WO2004092404A2 - Inhibiteurs de la chtitinase acide mammifere utilises en tant qu'agents therapeutiques contre l'asthme - Google Patents

Inhibiteurs de la chtitinase acide mammifere utilises en tant qu'agents therapeutiques contre l'asthme Download PDF

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WO2004092404A2
WO2004092404A2 PCT/US2004/011035 US2004011035W WO2004092404A2 WO 2004092404 A2 WO2004092404 A2 WO 2004092404A2 US 2004011035 W US2004011035 W US 2004011035W WO 2004092404 A2 WO2004092404 A2 WO 2004092404A2
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asthma
amcase
test agent
chitinase
antagonist
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PCT/US2004/011035
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WO2004092404A3 (fr
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Maximillian Todd Follettie
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Wyeth
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K49/00—Preparations for testing in vivo
    • A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7042—Compounds having saccharide radicals and heterocyclic rings
    • A61K31/7052—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides
    • A61K31/7056—Compounds having saccharide radicals and heterocyclic rings having nitrogen as a ring hetero atom, e.g. nucleosides, nucleotides containing five-membered rings with nitrogen as a ring hetero atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/7105—Natural ribonucleic acids, i.e. containing only riboses attached to adenine, guanine, cytosine or uracil and having 3'-5' phosphodiester links
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/74—Synthetic polymeric materials
    • 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
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/177—Receptors; Cell surface antigens; Cell surface determinants
    • A61K38/1793—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/24—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
    • C07K16/244—Interleukins [IL]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/564—Immunoassay; Biospecific binding assay; Materials therefor for pre-existing immune complex or autoimmune disease, i.e. systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, rheumatoid factors or complement components C1-C9
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the present invention relates generally to asthma therapeutics. Specifically, the invention relates to methods of screening for agents for treating asthma and methods for treating asthma.
  • Asthma is a chronic inflammatory disease of the airways characterized by recurrent episodes of reversible airway obstruction and airway hyperresponsiveness (AHR). Typical clinical manifestations include shortness of breath, wheezing, coughing and chest tightness that can become life threatening or fatal. While existing therapies focus on reducing the symptomatic bronchospasm and pulmonary inflammation, there is a growing awareness of the role of long term airway remodeling in accelerated lung deterioration in asthmatics.
  • Airway remodeling refers to a number of pathological features including epithelial smooth muscle and myofibroblast hyperplasia and/or metaplasia, subepithelial fibrosis and matrix deposition. The processes collectively result in up to about 300% thickening of the airway in cases of fatal asthma.
  • Asthma is the only chronic disease, besides AIDS and tuberculosis, with an increasing death rate. Each day, fourteen Americans die from asthma. The cost of asthma in 1998 was estimated to be $11.3 billion.
  • asthma results in about 500,000 hospitalizations annually, hospitalizations account for the single largest portion of this cost. Asthma further accounts for about 1.8 million emergency room visits and 10 million doctor's office visits each year.
  • OVA ovalbumin
  • This procedure generates a T H 2 immune reaction in the mouse lung and mimics four major pathophysiological responses seen in human asthma, including upregulated serum IgE (atopy), eosinophilia, excessive mucus secretion, and AHR.
  • the cytokine IL-13 expressed by basophils, mast cells, activated T cells and NK cells, plays a central role in the inflammatory response to OVA in mouse lungs.
  • IL-13 mediated signaling is sufficient to elicit all four asthma-related pathophysiological phenotypes and is required for the hypersecretion of mucus and induced AHR in the mouse model.
  • Biologically active IL-13 binds specifically to a low-affinity binding chain IL-13R ⁇ l and to a high-affinity multimeric complex composed of IL-
  • IL-13 also binds to an additional receptor chain, IL-13R ⁇ 2, expressed in both human and mouse with as yet undefined biological function.
  • the murine B -13R 2 binds IL-13 with approximately 100-fold greater affinity (Kd of 0.5 to
  • sIL-13R ⁇ 2-Fc a potent soluble IL-13 antagonist
  • the sIL-13R ⁇ 2-Fc has been used as an antagonist in a variety of disease models to demonstrate the role of EL- 13 in Schistosomiasis induced liver fibrosis and granuloma formation, tumor immune surveillance, as well as in the OVA-challenge asthma model.
  • the invention provides a method of screening for agents for treating asthma by (a) contacting a nucleotide sequence encoding a reporter gene product operably linked to an AMCase promoter with a test agent;
  • test agent inhibits production of the reporter gene product
  • Another embodiment of the method of treating asthma includes administering to a mammal in need thereof a therapeutic amount of an IL- 13 antagonist and detecting a decrease in the production or activity of AMCase, wherein a decrease in the production or activity levels of AMCase is indicative of an efficacious asthma treatment.
  • the invention also includes method for treating asthma by administering a therapeutic amount of a ribonucleic acid, which decreases the production of an AMCase protein.
  • the ribonucleic acid includes one or more small interfering RNAs ("siRNA").
  • the method includes administering to a test subject in need of a combination treatment for asthma using one or more inhibitors of AMCase and one or more antagonists of IL-13.
  • the inhibitor or antagonist can be administered simultaneously or each agent administered sequentially.
  • the inhibitor or antagonist can also be administered by different routes of administration.
  • the agent may be administered by a wide variety of routes. Exemplary routes of administration include oral, parenteral, transdermal, and pulmonary administration.
  • the inhibitors or antagonists may be administered intranasally, intramuscularly, subcutaneously, intraperitonealy, intravaginally and any combination thereof.
  • nebulizers, inhalers or aerosol dispensers may be used to deliver the therapeutic inhibitor or antagonist in an appropriate formulation (e.g., with an aerolizing agent).
  • the therapeutic amounts may also depend on the particular patient population and stage of asthma that the patient is experiencing.
  • the invention further includes methods for decreasing the production of AMCase by treating a mammal with an effective amount of an antagonist of IL- 13.
  • the antagonist blocks the ability of IL-13 to bind to the
  • the method includes the steps of (a) administering a test agent to a mammal; and (b) monitoring the expression of AMCase in the mammal.
  • a decrease in the expression or production of AMCase indicates that the test agent is useful in treating asthma.
  • the test agent is an antagonist of IL-13.
  • the antagonist blocks the ability of IL-13 to bind to the IL-13 receptor.
  • Nonlimiting examples of antagonists include sLL13R l-Fc, sBL-13R ⁇ 2-Fc, neutralizing antibodies, ribozymes, antisense oligonucleotides, RNA inhibitors such as siRNA, hormones, cytokines, small molecules and chemicals.
  • the method includes a method for determining the effective amount of a test agent useful for treating asthma in a mammal.
  • the method includes the steps of (a) identifying a test subject in need of such treatment; (b) administering a test agent to the test subject; (c) monitoring the expression of AMCase in the test subject after administration of the test agent; and (d) identifying the test agent dosage level at which expression of AMCase decreases.
  • the test agent is an antagonist of IL-13.
  • the antagonist blocks the ability of IL-13 to bind to the IL-13 receptor.
  • antagonists include, without limitation, soluble EL- 13 receptors (for example, extracellular domains of IL-13R ⁇ l and IL-13R ⁇ 2, alone or fused to a heterologous moiety, for example, Fc, such as 13R sEL-13R ⁇ l-Fc, sIL-13R 2-Fc); neutralizing antibodies against IL-13 or IL-13R or antigen- binding fragments thereof; ribozymes; antisense oligonucleotides; RNA inhibitors (for example, siRNA); hormones; cytokines; and chemicals (for example, small molecules).
  • Fc such as 13R sEL-13R ⁇ l-Fc, sIL-13R 2-Fc
  • Fc such as 13R sEL-13R ⁇ l-Fc, sIL-13R 2-Fc
  • neutralizing antibodies against IL-13 or IL-13R or antigen- binding fragments thereof ribozymes
  • antisense oligonucleotides for example, siRNA
  • hormones for example,
  • Figure 6 is a representation of the nucleotide sequence of the promoter for human acidic mammalian chitinase.
  • Figure 7 is a representation of the nucleotide sequence of the promoter for human acidic mammalian chitinase.
  • the invention is based, at least in part, upon the unexpected discovery that the mRNA of acidic mammalian chitinase ("AMCase”) is significantly increased in an animal model of asthma compared to control, non-asthmatic animals. Specifically, the mRNA encoding the AMCase protein is elevated by either intratracheal ovalbumin challenge or direct pulmonary instillation of IL-13. Moreover, blocking the ability of IL-13 to bind to the IL-13 receptor in turn inhibits expression of AMCase. Thus, it has been discovered that expression of AMCase is regulated through the IL-13 pathway.
  • a number of chitinase-related family members are human allergens.
  • AMCase catalyzes the hydrolysis of artificial chitin-like substrates with an acidic pH optimum.
  • AMCase has been shown to be expressed predominantly in the gastrointestinal tract and stomach and to a lesser extent in lung and is thought to play a digestive role or possibly an anti- parasitic defense mechanism.
  • AMCase an inhibitor of AMCase should be effective to treat asthma.
  • Asthma includes, but is not limited to, atopic asthma, nonatopic asthma, allergic asthma, exercise- induced asthma, drug-induced asthma, occupational asthma and late stage asthma.
  • Inhibition of an IL-13 receptor interaction has been shown to ameliorate airway hyperresponsiveness ("AHR") in allergen-immunized mice.
  • IL-13bc-IgG fusion proteins EL-13R ⁇ 2-Fc
  • IL-13R ⁇ 2-Fc which acts as an IL-13 antagonist
  • the test agent modulates (e.g., inhibits or increases) the activity of AMCase. In another aspect, the test agent modulates (e.g., inhibits or increases) the production or expression of chitinase.
  • a "test agent” is a putative "agent,” the modulating ability of which has not yet been confirmed. Once test agents are screened, they are classified as “agents,” if they are shown to modulate activity (for example, by inhibiting enzymatic activity) or production or expression (for example by modulating transcription or translation) of an AMCase protein. Accordingly, in additional embodiments, the agent may modify AMCase transcription, AMCase translation, or the EL- 13 pathway.
  • the agent reduces or inhibits the activity or expression of AMCase.
  • the agent binds to AMCase.
  • the agent interacts with (such as by chemically modifying) an inhibitor or activator of AMCase activity or expression.
  • an agent may bind to and inhibit an activator of AMCase or an agent may bind to and activate an inhibitor of AMCase activity.
  • AMCase The nucleotide and amino acid sequences of murine AMCase are set forth in SEQ ID NO: 3 and SEQ ID NO:4, as provided in Figures 3 and 4, respectively.
  • agents that inhibit the activity or expression of AMCase include, without limitation, allosamidin, argifin, argadin, antagonists of EL- 13 such as sIL-13R ⁇ l-Fc, sIL-13R 2-Fc, antagonists of the EL- 13 receptor, antagonists of the IL-4 receptor, antibodies such as AMCase antibodies, EL- 13 neutralizing antibodies, EL-13R antibodies, nucleic acids such as siRNA, aptamers (i.e., nucleic acid ligands), antisense oligonucleotides and ribozymes and small molecule chemical inhibitors.
  • nucleic acids such as siRNA, aptamers (i.e., nucleic acid ligands), antisense oligonucleotides and
  • the method includes, without limitation, a polyclonal antibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, a genetically engineered antibody, a bispecific antibody, antibody fragments (including but not limited to "Fv,” “F(ab') 2 ,” “F(ab),” and “Dab”) and single chains representing the reactive portion of the antibody.
  • a polyclonal antibody a monoclonal antibody, a chimeric antibody, a humanized antibody, a genetically engineered antibody, a bispecific antibody, antibody fragments (including but not limited to "Fv,” “F(ab') 2 ,” “F(ab),” and “Dab") and single chains representing the reactive portion of the antibody.
  • Such an antibody includes antibodies belonging to any of the immunoglobulin classes, such as IgM, IgG, IgD, IgE, IgA or their subclasses or mixtures thereof.
  • the invention further includes derivatives of these antibodies, such as those that retain their AMCase-binding activity while altering one or more other properties related to their use as a pharmaceutical agent, e.g., serum stability or efficiency of production.
  • the production of AMCase is inhibited.
  • the methods include, without limitation, determining if the test agent modulates (e.g., inhibits) the expression of AMCase and classifying the test agent as an agent for treating asthma if the test agent modulates (e.g., inhibits) the expression of AMCase.
  • agents that inhibit the expression of AMCase include, without limitation, antagonists selected from the group consisting of soluble EL- 13 receptors (for example, extracellular domains of IL-13R ⁇ l and IL-13R ⁇ 2, alone or fused to a heterologous moiety, for example, Fc, such as 13R ⁇ sEL-13R ⁇ l-Fc, sEL-13R ⁇ 2-Fc); neutralizing antibodies against EL-13 or EL-13R or antigen- binding fragments thereof; ribozymes; antisense oligonucleotides; RNA inhibitors (for example, siRNA); hormones; cytokines; and chemicals (for example, small molecules). See Wynn et al. U.S. Patent No. 6,664,227, which is herein incorporated in its entirety by reference.
  • AMCase useful in methods of identifying agents of the invention.
  • Such methods include assaying potential agents for the ability to inhibit AMCase activity and/or production or expression.
  • Polynucleotides and polypeptides useful in these assays include not only the genes and encoded polypeptides disclosed herein, but also variants thereof that have substantially the same activity as wild-type genes and polypeptides.
  • “Variants” as used herein includes polynucleotides or polypeptides containing one or more deletions, insertions or substitutions, as long as the variant retains substantially the same activity of the wild-type polynucleotide or polypeptide.
  • deletion variants are contemplated to include fragments lacking portions of the polypeptide not essential for biological activity
  • insertion variants are contemplated to include fusion polypeptides in which the wild-type polypeptide or fragment thereof has been fused to another polypeptide.
  • the AMCase utilized in the invention may be encoded by a nucleotide sequence that has at least about 60%, at least about 70%, at least about 80% or at least about 90% identity to the nucleotide sequence set forth in SEQ ID NO:l ( Figure 1) or SEQ ID NO:3 ( Figure 3). Percent identity may be determined, for example, by comparing sequence information using the advanced BLAST computer program, version 2.0.8, available from the National Institutes of Health.
  • the AMCase protein may be encoded by nucleotide sequences having substantial similarity to the nucleotide sequence set forth in SEQ ID NO:l ( Figure 1) or SEQ ED NO:3 ( Figure 3).
  • “Substantial similarity,” as used herein means that the nucleotide sequence is sufficiently similar to a reference nucleotide sequence that it will hybridize therewith under moderately stringent conditions. This method of determining similarity is well known in the art to which the invention pertains.
  • the hybrid length is that anticipated for the hybridized region(s) of the hybridizing polynucleotides.
  • the hybrid length is assumed to be that of the hybridizing polynucleotide.
  • the hybrid length can be determined by aligning the sequences of the polynucleotides and identifying the region or regions of optimal sequence complementarity.
  • An exemplary test agent is one that binds to or otherwise decreases the catalytic activity of an AMCase, although test agents that inhibit AMCase activity or expression by, for example, binding to the substrate for AMCase, or binding to a component of the signal pathway, such as EL- 13R, IL-4R or IL-13 are also envisioned.
  • An exemplary substrate for chitinase is chitin, although derivatives thereof that may participate in a reaction catalyzed by chitinase are also encompassed by the invention.
  • the amount of chitin remaining after contacting AMCase with the test agent as a function of time may be determined.
  • the amount of ⁇ -acetyl-D-glucosamine or 4- methylumbelliferyl or -nitrophenyl produced after contacting chitinase with the test agent in the presence of, for example, chitin as a function of time may be determined.
  • Various assays may be used to determine the quantity of these products and/or reactants. [0043] For example, colorimetric assays may be utilized to determine the quantity of ⁇ -acetyl-D-glucosamine as described in, for example, Reissig, J.L., /.
  • small molecule compounds known in the art, synthetic small molecule chemicals, nucleic acids such as antisense oligonucleotides, RNA inhibitors such as siRNA, ribozymes, and aptamers, peptides and proteins such as hormones, antibodies, and portions thereof, may act as test agents.
  • the three-dimensional structure of the active site of acidic mammalian chitinase is determined by crystallizing the complex formed by the enzyme and a known inhibitor. Rational drug design is then used to identify new test agents by making alterations in the structure of a known inhibitor or by designing small molecule compounds that bind to the active site of the enzyme.
  • test agents include inhibitors of chitinase enzymatic activity as well as inhibitors of chitinase expression or production.
  • inhibitors of chitinase activity include allosamidin argifin, argadin, and antibodies to AMCase, small molecule inhibitors, and proteins and peptides such as hormones and cytokines.
  • Inhibitors of chitinase production include, without limitation, test agents that inhibit the production of AMCase mRNA or protein.
  • test agents include antagonists of IL-13, siRNA, antisense nucleic acids, ribozymes, aptamers, neutralizing antibodies to EL-13, IL-13R and DL-4R.
  • Antagonists of L- 13 include without limitation test agents that block the ability of IL-13 to bind to either the EL-13 receptor or the EL- 4 receptor.
  • Such antagonists include without limitation EL-13R l-Fc, sEL-13R ⁇ 2-
  • the invention also provides a method of screening for agents for treating asthma in a mammal by screening for an agent that modulates (e.g.,, inhibits or activates) the activity of AMCase, including the expression level of AMCase.
  • the method includes contacting a nucleotide sequence encoding a reporter gene product operably linked to an AMCase promoter, with a test agent; determining if the test agent inhibits production of the reporter gene product; and classifying the test agent as an agent for treating asthma if the test agent inhibits production of the reporter gene product.
  • the mammal is a human.
  • the acidic mammalian chitinase promoter preferably includes a nucleotide sequence set forth in SEQ ID NO:5 or SEQ ED NO:6, as set forth in Figures 6 and 7. Nucleotide sequences having at least about 50%, at least about
  • the nucleotide sequence of the AMCase promoter is determined by art- recognized methods.
  • One nonlimiting example of such a method is to screen a genomic library (e.g., a YAC human genomic library) for the promoter sequence of interest using SEQ ID NO: 1 ( Figure 1) or SEQ ID NO:3 ( Figure 3) as a probe.
  • Another nonlimiting example of a method to determine the appropriate promoter sequence is to perform a Southern blot of the human genomic DNA by probing electrophoretically resolved human genomic DNA with a probe (e.g., a probe comprising SEQ ID NO: 1 or a portion thereof) and then determining where the cDNA probe (e.g., SEQ ID NO:l) hybridizes.
  • a probe e.g., a probe comprising SEQ ID NO: 1 or a portion thereof
  • the band can be isolated (e.g., cut out of the gel) and subjected to sequence analysis. This allows detection of the nucleotide fragment 5' of nucleotides 104-106 (i.e., the ATG site) of SEQ ID NO: 1.
  • the nucleotide fragment may be between approximately 500 to 1000 units in length.
  • the promoter sequence for murine AMCase set forth in SEQ ID NO: 3
  • Figure 3 may be determined by these methods as well. Nucleotide sequences having at least about 70%, at least about 80% and at least about 90% identity to such sequences and that function as promoter, for example, to direct expression of a gene encoding an AMCase protein described herein, are also encompassed in the invention.
  • reporter genes may be operably linked to the AMCase promoter described above.
  • Such genes may encode, for example, luciferase, ⁇ -galactosidase, chloramphenical acetyltransferase, ⁇ -glucuronidase, alkaline phosphatase, and green fluorescent protein, or other reporter gene products known to the art.
  • host cells may be utilized in the methods of screening in the present invention.
  • Exemplary host cells include, for example, Chinese hamster ovary, E. coli, COS and Bacillus.
  • nucleotide sequence encoding all or a portion of the chitinase gene may be utilized in the vector for the screening methods described herein.
  • chitinase may be isolated and purified by techniques well known to the skilled artisan, including chromatographic, electrophoretic and centrifugation techniques, as previously described herein. Additionally, chitinase may be quantified by methods known to the art.
  • test agent After contacting a nucleotide sequence encoding a reporter gene, or an AMCase gene, operably linked to an AMCase promoter with a test agent, it is determined if the test agent inhibits production of the reporter gene product.
  • This endpoint may be determined by quantifying either the amount or activity of the reporter gene product. The method of quantification will depend on the reporter gene that is used, but may involve use of an enzyme-linked immunosorbent assay with antibodies to the reporter gene product. Additionally, the assay may measure chemiluminescence, fluorescence or radioactive decay, or other methods known in the art. Assays for determining the activity or amount of the reporter gene products described herein are known to the art. If the test agent inhibits production of the reporter gene product, it is classified as an agent for treating asthma.
  • the screening methods of the invention are performed either in vitro (for example by monitoring AMCase activity or expression in a cell-based assay or in an enzymatic activity assay) or in vivo (for example by monitoring AMCase activity or expression in tissue samples such as BAL after administering a test agent to a mammal).
  • exemplary mammals include without limitation human, mouse, rat and dog.
  • a “therapeutic amount” represents an amount of an agent that is capable of inhibiting or decreasing the production of chitinase and causes a clinically significant response.
  • the clinical response includes an improvement in the condition treated or in the prevention of the condition.
  • the particular dose of the agent administered according to this invention will, of course, be determined by the particular circumstances surrounding the case, including the agent administered, the particular asthma being treated and similar conditions.
  • Agents that decrease the activity of an AMCase protein include those agents discovered in the screening assays described herein.
  • Additional agents are well known in the art and include, for example, allosamidin, argifin, argadin, proteins and peptides such as hormones, cytokines and antibodies and portions thereof, nucleic acids such as antisense oligonucleotides, siRNA, ribozymes, and aptamers, and small molecules. See, e.g., Houston, et al, PNAS
  • the detection or quantification of the AMCase in a sample can be carried out by an immunoassay utilizing the specific binding reaction between the monoclonal antibody and AMCase or IL-13.
  • immunoassays are well-known in the art and any of them can be employed. Examples of the immunoassays include sandwich methods employing the monoclonal antibody and another monoclonal antibody as primary and secondary antibodies, respectively, sandwich methods employing the monoclonal antibody and a polyclonal antibody as primary and secondary antibodies, staining methods employing gold colloid, agglutination methods, latex methods and chemical luminescence.
  • Antibody fragments may also be used for AMCase or IL-13 detection of cancer treatment.
  • Antibody fragments can be obtained, for example, by enzymatic means by eliminating the Fc part of the antibody with enzymes such as papain or pepsin, by chemical oxidation or by genetic manipulation of the antibody genes. It is also possible and advantageous to use genetically manipulated, non-truncated fragments. These antibodies or fragments thereof can be used alone or in mixtures. It is understood by those of skill in the art that antibodies against the EL-13 receptor or the IL-4 receptor are also useful as measures of treating asthma by decreasing activity or expression of AMCase.
  • Nonlimiting examples of soluble fusion proteins include DL- 13R l-Fc and EL-13R 2-Fc.
  • Exemplary test agents also include without limitation chemicals, including synthetic small molecules, nucleic acids such as antisense oligonucleotides, siRNA, ribozymes, and aptamers, peptides and proteins such as hormones, cytokines and antibodies or portions thereof, such as anti-IL13 neutralizing antibodies and anti-D -13R and EL-4R antibodies.
  • the invention includes a method for treating asthma by administering a nucleic acid.
  • Exemplary nucleic acids include, but are not limited to, a deoxyribonucleic acid or a ribonucleic acid.
  • the ribonucleic acid has a nucleotide sequence that is complementary to a portion of the nucleotide sequence set forth in SEQ ID NO:l or SEQ ID NO: 3, as set forth in Figures 1 and 3, encoding acidic mammalian protein.
  • small interfering RN As i.e., through the process of RNA interference
  • RNA interference relates to sequence-specific, posttranscriptional gene silencing brought about by double-stranded RNA that is homologous to the silenced gene target.
  • Exemplary agents that inhibit the activity or expression of FoxMl include, without limitation, allosamidin, argifin, argadin and anti- AMCase antibodies
  • the test agents and agents may be administered by a wide variety of routes.
  • Exemplary routes of administration include oral, parenteral, transdermal, and pulmonary administration.
  • the agents may be administered intranasally, intramuscularly, subcutaneously, inrraperitonealy, intravaginally and any combination thereof.
  • pulmonary administration nebulizers, inhalers or aerosol dispensers may be used to deliver the therapeutic agent in an appropriate formulation (i.e., with an aerolizing agent).
  • the agents may be administered alone or in combination with other agents or known drugs.
  • agents may be administered simultaneously or each agent may be administered at different times.
  • agents and drugs may be administered simultaneously or the agent can be administered before or after the drug(s).
  • the agents are administered in a pharmaceutically acceptable carrier. Any suitable carrier known in the art may be used. Carriers that efficiently solubilize the agents are preferred. Carriers include, but are not limited to a solid, liquid or a mixture of a solid and a liquid. The carriers may take the form of capsules, tablets, pills, powders, lozenges, suspensions, emulsions or syrups. The carriers may include substances that act as flavoring agents, lubricants, solubilizers, suspending agents, binders, stabilizers, tablet disintegrating agents and encapsulating materials.
  • the carrier is a finely divided solid, which is mixed with an effective amount of a finely divided agent.
  • mice were anesthetized with a mixture of ketamine and xylazine (45 and 8 mg/kg respectively) and challenged intratracheally with 50 ⁇ l of a 1.5% solution of OVA or an equivalent volume of PBS.
  • OVA soluble EL-13 receptor fusion protein
  • RNA frequencies were approximately 3.3 to 1000 parts per million (ppm) assuming an average transcript size of 2kb.
  • biotinylated standard curve fragments were synthesized by T7 polymerase driven FVT reactions from plasmid-based templates.
  • the spiked biotinylated RNA fragments serve both as an internal standard to assess chip sensitivity and as standard curve to convert measured fluorescent difference averages from individual genes into RNA frequencies in ppm. Average fluorescence difference between perfect match and single mismatch probe sets containing gene-specific oligonucleotides were used to determine frequency values with respect to spiked standard curve.
  • a second set of algorithms based primarily on the fraction of individual positive or negative responding probe pairs, is used to assess the absolute presence or absence of the gene product.
  • AMCase is identified as an EL-13 responsive gene downstream of the ovalbumin allergic challenge, which identifies it as a therapeutic agent in the target of asthma.
  • Balb/C mice (Jackson Laboratories, Bar Harbor, ME) were treated with multiple 5 ⁇ g dose (0, 24hr, and 48hr) lung instillation of recombinant mEL-13.
  • Stat6-/- is an additional control; it is a key intermediate in EL-13 signaling pathway, critical for mucus production and AHR; the absence of this EL-13 signaling transducer ameliorates asthmatic symptoms.
  • mice were challenged with ovalbumin according to the following regimen.
  • Female C57BL/6 mice were immunized by i.p. injection of 20 ⁇ g of OVA emulsified in 2.25 mg alum (Alumlnject; Pierce, Rockford, EL) in a total volume of 200 ⁇ l on days 0 and 14.
  • Mice were challenged via the airways with OVA (1% in PBS) for 20 min on days 28 and 29 by ultrasonic nebulization. All animals were sacrificed by CO 2 asphyxiation.
  • BAL was collected by flushing the lungs via the trachea 3X with 1 ml of warmed PBS.
  • mice received one i.p. injection of IgG control antibody (lOOO ⁇ g /mouse), hD -13RA2 (lOOO ⁇ g /mouse), mIL-13RA2 (lOOO ⁇ g / mouse) on day 28, 2 hours prior to the commencement of OVA challenge.
  • Chitinase enzyme activity was determined as described in Guo, L., et al, "Biochemical Characterization of Endogenously Formed Eosinophilic
  • Ketamine Twenty minutes prior to tranquilization with Ketamine (8-10 mg/Kg), animals were pretreated with Atropine (0.4 mg/Kg). A catheter was inserted in the saphenous vein and anesthesia was induced with a bolus injection of Propofol (3 mg/Kg). The animal was placed in a supine position and at 0 hours, a pediatric fiberoptic bronchoscope was introduced into the left lung. A baseline bronchoalveolar lavage (BAL) was performed with three separate 20 mL aliquots of warmed sterile saline in the left lung.
  • BAL baseline bronchoalveolar lavage
  • the bronchoscope was partially withdrawn and reintroduced in the right lung, and a bolus of 0.5 mg Ascaris suum antigen in 5 mL of saline was administered. The bronchoscope was removed and the animal was allowed to recover.
  • BAL fluid was filtered through a cell strainer and centrifuged at 1,500 RPM for 15 minutes. BAL fluid was removed from the cell pellet, aliquoted and frozen at -80°C. BAL fluid was thawed and a 15 mL aliquot was concentrated using Centriprep-YM3 concentrator tubes (Amicon). BAL fluid was centrifuged twice at 3,000xg until 0.9 mL was remaining (approximately 90 min, then 30 min). [0097] Lyophilized pig Ascaris suum allergenic extract (Greer Laboratories,
  • Lenoir, NC Lenoir, NC was reconstituted with sterile saline for injection. Dilutions were made and frozen at -80°C. Prior to obtaining BAL, an aliquot was thawed and 0.5 mg was removed and mixed with 5 mL sterile saline for injection using a 5 mL syringe.
  • hAMCase Human AMCase
  • E. coli Human AMCase
  • the partially purified enzyme preparation is assayed for hAMCase activity by bringing it in contact with 0.025mM 4-methylumbelliferyl ⁇ -D-N,N'N"-triacetylchitotiose (Sigma Chemical Corp.) as substrate in citrate/phosphate buffer (100rnM/200mM) at 37°C.
  • test agents will be screened by their ability to modulate (e.g., inhibit) the enzymatic reaction as determined by altered (e.g., decreased) amount of the fluorescent 4- methylumbelliferone product formed as function of time relative to control enzyme reactions.
  • An AMCase promoter is linked to a reporter gene, for example, a ' luciferase. Activation of the reporter gene is demonstrated by inducing with EL- 13, indicating transcriptional specificity. Test agents are screened to identify those that block the EL-13-induced reporter gene activity.
  • a therapeutically effective amount of a known AMCase inhibitor is administered to a subject diagnosed with asthma.
  • a control group also exhibiting asthmatic symptoms is treated will a placebo control.
  • Administration may be by a single treatment or treatment over a course of days.
  • Subjects are evaluated for asthma-related symptoms, such as AHR and mucus production. Effective treatment is determined by a reduction in asthma-related symptoms compared to the control group.

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Abstract

L'invention concerne des méthodes de criblage permettant de traiter l'asthme. Lesdites méthodes impliquent le criblage d'agents diminuant la production de l'activité d'une protéine AMCase, laquelle joue un rôle dans l'apparition des symptômes et des complications pathologiques associés à l'asthme. Lesdits agents comprennent des inhibiteurs de la AMCase, ainsi que des antagonistes de la IL-13 et du récepteur de la IL-13. L'invention concerne également des méthodes pour traiter l'asthme, ainsi que des procédés pour surveiller l'efficacité du traitement de l'asthme au moyen des inhibiteurs de la protéine AMCase et des antagonistes IL-13.
PCT/US2004/011035 2003-04-11 2004-04-12 Inhibiteurs de la chtitinase acide mammifere utilises en tant qu'agents therapeutiques contre l'asthme WO2004092404A2 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008144646A1 (fr) * 2007-05-18 2008-11-27 Wyeth Nouvelles chitinases de primate et leurs utilisations
WO2009076621A1 (fr) * 2007-12-13 2009-06-18 Wyeth Structures de haute résolution de chitinases mammifère acides et leurs utilisations

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WO2000036103A1 (fr) * 1998-12-14 2000-06-22 Genetics Institute, Inc. Chaine de recepteurs de cytokine
WO2003009808A2 (fr) * 2001-07-24 2003-02-06 Yale University Procedes, compositions et necessaires relatifs a des molecules chitinases et de type chitinase ainsi qu'a des maladies inflammatoires

Patent Citations (2)

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Publication number Priority date Publication date Assignee Title
WO2000036103A1 (fr) * 1998-12-14 2000-06-22 Genetics Institute, Inc. Chaine de recepteurs de cytokine
WO2003009808A2 (fr) * 2001-07-24 2003-02-06 Yale University Procedes, compositions et necessaires relatifs a des molecules chitinases et de type chitinase ainsi qu'a des maladies inflammatoires

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DONALDSON DEBRA D ET AL: "The murine IL-13 receptor alpha 2: molecular cloning, characterization, and comparison with murine IL-13 receptor alpha 1" JOURNAL OF IMMUNOLOGY, THE WILLIAMS AND WILKINS CO. BALTIMORE, US, vol. 161, no. 5, 1 August 1998 (1998-08-01), pages 2317-2324, XP002195293 ISSN: 0022-1767 *
GRÜNIG G ET AL: "Requirement for IL-13 independently of IL-4 in experimental Asthma" SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE,, US, vol. 282, 18 December 1998 (1998-12-18), pages 2261-2263, XP002966773 ISSN: 0036-8075 cited in the application *
HOUSTON DOUGLAS R ET AL: "High-resolution structures of a chitinase complexed with natural product cyclopentapeptide inhibitors: mimicry of carbohydrate substrate." PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA. 9 JUL 2002, vol. 99, no. 14, 9 July 2002 (2002-07-09), pages 9127-9132, XP002311245 ISSN: 0027-8424 cited in the application *
WILLS-KARP M ET AL: "INTERLEUKIN-13: CENTRAL MEDIATOR OF ALLERGIC ASTHMA" SCIENCE, AMERICAN ASSOCIATION FOR THE ADVANCEMENT OF SCIENCE,, US, vol. 282, 18 December 1998 (1998-12-18), pages 2258-2260, XP002948381 ISSN: 0036-8075 cited in the application *
ZHU ZHOU ET AL: "Acidic mammalian chitinase in asthmatic Th2 inflammation and IL-13 pathway activation." SCIENCE. 11 JUN 2004, vol. 304, no. 5677, 11 June 2004 (2004-06-11), pages 1678-1682, XP002311246 ISSN: 1095-9203 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008144646A1 (fr) * 2007-05-18 2008-11-27 Wyeth Nouvelles chitinases de primate et leurs utilisations
WO2009076621A1 (fr) * 2007-12-13 2009-06-18 Wyeth Structures de haute résolution de chitinases mammifère acides et leurs utilisations

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