Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/46—Hydrolases (3)
  • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
  • A61K38/482—Serine endopeptidases (3.4.21)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/46—Hydrolases (3)
  • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
  • A61K38/482—Serine endopeptidases (3.4.21)
  • A61K38/4826—Trypsin (3.4.21.4) Chymotrypsin (3.4.21.1)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides
  • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
  • A61K38/46—Hydrolases (3)
  • A61K38/48—Hydrolases (3) acting on peptide bonds (3.4)
  • A61K38/482—Serine endopeptidases (3.4.21)
  • A61K38/486—Elastase (3.4.21.36 or 3.4.21.37)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/06—Antiasthmatics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• This invention relates to the fields of cell biology, biochemistry, medicine, and pharmacology.
• serpins serine protease inhibitors
• related proteins constitute one of the earliest described protein superfamilies recognized by Hunt and Dayhoff in 1981 by computer analysis of a ino acid sequence identity (Hunt et al . (1981) ⁇ ochem. Biophys . Res . Co mun . 95:864-871).
• One group in this superfamily is the "Qv-familiy", which would not be recognised as such based on sequence identity alone (Rem ⁇ ld- O'Donnell, E. (1993) FEBS 315 : 105-108) .
• Ov-family members include human squamous cell antigen, human plasminogen activator (PAI-2) , human monocyte/neutrophil elastase inhibitor ( UJEI) , chicken ovalbumin (Oval), and chicken gene Y.
• PAI-2 human plasminogen activator
• UJEI human monocyte/neutrophil elastase inhibitor
• Oval chicken ovalbumin
• Cv-family proteins lack the N-terminal extension region and a cleavable hydrophobia signal sequence, two properties normally associated with serpin f mily members .
• Several members of this family appear to rely on a non-cleavable internal signal sequence, since they exist as dualistic molecules that can be either secreted or located ⁇ ytoplasmi ⁇ ally.
• Human monocyte/neutrophil elascase inhibitor (MNEI) (GenBank Accession No. P3Q740) is a serpin superfamily protein that rapidly inhibits the neutrophil granule proteases elastase (GenBank Accession No. AAD45239) , cathepsin G (cat G) (GenBank Accession No. XP_007318) . and pr ⁇ teinase-3 (P -35
• MNEI was first detected in secretions from cultured macrophages and is believed to play a central role in the control o£ extracellular serine protease activity in the lung. MNEI is found at elevated concentrations in the bronchoalveolar lavage (BAL) fluid of cystic fibrosis patients with severe lung inflammation, suggesting increased release in response to inflammatory conditions .
• BAL bronchoalveolar lavage
• MNEI is released from cells by way of a novel secretory pathway and that various agents are useful for stimulating the cellular secretion or release of MNEI .
• These discoveries have been utilised to provide the present invention, which includes methods for stimulating the release of MNEI from a cell and methods of identifying agents that stimulate MNEI release from a cell. These methods are useful in the treatment of inflammatory conditions and diseases.
• the invention provides a method of stimulating the release of MNEI from a cell.
• the method comprises contacting the cell with an agent that stimulates the release of MNEI from the cell.
• the agent is a protease.
• the protease is a neutrophil granule protease.
• the protease is selected from the group consisting of monocyte/neutrophil elastase, cathepsin G, proteinase-3, pancreatic elastase, and chymotrypsin.
• the protease is a analog of monocyte/neutrophil elastase, cathepsin G, proteinase-3, pancreatic elastase, or chymotrypsin.
• the agent is a small molecule agonist of MNEI release.
• the invention provides a method of stimulating the secretion of MNEI from a cell ,
• the method comprises contacting the cell with a priming agent followed by contacting the cell with an agent that stimulates the release of MNEI f om the cell .
• the agent is a protease.
• the protease is a neutrophil granule protease.
• the protease is selected from the group consisting of monocyte/neutrophil elastase, cathepsin G, proteinase-3, pancreatic elastase, and chymotrypsin.
• the protease is a analog of monocyte/neutrophil elastase, cathepsin G, proteinase-3, pancreatic elastase, or chymotrypsin.
• the priming agent is selected from the group consisting of lipop ⁇ lysaccaride (LPA) or ⁇ yclohe amide (CHX) .
• the agent is a small molecule agonist of MNEI release.
• the invention provides a method of stimulating the secretion of MNEI from a cell.
• the method comprises contacting the cell with a priming agent and an agent that stimulates the release of MNEI from the cell.
• the priming agent is etoposide (stop) .
• the agent that stimulates the release of MNEI from the cell is a protease.
• the protease is a neutrophil granule protease.
• the protease is selected from the group consisting of monocyte/neutrophil elastase, cathepsin G, proteinase-3, pancreatic elastase, and chymotrypsin.
• the protease is a analog of monocyte/neutrophil elastase, cathepsin G, proteinase-3, pancreatic elastase, or chymotrypsin.
• the agent is a small molecule agonist of MNEI release.
• the invention provides a method of identifying an agent that stimulates the release of MNEI from a cell.
• the method comprises contacting the cell with a test agent and assaying for MNEI release, wherein an increase in the release of MNEI compared to a control sample not treated with the test agent indicates that the test agent stimulates the release of MNEI from the cell.
• the test agent is a small molecule agonist of MNEI release.
• the invention provides a method for the treatment of inflammation.
• the method comprises contacting a cell with an agent that stimulates the release of MNEI, thereby reducing inflammation.
• the treatment of inflammation is directed to a patient with cystic fibrosis, emphysema, asthma, chronic bronchitis, sarcoidosis, bronchiectasis, respiratory distress syndrome, arthritis and certain skin diseases.
• the method is used for the treatment of inflammation in patients with cystic fibrosis.
• the invention provides a pharmaceutical composition useful for the treatment of inflammatory conditions and diseases.
• the composition comprises an agent that stimulates the release of MNEI and an acceptable pharmaceutical carrier.
• the agent that stimulates the release of MNEI from the cell is a protease.
• the protease is a neutrophil granule protease.
• the protease is selected from the group consisting of monocyte/neutrophil elastase, cathepsin G, proteinase-3, pancreatic elastase, and chymotrypsin.
• the protease is a analog of monocyte/neutrophil elastase, cathepsin G, proteinase-3, pancreatic elastase, or chymotrypsin.
• the agent is a small molecule agonist of MNEI release.
• the invention provides a kit comprising a pharmaceutical composition useful for the treatment of inflammatory conditions or diseases and printed instruction on the use of the composition for the treatment of inflammatorv conditions or diseases.
• the ⁇ ⁇ h r c u + "' ' ' , a'' composition of the kit comprising an agent that stimulates the release of MNEI.
• this agent is a protease.
• the protease is a neutrophil granule protease.
• the protease is selected from the group consisting of monocyte/neutrophil elastase, cathepsin G, proteinase-3, pancreatic elastase, and chymotrypsin.
• the protease is a analog of monocyte/neutrophil elastase, cathepsin G, proteinase-3, pancreatic elastase, or chymotrypsin.
• the agent is a small molecule agonist of MNEI release.
• the phrase “stimulates release of MNEI” refers to a property of an agent, e.g. , a protein or chemical compound, that causes an increase in the amount of MNEI secreted from the cell.
• the term “stimulate” may refer to the production and release of MNEI de ⁇ vo.
• protease refers to an endopeptidase that cleave proteins into small fragments. More specifically, the term refers to serine protease, a group of proteins which share a common reaction mechanism based on the formation of an acyl-enzyme intermediate on a specific active serine residue. Serine proteases are all irreversibly inactivated by a series of organophospho us esters, such as di-isopropylfluorophosphate (DFP) and by naturally-occurring inhibitors fe.gr., serpins) .
• DFP di-isopropylfluorophosphate
• cell is meant to encompass one that normally expresses MNEI.
• Non-limiting examples include monocytes and neutrophils .
• small molecule agonist refers to a compound having a molecular weight preferably less than 1000 Da, more preferably less than 800 Da, and most preferably less than 600 daltons (Da) , which is capable of stimulating the release of MNEI from a cell.
• analog refers tc a compound having a related structure and at least a measurable amount cf similar activity as the reference compound.
• neutrophil granule prot ⁇ asa tc encompass a protease normally occurring in neutrophil granules .
• Figure 1 is a general schematic presentation of the method of stimulating MNEI release from a cell.
• Figure 2A is a graphic representation showing the effect of different agents on MNEI secretion.
• Figure 2B is a graphic representation showing the viability of cells utilized in experiments performed to determine the affect of different agents on MNEI secretion.
• Figure 3A is a graphic representation showing the ability of human neutrophil elastase to stimulate the release of MNEI in a dose-dependent manner.
• Figure 3B is a graphic representation showing the results of control experiments designed to determine the percent of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) released by cells in experiments designed to demonstrate that human neutrophil elastase stimulates the release of MNEI in a dose-dependent manner.
• GPDH glyceraldehyde-3-phosphate dehydrogenase
• Figure 4 is a graphic representation showing that various neutrophil granule proteases stimulate the release of MNEI .
• Figure 5A is a graphic representation showing that pretreatment with cyclohexamide (CHX) increases cathespsin G- induced stimulation of MNEI release.
• CHX cyclohexamide
• Figure 5B is a graphic representation showing that pretreatment with lipopolysaccarhide (LPS) increases neutrophil elastase-induced stimulation of MNEI release.
• Figure 5C is a graphic representation showing that co- incubation with etoposide (etop) increases neutrophil elastase-indu ⁇ ed stimulation of MNEI release.
• LPS lipopolysaccarhide
• Figure 6 is a graphic representation showing the kenetics of elastase- and cathepsin G-induced MNEI release.
• Figure 7B is a representation of a Western blot showing that the release of MNEI is not affected by treatment with bradfeldin A, and indicating that this protein is not released by the endoplas ic reticulum/Golgi pathway.
• Figure 7B is a representation of a Western blot showing that the release of TNF- ⁇ is inhibited by treatment with bredfeldin A, indicating that this protein is released by the endoplasmic reticulum/Golgi pathway.
• the invention discloses a novel pathway for the release of MNEI from a cell.
• the ability to stimulate the release of MNEI from a cell provides a tool for the management of inflammatory conditions and diseases.
• the invention is directed, in part, to the management of cystic fibrosis by way of stimulating the release of MNEI from a cell.
• the invention provides a method of stimulating the release of MNEI from a cell.
• the method comprises contacting the cell with an agent that stimulates the release of MNEI from the cell.
• Calls useful in the method of the invention include, but are not limited to, monocytes and neutrophils. Stimulation of the cells utilized in the methods of the invention may be done in vitro or in vivo . The isolation and handling of such cells being well within the skill of those in the art.
• the agent is a protease.
• the protease is a neutrophil granule protease.
• the protease is monocyte/neutrophil elastase, cathepsin G, proteinase-3, pancreatic elastase, chymotrypsin, an analog of monocyte/neutrophil elastase, cathepsin G, proteinase-3, pancreatic elastase, or chymotrypsin.
• the agent may also be a small molecule agonist of MNEI release.
• the invention provides a method of stimulating the secretion the secretion of MNEI from a cell.
• the method comprises contacting the cell with a priming agent followed by contacting the cell with an agent that stimulates the release of MNEI from the cell .
• the agent may be a protease such as a neutrophil granule protease.
• the protease is monocyte/neutrophil elastase, cathepsin G, proteinase-3, pancreatic elastase, and chymotrypsin, or an analog of monocyte/neutrophil elastase, cathepsin G, proteinase-3, pancreatic elastase, or chymotrypsin.
• the priming agent is preferably selected from the group consisting of lipopolysaccaride (LPA) or cyclohexamide (CHX) , or a small molecule agonist of MNEI release.
• the invention provides a method of stimulating the secretion the secretion of MNEI from a cell.
• the method comprises contacting the cell with a priming agent and an agent that stimulates the release of MNEI from the cell.
• the priming agent is etoposide (etop) .
• the agent that stimulates the release of MNEI from the cell is a protease, such as a neutrophil granule protease or monocyte/neutrophil elastase, cathepsin G, proteinase-3, pancreatic elastase, or chymotrypsin or analog thereof.
• the agent is a small molecule agonist of MNEI release.
• the invention provides a method of identifying an agent that stimulates the release of MNEI from a cell.
• the method comprises contacting the cell with a test agent and assaying for MNEI release, wherein an increase in the release of MNEI compared to a control sample not treated with the test_ agent indicates that the test agent stimulates the release of MNEI from the cell.
• the test agent is a small molecule agonist of MNEI release.
• the invention disclosed herein encompasses the use of different libraries for the identification of small molecule agonists of MNEI secretion.
• Libraries useful for the purposes of the invention include, but are not limited to, (1) chemical libraries, (2) natural product libraries, and (3) combinatorial libraries comprised of random peptides, oligonucleotides and/or organic molecules.
• Chemical libraries consist of structural analogs of known compounds or compounds that are identified as “hits” or “leads” via natural product screening.
• monocyte/neutrophil elastase, cathepsin G, pr ⁇ teinase-3 , pancreatic elastase, and chymotrypsin are lead agents for the design of analogs that stimulate MNEI release.
• the protease is a analog of monocyte/neutrophil elastase, cathepsin G, proteinase-3, pancreatic elastase, or chymotrypsin.
• Natural product libraries are derived from collections of microorganisms, animals, plants, or marine organisms which are used to create mixtures for screening by: (1) fermentation and extraction of broths from soil, plant or marine microorganisms or (2) extraction of plants or marine organisms. Natural product libraries include polyketides, non-ribosomal peptides, and variants (non-naturally occurring) thereof. For a review, see, Cane, D.E., et al . (1998) Science 282:63-63. Combinatorial libraries are composed of large numbers of peptides, oligonucleotides or organic compounds as a mixture. They are relatively easy to prepare by traditional automated synthesis methods, PCR, cloning or proprietary synthetic methods.
• a combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks" such as reagents.
• a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (a ino acids) in every possible way for a given compound length ⁇ i . e . , the number of a ino acids in a polypeptide compound) . Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
• Still other libraries of interest include peptide, protein, peptido imetic, multiparallel synthetic collection, recombinatorial, and polypeptide libraries.
• Small molecule agonists of MNEI release are identified and isolated from the libraries described herein by any method known in the art, e.g., functional screening assays, and affinity binding methodologies.
• experiments detailed herein provide a general assay for the determination of whether an agent stimulates MNEI release from a cell.
• the screening methods utilized for the identification of small molecule agonists of MNEI secretion include high throughput assays.
• the invention provides a method for the treatment of inflammation.
• the method comprises contacting a cell with an agent that stimulates the release of MNEI, thereby reducing inflammation.
• the treatment of inflammation is directed to a patient with cystic fibrosis, emphysema, asthma, chronic bronchitis, sarcoidosis, bronchiectasis, respiratory distress syndrome, arthritis and certain skin diseases.
• the methods are useful for the treatment of inflammation in patients with cystic fibrosis.
• the invention provides a kit comprising a pharmaceutical composition useful for the treatment of inflammatory conditions or diseases and printed instruction on the use of the composition for the treatment of inflammatory conditions or diseases.
• the pharmaceutical composition of the kit comprising an agent that stimulates the release of MNEI .
• the agent that stimulates the release of MNEI from the cell is a protease, such as a neutrophil granule protease, In monocyte/neutrophil elastase, cathepsin G, proteinase-3, pancreatic elastase, chymotrypsin, and analogs thereof .
• the agent is a small molecule agonist of MNEI release.
• compositions of the invention are readily prepared using methods known in the art. For example,
• agents useful according the invention are included in pharmaceutical compositions of the invention at about 0.0001% to about 50% by weight, or more preferably at about 0.001% to about 10% by weight, or most preferably at about 0.05% to about 5% by weight.
• human monocyte-like THP-1 (ATCC NO. were obtained from the American Tissue Type Collection (ATCC) located in Manassas, VA. Cells were cultured in RPMI/5% endotoxin-free fetal calf serum (FCS) and incubated with various agents for the indicated times at 37°C in an incubator under 5% C0 2 . When incubated with proteases, FCS was omitted. Where indicated, cells were preincubated or ⁇ oincubated with a priming agent.
• ATCC NO. human monocyte-like THP-1
• FCS endotoxin-free fetal calf serum
• Cells were harvested by centrifugation and cytosolic and nuclear fractions were prepared by lysis with lOmMTris/HCl, pH 7.6, 0.5%NP-40, 25mM KC1, SmM MgCl2 0.5mg/ml EDTA, 50 ⁇ g/ml leupeptine, 2mM DFP (cytosol) and 10 mM Tris/HCl, pH8.0, 0.1% NP-40, 50 ⁇ g/ml leupeptine, 2mM DFP nuclear fractions) .
• the amount of MNEI in culture supernatant and cytosol was measured by ⁇ LISA and/or Western Blot.
• THP-1 cell culture and sample collection were done essentially as described in Example 1.
• THP-1 cells were incubated for 4 hours with MNEI or various agents known to affect non-classical secretion or to induce apoptosis.
• MNEI and GAPDH in culture supernatants were determined by ELISA and Western Blot, respectively.
• Cell viability was determined by Trypan Blue exclusion.
• Human neutrophil elastase (NE) stimulates MNEI release from THP-1 cells . Agents known to influence secretion of other proteins lacking N-terminal signal peptides do not affect MNEI secretion.
• NE Human Neutrophil Elastase Stimulates MNEI Release in THP-1 Cells in a Dose-Dependent Mannar THP-1 cell culture and sample collection were done essentially as described in Example 1. Briefly, THP-1 cells in serum-free medium were incubated with purified human neutrophil elastase (NE) at different concentrations for three hours . The amount of MNEI and GAPDH in culture supernatents was determined by Western blot as percent of total MNEI and GAPDH, respectively.
• THP-l cell culture and sample collection were done essentially as described in Example i. Briefly, THP-I cells in serum-free medium were incubated for 4 hours with various proteases at 5 ⁇ g/ml. MNEI release into culture supernatants was monitored by Western Blot and is graphed as release relative to control cells without protease treatment.
• THP-1 cell culture and sample collection were done essentially as described in Example 1. Briefly, after 3 hours activation with 25 ⁇ M CHX or 1 ⁇ g/ml LPS respectively, THP-1 cells in serum-free medium were incubated with cathepsin G (10 ⁇ g/ml) or human neutrophil elastase (NE, 10 ⁇ g/ml) for 3 hours. Alternatively, cells were co-incubated with etoposide (25 ⁇ M) and NE (10 ⁇ g/ml) for 4 hours. Control cells were not pre-incubated and not treated with protease. The release of MNEI into the culture supernatants was measured by Western blot (CHX and LPS) and by ELISA (etoposide) .
• CHX and LPS human neutrophil elastase
• THP-1 cell culture and sample collection were done essentially as described in Example 1. Briefly, after 3 hours activation with 1 ⁇ g/ml LPS, THP-1 cells in serum-free medium were incubated with human neutrophil elastase (NE, 1 ⁇ g/ml) or cathepsin 3 (1 ⁇ g/ml) for times indicated. Cells activated with LPS but without protease served as control . The release of MNEI into the culture supernatants was monitored by Western blot .
• human neutrophil elastase NE
• cathepsin 3 1 ⁇ g/ml
• THP-1 cell culture and sample collection were done essentially as described in Example 1. Briefly, after 3 hours pre-incubation with 1 ⁇ g/ml LPS, THP-l cells in serum-free medium were incubated for 3 hours with purified human neutrophil elastase (NE) at 10 ⁇ g/tt ⁇ l or brefeldin A (0.5 ⁇ g/ml) . The release of MNEI and TNF- ⁇ into the culture supernatants was monitored by Western blot . The results of the experiment are presented in Figure 7A and 7B and demonstrate that MNEI secretion is not inhibited by brefeldin A, a fungal metabolite that blocks protein translocation from the ER to the Golgi .
• NE human neutrophil elastase
• MNEI secretion does not occur via the classical ⁇ R/Golgi pathway.
• secretion of TNF- ⁇ a protein secreted via the ⁇ R/Golgi pathway, is completely blocked by brefeldin A.

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