosage may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, for example, gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base, such as lactose or starch.

The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with, optionally, an added preservative. The compositions may be suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing, and/or dispersing agents.

Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water-based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters such as ethyl oleate, triglycerides, or liposomes. Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents that increase the solubility of the active ingredients, to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., a sterile, pyrogen-free, water-based solution, before use.

The pharmaceutical composition of the present invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, for example, traditional binders and carriers such as triglycerides, microcrystalline cellulose, gum tragacanth or gelatin.

For directed internal topical applications, the pharmaceutical composition may be in the form of tablets or capsules, which can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose; a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate; or a glidant such as colloidal silicon dioxide. When the dosage unit form is a capsule, it can contain, in addition to materials of the above type, a liquid carrier such as oil. In addition, dosage unit forms can contain other materials which modify the physical form of the dosage unit, for example, coatings of sugar, shellac, or other enteric agents.

A peptide of the invention can be delivered in a controlled release system. For example, the peptide can be administered in combination with a biodegradable, biocompatible polymeric implant, which releases the peptide over a controlled period of time at a selected site. Examples of preferred polymeric materials include polyanhydrides, polyorthoesters, polyglycolic acid, polylactic acid, polyethylene vinyl acetate, copolymers and blends thereof (See, Medical applications of controlled release, Langer and Wise (eds.), 1974, CRC Pres., Boca Raton, Fla.). A controlled release system can be placed in proximity of the therapeutic target, thus requiring only a fraction of a systemic dose.

Uses of the peptides

The present invention provides uses of the peptides as disclosed herein above for the treatment, prophylaxis and/or inhibition of a respiratory disease associated with bronchospasm such as asthma or bronchitis in a subject in need of such treatment. The present invention further provides uses of the peptides as disclosed herein above for the inhibition of airway smooth muscle contractility. It will be appreciated that the term "treatment" as used herein includes both treatment and/or prophylactic use of the peptides disclosed. In the present invention prophylactic use of the peptides comprises administering to a subject in need of such treatment a therapeutically effective amount of a pharmaceutical composition comprising a peptide as disclosed herein above to prevent the onset of a respiratory disease associated with bronchospasm such as asthma or bronchitis or to prevent the onset of airway smooth muscle contraction; and to prevent the progression of said diseases or conditions.

The term a "therapeutically effective amount" means an amount of a peptide effective to prevent, alleviate, or ameliorate symptoms of a condition or disease associated with airway smooth muscle contraction in the subject being treated.

The term "airway smooth muscle" refers to the smooth muscle lining the bronchi or tracheal region. The peptides of the invention can be administered as therapeutic agents for the treatment or prevention of a respiratory disorder. The term "respiratory disorder" refers to any impairment of lung function which involves constriction of airways and changes in blood gas levels or lung function. Examples of respiratory diseases include, but are not limited to, asthma and bronchitis.

Asthma is a respiratory tract condition characterized by bronchial smooth muscle contractility and enhanced pulmonary vascular permeability. Increases in bronchial smooth muscle contractility leads to airway hyperactivity. Enhanced pulmonary vascular permeability causes extravasation of fluid into the extravascular space which acts as a barrier for the diffusion of oxygen from the airway into the blood.

Asthma is manifested physiologically by a widespread narrowing of the air passages which may be relieved spontaneously or as a result of therapy. Asthma is manifested clinically by paroxysms of dyspnea, cough, wheezing, shortness of breath, hypoxemia, and in severe cases, status asthmaticus, resulting in death. It is an episodic disease, acute exacerbations being interspersed with symptom-free periods. Typically, most attacks are short-lived, lasting minutes to hours, however, there can be a phase in which the patient experiences some degree of airway obstruction daily.

Asthma can be broadly divided into two groups: allergic and idiosyncratic. Allergic asthma is dependent upon an IgE response controlled by T and B lymphocytes and activated by the interaction of antigen with mast cell-bound IgE molecules. Allergic asthma is often associated with a personal and/or family history of allergic diseases such as rhinitis, urticaria, and eczema; positive wheal-and-flare skin reactions to intradermal injection of extracts of airborne antigens; increased levels of IgE in the serum; and/or positive response to provocation tests involving the inhalation of specific antigen.

A significant segment of the asthmatic population will present with negative family or personal histories of allergy, negative skin tests, and normal serum levels of IgE, and therefore cannot be classified on the basis of defined immunologic mechanisms. These are termed idiosyncratic asthma. Many of these will develop a typical symptom complex upon contracting an upper respiratory illness.

Although asthma is primarily a disease of airways, virtually all aspects of pulmonary function are compromised during an acute attack. The pathophysiologic hallmark of asthma is a reduction in airway diameter brought about by contraction of smooth muscle, edema of the bronchial wall, and thick tenacious secretions. The syndromes or disease conditions associated with asthma include an increase in airway resistance, decreased forced expiratory volumes and flow rates, hyperinflation of the lungs and thorax, increased work of breathing, changes in elastic recoil of the lung tissue, abnormal distribution of both ventilation and pulmonary blood flow, mismatched ratios, and altered arterial blood gases. In addition, in very symptomatic patients there frequently is electrocardiographic evidence of right ventricular hypertrophy.

For use in the treatment of asthma, the present invention encompasses asthma that is a member selected from the group consisting of atopic asthma, non-atopic asthma, allergic asthma, bronchial asthma, intrinsic asthma caused by pathophysiologic disturbances, extrinsic asthma caused by environmental factors, emphysematous asthma, exercise-induced asthma, cold air induced asthma, infective asthma caused by bacterial, fungal, protozoal, or viral infection, and non-allergic asthma.

The term "bronchospasm" means the state when the bronchial muscle undergoes tight contraction. The most common cause of bronchospasm is asthma, though other causes include, but are not limited to, respiratory infection, chronic lung disease (including emphysema and chronic bronchitis), anaphylactic shock, or an allergic reaction to chemicals. The time of treatment can be of importance. Administration can be before or after onset of a respiratory disease or an airway contraction has occurred. Administration before an airway contraction has occurred can be of value for prophylactic treatment, for example when the subject is considered to be at risk of airway contraction condition. Such conditions could be, for example in asthmatic patients or subjects at risk of bronchitis. The more common time of administration is after onset of an airway contraction has occurred or is suspected, for example in the conditions of treating a respiratory disease or condition, and in such cases it is desirable to make the administration as soon as possible after the event to get best results-- preferably within an hour or less, though administration later than that time can still be beneficial.

According to some embodiments, the subject is a mammal. According to a certain embodiment, the mammal is a human.

.Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

The precise amount of the peptide administered to a particular subject, preferably a mammal, more preferably a human being, in the method of treatment of the present invention will depend on a number of factors, for example the specific mode of administration and/or the use for which it is intended; the age, body mass and/or past clinical history of the patient to be treated, and always lies within the sound discretion of the person administering and/or supervising the treatment, for example a medical practitioner such as nurse and/or physician. Nevertheless, a suitable daily dose of the peptide for administration to a mammal is generally from about 0.01 mg/day per kg of the mammal's body mass to about 80 mg/kg/day, more usually 0.2-40 mg/kg/day given in a single dose and/or in divided doses at one or more times during the day. The pharmaceutical composition can contain from about 0.1% to about 99% by weight of the peptide and is generally prepared in unit dose form, a unit dose of a peptide generally being from about 0.1 mg to about 500 mg. The following examples are intended to be merely illustrative in nature and to be construed in a non-limitative fashion. EXAMPLE 1

Effect of PAs on tracheal contractility Smooth muscle cells (SMCs) are found in the proximal components of the respiratory tract where their contraction contributes to the development of airway disorders such as asthma. As the role of tPA or uPA in bronchial contractility has not yet been demonstrated, this study aimed at identifying whether plasminogen activators affect airway contractility and whether their effect can be modulated.

Dawley rats were killed by exanguination. The tracheas were removed with care to avoid damage to the epithelium, dissected free of fat and connective tissue, and cut into transverse rings 5mm in length. To record isometric tension, the tracheal rings were mounted in a 10-ml bath containing an oxygenated (95% 0₂, 5%C0₂) solution of Krebs- Henseliet (KH) buffer. The rings were equilibrated for 1.5 hrs at 37° C and maintained under a resting tension of 2 g throughout the experiment. Each ring was then contracted by adding acetylcholine (Ach) in stepwise increments from 10^"10 to 10^"5 M. The absolute maximal contraction was determined by adding 30 mM KC1. The term 100% contraction used in the graphs represents the maximal tension induced by Ach, defined by the concentration at which tension reached a plateau. Recombinant tPA or uPA

FIG. 1A shows that addition of uPA to isolated rat tracheal rings increased contractility induced by Ach. Addition of 10 nM uPA decreased the EC₅₀ of Ach by 5- fold, from 40 to 8 nM (FIG. 1A). The effect of uPA was dose-dependent and saturable with EC₅₀ of approximately 5 nM (FIG. IB). Addition of uPA alone had no effect on rat tracheal contractility (see also Nassar et al., Amer. J. Respir. Cell Mol. Biol. 43: 703- 71 1 , 2010, the content of which is incorporated by reference as if fully set forth herein).

FIG. 2 shows the effect of tPA on tracheal contractility induced by Ach. As shown in FIG. 2, tPA increased the contractility of isolated rat tracheal rings induced by Ach even when added at physiological concentrations. Addition of tPA alone had no effect on the tracheal contractility. While tPA was shown to exert opposing effects on the contraction of isolated aortic rings depending on its concentration, i.e. a low concentration (1 nM) of tPA inhibited, whereas a higher (20 nM) concentration of tPA simulated vascular tone, tPA enhanced tracheal contractility at both concentrations tested (FIG. 2). Previous studies showed that the effect of tPA and uPA on the contractility of systemic blood vessels was independent of their catalytic activity. To examine whether the catalytic activity of tPA or uPA is required for tracheal contraction induced by Ach, the effect of catalytically inactive variants, tPA-Ser⁴⁸¹Ala and uPA-Ser³⁵⁶Ala was examined. The tPA-Ser⁴⁸¹Ala and uPA-Ser³⁵⁶Ala variants were prepared as follows: cDNAs encoding mature human tPA or single-chain uPA were cloned into the pMT/BiP/V5-HisA plasmid (Invitrogen Corp., Carlsbad, CA). The catalytically inactive variants were generated by PCR with the QuickChange Mutagenesis kit (Stratagene, La Jolla, CA), and the complete sequences were verified. Proteins were expressed in S2 Drosophila Expression System (Invitrogen) according to the manufacturer's protocol, and were purified by antibody affinity chromatorgraphy with anti-tPA or anti-uPA coupled to cyanogens-Br-activated sepharose. The final products migrated as single bands on SDS-PAGE at the expected sizes, and their plasminogen activator activity was confirmed with the plasmin chromogenic substrate, Spectrazyme PL.

As shown in FIG.3, none of the catalytically inactive PA variants (designated uPA-m and tPA-m) expressed procontractile activity on tracheal rings (FIG. 3A). In contrast, both PA variants enhanced the contractility of isolated aortic rings (FIG. 3B).

EXAMPLE 2

Role of NMDA-Rs in tracheal contractility

The most extensively studied signal-transduction activity of tPA that is dependent on its catalytic activity involves cleavage of NMDA-R1. NMDA-Rs, including NMDA-R1, have been identified in the respiratory system, but their function and agonists have not been fully characterized. To determine whether the pro- contractile activity of the PAs is mediated through activation of NMDA-Rs, the effect of the NMDA-R antagonist MK-801 on tracheal contraction was examined. The NMDA- R antagonist MK-801 stimulated the contraction of tracheal rings induced by Ach (FIG. 4A), which suggests that NMDA-Rs contribute to the constitutive relaxation of tracheal SMC. To examine the role of NMDA-Rs in the contraction of tracheal rings, the effect of the NMDA-R agonist, glutamate, was determined. Glutamate (150 μΜ) inhibited tracheal contraction induced by Ach (FIG. 4A), supporting the hypothesis that activation of NMDA-Rs inhibits tracheal contractility or induces bronchodilation directly. EXAMPLE 3

Role of epithelial cells in tracheal contractility

Glutamate-induced activation of NMDA-Rs in lungs and in blood vessels induces pulmonary edema and vasodilation through a NOS dependent mechanism. tPA and uPA may therefore stimulate contractility by either: 1) inhibiting NMDA-R mediated induction of NOS activity lowering the threshold for contraction; or 2) acting directly on NMDA-Rs to initiate NOS-independent contractile mechanisms. To distinguish between these possibilities, the effect of the NOS inhibitor L-NAME was examined. L-NAME (50 μΜ) by itself stimulated the contraction of isolated tracheal rings induced by Ach (FIG. 4B), as was seen after the addition of the NMDA-R antagonist MK-801 (100 nM; FIG. 4A). MK-801 lost its procontractile effect (and indeed somewhat inhibited Ach-induced contractility) in the presence of L-NAME (FIG. 4B). In addition, in the presence of L-NAME, glutamate stimulated the contractile effects of Ach (FIG. 4B). L-NAME enhanced the procontractile effect of tPA and uPA, reducing the EC₅₀ of Ach from 3.4 to 2.2 nM and 5.8 to 2.5 nM respectively (FIG. 4B). Taken together, this data strongly suggests that the anti-contractile effect of NMDA-R activation is mediated through NOS, similar to the role of endothelial NOS in NMDA-R mediated vasorelaxation. These results also suggest that the contractile activity exerted by tPA and uPA is partially counteracted by NOS activity.

NOS is present in the airways epithelial cells. Therefore, to examine the role of NOS in NMDA-R mediated bronchial contractility in situ, tracheal rings were denuded of epithelium by rotating a stainless steel rod along the surface of the tracheal rings, and the denuded rings were then used. In contrast to the procontractile effect of L-NAME on intact rings, the NOS inhibitor had no effect on the contractility of denuded tracheal rings induced by Ach (FIG. 4C). Moreover, the procontractile activity of uPA, tPA and glutamate on denuded tracheal rings was inhibited by MK-801 (FIG. 4C).

EXAMPLE 4

Effect of PAI-1 derived peptides on tracheal contractility

The effect of an 18-mer peptide of the amino acid sequence: Ac- RMAPEEIIMDRPFLYVVR-amide, on PA-induced tracheal contractility was determined. Ac-RMAPEEIIMDRPFLYVVR-amide (1 μΜ) added 5 minutes before the addition of tPA or uPA inhibited the pro-contractile activity of both plasminogen activators (FIG. 5).

The foregoing description of the specific embodiments will so fully reveal the general nature of the invention that others can, by applying current knowledge, readily modify and/or adapt for various applications such specific embodiments without undue experimentation and without departing from the generic concept, and, therefore, such adaptations and modifications should and are intended to be comprehended within the meaning and range of equivalents of the disclosed embodiments. It is to be understood that the phraseology or terminology employed herein is for the purpose of description and not of limitation. The means, materials, and steps for carrying out various disclosed chemical structures and functions may take a variety of alternative forms without departing from the invention.

Claims

1. A method for treating asthma comprising administering to a subject in need of such treatment a pharmaceutical composition comprising a therapeutically effective amount of an isolated peptide of the amino acid sequence as set forth in

SEQ ID NO:2:

RrArg-Met-Ala-Pro-Xi^-Ile-Ile-Met-Xs-Arg-Pro-Phe-Leu^-Val-Val-Arg-^ wherein R_\ is selected from the group consisting of a hydrogen, acyl, alkyl, an amino blocking group, and 1 to 6 amino acid residues; Xi is selected from the group consisting of Asp, Glu, and Arg; X₂ is selected from the group consisting of Asp and Glu; X₃ is selected from the group consisting of Asp and Glu; X4 is selected from the group consisting of Phe and Tyr; and R₂ is selected from the group consisting of a carboxyl, amide, alcohol, ester, a carboxyl blocking group, and 1 to 6 amino acid residues; or a fragment thereof, wherein the fragment consists of 7 to 17 amino acid residues comprising the amino acid sequence Xi-X₂-Ile-Ile-Met-X3; the pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

2. The method according to claim 1, wherein Xi is Glu, X₂ is Glu, and X₃ is Asp.

3. The method according to claim 1, wherein Ri is acetyl and R₂ is amide.

4. The method according to claim 1, wherein the peptide is selected from the group consisting of SEQ ID NOs: l, 3 to 9.

5. The method according to claim 4, wherein the peptide is set forth in SEQ ID NO: l .

6. The method according to claim 4, wherein the peptide is set forth in SEQ ID NO:6.

7. The method according to claim 1 , wherein the pharmaceutical composition is administered by intravenous, intranasal, subcutaneous, intramuscular, intraperitoneal, oral, topical, intradermal, transdermal, epidural, ophthalmic, vaginal or rectal administration route.

8. The method according to claim 1, wherein the pharmaceutical composition is formulated in a form selected from the group consisting of a solution, spray, suspension, emulsion, tablet, capsule, gel, powder, cream, depot, and a sustained- release formulation.

9. A method for inhibiting airway smooth muscle contractility comprising administering to a subject in need of such treatment a pharmaceutical composition comprising a therapeutically effective amount of an isolated peptide of the amino acid sequence as set forth in SEQ ID NO:2:

Ri-Arg-Met-Ala-Pro-Xi-X₂-Ile-Ile-Met-X₃-Arg-Pro-Phe-Leu-X₄-Val-Val-Arg-R₂ wherein R_\ is selected from the group consisting of a hydrogen, actyl, alkyl, an amino blocking group, and 1 to 6 amino acid residues; Xi is selected from the group consisting of Asp, Glu, and Arg; X₂ is selected from the group consisting of Asp and Glu; X₃ is selected from the group consisting of Asp and Glu; X4 is selected from the group consisting of Phe and Tyr; and R₂ is selected from the group consisting of a carboxyl, amide, alcohol, ester, a carboxyl blocking group, and 1 to 6 amino acid residues; or a fragment thereof, wherein the fragment consists of 7 to 17 amino acid residues comprising the amino acid sequence Xi-X₂-Ile-Ile-Met-X₃; the pharmaceutical composition further comprising a pharmaceutically acceptable carrier.

10. The method according to claim 9, wherein Xi is Glu, X₂ is Glu, and X₃ is Asp.

1 1. The method according to claim 9, wherein Ri is acetyl and R₂ is amide.

12. The method according to claim 9, wherein the peptide is selected from the group consisting of SEQ ID NOs:l, 3 to 9.

13. The method according to claim 12, wherein the peptide is set forth in SEQ ID NO: l .

14. The method according to claim 12, wherein the peptide is set forth in SEQ ID NO:6.

15. The method according to claim 9, wherein the airway smooth muscle contractility is bronchial smooth muscle contractility.

16. The method according to claim 15, wherein bronchial smooth muscle contractility is associated with bronchitis.

17. A pharmaceutical composition comprising an isolated peptide of the amino acid sequence as set forth in SEQ ID NO:2:

R_!-Arg-Met-Ala-Pro-X_!^-Ile-Ile-Met-Xs-Arg-Pro-Phe-Leu^-Val-Val-Arg-Rz wherein R_\ is selected from the group consisting of a hydrogen, acyl, alkyl, an amino blocking group, and 1 to 6 amino acid residues; Xi is selected from the group consisting of Asp, Glu, and Arg; X₂ is selected from the group consisting of Asp and Glu; X₃ is selected from the group consisting of Asp and Glu; X4 is selected from the group consisting of Phe and Tyr; and R₂ is selected from the group consisting of a carboxyl, amide, alcohol, ester, a carboxyl blocking group, and 1 to 6 amino acid residues; or a fragment thereof, wherein the fragment consists of 7 to 17 amino acid residues comprising the amino acid sequence Xi- X₂-Ile-Ile-Met-X₃, for treating asthma.

18. A pharmaceutical composition comprising an isolated peptide of the amino acid sequence as set forth in SEQ ID NO:2:

R,-Arg-Met-Ala-Pro-Xi-X₂-Ile-Ile-Met-X₃-Arg-Pro-Phe-Leu-X₄-Val-Val-Arg-R2 wherein Ri is selected from the group consisting of a hydrogen, acyl, alkyl, an amino blocking group, and 1 to 6 amino acid residues; Xj is selected from the group consisting of Asp, Glu, and Arg; X₂ is selected from the group consisting of Asp and Glu; X₃ is selected from the group consisting of Asp and Glu; X4 is selected from the group consisting of Phe and Tyr; and R₂ is selected from the group consisting of a carboxyl, amide, alcohol, ester, a carboxyl blocking group, and 1 to 6 amino acid residues; or a fragment thereof, wherein the fragment consists of 7 to 17 amino acid residues comprising the amino acid sequence Xi- X₂-Ile-Ile-Met-X₃, for inhibiting airway smooth muscle contractility.