WO2011047287A1 - Methods of treating inflammatory and fibrotic lung diseases with substance p analogs - Google Patents

Abstract

The present invention relates to pulmonary inflammatory/fibrotic diseases. Methods of protecting the lungs from tissue damage caused by inflammation and the generation of fibrosis or loss of normal lung function in individuals are disclosed. The methods generally comprise administration of substance P, or an analog thereof.

Description

METHODS OF TREATING INFLAMMATORY AND FIBROTIC LUNG

DISEASES WITH SUBSTANCE P ANALOGS

FIELD OF THE INVENTION

The present invention relates to the field of pulmonary inflammatory/fibrotic diseases. Methods of protecting the lungs from tissue damage caused by inflammation and the generation of fibrosis or loss of normal lung function in individuals are disclosed. The methods generally comprise administration of substance P, or an analog thereof.

BACKGROUND OF THE INVENTION

The lung is a highly immunocompetent organ interfacing with external pathogens and antigenic compounds. Many pulmonary diseases are diseases of inflammatory origin resulting in inflammatory cell infiltrates, leakage of fluid into alveolar spaces, pulmonary tissue damage, development of fibrosis and loss of function of the lung and possible death of the individual. There is currently limited treatment of interstitial lung diseases.

SUMMARY OF THE INVENTION

Provided herein are methods for treating inflammatory and fibrotic pulmonary diseases, that may include interstitial lung diseases. In one embodiment, the methods provide for therapy where therapy entails oral administration of one or more Substance P analogs. In a preferred embodiment, the Substance P or analog thereof is administered by inhalation. In one embodiment, the Substance P or analog thereof is administered intravenously. In one embodiment the Substance P or analog thereof is administered subcutaneously. In one embodiment the Substance P or analog thereof is administered intranasally. In one embodiment the Substance P or analog thereof is administered intraperitoneally. In one embodiment the Substance P or analog thereof is administered intramuscularly. In one embodiment the Substance P or analog thereof is administered transmucosally.

In a preferred embodiment the pulmonary disease is idiopathic pulmonary fibrosis. In another embodiment the pulmonary disease is chronic neonatal lung disease/bronchopulmonary dysplasia. In another embodiment the pulmonary disease is silicosis. In another embodiment the pulmonary disease is asbestosis. In another embodiment the pulmonary disease is berylliosis. In another embodiment the pulmonary disease is hypersensitivity pneumonitis. In another embodiment the pulmonary disease is Pneumocystis pneumonia. In another embodiment the pulmonary disease is tuberculosis. In another embodiment the pulmonary disease is Hamman-Rich syndrome. In another embodiment, the pulmonary disease is chronic obstructive pulmonary disease. In another embodiment, the pulmonary disease is chronic neonatal lung disease. In another embodiment the pulmonary disease is sarcoidosis. In another embodiment the disease is asthma. In another embodiment the disease is cystic fibrosis.

DETAILED DESCRIPTION OF THE INVENTION

Introduction

There are many examples of pulmonary disease caused by or exacerbated by inflammation with increased inflammatory cell infiltration, accumulation of fluid into alveolar spaces, development of fibrosis and eventual loss of pulmonary function and possibly death. Examples include, but are not limited to chronic neonatal lung disease (CNLD), chronic obstructive pulmonary disease (COPD), Asthma, bronchopulmonary dysplasia (BPD), idiopathic pulmonary fibrosis, cystic fibrosis and sarcoidosis.

CNLD is a growing societal problem because of the increasing incidence of extremely low birth weight (<1000g) infants and the advances in Neonatal Intensive Care Medicine that allow these infants to survive. The disease was formerly known as Bronchopulmonary dysplasia (BPD). As many as 50% of infants born less than lOOOg are at risk to develop this syndrome. CNLD can be defined as an arrested pulmonary development as a consequence of pre-term delivery, hyperoxygen-mediated lung toxicities, and mechanical ventilator-mediated barotrauma/volutrauma. This disease can result in severe and debilitating long-term health effects including susceptibility to chronic pulmonary infections and chronic lung diseases such as asthma. Recent studies indicate that positive-high pressure full concentration oxygen mechanical ventilation therapy, while potentially life-saving, is a major risk factor for the development of CNLD ^{1 2} .

Clinical symptoms of CNLD include; poorly inflated lungs withreticulogranular opacities radiographically and tachypnea, alveolar retractions, and apnea upon examination. While postnatal corticosteroid therapy continues to be the mainstay of treatment for CNLD, there are a number of detrimental side effects associated with this treatment including periventricular leukomalacia. Therefore safer and more efficacious therapies for CNLD are needed.

Chronic obstructive pulmonary disease (COPD) is a highly prevalent disease, characterized by poorly reversible, obstructive airflow limitation. Risk factors include life-style features including smoking, a sedimentary lifestyle, obesity and diabetes. The pathogenesis of chronic obstructive pulmonary disease is related to fibrosis surrounding the small airways of affected lungs ³. COPD is increasingly seen as a systemic disease with inflammation playing in important role for morbitiy and mortality in these patients ⁴. Asthma has a well documented inflammatory component such that antiinflammatory therapies are the gold standard of contemporary therapies. Idiopathic Pulmonary Fibrosis (EPF) is characterized by injury and loss of lung epithelial cells, accumulation of fibroblasts and myofibroblasts and abnormal remodeling of the lung parenchyma. Recent studies indicate that the proinflammatory cytokine TGF-β plays a critical role if this disease ⁵. Cystic fibrosis is another common

¹ Fitzgerald et. al. 2000. Pulmonary outcomes in extremely low birth weight infants. Pediatrics 105: 1209-15

² Abman et. al. 1994. Pathophysiology and treatment of bronchopulmonary dysplasia. Ped. Clin. North Am. 41 :277- 315

³ Kohyama et. al. 2009. Procaterol Inhibits Lung Fibroblast Migration. Inflammation. 2009 Sep 2

⁴ Van Helvoort et. al., 2006. Systemic immunological response to exercise in patients with chronic obstructive pulmonary disease: what does it mean? Respiration. 73(2):255-64.

Gharaee-Kermani et. al. 2009. Recent advances in molecular targets and treatment of idiopathic pulmonary fibrosis: focus on TGFbeta signaling and the myofibroblast. Curr Med Chem. 16( 1 1): 1400-17. lung disease with well-established inflammatory and fibrotic components ⁶. Finally, systemic sarcoidosis is a disease of unknown or variable etiology often encompassing a pulmonary component to the disease. Pulmonary sarcoidosis is likely mediated by abnormal immune responses that often result in life- threatening pulmonary fibrosis ¹. Thus, the emerging view of many common lung diseases is dual expression of inflammatory and fibrotic properties with tissue destruction and loss of pulmonary function.

Mycoplasma pulmonis infection of pathogen-free F344 rats is an established model to induce long-term neurogenic inflammatory infection ⁸. Importantly, endogenous Substance P (SP) has been shown to modulate this chronic life-long Mycoplasma pulmonis infection in rats resulting in reduced infection and inflammatory damage ⁹. Studies such as these demonstrate the linkage between chronic infection and neuroimmune modulators like Substance P (and by extension all Substance P analogs which activate the same receptor) providing evidence that Substance P and analogs thereof will be useful as a therapy for inflammatory degenerative ling diseases.

The Neurokinin 1 receptor (NK-1R; the receptor predominately used by Substance P and analogs thereof) has been detected on many pulmonary cells including; pulmonary neurons, lung epithelial cells, infiltrating macrophages and other immune cells, and pulmonary smooth muscle and capillaries ¹⁰.

Substance P and an analog thereof have been shown to increase the number of progenitor blood cells derived from hematopoietic stem cells. Recent studies demonstrate this effect is likely mimicked on mesenchymal stem cells (MSCs) and differentiation products of MSCs because of the effect of SP on

⁶ Nicolis et. al. 2009. Modulation of expression of IL-8 gene in bronchial epithelial cells by 5-methoxypsoralen. Int Immunopharmacol. 2009 Nov;9(12): 1411-22

⁷ Toonkel et. al. 2009. Sarcoidosis-Associated Fibrosing Mediastinitis with Resultant Pulmonary Hypertension: A Case Report and Review of the Literature. Respiration. 2009 Oct 1

⁸ McDonald et. al. 1991 Mycoplasma pulmonis infections cause long-lasting potentiation of neurogenic inflammation in the respiratory tract of the rat. J Clin Invest. Mar;87(3):787-99.

⁹ Bowden at. Al. 1996 Sensory denervation by neonatal capsaicin treatment exacerbates Mycoplasma pulmonis infection in rat airways. Am J Physiol. Mar;270(3 Pt l):L393-403

¹⁰ Baluk et. al., 1997. Upregulation of substance P receptors in angiogenesis associated with chronic airway inflammation in rats. Am J Physiol. Sep;273(3 Pt 1):L565-71 cultured human MSCs ¹¹. These investigators showed that MSCs express the NK1R and when stimulated with the pro-inflammatory cytokine IL-la expressed high levels of substance P and importantly SP served as a paracrine growth factor for MCSs. Perhaps most relevantly MSCs have been shown to home to damaged tissues and demonstrate reparative and anti-inflammatory properties in a variety of disease models ¹². MSCs are in clinical investigation for a number of inflammatory/degenerative and fibrotic lung diseases because of their regenerative and anti-inflammatory properties ¹³. Thus Substance P and analogs thereof have the potential to be a therapy for CNLD, asthma, COPD and IPF likely through their anti-inflammatory and other unique properties ¹⁴.

Inflammation is intimately tied to CNLD since infants with fetal inflammatory response syndrome are at greater risk of developing CNLD ¹⁵. Substance P and an analog thereof have been shown to be anti-inflammatory in human lungs wherein pulmonary macrophages demonstrated statistically significant reduction of the proinflammatory cytokines EL- la and EL-Ι β ¹⁶. In fact, both SP demonstrated this significant anti-inflammatory property in damaged lung tissue. These investigators reported however differences between SP and analog in that the NK1 -receptor specific analog demonstrated increased nitric oxide production (a mediator of pro-inflammatory cytokines) by damaged both pulmonary macrophages and type II pneumocytes indicating that Substance P and analogs thereof can have both pro and anti-inflammatory properties depending on the exposed cell type and the disease condition of the tissue.

¹¹ Greco and Rameshwar. 2007. Enhancing effect of IL-1 alpha on neurogenesis from adult human mesenchymal stem cells: implication for inflammatory mediators in regenerative medicine. J Immunol. Sep l;179(5):3342-50.

² Bernardo et. al, 2009. Mesenchymal stromal cells. Ann N Y Acad Sci. Sep; l 176: 101-17.

¹³ Iyer et. al, 2009. Mesenchymal stem cells and inflammatory lung diseases. Panminerva Med. Mar;51(l):5-16.

¹⁴ Aslam et. al. 2009. Bone Marrow Stromal Cells Attenuate Lung Injury in a Murine Model of Neonatal Chronic Lung Disease. Am J Respir Crit Care Med. Aug 27

¹⁵ Nishimaki et. al. 2009. Comparison of markers for fetal inflammatory response syndrome: fetal blood interleukin- 6 and neonatal urinary beta(2)-microglobulin. J Obstet Gynaecol Res. Jun;35(3):472-6.

¹⁶ Sun et. al., 2007. In vitro Pro-inflammatory Role of Substance P in Alveolar Macrophages and Type II

Pneumocytes after JP-8 Exposure. J. Immunotox. 4:61-67. Lung fibrosis is a common phenotype of CNLD and infants who died of CNLD . These patients often displayed pulmonary fibrosis as well as obstructive bronchiolitis, and dysplastic changes in their lung tissue at autopsy indicating a common linkage of lung diseases of inflammation and fibrosis that extends to many different lung diseases with inflammatory and fibrotic components. Substance P and analogs thereof are therefore believed to be unique in their immunoregulatory property to increase inflammation in diseases or conditions that warrant augmented immunity and to decrease inflammation in over inflammatory diseases such as inflammatory/fibrotic pulmonary diseases including CNLD, COPD, Asthma and IPF. Accordingly, substance P and its analogs are believed to be useful in the methods and compositions described herein.

METHODS

Provided herein are methods for treating inflammatory and fibrotic pulmonary diseases, that may include interstitial lung diseases. In one embodiment, the methods provide for therapy where therapy entails oral administration of one or more Substance P analogs. In a preferred embodiment, the Substance P or analog thereof is administered by inhalation. In one preferred embodiment inhalation is

accomplished using a variety of nebulizing medical devises at doses about 100 to 400ug for periods of time ranging from 1.0 to 10 minutes. In one embodiment, the Substance P or analog thereof is administered intravenously. In one embodiment intravenous administration of substance P or an analog thereof is at a dose about 0.02 to 2.0mg/kg. In one embodiment the Substance P or analog thereof is administered subcutaneously. In one embodiment subcutaneous administration of substance P or an analog thereof is at a dose about 10 to lOOnmol/ml. In one embodiment the Substance P or analog thereof is administered intranasally. In one embodiment intranasally administration of substance P or an analog thereof is at a dose about 0.02 to 2.0 mg/kg. In one embodiment the Substance P or analog thereof is administered intraperitoneally. In one embodiment intraperitoneal administration of substance P or an analog thereof is at a dose about 0.02 to 2.0 mg kg. In one embodiment the Substance P or analog thereof

¹⁷ Phillip A. G., 2009. Chronic lung disease of prematurity: A short history. Semin Fetal Neonatal Med. Aug 19 is administered intramuscularly. In one embodiment intramuscularly administration of substance P or an analog thereof is at a dose about 0.02 to 2.0 mg/kg. In one embodiment the Substance P or analog thereof is administered transmucosally. In one embodiment intranasally administration of substance P or an analog thereof is at a dose about 0.02 to 2.0 mg/kg. In one embodiment intranasally or transmucosally administered substance P or an analog thereof may be combined with varying concentrations of powder or liquid bioadhesives or increased viscosity carriers known to practitioners of the art as products such as (but not restricted to) mucopolysaccharides, nanoparticle emulsions or Chitosan.

In a preferred embodiment the pulmonary disease is idiopathic pulmonary fibrosis. In another embodiment the pulmonary disease is bronchopulmonary dysplasia. In another embodiment the pulmonary disease is silicosis. In another embodiment the pulmonary disease is asbestosis. In another embodiment the pulmonary disease is berylliosis. In another embodiment the pulmonary disease is hypersensitivity pneumonitis. In another embodiment the pulmonary disease is Pneumocystis pneumonia. In another embodiment the puhnonary disease is tuberculosis. In another embodiment the pulmonary disease is Hamman-Rich syndrome. In another embodiment, the puhnonary disease is chronic obstructive pulmonary disease. In another embodiment, the pulmonary disease is chronic neonatal lung disease. In another embodiment the pulmonary disease is sarcoidosis. In another embodiment the disease is asthma. In another embodiment the disease is cystic fibrosis.

In certain embodiments, the substance P analog comprises SEQ ID NO 1. In certain embodiments, the substance P analog consists of SEQ ID NO 1.

In certain embodiments, the substance P analog is of Formula (I) (SEQ ID NO.: 12):

Zi-Xaa'-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Xaa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa^u -Z₂ (I) wherein:

Xaa¹ is Arg, Lys, 6-N methyllysine, or (6-N, 6-N) dimethyllysine; Xaa² is Pro or Ala;

Xaa³ is Lys, Arg, 6-N-methyllysine, or (6-N, 6-N) dimethyllysine; Xaa⁴ is Pro or Ala; Xaa⁵ is Gin or Asn; Xaa⁶ is Gin or Asn;

Xaa⁷ is Phe or Phe substituted with chlorine at position 2, 3 or 4;

Xaa⁸ is Tyr, Phe and Phe substituted with chlorine at position 2, 3 or 4;

Xaa⁹ is selected from the group consisting of Gly, Pro, Ala, and sarcosine (N-methylglycine);

Xaa¹⁰ is Leu, Val, He, Norleucine, Met, Met sulfoxide, Met sulfone, N-methylleucine, or N- methylvaline; and

Xaa¹¹ is Met, Met sulfoxide, Met sulfone, or Norleucine;

Zi is R₂N- or RCXO)NR-;

Z₂ is -C(0)NR₂ or -C(0)OR or a salt thereof; each R is independently R is— H, (Q -C₆) alkyl, (Q -C₆) alkenyl, (C] -Q) alkynyl, (C₅ -C₂₀) aryl, (C₆ -C₂6) alkaryl, 5-20 membered heteroaryl or 6-26 membered alkheteroaryl; and each "— " between residues Xaa¹ through Xaa¹¹ independently designates an amide linkage, a substitute amide linkage or an isostere of an amide.

In one embodiment, the substance P analog can be of Formula (I) as described herein, wherein Xaa¹ is Arg; Xaa² is Pro; Xaa³ is Lys; Xaa⁴ is Pro; Xaa⁵ is Gin; Xaa⁶ is Gin; Xaa⁷ is Phe or Phe substituted with chlorine at position 4; Xaa⁸ is Tyr, Phe, or Phe substituted with chlorine at position 4; Xaa⁹ is Gly, Pro or N-methylglycine; Xaa¹⁰ is Leu; and Xaa¹¹ is Met, Met sulfoxide, Met sulfone, or Norleucine.

In a preferred embodiment, the substance P analog can be of Formula (I) as described herein wherein the "— " between residues Xaa¹ through Xaa¹¹ designates

-C(0) H-; Z, is H₂N-; and Z₂ is -C(0) H₂.

In another preferred embodiment, the substance P analog can be selected from the group consisting of:

RP PQQFFGLM (SEQ ID NO.: 1);

RP PQQFFGLNle (SEQ ID NO.: 2);

RPKPQQFFPLM (SEQ ID NO.: 3);

RPKPQQFFMeGlyLM (SEQ ID NO.: 4);

RPKPQQFTGLM (SEQ ID NO.: 5);

RPKPQQF(4-C1)F(4-C1)GLM (SEQ ID NO.: 6);

RP PQQFFGLM(O) (SEQ ID NO.: 7);

RPKPQQFFMeGlyLM(O) (SEQ ID NO.: 8);

RPKPQQFFGLM(0₂) (SEQ ID NO.: 9); or

RPKPQQFFMeGlyLM(0₂) (SEQ ID NO.: 10).

In an even more preferred embodiment, the substance P analog can be

Zi-RPKPQQFFMeGlyM(0₂)-Z₂; wherein Z, is NH₂ and Z₂ is C(0)NH₂ (SEQ ID NO.: 1 1 ). As will be understood by those of skill in the art, substance P (SEQ ID NO. 1) refers to peptide sequence: Arg Pro Lys Pro Gin Gin Phe Phe Gly Leu Met, or the single letter representation

RPKPQQFFGLM (SEQ ID NO 1). In one particularly embodiment the peptide can be amidated at the carboxy terminus represented as RPKPQQFFGLM-NH₂.

DEFINITIONS

The term "alkyl" refers to a saturated branched, straight chain or cyclic hydrocarbon radical. Typical alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, t-butyl, pentyl, isopentyl, hexyl, and the like. In preferred embodiments, the alkyl groups are (Ci -Q) alkyl.

The term "alkenyl" refers to an unsaturated branched, straight chain or cyclic hydrocarbon radical having at least one carbon-carbon double bond. The radical may be in either the cis or trans conformation about the double bond(s). Typical alkenyl groups include, but are not limited to, ethenyl, propenyl, isopropehyl, butenyl, isobutenyl, tert-butenyl, pentenyl, hexenyl and the like. In preferred embodiments, the alkenyl group is (Ci -C₆) alkenyl.

The term "alkynyl" refers to an unsaturated branched, straight chain or cyclic hydrocarbon radical having at least one carbon-carbon triple bond. Typical alkynyl groups include, but are not limited to, ethynyl, propynyl, butynyl, isobutynyl, pentynyl, hexynyl and the like. In preferred embodiments, the alkynyl group is (Q -C₆) alkynyl.

The term "aryl" refers to an unsaturated cyclic hydrocarbon radical having a conjugated π electron system. Typical aryl groups include, but are not limited to, penta-2,4-diene, phenyl, naphthyl, anthracyl, azulenyl, chrysenyl, coronenyl, fluoranthenyl, indacenyl, idenyl, ovalenyl, perylenyl, phenalenyl, phenanthrenyl, picenyl, pleiadenyl, pyrenyl, pyranthrenyl, rubicenyl, and the like. In preferred embodiments, the aryl group is (C₅ -C₂o) aryl, with (C₅ -C_]0) being particularly preferred. The term "alkaryl" refers to a straight-chain alkyl, alkenyl or alkynyl group wherein one of the hydrogen atoms bonded to a terminal carbon is replaced with an aryl moiety. Typical alkaryl groups include, but are not limited to, benzyl, benzylidene, benzylidyne, benzenobenzyl, naphthenobenzyl and the like. In preferred embodiments, the alkaryl group is (C₆ -C₂6) alkaryl, i.e., the alkyl, alkenyl or alkynyl moiety of the alkaryl group is (Ci -C₆) and the aryl moiety is (C5 -C₂o). In particularly preferred embodiments, the alkaryl group is (C₆ -Q3) alkaryl, i.e., the alkyl, alkenyl or alkynyl moiety of the alkaryl group is ( -C₃) and the aryl moiety is (C5-C10).

The term "heteroaryl" refers to an aryl moiety wherein one or more carbon atoms is replaced with another atom, such as N, P, 0, S, As, Se, Si, Te, etc. Typical heteroaryl groups include, but are not limited to, acridarsine, acridine, arsanthridine, arsindole, arsindoline, carbazole, β-carboline, chromene, cinnoline, furan, imidazole, indazole, indole, indolizine, isoarsindole, isoarsinoline, isobenzofuran, isochromene, isoindole, isophosphoindole, isophosphinoline, isoquinoline, isothiazole, isoxazole, naphthyridine, perimidine, phenanthridine, phenanthroline, phenazine, phosphoindole, phosphinoline, phthalazine, pteridine, purine, pyran, pyrazine, pyrazole, pyridazine, pyridine, pyrimidine, pyrrole, pyrrolizine, quinazoline, quinoline, quinolizine, quinoxaline, selenophene, tellurophene, thiophene and xanthene. In preferred embodiments, the heteroaryl group is a 5-20 membered heteroaryl, with 5-10 membered aryl being particularly preferred.

The term "alkheteroaryl" refers to a straight-chain alkyl, alkenyl or alkynyl group where one of the hydrogen atoms bonded to a terminal carbon atom is replaced with a heteroaryl moiety. In preferred embodiments, the alkheteroaryl group is 6-26 membered alkheteroaryl, i.e., the alkyl, alkenyl or alkynyl moiety of the alkheteroaryl is (Q -C₆) and the heteroaryl is a 5-20-membered heteroaryl. In particularly preferred embodiments the alkheteroaryl is 6-13 membered alkheteroaryl, i.e., the alkyl, alkenyl or alkynyl moiety is a 5-10 membered heteroaryl. The term "substituted alkyl, alkenyl, alkynyl, aryl alkaryl, heteroaryl or alkheteroaryl" refers to an alkyl, alkenyl, alkynyl, aryl, alkaryl, heteroaryl or alkheteroaryl group in which one or more hydrogen atoms is replaced with another substituent. Preferred substituents include—OR,— SR,— NRR,— N0₂, — CN, halogen,— C(0)R,— C(0)OR and— C(0)NR, wherein each R is independently hydrogen, alkyl, alkenyl, alkynyl, aryl, alkaryl, heteroaryl or alkheteroaryl.

In one embodiment, the methods provide for stimulating fibroblast proliferation or collagen by use of a substance P analog. In one embodiment the substance P analog can be of Formula (I):

ZpXaa^Xaa^Xaa^Xaa^Xaa^Xaa^Xaa^Xaa^Xaa^Xaa^-Xaa¹¹^ (I) or a pharmaceutically acceptable salt thereof, wherein:

Xaa¹ is Arg, Lys, 6-N methyllysine, or (6-N, 6-N) dimethyllysine;

Xaa² is Pro or Ala;

Xaa³ is Lys, Arg, 6-N-methyllysine, or (6-N, 6-N) dimethyllysine; Xaa⁴ is Pro or Ala; Xaa⁵ is Gin or Asn; Xaa⁶ is Gin or Asn;

Xaa⁷ is Phe or Phe substituted with chlorine at position 2, 3 or 4; Xaa⁸ is Tyr, Phe, or Phe substituted with chlorine at position 2, 3 or 4; Xaa⁹ is Gly, Pro, Ala, or N-methylglycine; Xaa is Leu, Val, He, Norleucine, Met, Met sulfoxide, Met sulfone, N-methylleucine, or N- methylvaline;

Xaa¹¹ is Met, Met sulfoxide, Met sulfone, or Norleucine;

Zi is R₂N- or RC(0)NR-;

Z₂ is -C(0)NR₂ or -C(0)OR or a salt thereof; each R is independently R is— H, (C_t -C₆) alkyl, (Q -C₆) alkenyl, (Ci -C₆) alkynyl, (C₅ -C₂₀) aryl, (C₆ -C₂6) alkaryl, 5-20 membered heteroaryl or 6-26 membered alkheteroaryl; and each "— " between residues Xaa¹ through Xaa¹¹ independently designates an amide linkage, a substitute amide linkage or an isostere of an amide.

In a preferred embodiment the substance P analogs can be of Formula (I) wherein

Xaa¹ is Arg; Xaa² is Pro; Xaa³ is Lys; Xaa⁴ is Pro; Xaa⁵ is Gin; Xaa⁶ is Gin; Xaa⁷ is Phe or Phe substituted with chlorine at position 4; Xaa⁸ is Tyr, Phe, or Phe substituted with chlorine at position 4; Xaa⁹ is Gly, Pro or N-methylglycine; Xaa¹⁰ is Leu; and Xaa¹¹ is Met, Met sulfoxide, Met sulfone, or Norleucine. In a more preferred embodiment, the "— " between residues Xaa¹ through Xaa¹¹ of the substance P analogs can be -C(0)NH-; and Zi is H₂N-; and Z₂ is -C(0)NH₂.

In yet another preferred embodiment the substance P analogs can be selected from the group consisting of:

RP PQQFFGLM (SEQ ID NO.: 1);

RPKPQQFFGLNle (SEQ ID NO.: 2);

RPKPQQFFPLM (SEQ ID NO.: 3);

RPKPQQFFMeGlyLM (SEQ ID NO.: 4); RP PQQFTGLM (SEQ ID NO.: 5);

RPKPQQF(4-C1)F(4-C1)GLM (SEQ ED NO.: 6);

RPKPQQFFGLM(O) (SEQ ID NO.: 7);

RPKPQQFFMeGlyLM(O) (SEQ ID NO.: 8);

RP PQQFFGLM(0₂) (SEQ ED NO.: 9); and

RPKPQQFFMeGlyLM(0₂) (SEQ ED NO.: 10).

In another preferred embodiment, the substance P analog can be

Zi-RPKPQQFFMeGlyLM(0₂)-Z2; wherein ¾ is NH₂ and Z₂ is C(0)NH₂.

It will be apparent to one skilled in the art that the amino (designated herein as Z)) or carboxy terminus (designated herein as Z₂) of the substance P analogs can be modified. Included are "blocked" forms of the substance P analogs, i.e., forms of the substance P analogs in which the N- and/or C- terminus is blocked with a moiety capable of reacting with the N-terminal— NH₂ or C-terminal — C(0)OH. In some embodiments the N- and/or C-terminal charges of the substance P analogs can be an N-acylated peptide amide, ester, hydrazide, alcohol and substitutions thereof. In a preferred embodiment, either the N- and/or C-terminus (preferably both termini) of the substance P analogs are blocked. Typical N-terminal blocking groups include RC(O)— , where R is— H, (Ci -C₆) alkyl, (Q -C₆) alkenyl, (Q -C₆) alkynyl, (C₅ -C₂o) aryl, (C₆ -C₂6) alkaryl, 5-20 membered heteroaryl or 6-26 membered alkheteroaryl. Preferred N-terminal blocking groups include acetyl, formyl and dansyl. Typical C-terminal blocking groups include— C(0)NRR and— C(0)OR, where each R is independently defined as above. Preferred C- terminal blocking groups include those where each R is independently methyl. In another preferred embodiment the C-terminal group is amidated. Substituted amides generally include, but are not limited to, groups of the formula— C(0)NR— where R is (Q -C₆) alkyl, substituted (Q -C₆) alkyl, ( -Q) alkenyl, substituted (Ci -C₆) alkenyl, (Q - C₆) alkynyl, substituted (d -C₆) alkynyl, (C₅ -C₂o) aryl, substituted (C₅ -C₂₀) aryl, (C₆ -C₂e) alkaryl, substituted (C₆ -C₂6) alkaryl, 5-20 membered heteroaryl, substituted 5-20 membered heteroaryl, 6-26 membered alkheteroaryl and substituted 6-26 membered alkheteroaryl.

Amide isosteres generally include, but are not limited to,— CH₂ NH— ,— CH₂ S— ,— CH₂CH₂— , -CH=CH- (cis and trans), -C(0)CH₂ - -CH(OH)CH₂ - and -CH₂ SO-. Compounds having such non-amide linkages and methods for preparing such compounds are well-known in the art (see, e.g., Spatola, March 1983, Vega Data Vol. 1, Issue 3; Spatola, 1983, "Peptide Backbone Modifications" In: Chemistry and Biochemistry of Amino Acids Peptides and Proteins, Weinstein, ed., Marcel Dekker, New York, p. 267 (general review); Morley, 1980, Trends Pharm. Sci. 1 :463-468; Hudson et al, 1979, Int. J. Prot. Res. 14:177-185 (-CH₂ NH— , -CH₂ CH₂ -); Spatola et al, 1986, Life Sci. 38: 1243-1249 (-C¾ -S); Hann, 1982, J Chem. Soc. Perkin Trans. I. 1 :307-314 (-CF CH-, cis and trans); Almquist et al., mO, J. Med. Chem. 23: 1392-1398 (-COCH₂ -); Jennings-White et al, Tetrafiedron. Lett. 23:2533 (-COCH₂-); European Patent Application EP 45665 (1982) CA 97:39405 (-CH(OH)CH₂ -); Holladay et al., 1983, Tetrahedron Lett. 24:4401-4404 (-C(OH)CH₂ -); and Hruby, 1982, Life Sci. 31 :189-199 (-CH₂ -S-).

Additionally, one or more amide linkages can be replaced with peptidomimetic or amide mimetic moieties which do not significantly interfere with the structure or activity of the peptides. Suitable amide mimetic moieties are described, for example, in Olson et al., 1993, J. Med. Chem. 36:3039-3049.

In one embodiment the substance P analogs can have a modified methionine residue. In a preferred embodiment, the methionine residue side chain S can be oxidated. In one embodiment the methionine is methionine sulfoxide (-NH-CHa(CO)-CH₂-CH₂-S(0)CH₃). In one embodiment the methionine is methionine sulfone or methionine S, S, dioxide, (-NH-CHa (CO)-CH₂-CHa₂-S(0₂)CH₃) , also referred to herein as Met(0)₂. Various embodiments have been described. The descriptions and examples are intended to be illustrative of the invention and not limiting. Indeed, it will be apparent to those of skill in the art that modifications can be made to the various embodiments described without departing from the spirit of the invention or scope of the appended claims set forth below.

All references cited herein are incorporated herein by reference in their entireties for all purposes.

EXAMPLES

Example 1: Administration of a Substance P Analog in a Rat Model of Pulmonary Fibrosis

This example describes administration of a substance P analog, HOMSPERA^®, in a rat model of pulmonary fibrosis. In the study, Sprague-Dawley rats were exposed to the drug bleomycin and then treated with HOMSPERA^® (SEQ ID NO: 10). Intra-tracheal bleomycin administration an art-recognized rat model for pulmonary fibrosis. Since fibrotic development is a complex process influenced by immune factors, four differnt Homspera dose regimens were employed (oral administration at doses of 0.02 mg/kg/day, 0.2 mg/kg/day, and 2.0 mg/kg/day; and intranasal administration at 0.2 mg/kg/day). Experimental animals were sacrificed at various times and tissues subsequently removed for assessment of the development of pulmonary fibrosis.

The bleomycin-exposed but untreated animals during the induction and treatment phases showed physiologic changes consistent with the development of pulmonary fibrosis, as expected. There were gross lesion in the lungs in most animals.. They appeared as a group to lose weight, which likely reflected the disease process, as ill animals tend to eat less. In contrast, their lung weights apparently increased, which was believed to be due to inflammatory fluid accumulation, to generation of fibrotic tissue, or both. Importantly, both the decrease in whole animal weights and the increases in lung weights appear to be inhibited by treatment with HOMSPERA^®. Based on the data, the treated animals in all HOMSPERA® groups generally gained more body weight throughout the study as compared to the vehicle control group... Regarding lung weight, the data suggest that lungs in HOMSPERA^®-treated groups weighed slightly less as a group mean percent of terminal body weight than did vehicle control lungs. Specifically, animals receiving Homspera orally at all 3 dosing levels (although without the benefit of formulation which could enhance bioavailability) were observed to have 13-17% lower lung weights than the non- Homspera treated animals.

In further analysis, lungs are removed from experimental animals into formalin and subsequently transferred into paraffin. Lung sections are prepared onto microscope slides and are analyzed with hematoxylin-eosin to assess lung tissue structural changes and Masson's trichrome staining and evaluation to detect potential differences in collagen deposition and lung pathology between the control bleomycin-only animals and those treated with different dose regimens of HOMSPERA^®.

Example 2: Mitigation of Radiation-Induced Pulmonary Fibrosis

This example describes mitigation of pulmonary damage induced by radiation by an exemplary Substance P analog, HOMSPERA^® Short-term HOMSPERA^® treatment immediately following gamma radiation decreased pulmonary damage resulting from bacterial endotoxin exposure two months later. This has significant implications as it is evidence that HOMSPERA^® treatment has long-lasting effects and can mitigate the delayed sequelae following the insult (e.g., radiation) that results in pulmonary fibrosis.

In the study, Mice were irradiated with 5Gy gamma radiation at 4 days of age then treated with HOMSPERA^® intranasally (2 mg/kg) daily for 10 days. Mice were allowed 6 weeks for the pulmonary sequelae of the sub-lethal radiation to develop, and during this time did NOT receive HOMSPERA^®. They were then exposed to bacterial endotoxin (lipopolysacchide, LPS), a component of the cell wall of Gram-negative bacteria.

In mice exposed to HOMSPERA^® for 10 days immediately after irradiation, the response to LPS was blunted, specifically, there is an approximately 15% reduction in protein leaking into the lung, possibly reflecting a decrease in damage to lung capillary endothelium. There was a similar protective effect in non-radiated lung.

In a related study, mice were gamma irradiated with 5 Gy whole body and 10 Gy lung top off to reflect multiorgan failure induced by radiation. 10 days later, HOMSPERA^®was administered orally (2mg/kg preferred and 20 mg kg) daily for 10 days. 26 weeks later, animals were sacrificed and evaluated.

Following trichrome and hematoxylin/eosin staining, as well as enumeration of neutrophils, macrophages and lymphocytes, HOMSPERA^® treated animals showed fewer inflammatory cells, less pulmonary edema and less pulmonary fibrosis.

This activity was confirmed in neonate studies, in which 5 gy radiation at 4d post natal was followed 10 days later by HOMSPERA^® administered intranasally for 10 days. When assessed at 8 weeks post radiation, HOMSPERA^® animal lungs were less emphysematous, suggesting that HOMSPERA^® treatment prevented defective septation induced by the disruptive effects of radiation on lung development.

Claims

1. A method of treating a subject suffering from an inflammatory and fibrotic lung disease selected from the group consisting of Chronic Neonatal Lung Disease, Bronchial Pulmonary Dysplasia, Chronic Obstructive Pulmonary Disease (COPD), Asthma, and Idiopathic Pulmonary Fibrosis, comprising administering to the subject by inhalation one or more substance P analogs wherein said substance P analog is according to Formula (I):

ZrXaa'-Xaa^Xaa^Xaa^Xaa^Xaa^Xaa^Xaa^Xaa^Xaa^-Xaa¹ '-Z₂ (I) or a pharmaceutically acceptable salt thereof, wherein:

Xaa¹ is Arg, Lys, 6-N methyllysine, or (6-N, 6-N) dimethyllysine;

Xaa² is Pro or Ala;

Xaa³ is Lys, Arg, 6-N-methyllysine, or (6-N, 6-N) dimethyllysine; Xaa⁴ is Pro or Ala; Xaa⁵ is Gin or Asn; Xaa⁶ is Gin or Asn;

Xaa⁷ is Phe or Phe substituted with chlorine at position 2, 3 or 4; Xaa⁸ is Tyr, Phe, or Phe substituted with chlorine at position 2, 3 or 4; Xaa⁹ is Gly, Pro, Ala or -methylglycine;

Xaa¹⁰ is Leu, Val, He, Norleucine, Met, Met sulfoxide, Met sulfone, N-methylleucine, or N- methylvaline;

Xaa¹¹ is Met, Met sulfoxide, Met sulfone or Norleucine; Z, is R₂N- or RC(0)NR-;

Z₂ is -C(0)NR₂ or -C(0)OR or a salt thereof; each R is independently R is— H, (Ci -C₆) alkyl, (Ci -C₆) alkenyl, (Ci -C₆) alkynyl, (C₅ -C₂₀) aryl, (C₆ -C₂6) alkarvl, 5-20 membered heteroaryl or 6-26 membered alkheteroaryl; and each "— " between residues Xaa¹ through Xaa¹¹ independently designates an amide linkage, a substitute amide linkage or an isostere of an amide.

2. The method of claim 1 wherein

Xaa¹ is Arg;

Xaa² is Pro; Xaa³ is Lys; Xaa⁴ is Pro; Xaa⁵ is Gin; Xaa⁶ is Gin;

Xaa⁷ is Phe or Phe substituted with chlorine at position 4; Xaa⁸ is Tyr, Phe, or Phe substituted with chlorine at position 4; Xaa⁹ is Gly, Pro or N-methylglycine; Xaa¹⁰ is Leu; and

Xaa¹¹ is Met, Met sulfoxide, Met sulfone or Norleucine.

3. The method of claim 1 wherein the "— " between residues Xaa¹ through Xaa¹¹ designates

-C(0)NH-; Z, is H₂N-; and Z₂ is -C(0)NH₂.

4. The method of claim 1 wherein the substance P analog is selected from the group consisting of:

RP PQQFFGLM (SEQ ID NO.: 1);

RPKPQQFFGLNle (SEQ ID NO.: 2);

RP PQQFFPLM (SEQ ID NO. : 3);

RPKPQQFFMeGlyLM (SEQ ID NO.: 4);

RPKPQQFTGLM (SEQ ID NO.: 5);

RPKPQQF(4-C1)F(4-C1)GLM (SEQ ID NO.: 6);

RP PQQFFGLM(O) (SEQ ID NO.: 7);

RP PQQFFMeGlyLM(O) (SEQ ID NO.: 8);

RP PQQFFGLM(0₂) (SEQ ID NO.: 9); and

RPKPQQFFMeGlyLM(0₂) (SEQ ID NO.: 10).

5. The method of claim 1 wherein the substance P analog is Z_x— RPKPQQFFMeGlyLM(0₂)— Z₂; wherein

Z, is NH₂ and Z₂ is C(0)NH₂.

6. The method of claim 1 wherein the disease is Chronic Obstructive Pulmonary Disease (COPD).

7. The method of claim 1 wherein the disease is Chronic Neonatal Lung Disease.

8. The method of claim 1 wherein the disease is Bronchial Pulmonary Dysplasia.

9. The method of claim 1 wherein the disease is Asthma.

10. The method of claim 1 wherein the disease is Idiopathic Pulmonary Fibrosis.

1 1. The method of claim 1 wherein the lung disease is mediated by hyperoxygen positive-pressure mechanical ventilation therapy.

12. The method of claim 1 wherein the pulmonary disease is mediated by exposure to ionizing

radiation.

13. The method of claim 12 wherein the radiation exposure was therapeutic.

14. The method of claim 1 wherein the radiation exposure was non-therapeutic.

15. The method of claim I wherein the pulmonary disease is mediated by severe or prolonged

cytokine imbalances.

16. The method of claim 1 wherein the pulmonary disease is caused by pulmonary inflammation.

17. The method of claim 1 wherein the pulmonary disease is caused by expression of proinflammatory cytokines by pulmonary cells such as alveolar type I and Π cells.

18. The methodof claim 17, wherein the pulmonary cells are alveolar type I or II cells

19. One method of claim 1 wherein the subject is a human.

20. One method of claim 1 wherein the subject is a non-human animal.

21. A method of reducing the risk for the development of an inflammatory and fibrotic lung disease selected from the group consisting of Chronic Neonatal Lung Disease, Bronchial Pulmonary Dysplasia, Chronic Obstructive Pulmonary Disease (COPD), Asthma, and Idiopathic Pulmonary Fibrosis in a subject at risk for developing such a disease comprising administering to the subject by inhalation of one or more substance P analogs wherein said substance P analog is according to Formula (I):

Zi-Xaa¹-Xaa²-Xaa³-Xaa⁴-Xaa⁵-Xaa⁶-Xaa⁷-Xaa⁸-Xaa⁹-Xaa¹⁰-Xaa^{, 1}-Z₂ (I) or a pharmaceutically acceptable salt thereof, wherein: Xaa¹ is Arg, Lys, 6-N methyllysine, or (6-N, 6-N) dimethyllysine; Xaa² is Pro or Ala;

Xaa³ is Lys, Arg, 6-N-methyllysine, or (6-N, 6-N) dimethyllysine; Xaa⁴ is Pro or Ala; Xaa⁵ is Gin or Asn; Xaa⁶ is Gin or Asn;

Xaa⁷ is Phe or Phe substituted with chlorine at position 2, 3 or 4; Xaa⁸ is Tyr, Phe, or Phe substituted with chlorine at position 2, 3 or 4; Xaa⁹ is Gly, Pro, Ala or N-methylglycine;

Xaa¹⁰ is Leu, Val, He, Norleucine, Met, Met sulfoxide, Met sulfone, N-methylleucine, or N- methylvaline;

Xaa¹¹ is Met, Met sulfoxide, Met sulfone or Norleucine;

¾ is R₂N- or RC(0)NR-;

Z₂ is -C(0)NR₂ or -C(0)OR or a salt thereof; each R is independently R is— H, (d -C₆) alkyl, (C, -C₆) alkenyl, (C, -C₆) alkynyl, (C₅ -C₂₀) aryl, (C₆ -C_¾) alkaryl, 5-20 membered heteroaryl or 6-26 membered alkheteroaryl; and each "— " between residues Xaa¹ through Xaa¹¹ independently designates an amide linkage, a substitute amide linkage or an isostere of an amide.

22. The method of claim 21 wherein

Xaa' is Arg;

Xaa" is Pro;

Xaa³ is Lys;

Xaa⁴ is Pro;

Xaa is Gin; Xaa⁶ is Gin;

Xaa⁷ is Phe or Phe substituted with chlorine at position 4; Xaa⁸ is Tyr, Phe, or Phe substituted with chlorine at position 4; Xaa⁹ is Gly, Pro or N-methylglycine;

Xaa is Leu; and

Xaa is Met, Met sulfoxide, Met sulfone or Norleucine.

23. The method of claim 21 wherein the "— " between residues Xaa¹ through Xaa¹¹ designates -C(0)NH-;

Z, is ¾N-; and Z₂ is -C(0) H₂.

24. The method of claim 21 wherein the substance P analog is selected from the group consisting of:

RP PQQFFGLM (SEQ ID NO.: 1);

RPKPQQFFGLNle (SEQ ID NO.: 2);

RPKPQQFFPLM (SEQ ID NO.: 3); RPKPQQFFMeGlyLM (SEQ ID NO.: 4);

RPKPQQFTGLM (SEQ ID NO.: 5);

RPKPQQF(4-C1)F(4-C1)GLM (SEQ ID NO.: 6);

RPKPQQFFGLM(O) (SEQ ID NO.: 7);

RPKPQQFFMeGlyLM(O) (SEQ ID NO.: 8);

RPKPQQFFGLM(0₂) (SEQ ID NO.: 9); and

RPKPQQFFMeGlyLM(0₂) (SEQ ID NO.: 10).

25. The method of claim 21 wherein the substance P analog is Z₍— RPKPQQFFMeGlyM(0₂)— Z₂; wherein

Zj is NH₂ and Z₂ is C(0)N¾.

26. The method of claim 21 wherein the disease is Chronic Obstructive Pulmonary Disease (COPD).

27. The method of claim 21 wherein the disease is Chronic Neonatal Lung Disease.

28. The method of claim 21 wherein the disease is Chronic Obstructive Pulmonary Disease (COPD).

29. The method of claim 21 wherein the disease is Asthma.

30. The method of claim 21 wherein the disease is Idiopathic Pulmonary Fibrosis.

31. The method of claim 21 wherein the lung disease is mediated by hyperoxygen positive-pressure mechanical ventilation therapy.

32. The method of claim 21 wherein the lung disease is mediated by exposure to ionizing radiation.

33. The method of claim 32 wherein the radiation exposure was therapeutic.

34. The method of claim 32 wherein the radiation exposure was non-therapeutic.

35. The method of claim 32 wherein the lung disease is caused by severe or prolonged cytokine imbalances.

36. The method of claim 32 wherein the lung disease is mediated by pulmonary inflammation.

37. The method of claim 32 wherein the pulmonary disease is mediated by expression of proinflammatory cytokines by pulmonary cells.

38. The method of claim 37 wherein the pulmonary cells are alveolar type I or II cells.

39. One method of claim 21 wherein the subject is a human.

40. One method of claim 21 wherein the subject is a non-human animal.